CN120344270A

CN120344270A - Drug linker and antibody conjugate thereof

Info

Publication number: CN120344270A
Application number: CN202380084192.6A
Authority: CN
Inventors: J·王; N·克努森; L·斯基摩尔; Y·孙; J·Y·金
Original assignee: Ambrx Inc
Current assignee: Ambrx Inc
Priority date: 2022-10-07
Filing date: 2023-10-06
Publication date: 2025-07-18
Also published as: WO2024077277A1; EP4598586A1; JP2025535713A

Abstract

Disclosed herein are antibody drug conjugates (ADCs), drug linkers for ADCs, and adjustable phosphate-based linkers. The ADCs of the present disclosure include antibodies, such as anti-CD70 antibodies conjugated to a biscarbamycin analog via a phosphate-based unit. Also disclosed are methods and compositions for using ADCs to inhibit, prevent, or treat diseases or conditions such as cancer.

Description

Drug linkers and antibody conjugates thereof

Citation of related application

The present application claims priority from U.S. provisional application 63/378,852, filed on 7 at 10 at 2022, which is hereby incorporated in its entirety.

Sequence listing

The present application comprises a sequence listing that has been submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy was created at 2023, 10/6, named AMBX-024500PCT, and is 78,546 bytes in size.

Technical Field

The present invention relates to Antibody Drug Conjugates (ADC), cytotoxic bicubicin (duocarmycin) analog drugs and drug linkers. In particular, the invention relates to antibodies comprising unnatural amino acids conjugated to drug linkers comprising a bicubicin analog and pegylated phosphate-based linkers. The invention also relates to methods of using the ADC, drugs and drug linkers, including the treatment of cancer.

Background

Antibody Drug Conjugates (ADCs) are a class of effective therapeutic constructs that advance the field of cancer therapy by allowing targeted delivery of cytotoxic agents to target cells, such as cancer cells. Currently, only a few ADCs have been approved for therapeutic use, including octotuzumab (gemtuzumab ozogamicin) for AML (followed by market withdrawal), vitamin b uzumab (brentuximab vedotin) for ALCL and hodgkin's lymphoma, and enmtuzumab (trastuzumab emtansine) for HER 2-positive metastatic breast cancer (Verma et al, N Engl J Med367:1783-91,2012; bross et al, CLIN CANCER RES 7:1490-96,2001; frankisco et al, blood 102:1458-65,2003), and golic Sha Tuozhu mab (sacituzumab govitecan) for metastatic Triple Negative Breast Cancer (TNBC) (Zaman et al, oncoTargets AND THERAPY 12:1781-1790,2019). However, ADCs present challenges due to lack of therapeutic index and toxicity. The linker technology used to attach the cytotoxic drug to the antibody affects the stability of the ADC during systemic circulation. Thus, there is a need in the art to design improved linkers, such as phosphate-based linkers and drug designs for antibody conjugation.

The present disclosure provides phosphate-based linkers with tunable stability for intracellular delivery of drug payloads. Phosphate-based linkers have distinguishable and adjustable stability in blood relative to the intracellular environment, and may also include self-destructing linkers. Antibody drug conjugates comprising these linkers are stable in circulation (plasma/blood) but are reactive or cleavable in intracellular compartments such as lysosomal compartments, so that they can be used for intracellular delivery, the rate depending on the structure of the regulatory element.

Cluster of differentiation 70 (CD 70) is a member of the tumor necrosis factor superfamily and a ligand for CD27 (Goodwin, R.G. et al, cell,73:447-456 (1993); hintzen, R.Q. et al, int Immunol,6:477-480 (1994)). CD70 was first identified in activated T lymphocytes and B lymphocytes. Binding of CD70 to CD27 on activated lymphocytes emits co-stimulatory signals for T cells, B cells and Natural Killer (NK) cells (Grewal, i.s., expert Opin THER TARGETS,12 (3): 341-351 (2008); borst, j. Et al, curr Opin immunol, 17 (3): 275-281 (2005)) and modulates cell differentiation and T helper 1/2 conversion (Wajant, h., expert Opin THER TARGETS,20 (8): 959-973 (2016)). The primary amino acid sequence of CD70 predicts a transmembrane type II protein whose carboxy terminus is exposed outside the cell and whose amino terminus is present on the cytoplasmic side of the plasma membrane. Human CD70 consists of a cytoplasmic domain of 20 amino acids, a transmembrane domain of 18 amino acids and an extracellular domain of 155 amino acids, with two potential N-linked glycosylation sites (Bowman et al, J Immunol,152:1756-1761 (1994); goodwin et al, cell,73:447-456 (1993)).

CD70 expression has been reported in different types of cancers, including lymphomas, carcinomas and neurogenic tumors. In malignant B cells, 71% diffuse large B cell lymphoma, 33% follicular central lymphoma, 25% mantle lymphoma, and 50% B-CLL expression CD70 have been reported (Lens et al 1999,Br J Haematol,106:491-503). CD70 has also been detected on brain tumor cells, particularly glioma cell lines, human solid gliomas and meningiomas (Held-Feindt and Mentlein, int J Cancer,98:352-56 (2002); wischlusen et al, can Res,62:2592-2599 (2002)). CD70 is typically expressed in renal cell carcinoma (RCC; 87%) and non-hodgkin lymphoma (NHL; 77%), tannir, n.m. et al, INVEST NEW Drugs,32 (6): 1246-1257 (2014)), but minimally expressed in normal tissues (Nakae, r. Et al, am JObstet gynecol.,224 (2): 197 (2021)).

Anti-CD 70 antibodies and Antibody Drug Conjugates (ADCs), and methods of making and using them to treat diseases such as cancer, are disclosed in WO2013/192360Al, which is hereby incorporated by reference in its entirety.

Multiple clinical trials evaluating anti-CD 70 agents (e.g., antibodies, ADCs, and Chimeric Antigen Receptor (CAR) T cell therapies with enhanced antibody-dependent cell-mediated cytotoxicity) are being examined in malignancies that display high CD70 expression. Previous studies have shown that anti-CD 70 monoclonal antibodies (mabs) and anti-CD 70 ADCs exhibit anti-tumor effects in xenograft models of CD70 malignant diseases such as lymphoma, NHL and RCC (Israel, b.f. et al, mol Cancer ter., 4 (12): 2037-2044 (2005); law, c.l. et al, cancer res.,66:2328-2337 (2006); MCEARCHERN, J.A. et al, blood,109 (3): 1185-92 (2007)). Based on the results of preclinical studies, two separate phase 1 studies of SGN-75 (anti-CD 70 mAb conjugated to maleimidocaproyl-monomethyl requisitetin F (MMAF)) were performed in patients with CD70 positive relapsed/refractory NHL or metastatic RCC, however SGN-75 exhibited modest effects on these diseases with some intolerable side effects (Tannir, N.M. et al, INVEST NEW Drugs,32 (6): 1246-1257 (2014)). Additional anti-CD 70 ADC, SGN-CD70A (anti-CD 70 mAb conjugated to pyrrolobenzodiazepine dimer) was introduced into phase 1 clinical trials (Pal, S.K. et al, cancer,125 (7): 1124-1132 (2019)), but SGN-CD70A 1 study was discontinued in 2018.

Bicubicin SA is a highly potent cytotoxic natural product that binds to the minor groove of DNA and is capable of inducing sequence-selective alkylation of double-stranded DNA. The bicubicin-based ADC includes BMS-936561 (MDX-1203) containing an anti-CD 70 antibody conjugated to the bicubicin derivative MED-A via a maleimide-containing citrulline-valine dipeptide linker (Wang H. Et al (2016) Biopharm Drug Disp (2): 93-106;Owonikoko T.K. Et al, cancer Chemother Pharmacol (2016) 77 (1): 155-162). Termination of BMS-936561/MDX-1203 development exemplifies the challenges faced by bicubicin-based ADCs (Hang-Ping Y. Et al, drug Discov Today (2021) 26 (8): 1857-1874).

There remains a need for anti-CD 70 ADCs constructed in such a way as to be able to exert a clinically useful cytotoxic, cytostatic or immunosuppressive effect on CD70 expressing cells, in particular without exerting an undesirable effect on cells that do not express CD 70. Such ADCs would be useful therapeutic agents for CD70 expressing cancers or immune disorders mediated by CD70 expressing cells. The present invention provides such ADCs for use in immunology and oncology.

Disclosure of Invention

The present invention provides novel drugs and drug-linkers suitable for antibody conjugation, wherein the drug is a bicubicin analog and the drug-linker comprises a phosphate-based linker. The invention further provides ADCs comprising phosphate-based drug linkers. Drugs, drug linkers and ADCs are useful for treating diseases and conditions, including cancer, in human subjects in need thereof.

In some general aspects, there is provided a compound of formula (I),

Wherein:

R is H or L-W, wherein L is a linker and W is a reactive moiety, and

A is a bicyclic system selected from the group consisting of formulas (a), (b), (c) and (d) having the structure:

Wherein:

Each X ¹ is C (R ^1a)(R^1b), wherein each R ^1a and R ^1b is independently H, halogen, alkyl, alkenyl or alkynyl;

each X ² is C (R ^2a)(R^2b), wherein each R ^2a and R ^2b is independently H, halogen, alkyl, alkenyl or alkynyl;

Each X ³ is C;

Each X ⁴ is C (R ⁴) or N, wherein R ⁴ is H, halogen, -OH, -SH, -NO ₂、-CN、-N₃、-N(R^a)(R^b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl 、-C(O)R^c、-C(O)OR^c、-C(O)N(R^a)(R^b)、-C(S)R^c、-C(S)OR^c、-C(S)N(R^a)(R^b)、-C(O)SR^c, or-S (O) _m(R^s);

Each X ⁵ is C (R ⁵) or N, wherein R ⁵ is H, halogen, -OH, -SH, -NO ₂、-CN、-N₃、-N(R^a)(R^b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl 、-C(O)R^c、-C(O)OR^c、-C(O)N(R^a)(R^b)、-C(S)R^c、-C(S)OR^c、-C(S)N(R^a)(R^b)、-C(O)SR^c, or-S (O) _m(R^s);

Each X ⁶ is C (R ⁶) or N, wherein R ⁶ is H, halogen, -OH, -SH, -NO ₂、-CN、-N₃、-N(R^a)(R^b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl 、-C(O)R^c、-C(O)OR^c、-C(O)N(R^a)(R^b)、-C(S)R^c、-C(S)OR^c、-C(S)N(R^a)(R^b)、-C(O)SR^c, or-S (O) _m(R^s);

Each X ⁷ is C (R ⁷) or N, wherein R ⁷ is H, halogen, -OH, -SH, -NO ₂、-CN、-N₃、-N(R^a)(R^b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl 、-C(O)R^c、-C(O)OR^c、-C(O)N(R^a)(R^b)、-C(S)R^c、-C(S)OR^c、-C(S)N(R^a)(R^b)、-C(O)SR^c, or-S (O) _m(R^s);

Each X ⁸ is C, and

Each X ⁹, when present, is C (R ^9a)(R^9b), wherein each R ^9a and R ^9b is independently H, halogen, alkyl, alkenyl or alkynyl;

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

each R ^c is independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl;

each R ^s is independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl, and

Each m is independently 0,1, 2 or 3;

Or a salt thereof.

In some embodiments, a-H (the corresponding amine of part a) has a ClogP value of at least about 1.

In some aspects, a has the structure of formula (a), and the compound is a compound of formula (Ia) having the structure:

Or a salt thereof.

In some aspects, a has the structure of formula (b), and the compound is a compound of formula (Ib) having the structure:

Or a salt thereof.

In some aspects, a has the structure of formula (c), and the compound is a compound of formula (Ic) having the structure:

Or a salt thereof.

In some aspects, a has the structure of formula (d), which is a compound of formula (Id) having the structure:

Or a salt thereof.

In some aspects, there is provided a compound of formula (I), or formula (Ia), or formula (Ib), or formula (Ic), or formula I (d), wherein:

R is H or L-W, wherein L is a linker and W is a reactive moiety;

X ¹ is C (R ^1a)(R^1b), wherein each R ^1a and R ^1b is independently H, halogen, alkyl, alkenyl or alkynyl;

X ² is C (R ^2a)(R^2b), wherein each R ^2a and R ^2b is independently H, halogen, alkyl, alkenyl or alkynyl;

X ³ is C;

X ⁴ is C (R ⁴) or N, wherein R ⁴ is H, halogen, -OH, -SH, -NO ₂、-CN、-N₃、-N(R^a)(R^b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl 、-C(O)R^c、-C(O)OR^c、-C(O)N(R^a)(R^b)、-C(S)R^c、-C(S)OR^c、-C(S)N(R^a)(R^b)、-C(O)SR^c, or-S (O) _m(R^s);

X ⁵ is C (R ⁵) or N, wherein R ⁵ is H, halogen, -OH, -SH, -NO ₂、-CN、-N₃、-N(R^a)(R^b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl 、-C(O)R^c、-C(O)OR^c、-C(O)N(R^a)(R^b)、-C(S)R^c、-C(S)OR^c、-C(S)N(R^a)(R^b)、-C(O)SR^c, or-S (O) _m(R^s);

X ⁶ is C (R ⁶) or N, wherein R ⁶ is H, halogen, -OH, -SH, -NO ₂、-CN、-N₃、-N(R^a)(R^b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl 、-C(O)R^c、-C(O)OR^c、-C(O)N(R^a)(R^b)、-C(S)R^c、-C(S)OR^c、-C(S)N(R^a)(R^b)、-C(O)SR^c, or-S (O) _m(R^s);

X ⁷ is C (R ⁷) or N, where R ⁷ is H, halogen, -OH, -SH, -NO ₂、-CN、-N₃、-N(R^a)(R^b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl 、-C(O)R^c、-C(O)OR^c、-C(O)N(R^a)(R^b)、-C(S)R^c、-C(S)OR^c、-C(S)N(R^a)(R^b)、-C(O)SR^c or-S (O) _m(R^s), and

X ⁸ is C;

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3.

In some further aspects, there is provided a compound of formula (I), or formula (Ia), or formula (Ib), or formula (Ic), or formula I (d), wherein:

X ¹ is C (R ^1a)(R^1b), wherein each R ^1a and R ^1b is independently H, halogen or unsubstituted alkyl;

X ² is C (R ^2a)(R^2b), wherein each R ^2a and R ^2b is independently H, halogen or unsubstituted alkyl;

X ³ is C;

X ⁴ is C (R ⁴) or N, wherein R ⁴ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl;

X ⁵ is C (R ⁵) or N, wherein R ⁵ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl;

X ⁶ is C (R ⁶) or N, wherein R ⁶ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl;

X ⁷ is C (R ⁷) or N, wherein R ⁷ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl;

X ⁸ is C, and

X ⁹, when present, is C (R ^9a)(R^9b), wherein each R ^9a and R ^9b is independently H, halogen or unsubstituted alkyl.

X ¹ is C (R ^1a)(R^1b), wherein each of R ^1a and R ^1b is H;

X ² is C (R ^2a)(R^2b), wherein each of R ^2a and R ^2b is H;

X ³ is C;

x ⁴ is C (R ⁴) or N, wherein R ⁴ is H;

X ⁷ is C (R ⁷) or N, wherein R ⁷ is H;

X ⁸ is C, and

X ⁹, when present, is CH ₂.

In some aspects, each of the heteroalkyl groups is an alkoxy group.

X ¹ is C (R ^1a)(R^1b), wherein each of R ^1a and R ^1b is H;

X ² is C (R ^2a)(R^2b), wherein each of R ^2a and R ^2b is H;

X ³ is C;

x ⁴ is C (R ⁴) or N, wherein R ⁴ is H;

X ⁵ is C (R ⁵) or N, wherein R ⁵ is H, halogen or alkoxy;

X ⁶ is C (R ⁶) or N, wherein R ⁶ is H, halogen or alkoxy;

X ⁷ is C (R ⁷) or N, wherein R ⁷ is H;

X ⁸ is C, and

X ⁹, when present, is CH ₂.

In some aspects, X ⁵ is C (R ⁵) or N, wherein R ⁵ is H or alkoxy, and X ⁶ is C (R ⁶) or N, wherein R ⁶ is H or alkoxy.

In some aspects, X ⁴ is N, X ⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is C (R ⁷). In some other aspects, X ⁴ is C (R ⁴),X⁵ is N, X ⁶ is C (R ⁶) and X ⁷ is C (R ⁷). In some other aspects, X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is N and X ⁷ is C (R ⁷). In some other aspects, X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is C (R ⁷). In some other aspects, X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is N).

In some aspects, at least one of X ⁴ and X ⁷ is CH. In some aspects, each of X ⁴ and X ⁷ is CH.

In some aspects, at least one of R ⁵ and R ⁶ is alkoxy. In some further aspects, each of the alkoxy groups is independently-OR ^k, wherein each R ^k is independently alkyl optionally substituted with a heterocyclyl OR-N (R ^d)(R^e), wherein the heterocyclyl contains at least one nitrogen atom, and each R ^d and R ^e is independently H, alkyl, alkenyl, OR alkynyl. In some aspects, each alkoxy group is selected from the group consisting of -OCH₃、-OCH₂CH₃、-OCH₂CH₂CH₃、-OCH(CH₃)₂、-OCH₂CH₂N(CH₃)₂、A group of groups.

In some aspects, the compounds of the present disclosure are compounds of formula (I), wherein R is H. In some aspects, the compound is a compound of formula (Ia), wherein R is H. In some aspects, the compound is a compound of formula (Ib), wherein R is H. In some aspects, the compound is a compound of formula (Ic), wherein R is H. In some aspects, the compound is a compound of formula (Id), wherein R is H.

In some aspects, the compound is a compound of formula (Ia), wherein R is H, and the compound is selected from the group consisting of:

And salts thereof.

In some other aspects, the compound is a compound of formula (I), wherein R is L-W. In some aspects, the compound is a compound of formula (Ia), wherein R is L-W. In some aspects, the compound is a compound of formula (Ib), wherein R is L-W. In some aspects, the compound is a compound of formula (Ic), wherein R is L-W. In some aspects, the compound is a compound of formula (Id), wherein R is L-W.

In some aspects, L is a phosphate-based linker. In some aspects, the phosphate-based linker comprises a phosphate-based moiety having the structure:

Wherein represents a linkage to an-O-atom at position R of formula (I), or formula (Ia), or formula (Ib), or formula (Ic) or formula I (d); wherein L further comprises at least one additional moiety and the wavy line of phosphate-based moiety represents a linkage to one of the at least one additional moiety, wherein the at least one additional moiety is selected from the group consisting of unsubstituted alkylene, substituted alkylene, - (alkylene-O) -, optionally substituted arylene, -O-, -C (O) -, -N (R _w)-、-S(O)_0-2 -, water-soluble polymer, and amino acid, wherein each R _w is independently H or C ₁-C₈ alkyl, and combinations thereof.

In some aspects, L is selected from the group of linkers of table 6. In some other aspects, L is selected from the group of linkers of table 7. In some other aspects, L is selected from the group of linkers of table 8. In some aspects, L has the following structure:

Wherein represents a linkage to an-O-atom at position R of formula (I), or formula (Ia), or formula (Ib), or formula (Ic) or formula I (d), and +represents a linkage to W. In some other aspects, L has the following structure:

Wherein T is a water-soluble polymer, R ^t is H or methyl, represents a linkage to an-O-atom at position R of formula (I), or formula (Ia), or formula (Ib), or formula (Ic) or formula I (d), and +represents a linkage to W. In some aspects, the water-soluble polymer is a polyethylene glycol (PEG) moiety. In some aspects, the PEG moiety has a molecular weight in the range of about 100Da to about 100,000Da, about 100Da to about 10,000Da, about 100Da to about 5,000Da, or about 100Da to about 1,000 Da. In some aspects, the PEG moiety is- (CH ₂CH₂O)_nCH₃) wherein n is an integer from 1 to 24, in some aspects, the PEG moiety is- (CH ₂CH₂O)_nCH₃) wherein n is 8,9, 10, 11, or 12.

In some aspects, the reactive moiety W comprises-N ₃、-OH、-SH、-NH(R^j)、-C(O)R^q、-C(O)OR^x、-C(O)CH₂NH₂, an activated ester, -O-NH ₂, maleimide, tetrazine, alkyne, cyclooctyne, or (E) -cyclooctene, wherein R ^j is H or unsubstituted alkyl, R ^q is unsubstituted alkyl, and R ^x is H, unsubstituted alkyl, or a carboxylic acid protecting group. In some further aspects, the reactive moiety W is selected from the group consisting of:

-N ₃、-OH、-SH、-NH(R^j)、-C(O)R^q、-C(O)OR^x, an activated ester, -O-NH ₂ and an optionally substituted mono-or polycyclic group comprising said cyclooctyne, wherein R ^j is H or unsubstituted C ₁-C₆ alkyl, R ^q is unsubstituted C ₁-C₆ alkyl, R ^x is H, unsubstituted C ₁-C₆ alkyl or carboxylic acid protecting group, R ^f is H or unsubstituted C ₁-C₆ alkyl, s is 0, 1, 2, 3, 4,5 or 6 and t is 0, 1, 2, 3, 4,5 or 6.

In some aspects, W is-ONH ₂.

In some aspects, there is provided a compound of formula (Ia), wherein R is L-W, and the compound is selected from the group consisting of:

And salts thereof.

In some other general aspects, the present disclosure provides an Antibody Drug Conjugate (ADC) of formula (II):

Wherein:

Ab is an antibody, wherein Ab comprises one or more unnatural amino acids;

l is a linker;

E is a moiety linking Ab and L;

d is an integer of 1 to 10, and

A is selected from the group consisting of formulas (a), (b), (c) and (d) having the following structure:

Wherein:

Each X ³ is C;

Each X ⁸ is C, and

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3;

or a pharmaceutically acceptable salt thereof.

In some aspects, an ADC of formula (II) is provided, wherein a is formula (a). In some aspects, an ADC of formula (II) is provided, wherein a is formula (b). In some aspects, an ADC of formula (II) is provided, wherein a is formula (c). In some aspects, an ADC of formula (II) is provided, wherein a is formula (d).

In some aspects, an ADC of formula (II) is provided, wherein:

X ³ is C;

X ⁸ is C, and

In some aspects, an ADC of formula (II) is provided, wherein:

X ¹ is C (R ^1a)(R^1b), wherein each of R ^1a and R ^1b is H;

X ² is C (R ^2a)(R^2b), wherein each of R ^2a and R ^2b is H;

X ³ is C;

x ⁴ is C (R ⁴) or N, wherein R ⁴ is H;

X ⁷ is C (R ⁷) or N, wherein R ⁷ is H;

X ⁸ is C, and

X ⁹, when present, is CH ₂.

In some aspects, each of the heteroalkyl groups is an alkoxy group. Thus, in some aspects, there is provided an ADC of formula (II), wherein:

X ¹ is C (R ^1a)(R^1b), wherein each of R ^1a and R ^1b is H;

X ² is C (R ^2a)(R^2b), wherein each of R ^2a and R ^2b is H;

X ³ is C;

x ⁴ is C (R ⁴) or N, wherein R ⁴ is H;

X ⁵ is C (R ⁵) or N, wherein R ⁵ is H, halogen or alkoxy;

X ⁶ is C (R ⁶) or N, wherein R ⁶ is H, halogen or alkoxy;

X ⁷ is C (R ⁷) or N, wherein R ⁷ is H;

X ⁸ is C, and

X ⁹, when present, is CH ₂.

In some aspects, at least one of R ⁵ and R ⁶ is alkoxy.

In some aspects, d is 1,2, 3, or 4. In some aspects, d is 2. In some aspects, d is 4.

In some aspects, an ADC of formula (II) is provided, wherein L is a phosphate-based linker. In some aspects, the phosphate-based linker comprises a phosphate-based moiety having the structure: wherein L further comprises at least one further moiety, and the wavy line of phosphate-based moiety represents a connection to one of the at least one further moiety. In some aspects, each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, - (alkylene-O) -, -C (O) -, -N (R _w) -, a water-soluble polymer, and an amino acid, wherein each R _w is independently H or C ₁-C₈ alkyl, and combinations thereof. In some aspects, L is selected from the group of linkers of table 6. In some other aspects, L is selected from the group of linkers of table 7. In some other aspects, L is selected from the group of linkers of table 8. In some aspects, L has the following structure:

Wherein represents a linkage to an-O-atom at position L of formula (II), and +represents a linkage to E. In some other aspects, L has the following structure:

Wherein represents a linkage to an-O-atom at position L of formula (II), and +represents a linkage to E. In some other aspects, L has the following structure: Wherein T is a water-soluble polymer, R ^t is H or methyl, represents a linkage to the-O-atom at position L of formula (II), and +represents a linkage to E. In some aspects, the water-soluble polymer is a polyethylene glycol (PEG) moiety. In some aspects, the PEG moiety has a molecular weight in the range of about 100Da to about 100,000Da, about 100Da to about 10,000Da, about 100Da to about 5,000Da, or about 100Da to about 1,000 Da. In some aspects, the PEG moiety is- (CH ₂CH₂O)_nCH₃, where n is an integer from 1 to 24, in some aspects, the PEG moiety is- (CH ₂CH₂O)_nCH₃, where n is 8, 9, 10, 11, or 12, in some aspects, n is 8, in some aspects, n is 12.

In some aspects, E comprises an amide, an ester, a thioester, a pyrrolidine-2, 5-dione, an oxime, a1, 2, 3-triazole, or a1, 4-dihydropyridazine, wherein each of the 1,2, 3-triazole and the 1, 4-dihydropyridazine is optionally fused to an 8-membered ring. In some aspects, E is selected from the group consisting of:

Wherein each R ^j is independently H or unsubstituted C ₁-C₆ alkyl, each R ^q is independently unsubstituted C ₁-C₆ alkyl, each R ^f is independently H or unsubstituted C ₁-C₆ alkyl, each s is independently 0,1, 2, 3,4,5, or 6, each t is independently 0,1, 2, 3,4,5, or 6, each +represents a linkage to L, and each wavy line represents a linkage to Ab. In some aspects, E is: Wherein R ^q is unsubstituted C ₁-C₆ alkyl. In some aspects, R ^q is methyl.

In some aspects, E connects L to an unnatural amino acid of Ab.

In some aspects, R ^q is methyl encoding an unnatural amino acid into the Ab. In some embodiments, the unnatural amino acid is p-acetyl-L-phenylalanine (pAF), and R ^q is a methyl group of the pAF acyl group.

In some aspects, ab is configured to bind to an antigen. In some aspects, the antigen is selected from the group consisting of ：PD-1、PD-L1、PSMA、CD70、CD3、HER2、HER3、TROP2、GPC3、VEGFR、EGFR、c-Met(HGFR)、CD19、CD22、CD25(IL-2Rα)、CD30、CD33、CD37、CD46、CD48、CD56(NCAM-1)、CD71( transferrin R), CD74, CD79B, CD123 (IL-3Rα), CD138 (syndecan-1), CD142, CD166 (ALCAM), CD203c (ENPP 3), CD205 (LY 75), CD221 (IGF-1R), CD262 (TRAIL R2), CD276 (B7-H3), mesothelin, epCAM, CEACAM5, CEACAM6, DLL3, ROR1, ROR2, GPNMB, GCC, GUCY c, naPi2B, flt-1, flt-3, folate receptor α, tissue factor (TF)、CA6、MUC1、MUC16(CA-125)、BCMA、SLAMF7(CS1)、TIM1、CanAg、Ckit(CD117)、EphA2、Nectin4、SLTRK6、FGFR2、LYPD3(C4.4a)、 cadherin 3, 5T4 (TPBG), STEAP1, PTK7, hepsin-A4, V LI-1 (SLC 39A6 or ZIP 6), SLC1A5, TENB2, ETBR, integrin v3, crip, AGS-5 (SLC 44A 4), 6 RC E, AXL, LAMP, LRIX 15, and TNF-MN/αα. In some aspects, the antigen is TROP2, CD70, HER2, PSMA, HER3, or GPC3.

In some aspects, ab is an anti-CD 70 antibody comprising the sequences listed in table 2. In some aspects, the anti-CD 70 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO. 26. In some aspects, the anti-CD 70 antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO. 27. In some aspects, the anti-CD 70 antibody comprises a heavy chain of the amino acid sequence of the particular SEQ ID NO. 25. In some aspects, the anti-CD 70 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO. 20. In some aspects, the anti-CD 70 antibody comprises a light chain having the amino acid sequence of SEQ ID NO. 19. In some aspects, an anti-CD 70 antibody comprises two heavy chains each having the amino acid sequence of SEQ ID NO. 20 and two light chains each having the amino acid sequence of SEQ ID NO. 19. In some other aspects, an anti-CD 70 antibody comprises two heavy chains each having the amino acid sequence of SEQ ID NO. 25 and two light chains each having the amino acid sequence of SEQ ID NO. 19.

In some other aspects, ab is an anti-TROP 2 antibody comprising the sequences listed in table 1. In some aspects, the anti-TROP 2 antibody comprises a heavy chain of the amino acid sequence of particular SEQ ID NO. 5. In some aspects, the anti-TROP 2 antibody comprises a light chain having the amino acid sequence of SEQ ID No. 4.

In some other aspects, ab is an anti-HER 2 antibody comprising the sequences listed in table 3.

In some aspects, the anti-HER 2 antibody comprises a heavy chain of the amino acid sequence of particular SEQ ID NO. 29. In some aspects, the anti-HER 2 antibody comprises a light chain having the amino acid sequence of SEQ ID No. 30.

In some other aspects, ab is an anti-PSMA antibody comprising a sequence listed in table 4.

In some aspects, the anti-PSMA antibody comprises the heavy chain of the amino acid sequence of a particular SEQ ID NO. 39. In some aspects, the anti-PSMA antibody comprises a light chain having the amino acid sequence of SEQ ID No. 40.

In some other aspects, ab is an anti-HER 3 antibody comprising the sequences listed in table 5.

In some aspects, the anti-HER 3 antibody comprises a heavy chain of the amino acid sequence of the particular SEQ ID NO. 58. In some aspects, the anti-PSMA antibody comprises a light chain having the amino acid sequence of SEQ ID No. 47.

In some aspects, the antibody (Ab) comprises two heavy chains, and one unnatural amino acid is incorporated into each of the heavy chains.

In some aspects, the unnatural amino acid is a p-acetyl-L-phenylalanine.

In some other general aspects, the present disclosure provides a pharmaceutical composition comprising a compound of formula (I), or formula (Ia), or formula (Ib), or formula (Ic), or formula I (d), or formula (II) ADC, and at least one pharmaceutically acceptable adjuvant, binder, buffer, carrier, diluent, or excipient.

In some other general aspects, the present disclosure provides a method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or formula (Ia), or formula (Ib), or formula (Ic), or formula I (d), or ADC of formula (II), or a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or formula (Ia), or formula (Ib), or formula (Ic), or formula I (d), or ADC of formula (II). In some aspects, the disease or condition is cancer. In some aspects, the cancer is a CD70 expressing cancer. In some aspects, the cancer is renal cell carcinoma. In some other aspects, the cancer is a leukemia. In some aspects, the blood cancer is leukemia, lymphoma, or myeloma.

It is to be understood that the methods and compositions described herein are not limited to the particular methods, protocols, cell lines, constructs, and reagents described herein, and as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods and compositions described herein.

Incorporated by reference

All publications, patents, patent applications, and/or other documents mentioned herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, and/or other document was specifically and individually indicated to be incorporated by reference for all purposes, and for the purpose of describing and disclosing compositions and other methods described in, for example, publications, patents, patent applications, and/or other documents, which may be used in connection with the presently described application. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application.

Drawings

FIGS. 1A and 1B show the evaluation of the in vitro cytotoxic activity of the bicubicin analog compound and the anti-CD 70 ADC against the CD70 positive cell line 786-O (FIG. 1A) and the CD70 negative cell line NCI-H929 (FIG. 1B).

FIGS. 2A and 2B show the evaluation of the in vitro cytotoxic activity of the biscarmycin analog compound and the anti-GPC 3 ADC against the GPC3 positive cell line HepG2 (FIG. 2A), and against the GPC3 negative cell line SUN499 (FIG. 2B).

Detailed Description

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular methods or compositions or biological systems, which may, of course, vary. In addition, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

While various embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It will be appreciated that in practicing the invention, a variety of alternative forms of the embodiments of the invention described herein may be used. The following claims are intended to define the scope of the invention and to cover methods and results that fall within the scope of these claims and their equivalents.

Definition of the definition

Unless otherwise defined herein or in the remainder of the following description, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention described herein belongs. Various methods, materials, and the like, similar or equivalent to those described herein, can be used in the practice or testing of the invention described herein.

All publications and patents mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the chemistry, chemical synthesis, compositions and other methods described in the publications, which might be used in connection with the presently described application. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application.

Chemical terminology

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used herein, the term "acyl" denotes-C (O) -alkyl as defined herein, and is exemplified by acetyl (-C (O) CH ₃), trifluoroacetyl, propionyl, and butyryl. Exemplary unsubstituted acyl groups contain 1 to 6, 1 to 11, or 1 to 21 carbons.

As used herein, the term "alkyl" refers to a branched or straight chain monovalent saturated aliphatic hydrocarbon radical having 1 to 20 carbon atoms. Non-limiting examples of alkyl groups include aliphatic hydrocarbon groups having 1 to 16 carbon atoms (C _1-16 alkyl), 1 to 10 carbon atoms (C _1-10 alkyl), 1 to 6 carbon atoms (C _1-6 alkyl), four carbon atoms (e.g., n-butyl, isobutyl, sec-butyl, tert-butyl), three carbon atoms (e.g., isopropyl or n-propyl), two carbon atoms (ethyl), and 1 carbon atom (methyl). Alkylene is a divalent alkyl group.

The term "alkenyl" as used herein alone or in combination with other groups refers to a straight or branched hydrocarbon residue having a carbon-carbon double bond and having 2 to 20 carbon atoms (e.g., 2 to 16 carbon atoms, 2 to 10 carbon atoms, 2 to 6 or 2 carbon atoms).

The term "alkoxy" as used herein alone or in combination with other groups refers to an alkyl group having a single bond to oxygen. Non-limiting examples of alkoxy groups of the present disclosure include methoxy (-OMe) and ethoxy (-OEt). The alkoxy groups of the present disclosure are optionally substituted. In some embodiments, the alkoxy groups of the present disclosure are optionally substituted with heterocyclyl or-N (R ^d)(R^e), wherein each R ^d and R ^e is independently H, alkyl, alkenyl, or alkynyl.

The term "alkynyl" as used herein alone or in combination with other groups refers to a straight or branched hydrocarbon residue having a carbon-carbon triple bond and having 2 to 20 carbon atoms (e.g., 2 to 16 carbon atoms, 2 to 10 carbon atoms, 2 to 6 or 2 carbon atoms).

As used herein, the term "amino" means-N (R ^N1)₂, wherein each R ^N1 is independently H、OH、NO₂、N(R^N2)₂、SO₂OR^N2、SO₂R^N2、SOR^N2、N- protecting group, alkyl, alkoxy, aryl, arylalkyl, cycloalkyl, acyl (e.g., acetyl, trifluoroacetyl, or other groups described herein), wherein each of these enumerated R ^N1 groups may be optionally substituted, or two R ^N1 groups combine to form an alkylene or heteroalkylene, and wherein each R ^N2 is independently H, alkyl, or aryl.

As used herein, the term "aryl" refers to an aromatic mono-or multi-carbocyclic group of 6 to 12 carbon atoms having at least one aromatic ring. Examples of such groups include, but are not limited to, phenyl, naphthyl, 1,2,3, 4-tetrahydronaphthyl, 1, 2-dihydronaphthyl, indanyl, and 1H-indenyl.

As used herein, the term "arylalkyl" refers to an alkyl group substituted with an aryl group. Exemplary unsubstituted arylalkyl groups are 7 to 30 carbons (e.g., 7 to 16 or 7 to 20 carbons, such as C _1-6 alkyl C _6-10 aryl, C _1-10 alkyl C _6-10 aryl, or C _1-20 alkyl C _6-10 aryl), such as benzyl and phenethyl. In some embodiments, the alkyl and aryl groups may each be further substituted with 1, 2, 3, or 4 substituents as defined herein for the corresponding group.

The term "azido" as used herein denotes the-N ₃ group.

As used herein, the term "bicyclic ring system" refers to a bicyclic moiety or molecule containing two linked rings, wherein the two rings are linked by sharing two or more atoms. In some embodiments, the bicyclic system shares two atoms. In some embodiments, the bicyclic ring system contains at least one nitrogen atom.

As used herein, the term "cyano" represents a —cn group.

As used herein, the term "carbocyclyl" refers to a non-aromatic C _3-12 monocyclic, bicyclic, or tricyclic structure in which the rings are formed from carbon atoms. Carbocyclyl structures include cycloalkyl and unsaturated carbocyclyl.

As used herein, the term "cycloalkyl" refers to a saturated, non-aromatic, monovalent, mono-or multicyclic group of three to ten, preferably three to six carbon atoms. The term is further exemplified by groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, and adamantyl.

As used herein, the term "halogen" refers to a fluoro (fluoro), chloro (chloro), bromo (bromo) or iodo (iodo) group.

As used herein, the term "heteroalkyl" refers to an alkyl group as defined herein wherein one or more of the constituent carbon atoms have been replaced with nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkyl group may be further substituted with 1, 2,3, or 4 substituents as described herein for the alkyl group. Non-limiting examples of heteroalkyl groups include aminoalkyl and "alkoxy".

Heteroalkylene is a divalent heteroalkyl group.

As used herein, the term "heteroalkenyl" refers to an alkenyl group as defined herein wherein one or more of the constituent carbon atoms have been replaced with nitrogen, oxygen or sulfur. In some embodiments, the heteroalkenyl group may be further substituted with 1, 2,3, or 4 substituents as described herein for the alkenyl group. An example of a heteroalkenyl group is "alkenyloxy", which as used herein refers to alkenyl-O-. The heteroalkenylene group is a divalent heteroalkenyl group.

As used herein, the term "heteroalkynyl" refers to an alkynyl group as defined herein in which one or more of the constituent carbon atoms have been replaced with nitrogen, oxygen or sulfur. In some embodiments, the heteroalkynyl group may be further substituted with 1, 2,3, or 4 substituents as described herein for the alkynyl group. An example of a heteroalkynyl group is "alkynyloxy", which as used herein refers to alkynyl-O-. Heteroalkynylenes are divalent heteroalkynyl groups.

As used herein, the term "heteroaryl" refers to an aromatic mono-or polycyclic group of 5 to 12 atoms having at least one aromatic ring containing one, two or three ring heteroatoms selected from N, O and S, the remaining ring atoms being C. One or both ring carbon atoms of the heteroaryl group may be substituted with a carbonyl group. Examples of heteroaryl groups are pyridyl, pyrazinyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, imidazolyl, oxazolyl and thiazolyl.

As used herein, the term "heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group. Exemplary unsubstituted heteroarylalkyl groups are 7 to 30 carbons (e.g., 7 to 16 or 7 to 20 carbons, such as C _1-6 alkyl C _2-9 heteroaryl, C _1-10 alkyl C _2-9 heteroaryl, or C _1-20 alkyl C _2-9 heteroaryl). In some embodiments, the alkyl and heteroaryl groups may each be further substituted with 1, 2, 3, or 4 substituents as defined herein for the corresponding group.

As used herein, the term "heterocyclyl" means a monocyclic or polycyclic group having 3 to 12 atoms having at least one ring containing one, two, three or four ring heteroatoms selected from N, O or S, wherein no ring is aromatic. Examples of heterocyclyl groups include, but are not limited to, morpholinyl, thiomorpholinyl, furanyl, piperazinyl, piperidinyl, pyranyl, pyrrolidinyl, tetrahydropyranyl, tetrahydrofuranyl, and 1, 3-dioxanyl.

As used herein, the term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl group. Exemplary unsubstituted heterocyclylalkyl groups are 7 to 30 carbons (e.g., 7 to 16 or 7 to 20 carbons, such as C _1-6 alkyl C _2-9 heterocyclyl, C _1-10 alkyl C _2-9 heterocyclyl, or C _1-20 alkyl C _2-9 heterocyclyl). In some embodiments, the alkyl and heterocyclyl groups may each be further substituted with 1, 2, 3 or 4 substituents as defined herein for the corresponding group.

As used herein, the term "hydroxy" means an-OH group.

The term "nitro" as used herein means a-NO ₂ group.

As used herein, the term "thiol" means a-SH group.

Alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl (e.g., cycloalkyl), aryl, heteroaryl, and heterocyclyl groups may be substituted or unsubstituted. When substituted, there will typically be 1 to 4 substituents unless otherwise indicated. Substituents include, for example, aryl (e.g., substituted and unsubstituted phenyl), carbocyclyl (e.g., substituted and unsubstituted cycloalkyl), halogen (e.g., fluorine), hydroxy, heteroalkyl (e.g., substituted and unsubstituted methoxy, ethoxy, or thioalkoxy), heteroaryl, heterocyclyl, amino (e.g., NH ₂ or mono-or di-alkylamino), azido, cyano, nitro, or thiol. Aryl, carbocyclyl (e.g., cycloalkyl), heteroaryl, and heterocyclyl groups may also be substituted with alkyl groups (unsubstituted and substituted, such as arylalkyl groups (e.g., substituted and unsubstituted benzyl)).

The compounds of the invention may have one or more asymmetric carbon atoms and may exist as optically pure enantiomers, mixtures of enantiomers (e.g., racemates), optically pure diastereomers, mixtures of diastereomers, racemates of diastereomers or mixtures of racemates of diastereomers. Optically active forms can be obtained, for example, by resolution of the racemate, by asymmetric synthesis or asymmetric chromatography (chromatography using chiral adsorbents or eluents). That is, certain disclosed compounds may exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in the spatial arrangement. Enantiomers are pairs of stereoisomers whose mirror images are non-overlapping, most commonly because they contain asymmetrically substituted carbon atoms as chiral centers. "enantiomer" means one of a pair of molecules that are mirror images of each other and that are non-overlapping. Diastereomers are stereoisomers that are not related in mirror image form, most commonly because they contain two or more asymmetrically substituted carbon atoms and represent a substituent configuration around one or more chiral carbon atoms. Enantiomers of compounds may be prepared, for example, by separating enantiomers from racemates using one or more well-known techniques and methods, such as chiral chromatography and separation methods based thereon. Suitable techniques and/or methods for separating enantiomers of compounds described herein from a racemic mixture can be readily determined by one skilled in the art. "racemate" or "racemic mixture" means a compound containing two enantiomers, wherein such mixtures do not exhibit optical activity, i.e., they do not rotate the plane of polarized light. "geometric isomer" means an isomer that differs in orientation from the substituent atom associated with the carbon-carbon double bond, cycloalkyl ring, or bridged bicyclic ring system. The atoms on each side of the carbon-carbon double bond (except H) may be in the E (substituents on opposite sides of the carbon-carbon double bond) or Z (substituents oriented on the same side) configuration. "R", "S", "R", "E", "Z", "cis" and "trans" refer to configuration relative to the core molecule. Certain disclosed compounds may exist in atropisomeric forms. Atropisomers are stereoisomers obtained by hindered rotation about a single bond, wherein the spatial strain barrier to rotation is sufficiently high to allow conformational isomer separation. The compounds of the present invention may be prepared as single isomers by isomer specific synthesis or resolution from mixtures of isomers. Conventional resolution techniques include the use of optically active acids to form salts of the free base of each isomer of the isomer pair (followed by fractional crystallization and regeneration of the free base), the use of optically active amines to form salts of the acid form of each isomer of the isomer pair (followed by fractional crystallization and regeneration of the free acid), the use of optically pure acids, amines or alcohols to form esters or amides of each isomer of the isomer pair (followed by chromatographic separation and removal of chiral auxiliary), or the use of various well known chromatographic methods to resolve mixtures of isomers of the starting material or final product. when the stereochemistry of a disclosed compound is named or described by structure, the named or described stereoisomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight relative to the other stereoisomers. When a single enantiomer is named or described by structure, the enantiomer described or named is at least 60%, 70%, 80%, 90%, 99% or 99.9% optically pure by weight. When a single diastereomer is named or described by structure, the diastereomer described or named is at least 60%, 70%, 80%, 90%, 99% or 99.9% pure by weight. the percent optical purity is the ratio of the weight of an enantiomer to the weight of the enantiomer plus the weight of its optical isomer. Diastereomeric purity by weight is the ratio of the weight of one diastereomer to the weight of all diastereomers. When the stereochemistry of a disclosed compound is named or described by structure, the named or described stereoisomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% pure relative to the other stereoisomers by mole fraction. When a single enantiomer is named or described by structure, the enantiomer described or named is at least 60%, 70%, 80%, 90%, 99% or 99.9% pure by mole fraction. When a single diastereomer is named or described by structure, the diastereomer described or named is at least 60%, 70%, 80%, 90%, 99% or 99.9% pure by mole fraction. The percent purity in mole fraction is the ratio of the moles of enantiomer to the moles of enantiomer plus the moles of optical isomer thereof. Similarly, the percent purity in mole fraction is the ratio of the moles of diastereomers to the moles of diastereomers plus the moles of isomers thereof. When a disclosed compound is named or described by structure without indicating stereochemistry and the compound has at least one chiral center, it is to be understood that the name or structure includes the enantiomer of the compound that does not contain the corresponding optical isomer, a racemic mixture of the compound, or a mixture enriched in one enantiomer relative to its corresponding optical isomer. When a disclosed compound is named or described by structure without indicating stereochemistry and having two or more chiral centers, it is to be understood that the name or structure includes diastereomers that are free of other diastereomers, numerous diastereomers that are free of other pairs of diastereomers, mixtures of pairs of diastereomers, mixtures of diastereomers in which one diastereomer is enriched relative to the other diastereomer, or mixtures of diastereomers in which one or more diastereomers is enriched relative to the other diastereomer. The present invention includes all such forms.

In embodiments, novel amino acid sequences are provided. The term "amino acid" refers to naturally occurring and non-natural or unnatural amino acids, which may be referred to herein as synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine), pyrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example only, with an alpha-carbon bound to hydrogen, a carboxyl group, an amino group, and a functional R group. Such analogs can have modified R groups (e.g., norleucine) or can have modified peptide backbones while still retaining the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Amino acids may be referred to herein by their name, by their commonly known three letter symbols or by the single letter symbols recommended by the IUPAC-IUB biochemical naming convention. In addition, nucleotides may be represented by their commonly accepted single letter codes.

"Amino or carboxyl terminal modifying group" refers to any molecule that can be attached to a terminal amine group or terminal carboxyl group, respectively. By way of example, such terminal amine groups or terminal carboxyl groups may be located at the ends of polymeric molecules, wherein such polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminal modifying groups include, but are not limited to, various water-soluble polymers, peptides or proteins. By way of example only, the terminal modifying group includes polyethylene glycol or serum albumin. The terminal modifying groups can be used to modify the therapeutic properties of the polymer molecule, including but not limited to increasing the serum half-life of the peptide, polypeptide, or protein.

In some embodiments, the present disclosure provides novel antibodies and antibody variants. The term "antibody" herein refers to a protein consisting of one or more polypeptides that are encoded substantially by all or part of an antibody gene. Immunoglobulin genes include, but are not limited to, kappa, lambda, alpha, gamma (IgG 1, igG2, igG3, and IgG 4), delta, epsilon, and mu constant region genes, and myriad immunoglobulin variable region genes. Antibodies herein are also intended to include full length antibodies and antibody fragments, and include antibodies, antibody variants, engineered antibodies and antibody fragments that naturally occur in any organism. Antibodies herein are also intended to include whole antibodies, monoclonal antibodies, or polyclonal antibodies. Antibodies herein also encompass multispecific antibodies and/or bispecific antibodies. Antibodies of the present disclosure include human antibodies. Human antibodies typically consist of two light chains and two heavy chains each comprising a variable region and a constant region. The light chain variable region comprises 3 CDRs, identified herein as CDRL1, CDRL2 and CDRL3 flanked by framework regions. The heavy chain variable region comprises 3 CDRs, identified herein as CDRH1, CDRH2 and CDRH3 flanked by framework regions.

The term "antibody fragment" refers herein to any form of antibody other than the full length form. Antibody fragments herein include antibodies that are smaller components present in a full length antibody, as well as antibodies that have been engineered, such as antibody variants. Antibody fragments include, but are not limited to Fv, fc, fab and (Fab') ₂, single chain Fv (scFv), diabodies, triabodies, tetravalent antibodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains and variable regions, and alternative scaffold non-antibody molecules, bispecific antibodies, etc. (Maynard&Georgiou,Annu.Rev.Biomed.Eng.2:339-76,2000;Hudson,Curr.Opin.Biotechnol.,9:395-402,1998). another functional substructure is a single chain Fv (scFv) consisting of the variable regions of immunoglobulin heavy and light chains covalently linked by peptide linkers (Hu et al CANCER RESEARCH,56,3055-3061,1996). These small (mr 25,000) proteins generally retain specificity and affinity for antigens in a single polypeptide and can provide convenient building blocks for larger antigen-specific molecules. Unless specifically stated otherwise, statements and claims using the term "antibodies" or "antibodies" specifically include "antibody fragment (antibody fragment)" and "antibody fragment (antibody fragments)".

In embodiments, novel Antibody Drug Conjugates (ADCs) are disclosed. As used herein, the term "antibody-drug conjugate" or "ADC" refers to an antibody molecule or fragment thereof that is covalently bound to one or more bioactive molecules. The bioactive molecule can be conjugated to the antibody via a linker, polymer, or other covalent bond. ADCs are a class of potent therapeutic constructs that allow targeted delivery of cytotoxic agents to target cells, such as cancer cells. Due to the targeting function, these compounds show a much higher therapeutic index than the same systemically delivered agent. ADCs have been developed as whole antibodies or antibody fragments, such as scFv. The antibody or fragment is linked to one or more copies of the drug via a linker that is stable under physiological conditions but may be cleaved once within the target cell.

As used herein, the term "antigen binding fragment" refers to one or more fragments of an antibody that retain the ability to bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V _L、V_H、C_L and C _H1 domains, (ii) a F (ab') ₂ fragment, a bivalent fragment comprising two Fab fragments linked by disulfide bonds at the hinge region, (iii) an Fd fragment consisting of the V _H and C _H1 domains, (iv) an Fv fragment consisting of the V _L and V _H domains of a single arm of the antibody, (V) a dAb fragment consisting of the V _H domain (Ward et al, nature 341:544-546,1989), and (vi) an isolated Complementarity Determining Region (CDR), e.g., with or without additional sequences (linker), Framework region, etc.) V _H CDR3 and (V) a combination of two to six separate CDRs with or without additional sequences (linkers, framework regions, etc.). Furthermore, although the two domains of the Fv fragment, V _L and V _H, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made into a single polypeptide chain, in which the V _L and V _H regions pair to form a monovalent molecule (known as a single chain Fv (scFv); see, e.g., bird et al, science 242:423-426, 1988), and (Huston et al, proc. Natl. Acad. Sci. USA 85:5879-5883,1988). such single chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. In addition, antigen binding fragments include binding domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide (such as a heavy chain variable region, a light chain variable region, or a heavy chain variable region fused to a light chain variable region via a linker peptide) fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to a hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to a CH2 constant region. The hinge region may be modified to prevent dimerization by substituting one or more cysteine residues with serine residues. Such binding domain immunoglobulin fusion proteins are further disclosed in US 2003/0116992 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those skilled in the art and fragments are screened for utility in the same manner as intact antibodies.

A typical antigen binding site consists of a variable region formed by pairing light chain immunoglobulins and heavy chain immunoglobulins. The structure of the antibody variable regions is very consistent and exhibits very similar structure. These variable regions are typically composed of relatively homologous Framework Regions (FR) separated by three hypervariable regions called Complementarity Determining Regions (CDRs). The overall binding activity of an antigen binding fragment is generally determined by the sequence of the CDRs. FR generally plays a role in correctly positioning and aligning in three dimensions of the CDRs to achieve optimal antigen binding. Indeed, because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that exhibit the properties of a particular naturally occurring antibody by constructing expression vectors that include CDR sequences from a particular naturally occurring antibody grafted onto framework sequences of different antibodies having different properties (see, e.g., riechmann, L. Et al, nature332:323-327,1998; jones, P. Et al, nature 321:522-525,1986; and Queen, C. Et al, proc. Natl. Acad. USA 86:10029-10033,1989). Such framework sequences may be obtained from a public DNA database comprising germline antibody gene sequences. These germline sequences will differ from the mature antibody gene sequences in that they will not include the fully assembled variable genes formed by V (D) J ligation during B cell maturation. Germline gene sequences will also differ from the sequences of high affinity secondary pool antibodies that contain mutations throughout the variable genes, but are typically aggregated in CDRs. For example, somatic mutations are relatively unusual in the amino-terminal portion of framework region 1 and in the carboxy-terminal portion of framework region 4. In addition, many somatic mutations do not significantly alter the binding properties of antibodies. Thus, it is not necessary to obtain the complete DNA sequence of a specific antibody to reconstruct a complete recombinant antibody having similar binding properties as the original antibody. Portions of the heavy and light chain sequences spanning the CDR regions are generally sufficient for this purpose. The partial sequences are used to determine which germline variable gene segments and connecting gene segments contribute to the recombinant antibody variable gene. The germline sequence is then used to fill in the deleted portion of the variable region. The heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add deleted sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized to produce a fully synthesized variable region clone. This approach has certain advantages, such as elimination or inclusion of specific restriction sites, or optimization of specific codons. Of course, all or part of the framework regions of the antibodies described herein may be used in combination with CDRs to optimize affinity, specificity, or any other desired property of the antibody.

In some embodiments, the present disclosure relates to polymers, such as difunctional polymers. "bifunctional polymer", also referred to as a "bifunctional linker", refers to a polymer comprising two functional groups capable of specifically reacting with other moieties to form covalent or non-covalent bonds. Such moieties may include, but are not limited to, pendant groups on natural or unnatural amino acids or peptides containing such natural or unnatural amino acids. The other moieties that may be attached to the bifunctional linker or the bifunctional polymer may be the same or different moieties. By way of example only, the bifunctional linker may have a functional group that reacts with a group on the first peptide and another functional group that reacts with a group on the second peptide, thereby forming a conjugate comprising the first peptide, the bifunctional linker, and the second peptide. Many methods and linker molecules for attaching various compounds to peptides are known. See, for example, european patent application 0188256, U.S. Pat. Nos. 4,659,839, 4,414,148, 4,699,784, 4,680,338, and 4,569,789, which are incorporated herein by reference in their entirety. "multifunctional polymer", also referred to as "multifunctional linker", refers to a polymer that contains two or more functional groups capable of reacting with other moieties. Such moieties may include, but are not limited to, natural or unnatural amino acids or pendant groups on peptides that contain such natural or unnatural amino acids (including, but not limited to, pendant amino acid groups) to form covalent or noncovalent bonds. The difunctional or polyfunctional polymer may be of any desired length or molecular weight and may be selected to provide a particular desired spacing or conformation between one or more molecules attached to the compound and the molecule bound thereto or the molecule bound thereto.

As used herein, the term "bioavailability" refers to the rate and extent at which a substance or active portion thereof is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the systemic circulation. An increase in bioavailability refers to an increase in the rate and extent at which a substance or active portion thereof is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the systemic circulation. As an example, an increase in bioavailability may be expressed as an increase in the concentration of a substance or active portion thereof in the blood when compared to other substances or active portions.

The term "bioactive molecule," "bioactive moiety," or "bioactive agent" as used herein means any substance that can affect any physical or biochemical characteristic of a biological system, pathway, molecule, or interaction associated with an organism, including but not limited to viruses, bacteria, phages, transposons, prions, insects, fungi, plants, animals, and humans. Specifically, as used herein, bioactive molecules include, but are not limited to, any substance intended for use in diagnosing, curing, alleviating, treating or preventing a disease in a human or other animal, or otherwise enhancing the physical or mental well-being of a human or animal. Examples of bioactive molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles. Classes of bioactive agents suitable for use with the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, antivirals, anti-inflammatories, antitumor agents, cardiovascular agents, anxiolytics, hormones, growth factors, steroidal and non-steroidal drugs, microbiologically derived toxins, and the like.

By "modulating biological activity" is meant increasing or decreasing the reactivity of a polypeptide, altering the selectivity of a polypeptide, increasing or decreasing the substrate selectivity of a polypeptide. Analysis of modified biological activity can be performed by comparing the biological activity of the non-native polypeptide to the biological activity of the native polypeptide.

In some embodiments, the disclosure relates to amino acids that have been incorporated into antibodies by biosynthesis. As used herein, the term "biosynthesis" refers to any method that utilizes a translation system (cellular or non-cellular) including the use of at least one of a polynucleotide, a codon, a tRNA, and a ribosome. By way of example, unnatural amino acids can be "biosynthetically incorporated" into unnatural amino acid polypeptides using methods and techniques described herein and well known in the art. See, for example, WO 2010/01735 and WO2005/074650.

The term "conservatively modified variants" applies to both natural and unnatural amino acids as well as to both natural and unnatural nucleic acid sequences, and combinations thereof. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those natural and unnatural nucleic acids that encode identical or essentially identical natural and unnatural amino acid sequences, or essentially identical sequences where the natural and unnatural nucleic acids do not encode natural and unnatural amino acid sequences. By way of example, due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, both codons GCA, GCC, GCG and GCU encode the amino acid alanine. Thus, at each position of a codon designated alanine, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variations. Thus, by way of example, each natural or unnatural nucleic acid sequence herein encoding a natural or unnatural polypeptide also describes every possible silent variation of the natural or unnatural nucleic acid. One of ordinary skill in the art will recognize that each codon in a natural or unnatural nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a natural or non-natural nucleic acid which encodes a natural or non-natural polypeptide is implicit in each said sequence. With respect to amino acid sequences, a single substitution, deletion, or addition of a single natural and unnatural amino acid or a small percentage of natural and unnatural amino acids in a encoded sequence is a "conservatively modified variant" where the alteration results in the deletion of an amino acid, the addition of an amino acid, or the substitution of a natural and unnatural amino acid with a chemically similar amino acid. Conservative substitutions providing functionally similar natural amino acids are well known in the art. Conservative substitutions that provide functionally similar amino acids are known to those of ordinary skill in the art. The following eight groups each contain amino acids conservatively substituted for each other, 1) alanine (A), glycine (G), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), valine (V), 6) phenylalanine (F), tyrosine (Y), tryptophan (W), 7) serine (S), threonine (T), 8) cysteine (C), methionine (M) (see, e.g., cright on, proteins: structures and Molecular Properties (W H FREEMAN & Co.; 2 nd edition, 1993). Such conservatively modified variants are complements of, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the compositions described herein.

As used herein, the term "drug" refers to any substance used to prevent, diagnose, alleviate, treat, or cure a disease or condition such as cancer, including but not limited to oral cancer, colorectal cancer, gastric cancer, esophageal cancer, hepatocellular cancer, non-small cell lung cancer (NSCL), small cell lung cancer (SCL), ovarian cancer, breast cancer (including triple negative breast cancer), prostate cancer, pancreatic cancer, head and neck cancer, squamous carcinoma, renal cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, glioblastoma, or blood cancer (including leukemia, lymphoma, or myeloma).

As used herein, the term "drug to antibody ratio" ("DAR") refers to the average (mean) number of drugs conjugated to an antibody in an Antibody Drug Conjugate (ADC) composition. The DAR value reflects the homogeneity of the ADC population in the composition, and also indicates the amount of "payload" (e.g., drug or drug linker) that is loaded onto the antibody and that can be delivered to a target (e.g., a cell or diseased tissue). DAR can be determined by methods known to those of ordinary skill in the art, such as LC-MS (see, e.g., tang, Y. Et al ,Real-Time Analysis on Drug-Antibody Ratio of Antibody-Drug Conjugates for Synthesis,Process Optimization and Quality Control,Sci Rep 7,7763(2017).doi:10.1038/s41598-017-08151-2; and Chen,Y.Drug-to-antibody ratio(DAR)by UV/Vis spectroscopy,Methods Mol.Biol.,2013;1045:267-73.doi:10.1007/978-1-62703-541-5_16). in one non-limiting example, ADC can have a population distribution of 20% drug-loaded antibodies, where drug loading is two (2) drugs per antibody, 25% drug-loaded antibodies, where drug loading is three (3) drugs per antibody, and 55% drug-loaded antibodies, where drug loading is four (4) drugs per antibody, thus, in this example, DAR is [ (0.2X2) + (0.25X3) + (0.55X4) ]=3.35.

As used herein, the term "effective amount" refers to a sufficient amount of an agent, compound, or composition to be administered that will alleviate to some extent one or more symptoms of the disease or condition being treated. The result may be a reduction and/or alleviation of the signs, symptoms, or causes of the disease, or any other desired alteration of the biological system. By way of example, the administered agent, compound, or composition includes, but is not limited to, a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified non-amino acid polypeptide, or an antibody or variant thereof. Compositions containing such natural amino acid polypeptides, unnatural amino acid polypeptides, modified natural amino acid polypeptides, modified unnatural amino acid polypeptides, or antibodies or variants thereof can be administered for prophylactic, enhancing, and/or therapeutic treatment. The appropriate "effective" amount in any individual case can be determined using techniques such as dose escalation studies.

The term "increase" means to increase or prolong the effectiveness or duration of a desired effect. As an example, an "enhancing" the effect of a therapeutic agent refers to increasing or prolonging the ability of the effect of the therapeutic agent in terms of efficacy or duration during treatment of a disease, disorder, or condition. As used herein, "an effective enhancing amount" refers to an amount sufficient to enhance the effect of a therapeutic agent in treating a disease, disorder, or condition. When used in a patient, the amount effective for this use will depend on the severity and course of the disease, disorder or condition, previous treatments, the health of the patient and the response to the drug, and the judgment of the treating physician.

The term "humanized or chimeric antibody" refers to a molecule that is typically prepared using recombinant techniques, having an antigen binding site derived from an immunoglobulin of a non-human species (e.g., murine), as well as the remaining immunoglobulin structure of the molecule based on the structure and/or sequence of a human immunoglobulin. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework residues/regions (FR) are of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The humanized form of the rodent antibody will comprise substantially the same CDR sequences of the parent rodent antibody, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody or for other reasons. However, since CDR loop exchange does not consistently produce antibodies with the same binding characteristics as the source antibody, variations in Framework Residues (FR) (residues involved in CDR loop support) can also be introduced in humanized antibodies to maintain antigen binding affinity. The antigen binding site may comprise the complete variable domain fused to a constant domain or simply comprise Complementarity Determining Regions (CDRs) grafted onto appropriate framework regions in the variable domain. The antigen binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in a human individual, but the possibility of an immune response to an exogenous variable region still exists (LoBuglio, a.f. et al ,"Mouse/Human Chimeric Monoclonal Antibody in Man:Kinetics and Immune Response,"Proc.Natl.Acad.Sci.(USA)86:4220-4224,1989). another approach focuses not only on providing a constant region derived from humans, but also on modifying the variable region so as to remodel it as closely as possible to human morphology. when non-human antibodies are prepared with respect to a particular antigen, the variable regions can be "humanized" by grafting CDRs from the non-human antibody onto the FRs present in the human antibody to be modified. The use of this approach for various antibodies has been reported by Kettleborough, c.a. et al ,"Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting:The Importance Of Framework Residues On Loop Conformation,"Protein Engineering 4:773-3783,1991;Co,M.S., "Humanized Antibodies For ANTIVIRAL THERAPY," proc.Natl.Acad.Sci. (USA) 88:2869-2873,1991; carter, p. Et al ,"Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy,"Proc.Natl.Acad.Sci.(USA)89:4285-4289,1992; and Co, m.s. et al ,"Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen,"J.Immunol.148:1149-1154,1992. in some embodiments, humanized antibodies retain all CDR sequences (e.g., humanized mouse antibodies containing all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, six) that are altered relative to the original antibody, which is also referred to as "one or more CDRs derived from" one or more CDRs from the original antibody.

As used herein, the term "identical" refers to two or more identical sequences or subsequences. Furthermore, as used herein, the term "substantially identical" refers to two or more sequences having a percentage of identical consecutive units when compared and aligned within a comparison window or designated region to obtain maximum correspondence, as measured using a comparison algorithm or by manual alignment and visual inspection. For example only, two or more sequences may be "substantially identical" if the consecutive units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical within the designated region. Such percentages describe "percent identity" of two or more sequences. Sequence identity may exist within a region of at least about 75 to 100 consecutive units in length, within a region of about 50 consecutive units in length, or in the whole sequence if not specified. The definition also refers to the complement of the test sequence. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are identical, while two or more polypeptide sequences are "substantially identical" if the amino acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical within a designated region. Identity may exist over a region of at least about 75 to about 100 amino acids in length, over a region of about 50 amino acids in length, or in the entire sequence of a polypeptide sequence, if not specified. Further, by way of example only, two or more polynucleotide sequences are identical when the nucleic acid residues are identical, while two or more polynucleotide sequences are "substantially identical" if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical within the designated region. Identity may exist over a region of at least about 75 to about 100 nucleic acids in length, over a region of about 50 nucleic acids in length, or in the entire sequence of a polynucleotide sequence, if not specified.

As used herein, the term "immunogenicity" refers to an antibody response to administration of a therapeutic drug. Immunogenicity of therapeutic unnatural amino acid polypeptides can be obtained using quantitative and qualitative assays for detecting antibodies to unnatural amino acid polypeptides in biological fluids. Such assays include, but are not limited to, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), light-emitting immunoassays (LIA), and Fluorescent Immunoassays (FIA). Analysis of the immunogenicity of a therapeutic unnatural amino acid polypeptide includes comparing the antibody response when the therapeutic unnatural amino acid polypeptide is administered to the antibody response when the therapeutic natural amino acid polypeptide is administered.

As used herein, the term "isolated" refers to the separation and removal of a component of interest from a component not of interest. The isolated material may be in a dry or semi-dry state or in solution, including but not limited to an aqueous solution. The isolated component may be in a homogeneous state, or the isolated component may be part of a pharmaceutical composition comprising an additional pharmaceutically acceptable carrier and/or excipient. Purity and uniformity may be determined using analytical chemistry techniques including, but not limited to, polyacrylamide gel electrophoresis or high performance liquid chromatography. Furthermore, a component of interest is described herein as substantially purified when it is isolated and is the predominant species present in the formulation. As used herein, the term "purified" may refer to a component of interest that is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% pure, or higher. By way of example only, a nucleic acid or protein is "isolated" when the nucleic acid or protein does not contain at least some cellular components that bind to it in its native state, or when the nucleic acid or protein has been concentrated to a level above its in vivo or in vitro production concentration. Furthermore, a gene is isolated, for example, when isolated from an open reading frame flanking the gene and encoding a protein other than the gene of interest.

As used herein, the term "linkage" or "adduct moiety" refers to a bond or chemical moiety formed by a chemical reaction between a functional group of one group (such as a linker of the present disclosure) and another molecule. Such linkages may include, but are not limited to, covalent and non-covalent linkages, and such chemical moieties may include, but are not limited to, ester, carbonate, imine, phosphate, hydrazone, acetal, orthoester, peptide linkage, oxime, and oligonucleotide linkages. Hydrolytically stable bonds means that these bonds are substantially stable in water and do not react with water at useful pH values, including but not limited to long periods of time under physiological conditions, and possibly even indefinitely. Hydrolytically unstable or degradable bond means that the bond is degradable in water or aqueous solutions (including, for example, blood). Enzymatically labile or degradable bond means that the bond is degradable by one or more enzymes. By way of example only, PEG and related polymers may include degradable linkages in the polymer backbone or in the linking groups between the polymer backbone and one or more terminal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with alcohol groups on the bioactive agent, wherein such ester groups are typically hydrolyzed under physiological conditions to release the bioactive agent. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages, imine linkages resulting from the reaction of an amine and an aldehyde, phosphate linkages formed by the reaction of an alcohol with a phosphate group, hydrazone linkages as the reaction product of a hydrazide and an aldehyde, acetal linkages as the reaction product of an aldehyde and an alcohol, orthoester linkages as the reaction product of a formate and an alcohol, peptide linkages formed from carboxyl groups including, but not limited to, amine groups and peptides at the end of a polymer such as PEG, and oligonucleotide linkages formed from 5' hydroxyl groups including, but not limited to, phosphoramidite groups and oligonucleotides at the end of a polymer.

As used herein, the term "linker" refers to any multivalent group that connects or is capable of connecting a first group with at least one other group. Typically, the linker is a divalent or trivalent organic moiety that connects the drug (first group) to the bioactive agent (second group), for example via a bond or adduct moiety, or connects the drug (first group) to a reactive moiety (second group), wherein the reactive moiety is capable of reacting with the bioactive agent. The linker may be susceptible to cleavage (cleavable linker) under conditions where the drug and at least one other group remain active, such as acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, disulfide cleavage, and the like. Alternatively, the linker may be substantially cut-resistant (e.g., a stable linker or a non-cleavable linker).

In some embodiments, linker L is a divalent or trivalent group comprising or consisting of at least one moiety, wherein each at least one moiety is independently selected from the group consisting of a bond, an unsubstituted alkylene, a substituted alkylene, - (alkylene-O) _n -, an optionally substituted arylene, -O-, -C (O) -, -C (S) -, -N (R _w)-、-S(O)_0-2 - Methine (-CH) -, amino acids, peptides, disulfides (-S-S-), water-soluble polymers, and phosphate-based moieties, and combinations thereof, wherein each R _w is independently H, C ₁-C₈ alkyl or a bond, and each phosphate-based moiety is independently selected from the group consisting of phosphate, pyrophosphate, triphosphate, tetraphosphate, phosphonate, bisphosphonate, Phosphoramidate, jiao Anji phosphate a triamino phosphate, a tetraphosphoramidate phosphorothioate and dithio phosphorothioates. In some embodiments, the phosphate-based moiety is a phosphonate, a bisphosphonate, a tetraphosphate, or a dithiophosphate. In some embodiments, the phosphate-based moiety is a bisphosphonate. In some embodiments, the bisphosphonate moiety is conjugated to an oxygen atom of a drug (e.g., a biscardamycin of the disclosure) to provide a drug linker comprising a pyrophosphate. In some embodiments, the water-soluble polymer is polyethylene glycol (PEG) or modified PEG. In some embodiments, the water-soluble polymer is a polysaccharide. Unless explicitly indicated otherwise, the direction in which the chemical formula of the linking group is written does not imply the orientation of the linker. As an example of this, the number of devices, formula-C (O) CH ₂CH₂ -represents-C (O) CH ₂CH₂ -and-CH ₂CH₂ C (O) -. In another example, the formula-C (O) CH ₂CH₂ -represents-C (O) CH ₂CH₂ -and-C (O) CH ₂CH₂ -, wherein x represents a point of attachment, e.g., to a drug. In some embodiments, when the selected moiety occurs two or more times in the same linker, the two or more occurrences are not adjacent. in some embodiments, the linker is not a bond.

In some embodiments, the linker is a divalent moiety linking the first group and the second group. In some other embodiments, the linker is a trivalent moiety that connects the first group, the second group, and the third group. In one non-limiting example, the trivalent moiety is C (H) (i.e., methine) or N. In some other embodiments, the linker is a tetravalent moiety that connects the first group, the second group, and the third group.

In some embodiments, the linker connects at least the first group and the second group, wherein the first group is a drug and the second group is a biologically active polypeptide or protein. In some embodiments, the biologically active polypeptide or protein contains at least one unnatural amino acid. In some embodiments, the linker connects the drug to an unnatural amino acid of the biologically active polypeptide or protein. In some embodiments, the biologically active polypeptide or protein is an antibody. Thus, the antibody linked to the drug via the linker may be an Antibody Drug Conjugate (ADC), such as the ADC of the present disclosure.

In some other embodiments, the linker connects at least the first group and the second group, wherein the first group is a drug and the second group is a reactive moiety. In some embodiments, the second group is a reactive moiety capable of reacting with a biologically active polypeptide or protein. In some embodiments, the biologically active polypeptide or protein contains at least one unnatural amino acid. Thus, in some embodiments, the reactive moiety is capable of reacting with an unnatural amino acid of a biologically active polypeptide or protein. In some embodiments, the biologically active polypeptide or protein is an antibody.

In some embodiments, the first linker is attached to the second linker, and the combined linker (composite linker) attaches at least the first group and the second group. The composite linkers of the present disclosure may contain 2,3, 4, 5, 6, 7, 8, 9,10 or more linking groups. In one non-limiting example, the first, second, and third linking groups are linked together to provide a composite linker that can link the first group (e.g., a drug) to at least one other group, such as a reactive moiety and/or a biologically active polypeptide or protein (e.g., an antibody). In some embodiments, the biologically active polypeptide or protein (e.g., antibody) contains unnatural amino acids.

In some embodiments, the linker is linear. In other embodiments, the linker is branched.

In some embodiments, the linker is a phosphate-based linker.

As used herein, the term "phosphate-based linker" refers to a linker comprising a phosphate-based moiety, wherein the phosphate-based moiety is a phosphate, pyrophosphate, triphosphate, tetraphosphate, phosphonate, bisphosphonate, phosphoramidate, jiao Anji phosphate, triamino phosphate, tetraphosphoric acid ester, phosphorothioate, and/or phosphorodithioate.

As used herein, the term "metabolite" refers to a derivative of a compound (e.g., a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide) that is formed when the compound (e.g., a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide) is metabolized. The term "pharmaceutically active metabolite" or "active metabolite" refers to a biologically active derivative of a compound (e.g., a natural amino acid polypeptide, an unnatural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified unnatural amino acid polypeptide) that is formed when such a compound (e.g., a natural amino acid polypeptide, an unnatural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified unnatural amino acid polypeptide) is metabolized. The term "pharmaceutically active metabolite" or "active metabolite" also refers to biologically active derivatives of the compounds, such as metabolic phosphate linkages, including, but not limited to, mono-, di-, pyrophosphate and triphosphate esters.

As used herein, the term "metabolic" refers to an overview of the process by which a particular substance is altered by an organism. Such methods include, but are not limited to, hydrolysis reactions and reactions catalyzed by enzymes. Further information on metabolism can be obtained from The Pharmacological Basis of Therapeutics, 9 th edition, mcGraw-Hill (1996). By way of example only, a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a metabolite of a modified non-natural amino acid polypeptide may be identified by administering the natural amino acid polypeptide, the non-natural amino acid polypeptide, the modified natural amino acid polypeptide, or the modified non-natural amino acid polypeptide to a host and analyzing a tissue sample from the host, or by incubating the natural amino acid polypeptide, the non-natural amino acid polypeptide, the modified natural amino acid polypeptide, or the modified non-natural amino acid polypeptide with hepatocytes in vitro and analyzing the resulting compound.

As used herein, the term "modified" refers to the presence of an alteration to a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide, or a non-natural amino acid polypeptide. Such alterations or modifications may be obtained by post-synthesis modification of natural amino acids, unnatural amino acids, natural amino acid polypeptides or unnatural amino acid polypeptides, or by co-translation or post-translational modification of natural amino acids, unnatural amino acids, natural amino acid polypeptides or unnatural amino acid polypeptides.

"Unnatural amino acid" refers to an amino acid that is not one of the 20 common amino acids or a pyrolysine or selenocysteine. Other terms that may be used synonymously with the term "unnatural amino acid" are "non-naturally encoded amino acid", "unnatural amino acid", "non-naturally occurring amino acid", "synthetic amino acid", and various hyphenated and non-hyphenated forms thereof. The term "unnatural amino acid" includes, but is not limited to, an amino acid that occurs naturally by modification of naturally encoded amino acids (including, but not limited to, 20 common amino acids or pyrrolysine and selenocysteine) but is not itself incorporated into a growing polypeptide chain by a translation complex. Examples of non-naturally encoded naturally occurring amino acids include, but are not limited to, N-acetylglucosamine-L-serine, N-acetylglucosamine-L-threonine, and O-phosphotyrosine. In addition, the term "unnatural amino acid" includes, but is not limited to, amino acids that do not occur naturally and that are available synthetically or that are available through modification of unnatural amino acids.

As used herein, the term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides in either single-or double-stranded form, as well as polymers thereof. By way of example only, such nucleic acids and nucleic acid polymers include, but are not limited to, (i) analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides, (ii) oligonucleotide analogs, including but not limited to PNA (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.), and (iii) conservatively modified variants thereof, including but not limited to degenerate codon substitutions, as well as complementary sequences and the sequences explicitly indicated. As an example, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al, nucleic Acid Res.19:5081,1991; ohtsuka et al, J.biol. Chem.,260:2605-2608,1985; and Rossolini et al, mol. Cell. Probes 8:91-98,1994).

As used herein, the term "pharmaceutically acceptable" refers to materials that do not abrogate the biological activity or properties of the compound and that are relatively non-toxic, including, but not limited to, salts, binders, adjuvants, excipients, carriers, or diluents, i.e., the material may be administered to an individual without causing an undesirable biological effect or interacting in a deleterious manner with any of the components of the composition in which the material is contained.

In some embodiments, the present disclosure relates to polymers. As used herein, the term "polymer" refers to a molecule consisting of repeating subunits. Such molecules include, but are not limited to, polypeptides, polynucleotides or polysaccharides or polyalkylene glycols. The polymers of the present disclosure may be linear or branched polymeric polyether polyols including, but not limited to, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, and derivatives thereof. The polymer may be an activated polymer (e.g., activated PEG) that facilitates conjugation to another group, such as a polypeptide, linker, or drug linker molecule. The polymer may also terminate in a moiety, such as a non-reactive moiety, for example an alkyl (such as methyl) or an alkoxy (such as methoxy). For example, other exemplary embodiments are listed in the commercial supplier catalog, such as catalog "Polyethylene Glycol AND DERIVATIVES for Biomedical Applications" of SHEARWATER CORPORATION (2001). By way of example only, such polymers have an average molecular weight of about 0.1kDa to about 100 kDa. Such polymers include, but are not limited to, about 100Da to about 100,000Da or more. The molecular weight of the polymer may be from about 100Da to about 100,000Da, including but not limited to about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about, About 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800Da, about 700Da, about 600Da, about 500Da, 400Da, about 300Da, about 200Da, and about 100Da. In some embodiments, the molecular weight of the polymer is from about 100Da to about 50,000Da. In some embodiments, the molecular weight of the polymer is from about 100Da to about 40,000Da. In some embodiments, the molecular weight of the polymer is from about 1,000da to about 40,000da. In some embodiments, the molecular weight of the polymer is from about 2,000da to about 50,000da. In some embodiments, the molecular weight of the polymer is from about 5,000da to about 40,000da. In some embodiments, the molecular weight of the polymer is from about 10,000da to about 40,000da. In some embodiments, the molecular weight of the polymer is in the range of about 100Da to about 10,000 Da. In some embodiments, the molecular weight of the polymer is in the range of about 100Da to about 5,000 Da. In some embodiments, the molecular weight of the polymer is in the range of about 100Da to about 1,000 Da. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, the PEG is a linear PEG. In some embodiments, the PEG is branched PEG. The molecular weight of the linear or branched PEG may be about 1,000Da and about 100,000Da, including but not limited to about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, About 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, and about 1,000Da. In some embodiments, the molecular weight of the linear or branched PEG is about 1,000da to about 50,000da. In some embodiments, the molecular weight of the linear or branched PEG is about 1,000da to about 40,000da. In some embodiments, the molecular weight of the linear or branched PEG is about 5,000da to about 40,000da. In some embodiments, the molecular weight of the linear or branched PEG is about 5,000da to about 20,000da. In other embodiments, the molecular weight of the linear or branched PEG is from about 2,000da to about 50,000da. In some embodiments, the molecular weight of the linear or branched PEG is in the range of about 100Da to about 10,000 Da. In some embodiments, the molecular weight of the linear or branched PEG is in the range of about 100Da to about 5,000 Da. In some embodiments, the molecular weight of the linear or branched PEG is in the range of about 100Da to about 1,000 Da. In some embodiments, the PEG is a linear PEG. In some embodiments, the PEG comprises a defined number of repeating (-alkylene-O-) units, such as 2, 4, 6, 8, 10, 12, 14 or more units (e.g., PEG-2, PEG-4, PEG-6, PEG-8, PEG-10, PEG-12, PEG-14). In some embodiments, the PEG is branched PEG. The term "PEGylated (PEGYLATING)" or "PEGylated (PEGylated)" means covalent bonding of a specific moiety to a polyethylene glycol (PEG) molecule. In some embodiments, the moiety may be present in a drug, a drug linker, a linker, or a polypeptide or protein. In some embodiments, the moiety is a hydroxyl group, carboxylic acid, acyl, or amino group such as that present in a drug, drug linker, or polypeptide. In some embodiments, hydroxyl, carboxylic acid, acyl, or amino groups are present in the amino acid. In some embodiments, the amino acid bearing a hydroxyl group, carboxylic acid, acyl, or amino group is a natural or unnatural amino acid present in a polypeptide (e.g., an antibody), linker, or pharmaceutical linker. The method can include contacting an isolated polypeptide comprising a natural or synthetic amino acid, or contacting a drug linker comprising a natural or synthetic amino acid with a water-soluble polymer (e.g., PEG) comprising a moiety that reacts with the natural or synthetic amino acid. In one non-limiting example, the method can include contacting an isolated anti-TROP 2 polypeptide, an isolated anti-HER 2 polypeptide, an isolated anti-CD 70 polypeptide, an isolated anti-PSMA polypeptide, an isolated anti-HER 3 polypeptide, or an isolated anti-GPC 3 polypeptide, each comprising a natural or synthetic amino acid, with a water-soluble polymer comprising a moiety that reacts with the natural or synthetic amino acid.

The terms "polypeptide", "peptide" or "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, the description for polypeptides applies equally to the description for peptides and the description for proteins and vice versa. The term applies to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are unnatural amino acids. In addition, such "polypeptides", "peptides" and "proteins" include amino acid chains of any length, including full length proteins, including but not limited to antibodies, in which amino acid residues are linked by covalent peptide bonds.

The term "post-translationally modified" refers to any modification of a natural or unnatural amino acid that occurs after such amino acid has been translationally incorporated into a polypeptide chain. Such modifications include, but are not limited to, in vivo co-translational modifications, in vitro co-translational modifications (such as in a cell-free translation system), in vivo post-translational modifications, and in vitro post-translational modifications.

As used herein, the term "prodrug" or "pharmaceutically acceptable prodrug" refers to an agent that does not abrogate the biological activity or properties of the drug and is converted to the parent drug in vivo or in vitro, that is, the material may be administered to an individual without causing an undesirable biological effect or interacting in a deleterious manner with any of the components of the composition in which the material is contained. Prodrugs are typically prodrugs that, upon administration to a subject and subsequent absorption, are converted to the active or more active substance via some process, such as conversion by metabolic pathways. Some prodrugs have chemical groups present on the prodrug that render them less active and/or impart drug solubility or some other property. Once the chemical groups have been cleaved from the prodrug and/or modified, the active drug is produced. The prodrug is converted in vivo to the active drug by enzymatic or non-enzymatic reactions. Prodrugs can provide improved physiochemical properties such as better solubility, enhanced delivery properties such as specific targeting to specific cells, tissues, organs or ligands, and improved therapeutic value of drugs. The beneficial effects of such prodrugs include, but are not limited to, (i) ease of administration compared to the parent drug, (ii) prodrugs can be bioavailable by oral administration while the parent is not, and (iii) prodrugs can also have improved solubility in pharmaceutical compositions compared to the parent drug. Prodrugs include pharmacologically inactive or reduced activity derivatives of the active agent. Prodrugs can be designed to modulate the amount of a drug or bioactive molecule that reaches a desired site of action by manipulating a property of the drug, such as physiochemical, biomedical or pharmacokinetic properties. One non-limiting example of a prodrug is a non-natural amino acid polypeptide that is administered in the form of an ester ("prodrug") to facilitate transport across a cell membrane where water solubility is detrimental to mobility, and then metabolically hydrolyzes to a carboxylic acid (active entity) once inside the cell where water solubility is beneficial. Prodrugs can be designed as reversible drug derivatives that act as modifiers to enhance drug transport to site-specific tissues.

As used herein, the term "prophylactically effective amount" refers to an amount of a composition containing at least one unnatural amino acid polypeptide or at least one modified unnatural amino acid polypeptide that is prophylactically administered to a patient, which will alleviate one or more symptoms of the disease, condition, or disorder being treated to some extent. In such prophylactic applications, such amounts may depend on the health, weight, etc. of the patient. Determination of such prophylactically effective amounts by routine experimentation, including but not limited to, up-dosing clinical trials, is considered well known to those skilled in the art.

The term "recombinant host cell," also referred to as a "host cell," refers to a cell comprising an exogenous polynucleotide, wherein the method for inserting the exogenous polynucleotide into the cell includes, but is not limited to, direct uptake, transduction, f-mating, or other methods known in the art for producing recombinant host cells. By way of example only, such exogenous polynucleotides may be non-integrated vectors, including but not limited to plasmids, or may be integrated into the host genome.

As used herein, the term "spacer" or "spacer element" refers to an atom or functional group that connects a first group to a second group. In some non-limiting embodiments, the spacer is carbonyl (-C (O) -), -C (O) O-, -C (O) N (R) -, -O-, -S-, -N (R) -, wherein each R is H or alkyl. In some embodiments, the spacer is a divalent spacer.

As used herein, the term "subject" refers to an animal that is the subject of treatment, observation or experiment. For example only, the subject may be, but is not limited to, a mammal (including, but not limited to, a human).

As used herein, the term "substantially purified" refers to a component of interest that may be substantially or essentially free of other components that are typically accompanied by or interact with the component of interest prior to purification. By way of example only, a component of interest may be "substantially purified" when a formulation of the component of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about l% (on a dry weight basis) of contaminating components. Thus, a "substantially purified" component of interest may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. By way of example only, the native amino acid polypeptide or the unnatural amino acid polypeptide can be purified from a native cell, or from a host cell in the case of recombinantly produced native amino acid polypeptides or unnatural amino acid polypeptides. By way of example, a preparation of a natural amino acid polypeptide or a non-natural amino acid polypeptide may be "substantially purified" when the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about l% (on a dry weight basis) of contaminating substances. By way of example, when the natural or unnatural amino acid polypeptide is recombinantly produced by a host cell, the natural or unnatural amino acid polypeptide can be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cell. By way of example, when the natural or unnatural amino acid polypeptide is recombinantly produced by a host cell, the natural or unnatural amino acid polypeptide can be present in the culture medium at about 5g/L, about 4g/L, about 3g/L, about 2g/L, about 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L, or about 1mg/L or less of the dry weight of the cell. By way of example, a "substantially purified" natural amino acid polypeptide or non-natural amino acid polypeptide may have a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more, as determined by suitable methods, including, but not limited to, SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

As used herein, the term "therapeutically effective amount" refers to an amount of a composition comprising at least one unnatural amino acid polypeptide and/or at least one modified unnatural amino acid polypeptide that is administered to a patient who has had a disease, condition, or disorder, that is sufficient to cure or at least partially inhibit or to some extent alleviate one or more symptoms of the disease, condition, or disorder being treated. The effectiveness of such compositions depends on conditions including, but not limited to, the severity and course of the disease, disorder or condition, previous treatments, the health status and response of the patient to the drug, and the discretion of the treating physician. By way of example only, a therapeutically effective amount may be determined by routine experimentation, including but not limited to, up-dosing clinical trials.

As used herein, the terms "toxic," "toxic moiety" or "toxic group" or "cytotoxic payload" or "cytotoxic drug" or "drug" refer to a cytotoxic compound that can cause injury, disorder, or death. Toxic moieties include, but are not limited to, drugs comprising or consisting of bicubicin or an analog or derivative thereof.

As used herein, the term "treating" includes alleviating, preventing, alleviating or ameliorating the symptoms of a disease or condition, preventing additional symptoms, ameliorating or preventing the underlying metabolic cause of the symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, alleviating the disease or condition, causing the disease or condition to resolve, alleviating the condition caused by the disease or condition, or stopping the symptoms of the disease or condition. The term "treatment" includes, but is not limited to, prophylactic and/or therapeutic treatment. The term "treating" may refer to reducing, alleviating or ameliorating one or more symptoms or conditions or diseases associated with an antigen-related or associated cancer. The term "treating" may refer to administering an ADC of the present disclosure to a subject in need thereof to reduce, alleviate, ameliorate, alter, alleviate, affect or mitigate an antigen-associated or associated cancer or disease or symptom or condition, or susceptibility to a condition. The term "capable of specifically binding" refers to a protein or peptide (e.g., an antibody) that binds to a predetermined target substance (e.g., an antigen and/or antigen group), such as a target substance expressed on the surface of a cell, and thus the term "binds to a target cell" or "binds to a cancer cell" is understood to refer to a protein or peptide (e.g., an antibody) that binds to a predetermined target substance (e.g., one or more antigens) expressed on such a cell. Typically, a protein or peptide (e.g., an antibody) binds with an affinity of at least about lx10 ⁷ M1 and/or binds with an affinity that is at least twice greater than its affinity to a predetermined target substance or a non-specific control substance other than a closely related target substance (e.g., BSA, casein, non-cancerous cells).

As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in an aqueous solvent. Such water-soluble polymers include, but are not limited to, polyethylene glycol propionaldehyde, mono-C ₁-C₁₀ alkoxy or aryloxy derivatives thereof (described in U.S. patent No. 5,252,714, incorporated herein by reference), mono-methoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N- (2-hydroxypropyl) -methacrylamide, dextran derivatives (including dextran sulfate), polypropylene glycol, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives (including, but not limited to, methylcellulose and carboxymethylcellulose), serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycols and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ether and α - β -poly [ (2-hydroxyethyl) -DL-asparagine, and the like, or mixtures thereof. By way of example only, coupling of such water-soluble polymers to a natural amino acid polypeptide or non-natural polypeptide may result in alterations including, but not limited to, increased water solubility, increased or modulated serum half-life, increased or modulated therapeutic half-life relative to unmodified forms, increased bioavailability, modulated biological activity, prolonged circulation time, modulated immunogenicity, modulated physical related characteristics (including, but not limited to, aggregation and multimerization), altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerization. Furthermore, such water-soluble polymers may or may not have their own biological activity.

As used herein, the term "modulated serum half-life" refers to a positive or negative change in the circulatory half-life of a modified biologically active molecule relative to its unmodified form. By way of example, modified bioactive molecules include, but are not limited to, natural amino acids, unnatural amino acids, natural amino acid polypeptides, or unnatural amino acid polypeptides. As an example, serum half-life is measured by taking blood samples at different time points after administration of a bioactive molecule or modified bioactive molecule and determining the concentration of the molecule in each sample. The correlation of serum concentration with time allows calculation of serum half-life. As an example, the modulated serum half-life may be an extension of serum half-life, which may enable improved dosing regimens or avoid toxic effects. Such an increase in serum may be at least about two times, at least about three times, at least about five times, or at least about ten times. Methods for assessing serum half-life are known in the art and can be used to assess serum half-life of antibodies and antibody drug conjugates of the present disclosure.

As used herein, the term "modulated therapeutic half-life" refers to a positive or negative change in the half-life of a therapeutically effective amount of a modified biologically active molecule relative to its unmodified form. By way of example, modified bioactive molecules include, but are not limited to, natural amino acids, unnatural amino acids, natural amino acid polypeptides, or unnatural amino acid polypeptides. As an example, the therapeutic half-life is measured by measuring the pharmacokinetic and/or pharmacodynamic properties of the molecule at different time points after administration. The extended therapeutic half-life may enable a particular beneficial dosing regimen, a particular beneficial total dose, or avoid undesirable effects. As an example, an extended therapeutic half-life may result from increased potency, increased or decreased binding of the modified molecule to its target, increased or decreased another parameter or mechanism of action of the unmodified molecule, or increased or decreased decomposition of the molecule by an enzyme (such as, by way of example only, a protease). Methods for assessing the therapeutic half-life are known in the art and can be used to assess the therapeutic half-life of the antibodies and antibody drug conjugates of the present disclosure.

Introduction to the invention

Antibody-based therapies have become an important component of the therapy of an increasing number of human malignancies in areas such as oncology, immunology, inflammatory and infectious diseases. In most cases, the basis for therapeutic function is the high specificity and affinity that antibody-based drugs have for their target antigens. Equipping monoclonal antibodies with drugs, toxins or radionuclides is another strategy by which monoclonal antibodies may induce therapeutic effects. By combining the precise targeting specificity of antibodies with the tumor killing ability of toxic effector molecules, immunoconjugates allow sensitive differentiation between target and normal tissues, resulting in fewer side effects than most conventional chemotherapeutic drugs. The toxins used can be conjugated specifically, stably and irreversibly to unique sites in the antibody. This unique conjugation process allows for precise control of the location of the toxin on the antibody as well as the number of toxins conjugated to each antibody. These two features are critical to control the biophysical properties and toxicity associated with ADCs. (see, e.g., jackson D. Et al (2014) PLoS ONE 9 (11): e83865; tian F. Et al (2014) Proc. Natl. Acad. Sci. U.S. A.111 (15): 1766-1771).

Currently, ADCs are advancing the field of cancer treatment, and many ADCs targeting various agents have been approved or are in clinical trials. However, ADCs present challenges due to lack of therapeutic index and toxicity. The linker technology used to attach the cytotoxic drug to the antibody affects the stability of the ADC during systemic circulation. Release of free drug in the circulation, rather than within antigen-expressing cancer cells, can lead to loss of ADC potency, insufficient immunogenic cancer cell death, and increased toxicity. Thus, there is a need to design a stable linker, such as a phosphate-based linker, for drug design and antibody conjugation, as well as for selective release within cancer cells.

ADC antibodies and antibody sequences

The present invention provides novel ADCs comprising antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acids incorporated at any desired position in the heavy and/or light chain amino acid sequence. Furthermore, the invention provides ADCs comprising one or more antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acids that are site-specifically incorporated into heavy and/or light chain amino acid sequences conjugated to a drug via a phosphate-based linker. In some embodiments, the antibody, antibody fragment, or variant thereof binds to a tumor-associated antigen (TAA) selected from the group consisting of PD-1、PD-L1、PSMA、CD70、CD3、HER2、HER3、TROP2、VEGFR、GPC3、EGFR、c-Met(HGFR)、CD33、CD19、CD22、CD25(IL-2Rα)、CD30、CD37、CD46、CD48、CD56(NCAM-1)、CD71( transferrin R), CD74, CD79b, C-D123 (IL-3 Rα), CD138 (multi-ligand glycan-1), CD142, CD166 (ALCAM), CD203C (ENPP 3), CD205 (LY 75), CD221 (IGF-1R), CD262 (TRAIL R2), CD138 (multi-ligand glycan-1), CD276 (B7-H3), mesothelin, epCAM, CEACAM5, CEACAM6, DLL3, ROR1, ROR2, GPNMB, GCC, GUCY c, naPi2B, flt-1, flt-3, folate receptor alpha, tissue factor (TF)、CA6、MUC1、MUC16(CA-125)、BCMA、SLAMF7(CS1)、TIM1、CanAg、Ckit(CD117)、EphA2、Nectin4、SLTRK6、FGFR2、LYPD3(C4.4a)、 cadherin 3, 5T4 (TPBG), STEAP1, PTK7, ephrin-A4, LIV-1 (SLC 39A6 or ZIP 6), SLC1A5, TENB2, ETBR, integrin v3, cripto, AGS-5 (SLC 44A 4), LY6E, AXL, LAMP1, LRRC15, TNF-alpha and MN/CA IX antibodies, antibody fragments or variants. in some embodiments, the antibody, antibody fragment, or variant thereof is a TROP2 antibody, antibody fragment, or variant. In some embodiments, the antibody, antibody fragment, or variant thereof is a HER2 antibody, antibody fragment, or variant. In some embodiments, the antibody, antibody fragment, or variant thereof is a CD3 antibody, antibody fragment, or variant. In some embodiments, the antibody, antibody fragment, or variant thereof is a PSMA antibody, antibody fragment, or variant. In some embodiments, the antibody, antibody fragment, or variant thereof is a CD70 antibody, antibody fragment, or variant. In other embodiments, the invention provides an anti-TROP 2ADC comprising an antibody, antibody fragment, or variant thereof engineered to have one or more unnatural amino acids incorporated at any desired position in the heavy and/or light chain amino acid sequence. In some embodiments, the invention provides an anti-TROP 2ADC comprising one or more antibodies, antibody fragments, or variants thereof, engineered to have site-specific incorporation of one or more unnatural amino acids in heavy and/or light chain amino acid sequences conjugated to a drug via a phosphate-based linker. In other embodiments, the invention provides an anti-HER 2ADC comprising an antibody, antibody fragment, or variant thereof engineered to have one or more unnatural amino acids incorporated at any desired position in the heavy and/or light chain amino acid sequence. In some embodiments, the invention provides an anti-HER 2 ADC comprising one or more antibodies, antibody fragments, or variants thereof, engineered to have site-specific incorporation of one or more unnatural amino acids in the heavy and/or light chain amino acid sequences conjugated to a drug via a phosphate-based linker. In other embodiments, the invention provides anti-CD 3 ADCs comprising antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acids incorporated at any desired position in the heavy and/or light chain amino acid sequence. In some embodiments, the invention provides anti-CD 3 ADCs comprising one or more antibodies, antibody fragments, or variants thereof engineered to have site-specific incorporation of one or more unnatural amino acids in heavy and/or light chain amino acid sequences conjugated to drugs via phosphate-based linkers. In other embodiments, the invention provides anti-PSMA ADCs comprising antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acids incorporated at any desired position in the heavy and/or light chain amino acid sequence. in some embodiments, the invention provides anti-PSMA ADCs comprising one or more antibodies, antibody fragments, or variants thereof engineered to have site-specific incorporation of one or more unnatural amino acids in heavy and/or light chain amino acid sequences conjugated to a drug via a phosphate-based linker. In other embodiments, the invention provides anti-CD 70 ADCs comprising antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acids incorporated at any desired position in the heavy and/or light chain amino acid sequence. In some embodiments, the invention provides anti-CD 70 ADCs comprising one or more antibodies, antibody fragments, or variants thereof engineered to have site-specific incorporation of one or more unnatural amino acids in heavy and/or light chain amino acid sequences conjugated to a drug via a phosphate-based linker. In other embodiments, the invention provides an anti-HER 3 ADC comprising an antibody, antibody fragment, or variant thereof engineered to have one or more unnatural amino acids incorporated at any desired position in the heavy and/or light chain amino acid sequence. in some embodiments, the invention provides an anti-HER 3 ADC comprising one or more antibodies, antibody fragments, or variants thereof, engineered to have site-specific incorporation of one or more unnatural amino acids in the heavy and/or light chain amino acid sequences conjugated to a drug via a phosphate-based linker. In other embodiments, the invention provides anti-GPC 3 ADCs comprising antibodies, antibody fragments, or variants thereof, engineered to have one or more unnatural amino acids incorporated at any desired position in the heavy and/or light chain amino acid sequence. In some embodiments, the invention provides anti-GPC 3 ADCs comprising one or more antibodies, antibody fragments, or variants thereof, engineered to have site-specific incorporation of one or more unnatural amino acids in heavy and/or light chain amino acid sequences conjugated to a drug via a phosphate-based linker.

The antibodies or antibody fragments or variants of the present disclosure may be human, humanized, engineered, non-human, and/or chimeric antibodies or antibody fragments. An antibody or antibody fragment or variant provided herein may comprise two or more amino acid sequences. The first amino acid sequence may comprise a first antibody chain and the second amino acid sequence may comprise a second antibody chain. The first antibody chain may comprise a first amino acid sequence and the second antibody chain may comprise a second amino acid sequence. The chain of an antibody may refer to an antibody heavy chain, an antibody light chain, or a combination of a portion or all of an antibody heavy chain and a portion or all of an antibody light chain. As non-limiting examples, antibodies provided herein comprise a heavy chain or fragment or variant thereof, and a light chain or fragment or variant thereof. The two amino acid sequences of an antibody (including the two antibody chains) may be linked, attached, or linked by one or more disulfide bonds, chemical linkers, peptide linkers, or combinations thereof. Chemical linkers include linkers via unnatural amino acids. Chemical linkers include linkers via one or more unnatural amino acids. The chemical linker may comprise a chemical conjugate. Peptide linkers include any amino acid sequence that links two amino acid sequences. The peptide linker may comprise 1 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more amino acids. The peptide linker may be part of any antibody, including the domains of an antibody, such as variable domains, CDR1, CDR2, CDR3 and/or a combination of CDRs (complementarity determining regions). In some embodiments, the heavy and light chains are connected, attached, or linked, e.g., via a peptide linker. In some cases, the heavy and light chains are linked, for example, by one or more disulfide bonds.

Antibodies, antibody fragments, and antibody variants of the disclosure can interact or bind to antigens on effector cells. Effector cells may include, but are not limited to, immune cells, genetically modified cells with increased or decreased cytotoxic activity, cells involved in host defense mechanisms, anti-inflammatory cells, leukocytes, lymphocytes, macrophages, erythrocytes, platelets, neutrophils, monocytes, eosinophils, basophils, mast cells, NK cells, B cells or T cells. In some embodiments, the immune cells may be T cells, such as cytotoxic T cells or natural killer T cells. The antibody or antibody fragment may interact with a receptor on a T cell, such as, but not limited to, a T Cell Receptor (TCR). TCRs may include tcra, tcrp, tcrγ, and/or tcrδ or tcrζ. The antibodies or antibody fragments of the present disclosure can bind to receptors on lymphocytes, dendritic cells, B cells, macrophages, monocytes, neutrophils and/or NK cells. The antibodies or antibody fragments of the disclosure may bind to a cell surface receptor. The antibodies or antibody fragments of the disclosure can bind to an antigen receptor, such as a TROP2 antigen receptor, or a HER2 antigen receptor, or a CD70 antigen receptor. The antibodies or antibody fragments of the disclosure may be conjugated to a T cell surface antigen.

Some cell surface antigens have high overexpression patterns in a large number of tumors, making them excellent targets in ADC development. Accordingly, the present disclosure provides novel anti-TROP 2 antibodies, anti-HER 2 antibodies, anti-CD 3 antibodies, anti-PSMA antibodies, anti-CD 70 antibodies, anti-HER 3 antibodies, anti-GPC 3 antibodies, or their corresponding antibody fragments and antibody drug conjugates for use as therapeutic agents. Disclosed herein are novel anti-TROP 2 antibodies, antibody fragments, or variants thereof, anti-HER 2 antibodies, antibody fragments, or variants thereof, anti-CD 3 antibodies, antibody fragments, or variants thereof, anti-PSMA antibodies, antibody fragments, or variants thereof, anti-CD 70 antibodies, antibody fragments, or variants thereof, anti-HER 3 antibodies, antibody fragments, or variants thereof, each having at least one unnatural amino acid or an unnatural encoded amino acid. The present invention provides anti-TROP 2 antibodies, antibody fragments or variants thereof, anti-HER 2 antibodies, antibody fragments or variants thereof, anti-CD 3 antibodies, antibody fragments or variants thereof, anti-PSMA antibodies, antibody fragments or variants thereof, anti-CD 70 antibodies, antibody fragments or variants thereof, anti-HER 3 antibodies, antibody fragments or variants thereof, and anti-GPC 3 antibodies, antibody fragments or variants thereof, each of the foregoing having an unnatural amino acid that facilitates conjugation of the antibody to a drug or drug linker.

The antibodies, antibody fragments, or variants provided in the present disclosure may be human, humanized, engineered, non-human, and/or chimeric antibodies or antibody fragments that bind to the extracellular domain of a target antigen, which may be overexpressed in many cancers. Thus, novel antibodies, compositions and antibody drug conjugates for the treatment and/or diagnosis of cancers that express antigens are beneficial, including but not limited to cancers that express TROP2, HER2, CD3, PSMA, CD70, anti-HER 3, and GPC 3.

Antibodies or antibody fragments or variants disclosed herein include, but are not limited to, anti-TROP 2, anti-HER 2, anti-CD 3, anti-PSMA, anti-CD 70, anti-HER 3, and anti-GPC 3 analogs, isoforms, mimetics, fragments, or hybrids. The anti-TROP 2, anti-HER 2, anti-CD 3, anti-PSMA, anti-CD 70, anti-HER 3, and anti-GPC 3 antibodies or antibody fragments or variants of the present disclosure include, but are not limited to Fv, fc, fab and (Fab') ₂, single chain Fv (scFv), diabodies, trivalent antibodies, tetravalent antibodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, and the like.

Antibodies comprising unnatural amino acids are also disclosed herein. In certain embodiments, antibodies or antibody fragments or variants include, but are not limited to Fv, fc, fab and (Fab') ₂, single chain Fv (scFv), diabodies, trivalent antibodies, tetravalent antibodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, and the like. In some embodiments, an anti-TROP 2, anti-HER 2, anti-CD 3, anti-PSMA, anti-CD 70, or anti-GPC 3 antibody or antibody fragment or variant comprises one or more unnatural amino acids.

Non-limiting examples of antibodies or antibody fragments or variants of the present disclosure include the sequences listed in tables 1 to 5.

In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-TROP 2 antibodies or antibody fragments or variants thereof. In certain embodiments, an anti-TROP 2 antibody or antibody fragment or variant disclosed herein may be humanized. anti-TROP 2 antibodies or antibody fragments or variants disclosed herein include, but are not limited to, anti-TROP 2 analogs, isoforms, mimetics, fragments or hybrids. anti-TROP 2 antibodies or antibody fragments or variants of the disclosure include, but are not limited to Fv, fc, fab and (Fab') ₂, single chain Fv (scFv), diabodies, trivalent antibodies, tetravalent antibodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, and the like. The anti-TROP 2 antibodies or antibody fragments or variants of the present disclosure comprise the sequences of SEQ ID NOs 1 to 17 (table 1). The antibodies, fragments or variants of the disclosure may be anti-TROP 2 antibodies, fragments or variants. In certain embodiments, the anti-TROP 2 antibody comprises heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOs 1 to 17. In certain embodiments, the anti-TROP 2 antibody consists of heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOs 1 to 17.

The anti-TROP 2 antibody can comprise a heavy and/or light chain amino acid sequence selected from the sequences of SEQ ID NOs 1 to 17. In some embodiments, the anti-TROP 2 antibody consists of a heavy and/or light chain amino acid sequence selected from the sequences of SEQ ID NOs 1 to 17. In certain embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of any one of SEQ ID NOs 1, 2, 5 and 6 and the light chain amino acid sequence of any one of SEQ ID NOs 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17.

In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 1 and the light chain amino acid sequence of SEQ ID NO. 3. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 1 and the light chain amino acid sequence of SEQ ID NO. 4. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 1 and the light chain amino acid sequence of SEQ ID NO. 7. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 1 and the light chain amino acid sequence of SEQ ID NO. 8. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 1 and the light chain amino acid sequence of SEQ ID NO. 9. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 1 and the light chain amino acid sequence of SEQ ID NO. 10. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 1 and the light chain amino acid sequence of SEQ ID NO. 11. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 1 and the light chain amino acid sequence of SEQ ID NO. 12. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 1 and the light chain amino acid sequence of SEQ ID NO. 13. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 1 and the light chain amino acid sequence of SEQ ID NO. 14. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 1 and the light chain amino acid sequence of SEQ ID NO. 15. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 1 and the light chain amino acid sequence of SEQ ID NO. 16. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 1 and the light chain amino acid sequence of SEQ ID NO. 17.

In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 1 and the two light chain amino acid sequences of SEQ ID NO. 3. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 1 and the two light chain amino acid sequences of SEQ ID NO. 4. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 1 and the two light chain amino acid sequences of SEQ ID NO. 7. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 1 and the two light chain amino acid sequences of SEQ ID NO. 8. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 1 and the two light chain amino acid sequences of SEQ ID NO. 9. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 1 and the two light chain amino acid sequences of SEQ ID NO. 10. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 1 and the two light chain amino acid sequences of SEQ ID NO. 11. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 1 and the two light chain amino acid sequences of SEQ ID NO. 12. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 1 and the two light chain amino acid sequences of SEQ ID NO. 13. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 1 and the two light chain amino acid sequences of SEQ ID NO. 14. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 1 and the two light chain amino acid sequences of SEQ ID NO. 15. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 1 and the two light chain amino acid sequences of SEQ ID NO. 16. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 1 and the two light chain amino acid sequences of SEQ ID NO. 17.

In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence of SEQ ID NO. 3. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence of SEQ ID NO. 4. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence of SEQ ID NO. 7. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence of SEQ ID NO. 8. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence of SEQ ID NO. 9. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence of SEQ ID NO. 10. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence of SEQ ID NO. 11. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence of SEQ ID NO. 12. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence of SEQ ID NO. 13. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence of SEQ ID NO. 14. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence of SEQ ID NO. 15. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence of SEQ ID NO. 16. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence of SEQ ID NO. 17.

In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 2 and the two light chain amino acid sequences of SEQ ID NO. 3. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 2 and the two light chain amino acid sequences of SEQ ID NO. 4. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 2 and the two light chain amino acid sequences of SEQ ID NO. 7. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 2 and the two light chain amino acid sequences of SEQ ID NO. 8. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 2 and the two light chain amino acid sequences of SEQ ID NO. 9. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 2 and the two light chain amino acid sequences of SEQ ID NO. 10. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 2 and the two light chain amino acid sequences of SEQ ID NO. 11. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 2 and the two light chain amino acid sequences of SEQ ID NO. 12. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 2 and the two light chain amino acid sequences of SEQ ID NO. 13. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 2 and the two light chain amino acid sequences of SEQ ID NO. 14. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 2 and the two light chain amino acid sequences of SEQ ID NO. 15. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 2 and the two light chain amino acid sequences of SEQ ID NO. 16. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 2 and the two light chain amino acid sequences of SEQ ID NO. 17.

In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 5 and the light chain amino acid sequence of SEQ ID NO. 3. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 5 and the light chain amino acid sequence of SEQ ID NO. 4. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 5 and the light chain amino acid sequence of SEQ ID NO. 7. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 5 and the light chain amino acid sequence of SEQ ID NO. 8. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 5 and the light chain amino acid sequence of SEQ ID NO. 9. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 5 and the light chain amino acid sequence of SEQ ID NO. 10. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 5 and the light chain amino acid sequence of SEQ ID NO. 11. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 5 and the light chain amino acid sequence of SEQ ID NO. 12. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 5 and the light chain amino acid sequence of SEQ ID NO. 13. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 5 and the light chain amino acid sequence of SEQ ID NO. 14. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 5 and the light chain amino acid sequence of SEQ ID NO. 15. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 5 and the light chain amino acid sequence of SEQ ID NO. 16. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 5 and the light chain amino acid sequence of SEQ ID NO. 17.

In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 5 and the two light chain amino acid sequences of SEQ ID NO. 3. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 5 and the two light chain amino acid sequences of SEQ ID NO. 4. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 5 and the two light chain amino acid sequences of SEQ ID NO. 7. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 5 and the two light chain amino acid sequences of SEQ ID NO. 8. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 5 and the two light chain amino acid sequences of SEQ ID NO. 9. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 5 and the two light chain amino acid sequences of SEQ ID NO. 10. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 5 and the two light chain amino acid sequences of SEQ ID NO. 11. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 5 and the two light chain amino acid sequences of SEQ ID NO. 12. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 5 and the two light chain amino acid sequences of SEQ ID NO. 13. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 5 and the two light chain amino acid sequences of SEQ ID NO. 14. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 5 and the two light chain amino acid sequences of SEQ ID NO. 15. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 5 and the two light chain amino acid sequences of SEQ ID NO. 16. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 5 and the two light chain amino acid sequences of SEQ ID NO. 17.

In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO. 3. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO. 4. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO. 7. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO. 8. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO. 9. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO. 10. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO. 11. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO. 12. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO. 13. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO. 14. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO. 15. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO. 16. In some embodiments, the anti-TROP 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO. 17.

In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 6 and the two light chain amino acid sequences of SEQ ID NO. 3. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 6 and the two light chain amino acid sequences of SEQ ID NO. 4. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 6 and the two light chain amino acid sequences of SEQ ID NO. 7. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 6 and the two light chain amino acid sequences of SEQ ID NO. 8. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 6 and the two light chain amino acid sequences of SEQ ID NO. 9. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 6 and the two light chain amino acid sequences of SEQ ID NO. 10. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 6 and the two light chain amino acid sequences of SEQ ID NO. 11. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 6 and the two light chain amino acid sequences of SEQ ID NO. 12. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 6 and the two light chain amino acid sequences of SEQ ID NO. 13. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 6 and the two light chain amino acid sequences of SEQ ID NO. 14. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 6 and the two light chain amino acid sequences of SEQ ID NO. 15. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 6 and the two light chain amino acid sequences of SEQ ID NO. 16. In some embodiments, the anti-TROP 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 6 and the two light chain amino acid sequences of SEQ ID NO. 17.

In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-CD 70 antibodies or antibody fragments or variants thereof. In certain embodiments, an anti-CD 70 antibody or antibody fragment or variant disclosed herein may be humanized. anti-CD 70 antibodies or antibody fragments or variants disclosed herein include, but are not limited to, anti-CD 70 analogs, isoforms, mimetics, fragments, or hybrids. anti-CD 70 antibodies or antibody fragments or variants of the present disclosure include, but are not limited to Fv, fc, fab and (Fab') ₂, single chain Fv (scFv), diabodies, trivalent antibodies, tetravalent antibodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, and the like.

The anti-CD 70 antibodies or antibody fragments or variants of the present disclosure comprise one or more of the sequences of SEQ ID NOS: 18 to 27 (Table 2). The antibodies, fragments or variants of the disclosure may be anti-CD 70 antibodies, fragments or variants. In certain embodiments, the anti-CD 70 antibody comprises heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOS: 18 to 27. In certain embodiments, the anti-CD 70 antibody consists of heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOS: 18 to 27.

In some embodiments, an anti-CD 70 antibody comprises a heavy chain, wherein the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO. 26, and a light chain, wherein the light chain comprises a variable region having the amino acid sequence of SEQ ID NO. 27. In some embodiments, an anti-CD 70 antibody comprises two heavy chains, wherein each heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO. 26, and two light chains, wherein each light chain comprises a variable region having the amino acid sequence of SEQ ID NO. 27.

In some embodiments, the anti-CD 70 antibody comprises a heavy chain and/or light chain amino acid sequence selected from the sequences of SEQ ID NOS: 18 to 27. In some embodiments, the anti-CD 70 antibody consists of heavy and/or light chain amino acid sequences selected from the sequences of SEQ ID NOS: 18 to 27. In some embodiments, an anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 18 or 20 and the light chain amino acid sequence of any of SEQ ID NO. 19, 21, 22, 23 and 24. In certain embodiments, an anti-CD 70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO. 18, 20 or 25 and two light chain amino acid sequences of any of SEQ ID NO. 19, 21, 22, 23 and 24.

In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 18 and the light chain amino acid sequence of SEQ ID NO. 19. In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 18 and the light chain amino acid sequence of SEQ ID NO. 21. In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 18 and the light chain amino acid sequence of SEQ ID NO. 22. In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 18 and the light chain amino acid sequence of SEQ ID NO. 23. In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 18 and the light chain amino acid sequence of SEQ ID NO. 24.

In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 18 and the two light chain amino acid sequences of SEQ ID NO. 19. In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 18 and the two light chain amino acid sequences of SEQ ID NO. 21. In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 18 and the two light chain amino acid sequences of SEQ ID NO. 22. In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 18 and the two light chain amino acid sequences of SEQ ID NO. 23. In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 18 and the two light chain amino acid sequences of SEQ ID NO. 24.

In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 20 and the light chain amino acid sequence of SEQ ID NO. 19. In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 20 and the light chain amino acid sequence of SEQ ID NO. 21. In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 20 and the light chain amino acid sequence of SEQ ID NO. 22. In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 20 and the light chain amino acid sequence of SEQ ID NO. 23. In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 20 and the light chain amino acid sequence of SEQ ID NO. 24.

In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 20 and the two light chain amino acid sequences of SEQ ID NO. 19. In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 20 and the two light chain amino acid sequences of SEQ ID NO. 21. In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 20 and the two light chain amino acid sequences of SEQ ID NO. 22. In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 20 and the two light chain amino acid sequences of SEQ ID NO. 23. In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 20 and the two light chain amino acid sequences of SEQ ID NO. 24.

In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 25 and the light chain amino acid sequence of SEQ ID NO. 19. In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 25 and the light chain amino acid sequence of SEQ ID NO. 21. In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 25 and the light chain amino acid sequence of SEQ ID NO. 22. In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 25 and the light chain amino acid sequence of SEQ ID NO. 23. In some embodiments, the anti-CD 70 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 25 and the light chain amino acid sequence of SEQ ID NO. 24.

In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 25 and the two light chain amino acid sequences of SEQ ID NO. 19. In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 25 and the two light chain amino acid sequences of SEQ ID NO. 21. In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 25 and the two light chain amino acid sequences of SEQ ID NO. 22. In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 25 and the two light chain amino acid sequences of SEQ ID NO. 23. In some embodiments, the anti-CD 70 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 25 and the two light chain amino acid sequences of SEQ ID NO. 24.

In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-HER 2 antibodies or antibody fragments or variants thereof. In certain embodiments, an anti-HER 2 antibody or antibody fragment or variant disclosed herein may be humanized. anti-HER 2 antibodies or antibody fragments or variants disclosed herein include, but are not limited to, anti-HER 2 analogs, isoforms, mimetics, fragments, or hybrids. anti-HER 2 antibodies or antibody fragments or variants of the present disclosure include, but are not limited to Fv, fc, fab and (Fab') ₂, single chain Fv (scFv), diabodies, trivalent antibodies, tetravalent antibodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, and the like.

The anti-HER 2 antibodies or antibody fragments or variants of the disclosure comprise one or more of the sequences of SEQ ID NOS 28-31 (Table 3). The antibody, fragment or variant of the disclosure may be an anti-HER 2 antibody, fragment or variant. In certain embodiments, the anti-HER 2 antibody comprises heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOS: 28-31. In certain embodiments, the anti-HER 2 antibody consists of heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOS: 28-31.

The anti-HER 2 antibody may comprise a heavy and/or light chain amino acid sequence selected from the sequences of SEQ ID NOs 28 to 31. In some embodiments, the anti-HER 2 antibody consists of heavy and/or light chain amino acid sequences selected from the sequences of SEQ ID NOS: 28-31. In certain embodiments, an anti-HER 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 28 or 29 and the light chain amino acid sequence of SEQ ID NO. 30 or 31.

In some embodiments, the anti-HER 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 28 and the light chain amino acid sequence of SEQ ID NO. 30. In some embodiments, the anti-HER 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 28 and the light chain amino acid sequence of SEQ ID NO. 31.

In some embodiments, the anti-HER 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 28 and the two light chain amino acid sequences of SEQ ID NO. 30. In some embodiments, the anti-HER 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 28 and the two light chain amino acid sequences of SEQ ID NO. 31.

In some embodiments, the anti-HER 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 29 and the light chain amino acid sequence of SEQ ID NO. 30. In some embodiments, the anti-HER 2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 29 and the light chain amino acid sequence of SEQ ID NO. 31.

In some embodiments, the anti-HER 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 29 and the two light chain amino acid sequences of SEQ ID NO. 30. In some embodiments, the anti-HER 2 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 29 and the two light chain amino acid sequences of SEQ ID NO. 31.

In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-PSMA antibodies or antibody fragments, or variants thereof. In certain embodiments, an anti-PSMA antibody or antibody fragment or variant disclosed herein may be humanized. anti-PSMA antibodies or antibody fragments or variants disclosed herein include, but are not limited to, anti-PSMA analogs, isoforms, mimetics, fragments, or hybrids. anti-PSMA antibodies or antibody fragments or variants of the disclosure include, but are not limited to Fv, fc, fab and (Fab') ₂, single chain Fv (scFv), diabodies, trivalent antibodies, tetravalent antibodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, and the like.

The anti-PSMA antibodies or antibody fragments or variants of the present disclosure comprise one or more sequences of SEQ ID NOs 32 to 45 (table 4). The antibodies, fragments, or variants of the disclosure may be anti-PSMA antibodies, fragments, or variants. In certain embodiments, the anti-PSMA antibody comprises heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOS: 39 to 45. In certain embodiments, the anti-PSMA antibody consists of heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOS: 39 to 45.

The anti-PSMA antibody may comprise heavy and/or light chain amino acid sequences selected from the sequences of SEQ ID NOs 39 to 45. In some embodiments, the anti-PSMA antibody consists of heavy and/or light chain amino acid sequences selected from the sequences of SEQ ID NOS: 39 to 45. In some embodiments, the anti-PSMA antibody comprises the heavy chain amino acid sequence of SEQ ID NO:39 and the light chain amino acid sequence of any of SEQ ID NO:40, 41, 42 or 43. In certain embodiments, the anti-PSMA antibody comprises the two heavy chain amino acid sequences of SEQ ID NO:39 and the two light chain amino acid sequences of any of SEQ ID NO:40, 41, 42 or 43.

In some embodiments, the anti-PSMA antibody comprises the heavy chain amino acid sequence of SEQ ID NO:39 and the light chain amino acid sequence of SEQ ID NO: 40. In some embodiments, the anti-PSMA antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 39 and the light chain amino acid sequence of SEQ ID NO. 41. In some embodiments, the anti-PSMA antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 39 and the light chain amino acid sequence of SEQ ID NO. 43. In some embodiments, the anti-PSMA antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 39 and the light chain amino acid sequence of SEQ ID NO. 43.

In some embodiments, the anti-PSMA antibody comprises the two heavy chain amino acid sequences of SEQ ID NO:39 and the two light chain amino acid sequences of SEQ ID NO: 40. In some embodiments, the anti-PSMA antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 39 and the two light chain amino acid sequences of SEQ ID NO. 41. In some embodiments, the anti-PSMA antibody comprises the two heavy chain amino acid sequences of SEQ ID NO:39 and the two light chain amino acid sequences of SEQ ID NO: 42. In some embodiments, the anti-PSMA antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 39 and the two light chain amino acid sequences of SEQ ID NO. 43.

In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-HER 3 antibodies or antibody fragments or variants thereof. In certain embodiments, an anti-HER 3 antibody or antibody fragment or variant disclosed herein may be humanized. The anti-HER 3 antibodies or antibody fragments or variants disclosed herein include, but are not limited to, anti-HER 3 analogs, isoforms, mimetics, fragments, or hybrids. anti-HER 3 antibodies or antibody fragments or variants of the disclosure include, but are not limited to Fv, fc, fab and (Fab') ₂, single chain Fv (scFv), diabodies, trivalent antibodies, tetravalent antibodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, and the like. The anti-HER 3 antibodies or antibody fragments or variants of the disclosure comprise the sequences of SEQ ID NOS: 46 to 58 (Table 5). The antibody, fragment or variant of the disclosure may be an anti-HER 3 antibody, fragment or variant. In certain embodiments, the anti-HER 3 antibody comprises heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOS: 46 to 58. In certain embodiments, the anti-HER 3 antibody consists of heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOS: 46 to 58.

The anti-HER 3 antibody may comprise a heavy and/or light chain amino acid sequence selected from the sequences of SEQ ID NOs 46 to 58. In some embodiments, the anti-HER 3 antibody consists of heavy and/or light chain amino acid sequences selected from the sequences of SEQ ID NOS: 46 to 58. In some embodiments, an anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 46 or 58 and the light chain amino acid sequence of any one of SEQ ID NO. 47 to 57.

In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 46 and the light chain amino acid sequence of SEQ ID NO. 47. In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 46 and the light chain amino acid sequence of SEQ ID NO. 48. In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 46 and the light chain amino acid sequence of SEQ ID NO. 49. In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 46 and the light chain amino acid sequence of SEQ ID NO. 50. In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 46 and the light chain amino acid sequence of SEQ ID NO. 51. In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 46 and the light chain amino acid sequence of SEQ ID NO. 52. In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 46 and the light chain amino acid sequence of SEQ ID NO. 53. In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 46 and the light chain amino acid sequence of SEQ ID NO. 54. In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 46 and the light chain amino acid sequence of SEQ ID NO. 55. In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 46 and the light chain amino acid sequence of SEQ ID NO. 56. In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 46 and the light chain amino acid sequence of SEQ ID NO. 57.

In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 46 and the two light chain amino acid sequences of SEQ ID NO. 47. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 46 and the two light chain amino acid sequences of SEQ ID NO. 48. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 46 and the two light chain amino acid sequences of SEQ ID NO. 49. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 46 and the two light chain amino acid sequences of SEQ ID NO. 50. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 46 and the two light chain amino acid sequences of SEQ ID NO. 51. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 46 and the two light chain amino acid sequences of SEQ ID NO. 52. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 46 and the two light chain amino acid sequences of SEQ ID NO. 53. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 46 and the two light chain amino acid sequences of SEQ ID NO. 54. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 46 and the two light chain amino acid sequences of SEQ ID NO. 55. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 46 and the two light chain amino acid sequences of SEQ ID NO. 56. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 46 and the two light chain amino acid sequences of SEQ ID NO. 57.

In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 58 and the light chain amino acid sequence of SEQ ID NO. 47. In some embodiments, an anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 58 and the light chain amino acid sequence of SEQ ID NO. 48. In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 58 and the light chain amino acid sequence of SEQ ID NO. 49. In some embodiments, an anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 58 and the light chain amino acid sequence of SEQ ID NO. 50. In some embodiments, an anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 58 and the light chain amino acid sequence of SEQ ID NO. 51. In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 58 and the light chain amino acid sequence of SEQ ID NO. 52. In some embodiments, an anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 58 and the light chain amino acid sequence of SEQ ID NO. 53. In some embodiments, an anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 58 and the light chain amino acid sequence of SEQ ID NO. 54. In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 58 and the light chain amino acid sequence of SEQ ID NO. 55. In some embodiments, an anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 58 and the light chain amino acid sequence of SEQ ID NO. 56. In some embodiments, the anti-HER 3 antibody comprises the heavy chain amino acid sequence of SEQ ID NO. 58 and the light chain amino acid sequence of SEQ ID NO. 57.

In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 58 and the two light chain amino acid sequences of SEQ ID NO. 47. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 58 and the two light chain amino acid sequences of SEQ ID NO. 48. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 58 and the two light chain amino acid sequences of SEQ ID NO. 49. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 58 and the two light chain amino acid sequences of SEQ ID NO. 50. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 58 and the two light chain amino acid sequences of SEQ ID NO. 51. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 58 and the two light chain amino acid sequences of SEQ ID NO. 52. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 58 and the two light chain amino acid sequences of SEQ ID NO. 53. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 58 and the two light chain amino acid sequences of SEQ ID NO. 54. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 58 and the two light chain amino acid sequences of SEQ ID NO. 55. In some embodiments, an anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 58 and the two light chain amino acid sequences of SEQ ID NO. 56. In some embodiments, the anti-HER 3 antibody comprises the two heavy chain amino acid sequences of SEQ ID NO. 58 and the two light chain amino acid sequences of SEQ ID NO. 57.

In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-GPC 3 antibodies or antibody fragments, or variants thereof. In certain embodiments, an anti-GPC 3 antibody or antibody fragment or variant disclosed herein can be humanized. anti-GPC 3 antibodies or antibody fragments or variants disclosed herein include, but are not limited to, anti-GPC 3 analogs, isoforms, mimetics, fragments, or hybrids. anti-GPC 3 antibodies or antibody fragments or variants of the present disclosure include, but are not limited to Fv, fc, fab and (Fab') ₂, single chain Fv (scFv), diabodies, trivalent antibodies, tetravalent antibodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, and the like.

In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-CD 3 antibodies or antibody fragments or variants thereof. In certain embodiments, an anti-CD 3 antibody or antibody fragment or variant disclosed herein may be humanized. anti-CD 3 antibodies or antibody fragments or variants disclosed herein include, but are not limited to, anti-CD 3 analogs, isoforms, mimetics, fragments, or hybrids. anti-CD 3 antibodies or antibody fragments or variants of the present disclosure include, but are not limited to Fv, fc, fab and (Fab') ₂, single chain Fv (scFv), diabodies, trivalent antibodies, tetravalent antibodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, and the like. The anti-CD 3 antibodies or antibody fragments or variants of the present disclosure comprise one or more sequences as disclosed, for example, in WO2020/047176, the contents of which are hereby incorporated by reference in their entirety.

Table 1. Anti-TROP 2 Heavy (HC) and Light (LC) chain amino acid sequences, including sequences having amber sites for unnatural amino acid incorporation. Also disclosed are all of the sequences in Table 1 wherein X is substituted with any unnatural amino acid, all of the sequences in Table 1 wherein any amino acid is substituted with any unnatural amino acid, all of the sequences in Table 1 wherein X is pAF, all of the heavy chain sequences in Table 1 wherein the unnatural amino acid is site-specifically incorporated at position 114 according to Kabat numbering, as is well known to the skilled artisan, and all of the heavy chain sequences in Table 1 wherein EEM is substituted with DEL. WT wild type, HC heavy chain, LC light chain, X represents unnatural amino acid.

Table 2. Anti-CD 70 Heavy Chain (HC) and Light Chain (LC) amino acid sequences, including sequences having amber sites for unnatural amino acid incorporation. Also disclosed are all of the sequences in Table 2 wherein X is substituted with any unnatural amino acid, all of the sequences in Table 2 wherein any amino acid is substituted with any unnatural amino acid, all of the sequences in Table 2 wherein X is pAF, all of the heavy chain sequences in Table 2 wherein the unnatural amino acid is site-specifically incorporated at position 114 according to Kabat numbering, as is well known to the skilled artisan, and all of the heavy chain sequences in Table 2 wherein EEM is substituted with DEL. WT wild type, HC heavy chain, LC light chain, X represents unnatural amino acid.

Table 3. Anti-HER 2 Heavy (HC) and Light (LC) chain amino acid sequences, including sequences with amber sites for unnatural amino acid incorporation. Also disclosed are all of the sequences in Table 3 wherein X is substituted with any unnatural amino acid, all of the sequences in Table 3 wherein any amino acid is substituted with any unnatural amino acid, all of the sequences in Table 3 wherein X is pAF, all of the heavy chain sequences in Table 3 wherein the unnatural amino acid is site-specifically incorporated at position 114 according to Kabat numbering, as is well known to the skilled artisan, and all of the heavy chain sequences in Table 3 wherein DEL is substituted with EEM. WT wild type, HC heavy chain, LC light chain, X represents unnatural amino acid.

Table 4. Anti-PSMA Heavy (HC) and Light (LC) chain amino acid sequences, including sequences with amber sites for unnatural amino acid incorporation. Also disclosed are all of the sequences in Table 4 wherein X is substituted with any unnatural amino acid, all of the sequences in Table 4 wherein any amino acid is substituted with any unnatural amino acid, all of the sequences in Table 4 wherein X is pAF, all of the heavy chain sequences in Table 4 wherein the unnatural amino acid is site-specifically incorporated at position 114 according to Kabat numbering, as is well known to the skilled artisan, and all of the heavy chain sequences in Table 4 wherein DEL is substituted with EEM. WT wild type, HC heavy chain, LC light chain, X represents unnatural amino acid.

Table 5. Anti-HER 3 Heavy (HC) and Light (LC) chain amino acid sequences, including sequences with amber sites for unnatural amino acid incorporation. Also disclosed are all of the sequences in Table 5 wherein X is substituted with any unnatural amino acid, all of the sequences in Table 5 wherein any amino acid is substituted with any unnatural amino acid, all of the sequences in Table 5 wherein X is pAF, all of the heavy chain sequences in Table 5 wherein the unnatural amino acid is site-specifically incorporated at position 114 according to Kabat numbering, as is well known to the skilled artisan, and all of the heavy chain sequences in Table 5 wherein EEM is substituted with DEL. WT wild type, HC heavy chain, LC light chain, X represents unnatural amino acid.

Unnatural amino acids

The present disclosure provides antibodies, antibody fragments, or variants comprising at least one unnatural amino acid. The introduction of at least one unnatural amino acid into an antibody can allow for the use of conjugation chemistry that involves specific chemical reactions with one or more unnatural amino acids, but not with the 20 commonly found.

Unnatural amino acid site selection is based on surface exposure/site accessibility within the antibody, and hydrophobic or neutral amino acid sites are selected to preserve charge on the antibody. Methods for inserting unnatural amino acids into a site in a protein are described, for example, in WO 2010/01735 and WO 2005/074650. The present disclosure employs such methods and techniques. The unnatural amino acids used in the methods and compositions described herein have at least one of four properties, (1) at least one functional group on the side chain of the unnatural amino acid has at least one property and/or activity that is substantially chemically inert to 20 common genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), and (4) the unnatural amino acid can be stably incorporated into polypeptides, preferably with stability comparable to naturally occurring amino acids or stability under typical physiological conditions, and further preferably such incorporation can occur via in vivo systems, and (4) the unnatural amino acid comprises at least one property and/or activity that is orthogonal to the chemical reactivity of naturally occurring amino acids found in polypeptides comprising the unnatural amino acid, (2) the introduced unnatural amino acid is substantially chemically inert to 20 common genetically encoded amino acids, preferably with stability comparable to naturally occurring amino acids or stability under typical physiological conditions, and further preferably such incorporation can occur via in vivo systems, and (4) the unnatural amino acid comprises an oxime functional group that is not at about pH or can be modified by the nature of course at a biologically preferred amino acid at a site of interest of about pH 8 or a non-labile amino acid group. Any number of unnatural amino acids can be introduced into a polypeptide. The unnatural amino acid can also include a protected or masked oxime or a protected or masked group that can be converted to an oxime group after the protected group is deprotected or the masked group is unmasked. Unnatural amino acids can also include protected or masked carbonyl or dicarbonyl groups that can be converted to carbonyl or dicarbonyl groups after deprotection of the protected groups or unmasking of the masked groups, and thus can be used to react with hydroxylamine or oxime to form oxime groups. Oxime-based unnatural amino acids can be synthesized by methods well known in the art (see, e.g., WO2013/185117 and WO 2005/074650) including (a) reacting a hydroxylamine-containing unnatural amino acid with a carbonyl-or dicarbonyl-containing reagent, (b) reacting a carbonyl-or dicarbonyl-containing unnatural amino acid with a hydroxylamine-containing reagent, or (c) reacting an oxime-containing unnatural amino acid with certain carbonyl-or dicarbonyl-containing reagents.

In some embodiments, the selection of non-naturally encoded amino acid sites is based on surface exposure. For example, one possible site is an amino acid having a solvent accessible surface area ratio of 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more. In some embodiments, one possible site is an amino acid having a solvent accessible surface area ratio of about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% or more. Solvent accessible surface areas can be calculated based on DSSP programs [ Biopolymers,22,2577-2637 (1983) ], using crystal structure analysis data files of antibodies or antibody fragments registered in the Protein Database (PDB).

The ratio of solvent accessible surface area of an amino acid residue of interest can be calculated by dividing the solvent accessible surface area of the antibody structure calculated above by the solvent accessible surface area of alanine-X-alanine (X represents the amino acid residue of interest). In this regard, there are cases in which two or more PDB files exist on one protein, and any of them may be used in the present invention.

Alternatively, the solvent accessibility of an amino acid may be determined by a solvent accessibility test, wherein the functional group (sulfhydryl, amino or carbonyl group) on the amino acid is functionalized when treated with an electrophile or nucleophile or the like. Based on the test results, when at least 50% of the functional groups are functionalized in the test, the functional groups (i.e., thiol, amino, or carbonyl groups) may be referred to as being at least 50% solvent accessible, for example. In some embodiments, the unnatural amino acid site is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% solvent accessible. Examples of solvent accessibility tests include, but are not limited to, propargylation of surface thiol groups, or reaction of alpha-bromopyruvate with surface thiol groups, and the like.

Unnatural amino acids that can be used in the methods and compositions described herein include, but are not limited to, amino acids comprising amino acids with new functionalities, amino acids that interact covalently or non-covalently with other molecules, glycosylated amino acids (such as sugar-substituted serine), other carbohydrate-modified amino acids, ketone-containing amino acids, aldehyde-containing amino acids, amino acids comprising polyethylene glycol or other polyethers, heavy atom-substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with extended side chains compared to natural amino acids (including, but not limited to polyethers or long chain hydrocarbons, including, but not limited to, greater than about 5 or greater than about 10 carbons), carbon-linked sugar-containing amino acids, redox-active amino acids, amino-thio acid-containing amino acids, and amino acids comprising one or more toxic moieties.

In some embodiments, disclosed herein are antibodies comprising one or more unnatural amino acids. The one or more unnatural amino acids can be encoded by a codon that does not encode one of the twenty natural amino acids. One or more unnatural amino acids can be encoded by a nonsense codon (stop codon). The stop codon may be an amber codon. The amber codon may comprise the UAG sequence. The stop codon may be an ocher codon. The ocher codon may comprise a UAA sequence. The stop codon may be an opal or an amber codon. The opal or amber codon may comprise the UGA sequence. One or more unnatural amino acids can be encoded by a four base codon.

Unnatural amino acids of the present disclosure include, but are not limited to, 1) substituted phenylalanine and tyrosine analogs such as 4-amino-L-phenylalanine, 4-acetyl-L-phenylalanine, 4-azido-L-phenylalanine, 4-nitro-L-phenylalanine, 3-methoxy-L-phenylalanine, 4-isopropyl-L-phenylalanine, 3-nitro-L-tyrosine, O-methyl-L-tyrosine, and O-phosphotyrosine, 2) photocrosslinkable amino acids, e.g., amino acids having aryl azide or benzophenone groups such as 4-azidophenylalanine or 4-benzoylphenylalanine, 3) amino acids having unique chemical reactivity such as 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine, O-allyl-L-tyrosine, O-2-propyn-1-yl-L-tyrosine, N- (ethylsulfanyl) thiocarbonyl-L-phenylalanine and p- (3-oxobutanoyl) -L-phenylalanine, 4) amino acids containing heavy atoms, for example for phasing in X-ray crystallography, such as 4-iodo-L-phenylalanine or 4-bromo-L-phenylalanine, 5) redox active amino acids, such as 3, 4-dihydroxy-L-phenylalanine, 6) fluorinated amino acids, such as 2-fluorophenylalanine (e.g., 2-fluoro-L-phenylalanine), 3-fluorophenylalanine (e.g., 3-fluoro-L-phenylalanine) or 4-fluorophenylalanine (e.g., 4-fluoro-L-phenylalanine), 7) fluorescent amino acids, such as those containing naphthyl, Dansyl or an amino acid of the 7-aminocoumarin side chain, 8) photocleavable or photoisomerizable amino acids, such as an amino acid containing an azobenzyl or nitrobenzyl group, for example, an azobenzyl-or nitrobenzyl-containing cysteine, Serine or tyrosine, 9) beta-amino acids (e.g., beta ² or beta ³ amino acids), 10) homoamino acids such as homoglutamine (e.g., beta-homoglutamine) or homophenylalanine (e.g., beta-homophenylalanine), 11) proline or pyruvic acid derivatives, 12) 3-substituted alanine derivatives, 14) glycine derivatives, 15) linear core amino acids, 16) diamino acids, 17) D-amino acids, 18) N-methyl amino acids, 19) phosphotyrosine mimetics such as carboxymethylphenylalanine (pCmF) (e.g., 4-carboxymethyl-L-phenylalanine), 20) 2-aminocaprylic acid, and 21) amino acids containing a sugar moiety such as N-acetyl-L-glucosamine-L-serine, beta-N-acetylglucosamine-O-serine, N-acetyl-L-galactosamine-L-serine, alpha-N-acetylgalactosamine-O-serine, O- (3-O-D-galactosyl-N-acetyl-beta-D-galactosamine) -L-serine, N-acetyl-L-glucosamine-L-threonine, alpha-N-acetylgalactosamine-O-threonine, 3-O- (N-acetyl-beta-D-glucosamine) -L-threonine, N-acetyl-L-glucosamine-L-asparagine, N4- (beta-N-acetyl-D-glucosamine) -L-asparagine, and O- (mannosyl) -L-serine, wherein the naturally occurring N-bond or O-bond between amino acid and sugar is covalently bound by a covalent bond not normally found in nature (including but not limited to olefins, Oxime, thioether, amide, etc.), or amino acids containing sugars that are unusual in naturally occurring polypeptides, such as 2-deoxy-glucose, 2-deoxy-galactose, etc. Specific examples of unnatural amino acids include, but are not limited to, p-acetylphenylalanine (4-acetylphenylalanine) (including 4-acetyl-L-phenylalanine, also referred to herein as p-acetyl-L-phenylalanine (pAF)), 4-borophenylalanine (pBoF) (e.g., 4-boro-L-phenylalanine), 4-propargyloxyphenylalanine (pPrF) (e.g., 4-propargyloxy-L-phenylalanine), O-methyltyrosine (e.g., O-methyl-L-tyrosine), 3- (2-naphthyl) alanine (NapA) (e.g., 3- (2-naphthyl) -L-alanine), and, 3-methylphenylalanine (e.g., 3-methyl-L-phenylalanine), O-allyltyrosine (e.g., O-allyl-L-tyrosine), O-isopropyltyrosine (e.g., O-isopropyl-L-tyrosine), dopamine (e.g., L-dopa), 4-isopropylphenylalanine (e.g., 4-isopropyl-L-phenylalanine), 4-azidophenylalanine (pAz) (e.g., 4-azido-L-phenylalanine), 4-benzoylphenylalanine (pBpF) (e.g., 4-benzoyl-L-phenylalanine), O-phosphoserine (e.g., O-phospho-L-serine), O-phosphotyrosine (e.g., O-phospho-L-tyrosine), 4-iodophenylalanine (pIF) (e.g., 4-iodo-L-phenylalanine, 4-bromophenylalanine (e.g., 4-bromo-L-phenylalanine), 4-aminophenylalanine (e.g., 4-amino-L-phenylalanine), 4-cyanophenylalanine (pCNF) (e.g., 4-cyano-L-phenylalanine), (8-hydroxyquinolin-3-yl) alanine (HQA) (e.g., (8-hydroxyquinolin-3-yl) -L-alanine), and (2, 2-bipyridin-5-yl) alanine (BipyA) (e.g., (2, 2-bipyridin-5-yl) -L-alanine), etc. Additional unnatural amino acids are disclosed in Liu et al (2010) Annu Rev Biochem,79:413-44, wang et al (2005) ANGEW CHEMINT ED,44:34-66, and published International applications ：WO 2012/166560、WO 2012/166559、WO 2011/028195、WO 2010/037062、WO 2008/083346、WO 2008/077079、WO 2007/094916、WO 2007/079130、WO 2007/070659 and WO 2007/059312, the entire contents of each of which are hereby incorporated by reference in their entirety. In some embodiments, the one or more unnatural amino acids can be p-acetylphenylalanine. in some more specific embodiments, the one or more unnatural amino acids can be p-acetyl-L-phenylalanine (pAF).

In some embodiments, the one or more unnatural amino acids are selected from the group consisting of: 4-acetylphenylalanine, 3-O- (N-acetyl-beta-D-glucosamine) threonine, N4- (beta-N-acetyl-D-glucosamine) asparagine, O-allyltyrosine, alpha-N-acetylgalactosamine-O-serine, alpha-N-acetylgalactosamine-O-threonine, 2-aminocaprylic acid, 2-aminophenylalanine, 3-aminophenylalanine, 4-aminophenylalanine, 2-aminotyrosine, 3-aminotyrosine, 4-azidophenylalanine, 4-benzoylphenylalanine, (2, 2-bipyridyl-5-yl) alanine, 3-borophenylalanine, 4-bromophenylalanine, p-carboxymethylphenylalanine, 4-carboxyphenylalanine, p-cyanophenylalanine, 3, 4-dihydroxyphenylalanine, 4-ethynylphenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, O- (3-O-D-galactosyl-N-acetyl-beta-D-galactosamine), homoserine, (3-hydroxy-8-phenylalanine, 3-hydroxy-isopropyl-phenylalanine, 3-hydroxy-8-phenylalanine, 6-hydroxy-isopropyl-phenylalanine, 3-phenyl-hydroxy-phenylalanine, L-isopropyl-8-phenylalanine, O-isopropyl tyrosine, 3-isopropyl tyrosine, O-mannopyranosylserine, 2-methoxyphenylalanine, 3-methoxyphenylalanine, 4-methoxyphenylalanine, 3-methylphenylalanine, O-methyltyrosine, 3- (2-naphthyl) alanine, 5-nitrohistidine, 4-nitroleucine, 2-nitrophenylalanine, 3-nitrophenylalanine, 4-nitrotryptophan, 5-nitrotryptophan, 6-nitrotryptophan, 7-nitrotryptophan, 2-nitrotyrosine, 3-nitrotyrosine, O-phosphoserine, O-phosphotyrosine, 4-propargyloxyphenylalanine, O-2-propargyl-1-yl tyrosine, 4-sulfophenylalanine and O-sulfotyrosine.

In some further embodiments, the one or more unnatural amino acids are selected from the group consisting of 4-acetyl-L-phenylalanine (para-acetyl-L-phenylalanine (pAF)), 3-O- (N-acetyl-beta-D-glucosamine) -L-threonine, N4- (beta-N-acetyl-D-glucosamine) -L-asparagine, O-allyl-L-tyrosine, alpha-N-acetylgalactosamine-O-L-serine, alpha-N-acetylgalactosamine-O-L-threonine, 2-aminocaprylic acid, 2-amino-L-phenylalanine, 3-amino-L-phenylalanine, 4-amino-L-phenylalanine, 2-amino-L-tyrosine, 3-amino-L-tyrosine, 4-azido-L-phenylalanine, 4-benzoyl-L-phenylalanine, (2, 2-bipyridyl-5-yl) -L-alanine, 3-boron-L-phenylalanine, 4-bromo-L-phenylalanine, carboxy-L-phenylalanine, p-cyano-phenylalanine, p-amino-L-phenylalanine, carboxy-L-phenylalanine, and combinations thereof, 3, 4-dihydroxy-L-phenylalanine (L-DOPA), 4-ethynyl-L-phenylalanine, 2-fluoro-L-phenylalanine, 3-fluoro-L-phenylalanine, 4-fluoro-L-phenylalanine, O- (3-O-D-galactosyl-N-acetyl-beta-D-galactosamine) -L-serine, L-homoglutamine, (8-hydroxyquinoline-3-yl) -L-alanine, 4-iodo-L-phenylalanine, 4-isopropyl-L-phenylalanine, O-isopropyl-L-tyrosine, 3-isopropyl-L-tyrosine, O-mannopyranosyl-L-serine, 2-methoxy-L-phenylalanine, 3-methoxy-L-phenylalanine, 4-L-phenylalanine, 3-methyl-L-phenylalanine, 3- (2-naphtyl) -L-alanine, 5-nitro-L-histidine, 4-nitro-L-leucine, 2-nitro-L-phenylalanine, 4-nitro-L-phenylalanine, 4-nitro-L-tryptophan, 5-nitro-L-tryptophan, 6-nitro-L-tryptophan, 7-nitro-L-tryptophan, 2-nitro-L-tyrosine, 3-nitro-L-tyrosine, O-phospho-L-serine, O-phospho-L-tyrosine, 4-propargyloxy-L-phenylalanine, O-2-propargyl-1-yl-L-tyrosine, 4-sulfo-L-phenylalanine and O-sulfo-L-tyrosine. In some embodiments, the one or more unnatural amino acids can be p-acetyl-L-phenylalanine (pAF). Thus, in some embodiments, each of the one or more unnatural amino acids is a pAF.

In certain embodiments of the present disclosure, an antibody having at least one unnatural amino acid comprises at least one post-translational modification. In one embodiment, the at least one post-translational modification includes attaching a molecule comprising a second reactive group (including but not limited to a water-soluble polymer, a derivative of polyethylene glycol, a drug, a second protein or polypeptide analog, an antibody or antibody fragment, a bioactive agent, a small molecule, or any combination of the foregoing or any other desired compound or substance) to the at least one unnatural amino acid comprising the first reactive group using chemical methods known to those of ordinary skill in the art as being suitable for the particular reactive group. For example, the first reactive group is an alkynyl moiety (including but not limited to the unnatural amino acid p-propargyloxyphenylalanine, where propargyl groups are sometimes also referred to as acetylene moieties), and the second reactive group is an azido moiety, and utilizes [3+2] cycloaddition chemistry. In another example, the first reactive group is an azido moiety (including but not limited to unnatural amino acid para-azido-L-phenylalanine) and the second reactive group is an alkynyl moiety. In certain embodiments of the modified antibody polypeptides of the present disclosure, at least one unnatural amino acid (including, but not limited to, one that contains a ketone functional group) is used that comprises at least one post-translational modification, where the at least one post-translational modification comprises a sugar moiety. In certain embodiments, the post-translational modification is performed in vivo in a eukaryotic cell or a non-eukaryotic cell. In other embodiments, the post-translational modification is performed in vitro. In another embodiment, the post-translational modification is performed in vitro and in vivo.

In some embodiments, unnatural amino acids can be modified to incorporate chemical groups. In some embodiments, unnatural amino acids can be modified to incorporate ketone groups. The one or more unnatural amino acids can comprise at least one oxime, carbonyl, dicarbonyl, hydroxylamine group, or a combination thereof. The one or more unnatural amino acids can comprise at least one carbonyl, dicarbonyl, alkoxy-amine, hydrazine, acyclic alkene, acyclic alkyne, cyclooctyne, aryl/alkyl azide, norbornene, cyclopropene, trans-cyclooctene, or tetrazine functional group, or a combination thereof.

In some embodiments disclosed herein, the unnatural amino acid is site-specifically incorporated into an antibody, antibody fragment, or variant. In some embodiments, the unnatural amino acid is site-specifically incorporated into an antibody, antibody fragment, or variant. Methods for incorporating unnatural amino acids into molecules (e.g., proteins, polypeptides, or peptides) are disclosed in U.S. Pat. Nos. 7,332,571, 7,928,163, 7,696,312, 8,008,456, 8,048,988, 8,809,511, 8,859,802, 8,791,231, 8,476,411, or 9,637,411 (each of which is incorporated herein by reference in its entirety) and in the examples herein. One or more unnatural amino acids can be incorporated by methods known in the art. For example, a cell-based system or a cell-free system can be used, and an auxotrophic strain can also be used in place of the engineered tRNA and synthetase. In certain embodiments, orthogonal tRNA synthetases are used as disclosed, for example, in WO2002085923A2, WO2002086075A2, WO2004035743A2, WO2007021297A1, WO2006068802A2, and WO2006069246A2, the entire contents of each of which are hereby incorporated by reference in their entirety. Incorporating one or more unnatural amino acids into an antibody or antibody fragment or variant can include modifying one or more amino acid residues in the antibody or antibody fragment or variant. Modifying one or more amino acid residues in an antibody or antibody fragment or variant may include mutating one or more nucleotides in a nucleotide sequence encoding the antibody or antibody fragment or variant. Mutating one or more nucleotides in the nucleotide sequence encoding the antibody or antibody fragment or variant may comprise changing the codon encoding the amino acid to a nonsense codon. Incorporating one or more unnatural amino acids into an antibody or antibody fragment or variant can include modifying one or more amino acid residues in the antibody or antibody fragment or variant to produce one or more amber codons in the antibody or antibody fragment or variant. One or more unnatural amino acids can be incorporated into an antibody or antibody fragment or variant in response to an amber codon. One or more unnatural amino acids can be site-specifically incorporated into an antibody or antibody fragment or variant. Incorporation of one or more unnatural amino acids into an antibody or antibody fragment or variant can comprise one or more genetically encoded unnatural amino acids that have orthogonal chemical reactivity with respect to the typical twenty amino acids to site-specifically modify the biologically active molecule or targeting agent. The incorporation of the one or more unnatural amino acids can include using the tRNA/aminoacyl-tRNA synthetase pair to site-specifically incorporate the one or more unnatural amino acids at a defined site in the biologically active molecule or the targeting agent in response to the one or more amber nonsense codons. Additional methods for incorporating unnatural amino acids include, but are not limited to, the methods disclosed in Chatterjee et al ,A Versatile Platform for Single-and Multiple-Unnatural Amino Acid Mutagenesis in Escherichia coli,Biochemistry,2013;Kazane, J Am Chem Soc,135 (1): 340-6,2013; kim et al, J Am Chem Soc,134 (24): 9918-21,2012; johnson et al, nat Chem Biol,7 (11): 779-86,2011; and Hutchins et al, J MolBiol,406 (4): 595-603, 2011. The one or more unnatural amino acids can be produced by selective reaction of the one or more natural amino acids. The selective reaction may be mediated by one or more enzymes. In a non-limiting example, selective reaction of one or more cysteines with Formylglycine Generating Enzymes (FGEs) may produce one or more formylglycines, as described in Rabuka et al, nature Protocols 7:1052-1067,2012. The one or more unnatural amino acids can be involved in a chemical reaction for forming the linker. The chemical reaction for forming the linker may include a bio-orthogonal reaction. The chemical reaction for forming the linker may include click chemistry. see, for example, WO2006/050262, which is incorporated herein by reference in its entirety.

Any position of the antibody or antibody fragment is suitable for selection to incorporate unnatural amino acids, and selection can be based on rational design or by random selection for any or no particular desired purpose. The selection of the desired site may be based on producing an unnatural amino acid polypeptide (which may be further modified or remain unmodified) with any desired property or activity, including, but not limited to, a receptor binding modulator, a receptor activity modulator, a modulator of binding to a binding partner, a modulator of binding partner activity, a binding partner conformational modulator, dimer or multimer formation, no change in activity or property compared to the native molecule, or manipulation of any physical or chemical property of the polypeptide such as solubility, aggregation or stability. Alternatively, a site identified as critical to biological activity may also be a good candidate site for substitution with an unnatural amino acid, again depending on the desired activity sought for the polypeptide. Another alternative is to simply make successive substitutions with unnatural amino acids at each position on a polypeptide chain, and observe the effect on the activity of the polypeptide. Any means, technique or method for selecting a position of any polypeptide substituted with an unnatural amino acid is suitable for use in the methods, techniques and compositions described herein.

Naturally occurring mutants containing deleted polypeptides can also be examined for structure and activity to determine protein regions that are likely to tolerate unnatural amino acid substitutions. Once residues that may be intolerant to unnatural amino acid substitutions are eliminated, methods including, but not limited to, the three-dimensional structure of the relevant polypeptide and any relevant ligand or binding protein can be used to examine the effect of the proposed substitutions at each of the remaining positions. The X-ray crystallography and NMR structures of many polypeptides are available in protein databases (PDB, see the world wide web of rcsb. Org), which are centralized databases of three-dimensional structural data of macromolecules containing proteins and nucleic acids, and can be used to identify amino acid positions that can be substituted with unnatural amino acids. In addition, if three-dimensional structural data is not available, models can be made to study the secondary and tertiary structure of the polypeptide. Thus, the identity of amino acid positions that may be substituted with unnatural amino acids can be determined by the skilled artisan.

Exemplary sites for incorporation of unnatural amino acids include, but are not limited to, those that are excluded from potential receptor binding domains, or regions that bind to binding proteins or ligands may be fully or partially exposed to solvents, have minimal or no hydrogen bond interactions with nearby residues, may be minimally exposed to nearby reactive residues, and/or may be in highly flexible regions as predicted by the three-dimensional crystal structure of the particular polypeptide and its associated receptor, ligand, or binding protein.

A variety of unnatural amino acids can be substituted or incorporated into a polypeptide at a given position. As an example, specific unnatural amino acids for incorporation, preferably conservative substitutions, can be selected based on examination of the three-dimensional crystal structure of the polypeptide with its associated ligand, receptor, and/or binding protein.

Joint

In some aspects, the disclosure relates to linkers for intracellular delivery of drug conjugates. Many methods and linker molecules for attaching various compounds to peptides are known. See, for example, european patent application 0188256, U.S. patents 4,671,958, 4,659,839, 4,414,148, 4,699,784, 4,680,338, 4,569,789 and 10,550,190, PCT application publications WO 2012/166559 A1, WO 2012/166560A1, WO 2013/185117A1, WO 2013/192360A1 and WO 2022/040596A1, and U.S. patent application publication US2017/0182181 A1, the contents of each of which are hereby incorporated by reference in their entirety.

In some embodiments, the present disclosure relates to phosphate-based groups for intracellular delivery of drug conjugates (see, e.g., U.S. patent 10,550,190). Phosphate-based linkers of the present disclosure include mono-, di-, tri-or tetraphosphate groups (phosphate groups) and linker arms, as well as optional spacers. The drug linker may be covalently linked to a reactive functional group, which may be covalently linked to a cell-specific targeting ligand (such as an antibody or antibody fragment). Phosphate-based linkers have differentiated and tunable stability in blood compared to the intracellular environment (e.g., lysosomal compartments). Thus ADCs comprising these phosphate-based linkers are stable in circulation (plasma/blood), but are reactive or cleavable in intracellular compartments (lysosomes), making them useful for intracellular delivery of drug conjugates. The phosphate-based linker can be conjugated to a drug and the reactive functional group can be conjugated to a cell-specific targeting ligand (such as an anti-TROP 2 antibody, an anti-HER 2 antibody, an anti-CD 70 antibody, an anti-CD 3 antibody, or an anti-PSMA antibody). The phosphate-based linkers of the present disclosure are designed to engineer ADCs such that the likelihood of the conjugates forming aggregates is reduced compared to conjugates in which the same drug is conjugated to an antibody or targeting ligand using linkers that are not phosphate-based linkers. In addition, phosphate-based linker design, stability, pH, redox sensitivity, and protease sensitivity affect cycling stability and drug release.

Methods for selecting and designing linkers are well known in the art. The linker may be redesigned, including by way of example only, as part of a high throughput screening process (in which case many polypeptides may be designed, synthesized, characterized, and/or tested) or based on the interest of the researcher. Linkers can also be designed based on the structure of the known or partially characterized polypeptides. The principle for selecting amino acids to be substituted and/or modified and for selecting modifications to be used is described, for example, in WO 2013/185117. The joint may be designed to meet the needs of the experimenter or end user. Such needs may include, but are not limited to, manipulating the therapeutic effectiveness of a polypeptide, improving the safety profile of a polypeptide, modulating the pharmacokinetics, pharmacology, and/or pharmacodynamics of a polypeptide, such as, by way of example only, increasing water solubility, bioavailability, extending serum half-life, extending therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending circulation time. Further, such modifications include, by way of example only, providing additional functionality to the polypeptide, incorporating antibodies, and any combination of the foregoing.

In general, a linker of the present disclosure may be a unit that may be combined with one or more additional units such that the combined linker unit may be combined with one or more drugs. Each joint unit may be made up of one or more sections, each of which may occur one or more times.

In some embodiments, the linker of the present disclosure comprises at least one phosphate-based moiety, as disclosed herein. In some embodiments, the linker comprises a phosphate-based moiety and further comprises at least one moiety or unit that is not phosphate-based. In some embodiments, the linker is a bivalent linker. In some embodiments, the linker is a trivalent linker. In some embodiments, the linker is a tetravalent linker.

Thus, in some aspects, the present disclosure provides phosphate-based linkers. The phosphate-based linker of the present disclosure or the drug linker of the present disclosure may comprise a phosphate-based moiety, wherein the phosphate-based moiety is a phosphate, pyrophosphate, triphosphate, tetraphosphate, phosphonate, bisphosphonate, phosphoramidate, jiao Anji phosphate, triamino phosphate, tetraphosphite, phosphorothioate, and/or phosphorodithioate. Thus, the phosphate-based linker or drug linker of the present disclosure may comprise:

Has the structure of Phosphate esters of (a);

Has the structure of Is a phosphonate of (2);

Has the structure of Pyrophosphate of (2);

Has the structure of Is a bisphosphonate of (2);

Has the structure of Is a triphosphate of (2);

Has the structure of Is a tetraphosphate of (2);

Has the structure of Is a phosphorothioate of (2);

Has the structure of Is a dithiophosphate of (C);

Has the structure of Phosphoramidates of (2);

Has the structure of Jiao Anji phosphate of (a);

Has the structure of And/or (C)

Has the structure ofIs a tetra phosphoramidate of (2).

In some embodiments, the phosphate-based linkers of the present disclosure comprise a phosphate-based moiety selected from the group consisting of pyrophosphates and diphosphonates. In some embodiments, the pharmaceutical linker of the present disclosure comprises a phosphate-based moiety selected from the group consisting of pyrophosphates and diphosphonates.

In some embodiments, the phosphate-based linker or drug linker of the present disclosure comprises pyrophosphate. For example, in some embodiments, a drug (e.g., a biscardamycin compound of the disclosure) comprises an oxygen atom (-O-) attached to a phosphorus atom of a bisphosphonate moiety, thereby providing a drug linker comprising a pyrophosphate.

In some other embodiments, the phosphate-based linker or pharmaceutical linker of the present disclosure comprises a bisphosphonate.

In some embodiments, the phosphate-based linker is a divalent linker.

In some embodiments, the phosphate-based moiety is covalently bonded to the-O-atom of the ADC of formula (II), via a phosphorus atom of the phosphate-based moiety, e.g., formula (X), or formula (I), or formula (Ia), or formula (Ib), or formula (Ic), or formula (Id), or formula (IL), or formula (ILa), or formula (ILb), or formula (ILc), or formula (ILd), as disclosed herein.

In some embodiments, the phosphate-based linker further comprises at least one additional moiety. In some embodiments, each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, substituted alkylene, - (alkylene-O) -, optionally substituted arylene, -O-, -C (O) -, -N (R _w)-、-S(O)_0-2 -, water soluble polymer, and amino acid; wherein each R _w is independently H or alkyl, alkenyl or alkynyl; in some embodiments, each R _w is independently H or unsubstituted C ₁-C₈ alkyl, C ₁-C₈ alkenyl, or C ₁-C₈ alkylalkynyl, in some further embodiments, each R _w is independently H or unsubstituted C ₁-C₈ alkyl, in still other embodiments, each R _w is independently H or methyl.

In some embodiments, each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, - (alkylene-O) -, -C (O) -, -N (R _w) -, a water-soluble polymer, and an amino acid, wherein each R _w is independently H or alkyl, alkenyl, or alkynyl, and combinations thereof. In some embodiments, each R _w is independently H or unsubstituted C ₁-C₈ alkyl, C ₁-C₈ alkenyl, or C ₁-C₈ alkyl alkynyl. In some further embodiments, each R _w is independently H or unsubstituted C ₁-C₈ alkyl. In still other embodiments, each R _w is independently H or methyl.

It is to be understood that each at least one additional moiety that may be present in the phosphate-based linkers of the present disclosure may be present one or more times in the linker. In one non-limiting example, the phosphate-based linkers of the present disclosure may comprise one or more unsubstituted alkylene groups, wherein each of the unsubstituted alkylene groups may be the same or different. In another non-limiting example, the phosphate-based linkers of the present disclosure may comprise one or more amino acids, wherein each amino acid is the same or different.

In some embodiments, the linker comprises at least one alkylene group.

In some embodiments, the linker comprises an amino acid. In some embodiments, the amino acid is selected from the group consisting of serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and N _ε -methyl-lysine. In some embodiments, the amino acid is lysine or N _ε -methyl-lysine. In some embodiments, the amino acid is lysine. In some embodiments, the amino acid is N _ε -methyl-lysine.

In some embodiments, the linker comprises a water-soluble polymer.

In some embodiments, the linker comprises a water-soluble polymer and an amino acid, wherein the water-soluble polymer is conjugated to the amino acid. In some embodiments, the water-soluble polymer is conjugated to a side chain of an amino acid. In some embodiments, the water-soluble polymer is conjugated to the amino acid via a spacer element.

In some embodiments, the phosphate-based linkers of the present disclosure comprise a water-soluble polymer and an amino acid that is serine, threonine, or tyrosine, wherein the water-soluble polymer is conjugated to a side chain-OH group of serine, threonine, or tyrosine. In some embodiments, the water-soluble polymer is conjugated to the amino acid via a spacer element.

In some embodiments, the phosphate-based linkers of the present disclosure comprise a water-soluble polymer and an amino acid that is a cysteine, wherein the water-soluble polymer is conjugated to a side chain-SH group of the cysteine. In some embodiments, the water-soluble polymer is conjugated to the amino acid via a spacer element.

In some embodiments, the phosphate-based linkers of the present disclosure comprise a water-soluble polymer and an amino acid that is aspartic acid or glutamic acid, wherein the water-soluble polymer is conjugated to a pendant carboxylate group of aspartic acid or glutamic acid. In some embodiments, the water-soluble polymer is conjugated to the amino acid via a spacer element.

In some embodiments, the phosphate-based linkers of the present disclosure comprise a water-soluble polymer and an amino acid that is lysine and N _ε -methyl-lysine, wherein the water-soluble polymer is conjugated to a side chain-N _ε H (R) group of lysine or N _ε -methyl-lysine, wherein R is H or methyl, respectively. In some embodiments, the water-soluble polymer is conjugated to the amino acid via a spacer element.

In some embodiments, the water-soluble polymer is a polysaccharide.

In some embodiments, the water-soluble polymer is a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety has a molecular weight in the range of about 100Da to about 100,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100Da to about 10,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100Da to about 5,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100Da to about 1,000 Da.

In some embodiments, the PEG moiety is- (CH ₂CH₂O)_nCH₃, where n is an integer from 1 to 100, in some embodiments, the PEG moiety is- (CH ₂CH₂O)_nCH₃, where n is an integer from 1 to 24, in some embodiments, the PEG moiety is- (CH ₂CH₂O)_nCH₃, where n is an integer from 6 to 12, in some embodiments, the PEG moiety is- (CH ₂CH₂O)_nCH₃, where n is an integer from 8 to 12, in some embodiments, the PEG moiety is- (CH ₂CH₂O)_nCH₃, where n is 8, in some embodiments, the PEG moiety is- (CH ₂CH₂O)_nCH₃, where n is 12).

In some embodiments, PEG is linear. In some embodiments, PEG is branched, multi-armed, or dendritic.

In some embodiments, the phosphate-based linker of the present disclosure is a linker selected from the group of linkers listed in table 6.

Table 6. Non-limiting examples of joints of the present disclosure.

In some embodiments, each i of table 6 is 1. In some other embodiments, each i of table 6 is 0.

In some embodiments, each U is independently optionally substituted with a water soluble polymer.

In some embodiments, each n of table 6 is independently an integer from 1 to 10. In some embodiments, each n of table 6 is independently 1, 2, or 3.

In some embodiments, each alkylene of table 6 is independently- (CH ₂)-、-(CH2)₂ -or- (CH 2) ₃ -.

In some embodiments, each linker of table 6 is substituted with one or more water-soluble polymers. In some embodiments, each U of table 6 is substituted with one or more water-soluble polymers. In some embodiments, each U of table 6 is substituted with a water soluble polymer.

In some embodiments, the water-soluble polymer is conjugated to the amino acid side chain of group U. In some embodiments, the water-soluble polymer is conjugated to the amino acid side chain of group U via a spacer element.

In some embodiments, the water-soluble polymer is a polysaccharide.

It is to be understood that the direction in which the chemical formula of the linking group is written does not imply the orientation of the linker unless explicitly stated otherwise. By way of example only, the formula-alkylene-O-P (=o) (OH) - (O) _i -represents

-Both alkylene-O-P (=o) (OH) - (O) _i -and- (O) _i -P (=o) (OH) -O-alkylene-. In another example, the formula-alkylene-O-P (=o) (OH) - (O) _i -represents-alkylene-O-P (=o) (OH) - (O) _i -and-alkylene-O-P (=o) (OH) - (O) _i -, wherein x represents a point of attachment, e.g., to a drug.

It will also be appreciated that where each alkylene (or other variable) of a linker is independently selected from a set of variables, the independent selection may be made within a given linker. By way of example only, the formula-alkylene-O-P (=o) (OH) - (O) _i -alkylene- (O-alkylene) _n - (wherein each alkylene is independently- (CH ₂)-、-(CH₂)₂ -or- (CH ₂)₃ -)) includes but is not limited to the following species:

*–(CH₂)-O-P(＝O)(OH)-O-P(＝O)(OH)-(O)_i-(CH₂)₂-(O-(CH₂)₃)_n–、

*–(CH₂)₂-O-P(＝O)(OH)-O-P(＝O)(OH)-(O)_i-(CH₂)₃-(O-CH₂)_n–、

*–(CH₂)₂-O-P(＝O)(OH)-O-P(＝O)(OH)-(O)_i-(CH₂)₂-(O-(CH₂)₂)_n–、

*–(CH₂)₃-O-P(＝O)(OH)-O-P(＝O)(OH)-(O)_i-(CH₂)-(O-(CH₂)₂)_n– And

*–(CH₂)₂-O-P(＝O)(OH)-O-P(＝O)(OH)-(O)_i-(CH₂)-(O-(CH₂)₂)_n–.

Furthermore, by way of example only, the group-alkylene-O-P (=o) (OH) - (O) _i -alkylene- (O-alkylene) _n - (wherein each alkylene is independently- (CH ₂)-、-(CH₂)₂ -or- (CH ₂)₃ -) can be rewritten as: -alkylene-O-P (=o) (OH) - (O) _i -alkylene' - (O-alkylene ") _n -, wherein each alkylene, alkylene' and alkylene "are independently- (CH ₂)-、-(CH₂)₂ -or- (CH ₂)₃ -). Similarly, - (alkylene-O) _n-P(＝O)(OH)-O-P(＝O)(OH)-(O)_i - (alkylene-O) _n -J-alkylene- (alkylene-O) _n - (wherein each alkylene is independently- (CH ₂)-、-(CH2)₂ -or- (CH 2) ₃ -and each n is independently 1, 2 or 3) is rewriteable as- (alkylene-O) _n-P(＝O)(OH)-O-P(＝O)(OH)-(O)_i - (alkylene '-O) _n′ -J-alkylene "- (alkylene"' -O) _n″ -, wherein each alkylene, alkylene ', alkylene "', and alkylene" '' is independently- (CH ₂)-、-(CH₂)₂ -or- (CH ₂)₃ -; and each n, n' and n″ are independently 1, 2 or 3.

In some embodiments, the phosphate-based linker of the present disclosure is a linker selected from the group of linkers listed in table 7.

Table 7. Non-limiting examples of joints of the present disclosure.

In some embodiments, each i of table 7 is 1. In some other embodiments, each i of table 7 is 0.

In some embodiments, each n of table 7 is independently an integer from 1 to 10. In some embodiments, each n of table 7 is independently 1, 2, or 3.

In some embodiments, each alkylene of table 7 is independently- (CH ₂)-、-(CH2)₂ -or- (CH 2) ₃ -.

In some embodiments, each linker of table 7 is substituted with one or more water-soluble polymers. In some embodiments, each U of table 7 is substituted with one or more water-soluble polymers. In some embodiments, each U of table 7 is substituted with a water soluble polymer.

In some embodiments, the water-soluble polymer is a polysaccharide.

In some further embodiments, the phosphate-based linkers of the present disclosure are linkers selected from the group of linkers listed in table 8.

Table 8. Non-limiting examples of joints of the present disclosure.

In some embodiments, each n of table 8 is independently an integer from 1 to 10. In some embodiments, each n of table 8 is independently 1, 2, or 3.

In some embodiments, each alkylene of table 8 is independently- (CH ₂)-、-(CH2)₂ -or- (CH 2) ₃ -.

In some embodiments, each linker of table 8 is substituted with one or more water-soluble polymers. In some embodiments, each U of table 8 is substituted with one or more water-soluble polymers. In some embodiments, each U of table 8 is substituted with a water soluble polymer.

In some embodiments, the linker is a-P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +,

Wherein:

u is selected from the group consisting of:

Each alkylene is independently selected from the group consisting of:

-(CH₂)-、-(CH₂)₂-、-(CH₂)₃-、-(CH₂)₄-、-(CH₂)₅-、-(CH₂)₆-、-(CH₂)₇-、

- (CH ₂)₈–、–(CH₂)₉–、–(CH₂)₁₀–、–(CH₂)₁₁ -and- (CH ₂)₁₂ -;

* Represents the point of attachment to the drug, for example to an-O-atom of formula (I), or formula (Ia), or formula (Ib), or formula (Ic) or formula I (d), and

+ Represents the point of attachment to a moiety (such as a reactive moiety);

wherein each of said linkers is optionally substituted with one or more water soluble polymers.

In some embodiments, the linker is substituted with one or more water soluble polymers.

In some embodiments, the linker comprises a water soluble polymer. In some embodiments, a water-soluble polymer is conjugated to the amino acid side chain of group U. In some embodiments, a water-soluble polymer is conjugated to the amino acid side chain of group U via a spacer element.

In some embodiments, the water-soluble polymer is a polysaccharide.

In some embodiments, the water-soluble polymer is a PEG moiety. In some embodiments, the PEG moiety has a molecular weight in the range of about 100Da to about 100,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100Da to about 10,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100Da to about 5,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100Da to about 1,000 Da.

In some embodiments, U is:

In some embodiments, U is In some other embodiments, U is

In some embodiments, each of the U is conjugated to a water soluble polymer. In some embodiments, the water-soluble polymer is conjugated to the amino acid side chain of group U.

In some embodiments, the water-soluble polymer is conjugated to the amino acid side chain of group U via a spacer element. In some embodiments, when the water-soluble polymer is conjugated to the amino acid side chain of group U via a spacer element, the spacer element is a carbonyl group.

In some embodiments, the linker has the following structure:

wherein represents the point of attachment to the drug, for example, to an-O-atom of formula (I), or formula (Ia), or formula (Ib), or formula (Ic) or formula (d), and +represents the point of attachment to the reactive moiety.

In some embodiments, the linker has the following structure:

In some other embodiments, the linker has the following structure:

Wherein T is the water-soluble polymer, R ^t is H or methyl, represents the point of attachment to the drug, for example, to an-O-atom of formula (I), or formula (Ia), or formula (Ib), or formula (Ic) or formula (d), and +represents the point of attachment to the reactive moiety.

In some embodiments, the one or more water-soluble polymers conjugated to the linker are polyethylene glycol (PEG) moieties.

In some embodiments, the linker (a water-soluble polymer) is conjugated to the amino acid side chain of the linker comprising group U. In some embodiments, a water-soluble polymer is conjugated to the amino acid side chain of group U via a spacer element.

In some embodiments, the water-soluble polymer is a polysaccharide.

In some embodiments, the phosphate-based linker of the present disclosure is linked to a drug, and is also linked to a reactive moiety. Thus, the linker bridges the drug and the reactive moiety. The reactive moiety may be a reactive moiety that is reactive with another portion of a natural amino acid or a non-natural amino acid of a polypeptide (such as an antibody, antibody fragment, or variant thereof of the present disclosure), as disclosed herein.

In some other embodiments, the phosphate-based linker as disclosed herein is linked to the drug via a bond or an adduct moiety, and is also linked to an antibody, antibody fragment, or variant thereof. Thus, the linker bridges the drug and the antibody, antibody fragment, or variant thereof.

Drug and drug linker

In some aspects, the present disclosure provides a drug or drug linker, wherein the drug is a cytotoxic drug or agent. In some aspects of the disclosure, the cytotoxic drug is bicubicin. In some aspects, the cytotoxic drug is a bicubicin analog. In some embodiments, the drug or drug linker is a drug or drug linker generated as described in the examples herein, wherein the linker (when present) may be derivatized with a reactive or other moiety, or a metabolite thereof.

In some aspects, there is provided a cytotoxic drug that is a bicubicin analog of formula (X) having the general structure:

Wherein:

A is an optionally substituted bicyclic ring system containing one or more nitrogen ring atoms, B is a carbonyl group, and R is H or L-W, wherein L is a linker and W is a reactive moiety;

Or a salt thereof. In some embodiments, the salt may be a pharmaceutically acceptable salt. In some embodiments, bicyclic system a is attached to carbonyl group B via one of the one or more nitrogen ring atoms of bicyclic system a.

In some embodiments, bicyclic system a may contain 9 ring atoms.

In some other embodiments, bicyclic system a may contain 10 ring atoms.

In some embodiments, bicyclic system a may contain 9 or 10 ring atoms, wherein the ring atoms are selected from the group consisting of carbon atoms and nitrogen atoms.

In some embodiments, bicyclic system a contains a 5-membered ring fused to a 6-membered ring, wherein the 5-membered ring contains a nitrogen atom that connects bicyclic system a to carbonyl group B. In some embodiments, the 5-membered ring is a pyrrolidine ring.

In some embodiments, the 6 membered ring is an aromatic ring containing 0 or 1 nitrogen atoms.

In some embodiments, bicyclic system a comprises a first 6 membered ring fused to a second 6 membered ring, wherein the first 6 membered ring comprises a nitrogen atom that connects bicyclic system a to carbonyl group B. In some embodiments, the first 6-membered ring is a piperidine ring. In some embodiments, the second 6-membered ring is an aromatic ring containing 0 or 1 nitrogen atoms.

In some embodiments, the bicyclic system a is hydrophobic. As will be appreciated by those of ordinary skill in the art, the hydrophobicity of a compound can be estimated from its CLogP value, which can be calculated from its structure, lower CLogP values indicating more hydrophilic molecules, while higher CLogP values indicate more hydrophobic molecules. Thus, in some embodiments, the hydrophobicity of a is characterized by its ClogP value. In some embodiments, the hydrophobicity of a is characterized by the ClogP value of its corresponding amine "a-H".

In some embodiments, a-H (the corresponding amine of a) has a ClogP value of at least about 1. The CLogP value may be calculated using tools such as ChemDraw Professional software (PERKINELMER INFORMATICS). Some non-limiting examples of the a-H groups of the present disclosure reported below were calculated using ChemDraw Professional version 20.1.1.125.

In some embodiments, R is hydrogen.

In some embodiments, the compound is a drug linker compound, and R is L-W. In some embodiments, L is a phosphate-based linker.

Accordingly, in some embodiments, there is provided a compound of formula (I) having the structure:

Wherein:

R is H or L-W, wherein L is a linker and W is a reactive moiety;

Wherein:

Each X ³ is C;

Each X ⁸ is C, and

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3;

Or a salt thereof.

In some embodiments, a-H (the corresponding amine of part a) has a ClogP value of at least about 1. In some embodiments, a has the structure of formula (a), and a-H have a ClogP value of at least about 1. In some embodiments, a has the structure of formula (b), and a-H have a ClogP value of at least about 1. In some embodiments, a has the structure of formula (c), and a-H have a ClogP value of at least about 1. In some embodiments, a has the structure of formula (d), and a-H have a ClogP value of at least about 1.

In some embodiments, X ¹ is C (R ^1a)(R^1b), wherein each R ^1a and R ^1b is independently H, Halogen or unsubstituted alkyl, X ² is C (R ^2a)(R^2b), wherein each R ^2a and R ^2b is independently H, Halogen or unsubstituted alkyl, X ³ is C, X ⁴ is C (R ⁴) or N, wherein R ⁴ is H, Halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ⁵ is C (R ⁵) or N, wherein R ⁵ is H, halogen, alkyl, Alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ⁶ is C (R ⁶) or N, wherein R ⁶ is H, halogen, alkyl, alkenyl, alkynyl, Carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ⁷ is C (R ⁷) or N, wherein R ⁷ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, Carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, Heterocyclylalkyl or heteroarylalkyl, X ⁸ is C, and X ⁹, when present, is C (R ^9a)(R^9b), wherein each R ^9a and R ^9b is independently H, halogen or unsubstituted alkyl.

In some embodiments, each of X ¹ and X ² is CH ₂, and X ⁹, when present, is CH ₂.

In some embodiments, X ⁴ is C (R ⁴) or N, wherein R ⁴ is H, halogen, or heteroalkyl, X ⁵ is C (R ⁵) or N, wherein R ⁵ is H, halogen, or heteroalkyl, X ⁶ is C (R ⁶) or N, wherein R ⁶ is H, halogen, or heteroalkyl, and X ⁷ is C (R ⁷) or N, wherein R ⁷ is H, halogen, or heteroalkyl.

In some embodiments, X ⁴ is C (R ⁴) or N, wherein R ⁴ is H or heteroalkyl, X ⁵ is C (R ⁵) or N, wherein R ⁵ is H or heteroalkyl, X ⁶ is C (R ⁶) or N, wherein R ⁶ is H or heteroalkyl, and X ⁷ is C (R ⁷) or N, wherein R ⁷ is H or heteroalkyl.

In some embodiments, each heteroalkyl group is an alkoxy group. In some embodiments, each of the alkoxy groups is independently-OR ^k, wherein each R ^k is independently alkyl optionally substituted with-N (R ^d)(R^e) OR heterocyclyl, wherein each R ^d and R ^e is independently H, alkyl, alkenyl, OR alkynyl.

In some embodiments, each heterocyclyl contains at least one nitrogen atom.

In some embodiments, X ⁴ is N, X ⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is C (R ⁷). In some embodiments, X ⁷ is CH.

In some embodiments, X ⁴ is C (R ⁴),X⁵ is N, X ⁶ is C (R ⁶) and X ⁷ is C (R ⁷). In some embodiments, at least one of X ⁴ and X ⁷ is CH.

In some embodiments, X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is N and X ⁷ is C (R ⁷). In some embodiments, at least one of X ⁴ and X ⁷ is CH.

In some embodiments, X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is n. in some embodiments, X ⁴ is CH).

In some embodiments, X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is C (R ⁷).

In some embodiments, X ⁷ is CH. In some embodiments, X ⁴ is CH. In some embodiments, each of X ⁴ and X ⁷ is CH.

In some embodiments, R is hydrogen.

In some embodiments, there is provided a compound of formula (Ia) having the structure:

Wherein:

R is H or L-W, wherein L is a linker and W is a reactive moiety;

X ³ is C;

X ⁸ is C;

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3.

In some embodiments, each heterocyclyl contains at least one nitrogen atom.

In some embodiments, R is hydrogen.

In some embodiments, there is provided a compound of formula (Ib) having the structure:

Wherein:

R is H or L-W, wherein L is a linker and W is a reactive moiety;

X ³ is C;

X ⁸ is C;

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3.

In some embodiments, each heterocyclyl contains at least one nitrogen atom.

In some embodiments, R is hydrogen.

In some embodiments, there is provided a compound of formula (Ic) having the structure:

Wherein:

R is H or L-W, wherein L is a linker and W is a reactive moiety;

X ³ is C;

X ⁷ is C (R ⁷) or N, wherein R ⁷ is H, halogen, -OH, -SH, -NO ₂、-CN、-N₃、-N(R^a)(R^b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl 、-C(O)R^c、-C(O)OR^c、-C(O)N(R^a)(R^b)、-C(S)R^c、-C(S)OR^c、-C(S)N(R^a)(R^b)、-C(O)SR^c, or-S (O) _m(R^s);

X ⁸ is C, and

X ⁹ is C (R ^9a)(R^9b), wherein each R ^9a and R ^9b is independently H, halogen, alkyl, alkenyl or alkynyl;

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3.

In some embodiments, each heterocyclyl contains at least one nitrogen atom.

In some embodiments, R is hydrogen.

In some embodiments, there is provided a compound of formula (Id) having the structure:

Wherein:

R is H or L-W, wherein L is a linker and W is a reactive moiety;

X ³ is C;

X ⁸ is C, and

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3.

In some embodiments, each heterocyclyl contains at least one nitrogen atom.

In some embodiments, R is hydrogen.

In some embodiments, a compound of formula (I) is provided, wherein R is H. In some embodiments, a compound of formula (Ia) is provided, wherein R is H. In some embodiments, the compound is selected from the group consisting of:

And salts thereof. In some embodiments, the salt is a pharmaceutically acceptable salt. In some embodiments, there is provided a compound of formula (Ia) having the structure:

Or a salt thereof.

In some aspects, a pharmaceutical linker compound of formula (I) is provided, wherein R is L-W. Accordingly, in some embodiments, there is provided a compound of formula (IL) having the structure:

Wherein:

r is L-W, wherein L is a linker and W is a reactive moiety;

Wherein:

Each X ³ is C;

Each X ⁸ is C, and

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3;

Or a salt thereof.

In some embodiments, each heterocyclyl contains at least one nitrogen atom.

In some embodiments, L is a phosphate-based linker comprising a phosphate-based moiety. In some embodiments, the phosphate-based moiety is selected from the group consisting of phosphate, pyrophosphate, triphosphate, tetraphosphate, phosphonate, bisphosphonate, phosphoramidate, jiao Anji phosphate, triamino phosphate, tetraphosphoric acid, phosphorothioate, and phosphorodithioate.

In some embodiments, the drug linker comprises a phosphate-based moiety. In some embodiments, the phosphate-based moiety is selected from the group consisting of phosphate, pyrophosphate, triphosphate, tetraphosphate, phosphonate, bisphosphonate, phosphoramidate, jiao Anji phosphate, triamino phosphate, tetraphosphoric acid, phosphorothioate, and phosphorodithioate. In some embodiments, the phosphate-based moiety is a pyrophosphate. In some embodiments, the phosphate-based moiety is a bisphosphonate.

In some embodiments, the phosphate-based linker is a divalent linker.

In some embodiments, the phosphate-based moiety of the phosphate-based linker is covalently bonded to the-O-atom of the drug. Thus, in some embodiments, the phosphate-based moiety of the phosphate-based linker is covalently bonded to the-O-atom of the compound of formula (X) or formula (I) as disclosed herein via the phosphorus atom of the phosphate-based moiety.

In some embodiments, L comprises at least one alkylene group.

In some embodiments, L comprises at least one amino acid. In some embodiments, L comprises one amino acid. In some embodiments, the amino acid is selected from the group consisting of serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and N _ε -methyl-lysine. In some embodiments, the amino acid is lysine or N _ε -methyl-lysine.

In some embodiments, L comprises one or more water-soluble polymers. In some embodiments, L comprises a water-soluble polymer.

In some embodiments, L comprises a water-soluble polymer and an amino acid, wherein the water-soluble polymer is conjugated to the amino acid. In some embodiments, the water-soluble polymer is conjugated to a side chain of an amino acid. In some embodiments, the water-soluble polymer is conjugated to the amino acid via a spacer element.

In some embodiments, L is selected from the group of linkers listed in table 6.

In some embodiments, L is selected from the group of linkers listed in table 7.

In some embodiments, L is selected from the group of linkers listed in table 8.

In some embodiments, L is selected from the group consisting of:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) - (alkylene-O) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene- (O-alkylene) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J- (alkylene-O) _n -alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +,

* -P (=O) (OH) -O-P (=O) (OH) - (O) -alkylene- (O-alkylene) _n -U-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene- (O-alkylene) _n -U-alkylene- +, and

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U- (alkylene-O) _n -alkylene- +;

Wherein:

each U is independently selected from the group consisting of:

Each J is independently

Each alkylene is independently selected from the group consisting of:

Each n is independently an integer from 1 to 100;

* Represents a linkage to an-O-atom of the formula (IL), and

+ Represents a connection to W;

wherein each linker L is optionally substituted with one or more water soluble polymers.

In some embodiments, each n is independently an integer from 1 to 10. In some embodiments, each n is independently 1, 2, or 3.

In some embodiments, L is substituted with one or more water-soluble polymers.

In some embodiments, L comprises a group U, and one water-soluble polymer is conjugated to the amino acid side chain of the group U. In some embodiments, the water-soluble polymer is conjugated to the amino acid side chain of group U via a spacer element.

In some embodiments, L is:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +, wherein:

u is selected from the group consisting of:

Each alkylene is independently selected from the group consisting of:

* Represents a linkage to an-O-atom of the formula (IL), and

+ Represents a connection to W;

wherein L is optionally substituted with one or more water soluble polymers.

In some embodiments, L is substituted with one or more water-soluble polymers. In some embodiments, a water-soluble polymer is conjugated to the amino acid side chain of group U. In some embodiments, a water-soluble polymer is conjugated to the amino acid side chain of group U via a spacer element. In some embodiments, the spacer element is a carbonyl group.

In some embodiments, U is:

In some embodiments, L has the following structure:

Wherein represents a linkage to an-O-atom of formula (IL), and +represents a linkage to W.

In some other embodiments, L has the following structure:

wherein T is the water-soluble polymer, R ^t is H or methyl, represents a linkage to an-O-atom of formula (IL), and +represents a linkage to W.

In some embodiments, the water-soluble polymer is a polysaccharide.

In some embodiments, the reactive moiety W comprises-N ₃、-OH、-SH、-NH(R^j)、-C(O)R^q、-C(O)OR^x、-C(O)CH₂NH₂, an activated ester, -O-NH ₂, maleimide, tetrazine, alkyne, cyclooctyne, or (E) -cyclooctene, wherein R ^j is H or unsubstituted alkyl, R ^q is unsubstituted alkyl, and R ^x is H, unsubstituted alkyl, or a carboxylic acid protecting group.

In some embodiments, the reactive moiety W is selected from the group consisting of:

-N ₃、-OH、-SH、-NH(R^j)、-C(O)R^q、-C(O)OR^x, activated esters, -O-NH ₂ and optionally substituted mono-or polycyclic groups comprising said cyclooctyne;

Wherein:

r ^j is H or unsubstituted C ₁-C₆ alkyl,

R ^q is unsubstituted C ₁-C₆ alkyl,

R ^x is H, unsubstituted C ₁-C₆ alkyl or a carboxylic acid protecting group,

R ^f is H or unsubstituted C ₁-C₆ alkyl,

S is 0,1, 2, 3,4, 5 or 6, and

T is 0,1, 2, 3, 4, 5 or 6.

In some embodiments, the optionally substituted monocyclic or polycyclic group comprising cyclooctyne is selected from the group consisting of:

In some embodiments, W is-ONH ₂.

In some aspects, a pharmaceutical linker compound of formula (Ia) is provided, wherein R is L-W. Accordingly, in some embodiments, there is provided a compound of formula (ILa) having the structure:

Wherein:

r is L-W, wherein L is a linker and W is a reactive moiety;

X ³ is C;

X ⁸ is C;

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3.

In some embodiments, each heterocyclyl contains at least one nitrogen atom.

In some embodiments, L is a phosphate-based linker comprising a phosphate-based moiety. In some embodiments, the phosphate-based moiety is selected from the group consisting of phosphate, pyrophosphate, triphosphate, tetraphosphate, phosphonate, bisphosphonate, phosphoramidate, jiao Anji phosphate, triamino phosphate, tetraphosphoric acid, phosphorothioate, and phosphorodithioate. In some embodiments, the phosphate-based moiety is a pyrophosphate. In some other embodiments, the phosphate-based moiety is a bisphosphonate.

In some embodiments, the phosphate-based linker is a divalent linker.

In some embodiments, L comprises at least one alkylene group.

In some embodiments, L is selected from the group of linkers listed in table 6.

In some embodiments, L is selected from the group of linkers listed in table 7.

In some embodiments, L is selected from the group of linkers listed in table 8.

In some embodiments, L is selected from the group consisting of:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) - (alkylene-O) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene- (O-alkylene) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J- (alkylene-O) _n -alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +,

* -P (=O) (OH) -O-P (=O) (OH) - (O) -alkylene- (O-alkylene) _n -U-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U- (alkylene-O) _n -alkylene- +;

Wherein:

each U is independently selected from the group consisting of:

Each J is independently

Each alkylene is independently selected from the group consisting of:

Each n is independently an integer from 1 to 100;

* Represents a linkage to an-O-atom of formula (ILa), and

+ Represents a connection to W;

In some embodiments, L is substituted with one or more water-soluble polymers.

In some embodiments, L is:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +, wherein:

u is selected from the group consisting of:

Each alkylene is independently selected from the group consisting of:

* Represents a linkage to an-O-atom of formula (ILa), and

+ Represents a connection to W;

wherein L is optionally substituted with one or more water soluble polymers.

In some embodiments, U is:

In some embodiments, L has the following structure:

wherein represents a linkage to an-O-atom of formula (ILa), and +represents a linkage to W.

In some other embodiments, L has the following structure:

wherein T is the water-soluble polymer, R ^t is H or methyl, represents a linkage to an-O-atom of formula (ILa), and +represents a linkage to W.

In some embodiments, the water-soluble polymer is a polysaccharide.

Wherein:

r ^j is H or unsubstituted C ₁-C₆ alkyl,

R ^q is unsubstituted C ₁-C₆ alkyl,

R ^f is H or unsubstituted C ₁-C₆ alkyl,

S is 0,1, 2, 3,4, 5 or 6, and

T is 0,1, 2, 3, 4, 5 or 6.

In some embodiments, W is-ONH ₂.

In some aspects, a pharmaceutical linker compound of formula (Ib) is provided, wherein R is L-W. Accordingly, in some embodiments, there is provided a compound of formula (ILb) having the structure:

Wherein:

r is L-W, wherein L is a linker and W is a reactive moiety;

X ³ is C;

X ⁸ is C;

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3.

In some embodiments, each heterocyclyl contains at least one nitrogen atom.

In some embodiments, the phosphate-based linker is a divalent linker.

In some embodiments, L comprises at least one alkylene group.

In some embodiments, L is selected from the group of linkers listed in table 6.

In some embodiments, L is selected from the group of linkers listed in table 7.

In some embodiments, L is selected from the group of linkers listed in table 8.

In some embodiments, L is selected from the group consisting of:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) - (alkylene-O) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene- (O-alkylene) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J- (alkylene-O) _n -alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +,

* -P (=O) (OH) -O-P (=O) (OH) - (O) -alkylene- (O-alkylene) _n -U-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U- (alkylene-O) _n -alkylene- +;

Wherein:

each U is independently selected from the group consisting of:

Each J is independently

Each alkylene is independently selected from the group consisting of:

Each n is independently an integer from 1 to 100;

* Represents a linkage to an-O-atom of formula (ILb), and

+ Represents a connection to W;

In some embodiments, L is substituted with one or more water-soluble polymers.

In some embodiments, L is:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +, wherein:

u is selected from the group consisting of:

Each alkylene is independently selected from the group consisting of:

* Represents a linkage to an-O-atom of formula (ILb), and

+ Represents a connection to W;

wherein L is optionally substituted with one or more water soluble polymers.

In some embodiments, U is:

In some embodiments, L has the following structure:

Wherein represents a linkage to an-O-atom of formula (ILb), and +represents a linkage to W.

In some other embodiments, L has the following structure:

wherein T is the water-soluble polymer, R ^t is H or methyl, represents a linkage to an-O-atom of formula (ILb), and +represents a linkage to W.

In some embodiments, the water-soluble polymer is a polysaccharide.

Wherein:

r ^j is H or unsubstituted C ₁-C₆ alkyl,

R ^q is unsubstituted C ₁-C₆ alkyl,

R ^f is H or unsubstituted C ₁-C₆ alkyl,

S is 0,1, 2, 3,4, 5 or 6, and

T is 0,1, 2, 3, 4, 5 or 6.

In some embodiments, W is-ONH ₂.

In some aspects, a pharmaceutical linker compound of formula (Ic) is provided, wherein R is L-W. Accordingly, in some embodiments, there is provided a compound of formula (ILc) having the structure:

Wherein:

r is L-W, wherein L is a linker and W is a reactive moiety;

X ³ is C;

X ⁸ is C, and

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3.

In some embodiments, each heterocyclyl contains at least one nitrogen atom.

In some embodiments, the phosphate-based linker is a divalent linker.

In some embodiments, L comprises at least one alkylene group.

In some embodiments, L is selected from the group of linkers listed in table 6.

In some embodiments, L is selected from the group of linkers listed in table 7.

In some embodiments, L is selected from the group of linkers listed in table 8.

In some embodiments, L is selected from the group consisting of:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) - (alkylene-O) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene- (O-alkylene) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J- (alkylene-O) _n -alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene- (O-alkylene) _n -U-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene- (O-alkylene) _n -U-alkylene- + and

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U- (alkylene-O) _n -alkylene- +;

Wherein:

each U is independently selected from the group consisting of:

Each J is independently

Each alkylene is independently selected from the group consisting of:

Each n is independently an integer from 1 to 100;

* Represents a linkage to an-O-atom of formula (ILc), and

+ Represents a connection to W;

In some embodiments, L is substituted with one or more water-soluble polymers.

In some embodiments, L is:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +, wherein:

u is selected from the group consisting of:

Each alkylene is independently selected from the group consisting of:

* Represents a linkage to an-O-atom of formula (ILc), and

+ Represents a connection to W;

wherein L is optionally substituted with one or more water soluble polymers.

In some embodiments, U is:

In some embodiments, L has the following structure:

Wherein represents a linkage to an-O-atom of formula (ILc), and +represents a linkage to W.

In some other embodiments, L has the following structure:

Wherein T is the water-soluble polymer, R ^t is H or methyl, represents a linkage to an-O-atom of formula (ILc), and +represents a linkage to W.

In some embodiments, the water-soluble polymer is a polysaccharide.

Wherein:

r ^j is H or unsubstituted C ₁-C₆ alkyl,

R ^q is unsubstituted C ₁-C₆ alkyl,

R ^f is H or unsubstituted C ₁-C₆ alkyl,

S is 0,1, 2, 3,4, 5 or 6, and

T is 0,1, 2, 3, 4, 5 or 6.

In some embodiments, W is-ONH ₂.

In some aspects, a pharmaceutical linker compound of formula (Id) is provided, wherein R is L-W. Accordingly, in some embodiments, there is provided a compound of formula (ILd) having the structure:

Wherein:

r is L-W, wherein L is a linker and W is a reactive moiety;

X ³ is C;

X ⁸ is C, and

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3.

In some embodiments, each heterocyclyl contains at least one nitrogen atom.

In some embodiments, the phosphate-based linker is a divalent linker.

In some embodiments, L comprises at least one alkylene group.

In some embodiments, L is selected from the group of linkers listed in table 6.

In some embodiments, L is selected from the group of linkers listed in table 7.

In some embodiments, L is selected from the group of linkers listed in table 8.

In some embodiments, L is selected from the group consisting of:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) - (alkylene-O) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene- (O-alkylene) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J- (alkylene-O) _n -alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene- (O-alkylene) _n -U-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U- (alkylene-O) _n -alkylene- +;

Wherein:

each U is independently selected from the group consisting of:

Each J is independently

Each alkylene is independently selected from the group consisting of:

Each n is independently an integer from 1 to 100;

* Represents a linkage to an-O-atom of formula (ILd), and

+ Represents a connection to W;

In some embodiments, L is substituted with one or more water-soluble polymers.

In some embodiments, L is:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +, wherein:

u is selected from the group consisting of:

Each alkylene is independently selected from the group consisting of:

* Represents a linkage to an-O-atom of formula (ILd), and

+ Represents a connection to W;

wherein L is optionally substituted with one or more water soluble polymers.

In some embodiments, U is:

In some embodiments, L has the following structure:

Wherein represents a linkage to an-O-atom of formula (ILd), and +represents a linkage to W.

In some other embodiments, L has the following structure:

Wherein T is the water-soluble polymer, R ^t is H or methyl;

* Represents a linkage to an-O-atom of formula (ILd), and +represents a linkage to W.

In some embodiments, the water-soluble polymer is a polysaccharide.

Wherein:

r ^j is H or unsubstituted C ₁-C₆ alkyl,

R ^q is unsubstituted C ₁-C₆ alkyl,

R ^f is H or unsubstituted C ₁-C₆ alkyl,

S is 0,1, 2, 3,4, 5 or 6, and

T is 0,1, 2, 3, 4, 5 or 6.

In some embodiments, W is-ONH ₂.

Table 9 provides exemplary drug linker compounds that can be used or conjugated to any targeting ligand (such as an antibody or antibody fragment) selected based on its specificity for an antigen expressed on a target cell or at a target site of interest. The drug linkers of the invention may be used with antibodies or antibody fragments directed against a variety of antigens including, but not limited to, tumor-associated antigens, tumor-specific antigens, cancer antigens, or disease-specific antigens. Non-limiting examples of such antigens include PD-1、PD-L1、PSMA、CD70、CD3、HER2、HER3、TROP2、GPC3、VEGFR、EGFR、c-Met(HGFR)、CD4、CD33、CD19、CD22、CD25(IL-2Rα)、CD30、CD37、CD38、CD40L、CD44、CD46、CD47、CD48、CD52、CD56(NCAM-1)、CD71( transferrin R), CD74, CD79b, CD80, CD123 (IL-3 Rα), CD138 (syndecan -1)、CD142、CD163、CD166(ALCAM)、CD203c(ENPP3)、CD205(LY75)、CD221(IGF-1R)、CD262(TRAIL R2)、CD276(B7-H3)、CTLA4、 integrin, mesothelin, epCAM, CEACAM5, CEACAM6, DLL3, FOLR1, ROR2, GPNMB, GCC, GUCY c, naPi2b, flt-1, flt-3, folate receptor α, tissue factor (TF)、CA6、MUC1、MUC16(CA-125)、BCMA、SLAMF7(CS1)、TIM1、CanAg、Ckit(CD117)、EphA2、Nectin4、SLTRK6、FGFR2、LYPD3(C4.4a)、 cadherin 3, 5T4 (TPBG), STEAP1, PTK7, ephrin-A4, LIV-1 (SLC 39A6 or ZIP 6), SLC1A5, TENB2, ETBR, integrin v3, cripto, AGS-5 (SLC 44A 4), LY6E, AXL, LAMP1, LRRC15, TNF- α, and MN/CA IX.

In some embodiments, the drug linker compounds disclosed in table 9 may be used with the anti-HER 2 antibodies, antibody fragments, or antibody drug conjugates of the invention. In some embodiments, the drug linker compounds disclosed in table 9 may be used with the anti-CD 3 antibodies, antibody fragments, or antibody drug conjugates of the present disclosure. In some embodiments, the drug linker compounds disclosed in table 9 may be used with the anti-CD 70 antibodies, antibody fragments, or antibody drug conjugates of the invention. In some embodiments, the drug linker compounds disclosed in table 9 may be used with the anti-PSMA antibodies, antibody fragments, or antibody drug conjugates of the invention. In some embodiments, the drug linker compounds disclosed in table 9 can be used with the anti-TROP 2 antibodies, antibody fragments, or antibody drug conjugates of the disclosure. In some embodiments, the drug linker compounds disclosed in table 9 may be used with the anti-HER 3 antibodies, antibody fragments, or antibody drug conjugates of the present disclosure. In some embodiments, the drug linker compounds disclosed in table 9 may be used with the anti-GPC 3 antibodies, antibody fragments, or antibody drug conjugates of the present disclosure.

TABLE 9 non-limiting drug linker compounds of the invention

In some embodiments, the present disclosure provides a compound selected from the group consisting of the compounds listed in table 9 and salts thereof. In some further embodiments, a compound selected from the group consisting of compounds 12, 14, 16, 18, and 22, and salts thereof, is provided. In further embodiments, a compound selected from the group consisting of compounds 12, 14, 16, and 18, and salts thereof, is provided.

In some embodiments, the invention provides additional drug linkers prepared using similar procedures as described herein, including the protocols disclosed in the examples. Additional drug linker compounds are engineered by attachment of any possible linking group known in the art or elsewhere. The drug linker compound is engineered by linking one or more phosphate-based linkers through any chemically or functionally reactive site in the drug (e.g., nitrogen, halogen, boron, phosphorus, silicon, carbon, or oxygen of the cytotoxic agent). As disclosed elsewhere herein, selection of nitrogen, halogen, boron, phosphorus, silicon, sulfur, carbon, or oxygen sites in a drug for attachment to a phosphate-based linker is assessed based on the structure of the cytotoxic agent and using methods known in the art or elsewhere for generating phosphate-drug bonds. In some embodiments, the drug linkers of the invention include phosphate-based linkers attached or linked at the hydroxyl group of a cytotoxic agent or analog thereof (such as a topoisomerase inhibitor). In other embodiments, the drug linkers of the invention include phosphate-based linkers attached or linked at the methyl or methylene group of a cytotoxic agent or analog thereof. In some embodiments, such additional drug linker compounds may comprise a branched linker attached to two identical or different drugs. In some embodiments, the drug linkers of the invention include drug linkers generated via the attachment of one or more phosphate-based linkers at one or more of nitrogen, halogen, boron, phosphorus, silicon, sulfur, carbon, or oxygen of a cytotoxic agent.

The present disclosure provides drug moieties having linkers that reduce toxicity of the moiety in vivo while retaining pharmacological activity. In some embodiments, the toxicity of the linked drug is reduced or eliminated when administered to an animal or human, as compared to the free toxic group or toxic group derivative comprising an labile bond, while retaining pharmacological activity. In some embodiments, an increased dose of the linked toxic groups may be administered more safely to animals or humans. In certain embodiments, the non-natural amino acid polypeptide linked to a drug moiety (e.g., bicubicin or a bicubicin derivative or analog) provides in vitro and in vivo stability. In some embodiments, the unnatural amino acid polypeptide linked to the drug moiety is effective and less toxic than the free drug moiety.

In some embodiments, at least one post-translational modification may occur at some position on the polypeptide. In some embodiments, the co-translational or post-translational modification occurs via cellular mechanisms (e.g., glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitic acid addition, phosphorylation, glycolipid-bond modification, etc.), and in many cases, such co-translational or post-translational modification based on cellular mechanisms occurs at a naturally occurring amino acid site on the polypeptide, however, in some embodiments, the co-translational or post-translational modification based on cellular mechanisms occurs at an unnatural amino acid site on the polypeptide.

In other embodiments, the post-translational modification does not utilize cellular mechanisms, but rather utilizes chemical methods described herein or other methods suitable for the particular reactive group to provide functionality through attachment of a molecule (polymer; water soluble polymer; derivative of polyethylene glycol; second protein or polypeptide analog; antibody or antibody fragment; and any combination thereof) comprising a second reactive group to at least one unnatural amino acid comprising a ketone, aldehyde, acetal, hemiacetal, alkyne, cycloalkyne, azide, oxime, or hydroxylamine functional group. In certain embodiments, the co-translation or post-translational modification is performed in vivo in a eukaryotic cell or a non-eukaryotic cell. In certain embodiments, the post-translational modification is performed in vitro, without utilizing cellular mechanisms. This aspect also includes methods for producing, purifying, characterizing, and using such a drug linker containing at least one such co-or post-translationally modified unnatural amino acid.

Also included within the scope of the methods, compositions, strategies, and techniques described herein are reagents that are capable of reacting with a drug linker (containing a carbonyl or dicarbonyl group, alkynyl, cycloalkynyl, azide, hydroxylamine group, or masked or protected forms thereof) as part of a polypeptide to produce any of the above post-translational modifications. In certain embodiments, the resulting post-translationally modified drug linker will contain at least one oxime group, and the resulting modified oxime-containing drug linker may be subjected to subsequent modification reactions. This aspect also includes methods for producing, purifying, characterizing, and using such agents that are capable of any such post-translational modification of such drug linkers.

In certain embodiments, the polypeptide or unnatural amino acid linked composition comprises at least one co-translational or post-translational modification made in vivo by one host cell, where the post-translational modification is not typically made by another host cell type. In certain embodiments, the polypeptide comprises at least one co-translational or post-translational modification made in vivo by a eukaryotic cell, wherein the co-translational or post-translational modification is not typically made by a non-eukaryotic cell. Examples of such co-translational or post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitic acid addition, phosphorylation, glycolipid-bond modification, and the like. In one embodiment, co-translation or post-translational modification includes attachment of an oligosaccharide to an asparagine by a GlcNAc-asparagine linkage (including, but not limited to, wherein the oligosaccharide includes (GlcNAc-Man) ₂ -Man-GlcNAc, etc.). In another embodiment, co-translation or post-translational modification includes attachment of an oligosaccharide (including but not limited to Gal-GalNAc, gal-GlcNAc, etc.) to serine or threonine by a GalNAc-serine, galNAc-threonine, glcNAc-serine, or GlcNAc-threonine linkage. In certain embodiments, the protein or polypeptide may comprise a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, and the like. This aspect also includes methods for producing, purifying, characterizing, and using such polypeptides that contain at least one such co-translational or post-translational modification. In other embodiments, the glycosylated unnatural amino acid polypeptide is produced in a non-glycosylated form. Such non-glycosylated forms of glycosylated non-natural amino acids may be produced by a method comprising the steps of chemically or enzymatically removing oligosaccharide groups from an isolated or substantially purified or unpurified glycosylated non-natural amino acid polypeptide, producing the non-natural amino acid in a host that does not glycosylate the non-natural amino acid polypeptide, such host comprising a prokaryote or eukaryote engineered or mutated to not glycosylate the polypeptide, introducing a glycosylation inhibitor into a cell culture medium in which the non-natural amino acid polypeptide is produced by a eukaryote that normally glycosylates the polypeptide, or a combination of any such methods. Also described herein are such non-glycosylated forms of normally glycosylated non-natural amino acid polypeptides (normal glycosylation refers to polypeptides that will be glycosylated when produced under conditions in which naturally occurring polypeptides are glycosylated). Of course, such non-glycosylated forms of normally glycosylated non-natural amino acid polypeptides (or indeed any of the polypeptides described herein) may be in an unpurified form, a substantially purified form, or an isolated form.

In some cases, the incorporation of unnatural amino acids into antibodies or antibody fragments will be combined with other additions, substitutions, or deletions within polypeptides to affect other chemical, physical, pharmacological, and/or biological properties. In some cases, other additions, substitutions, or deletions may increase the stability of the polypeptide (including, but not limited to, resistance to proteolytic degradation) or increase the affinity of the polypeptide for its appropriate receptor, ligand, and/or binding protein. In some cases, other additions, substitutions, or deletions may increase the solubility of the polypeptide (including but not limited to when expressed in E.coli or other host cells). In some embodiments, to increase the solubility of the polypeptide after expression in E.coli or other recombinant host cells, a site for substitution with a naturally encoded or unnatural amino acid is selected in addition to another site for incorporation of the unnatural amino acid. In some embodiments, the polypeptide comprises another addition, substitution, or deletion that modulates affinity for the relevant ligand, binding protein, and/or receptor, modulates (including but not limited to increasing or decreasing) receptor dimerization, stabilizes receptor dimers, modulates circulatory half-life, modulates release or bioavailability, facilitates purification, or improves or alters a particular route of administration. Similarly, the unnatural amino acid polypeptide can comprise a chemical or enzymatic cleavage sequence, a protease cleavage sequence, a reactive group, an antibody binding domain (including but not limited to FLAG or poly-His) or other affinity-based sequence (including but not limited to FLAG, poly-His, GST, etc.) or a linked molecule (including but not limited to biotin), which improves detection (including but not limited to GFP), purification, transport through tissue or cell membranes, prodrug release or activation, size reduction, or other properties of the polypeptide.

Antibody Drug Conjugates (ADC)

The Antibody Drug Conjugates (ADCs) of the present disclosure provide novel therapeutic or anticancer agents by combining the selectivity of antibodies comprising one or more unnatural amino acids with cytotoxic agents. Targeted delivery of cytotoxic drugs into tumor tissue significantly increases the therapeutic window of these agents. The ADC of the present disclosure comprises an antibody that binds to a cytotoxic drug via a linker. The stability of the linker between the antibody and the cytotoxic drug is critical to the integrity of the ADC in the circulation. Successful ADC development for a given target antigen depends on optimization of antibody selection, linker design and stability, drug potency and drug pattern, and linker conjugation to the antibody. The pH and redox-sensitive, protease-sensitive linker properties influence the cycling stability and release of the drug moiety.

In some embodiments of the disclosure, the antibody of the ADC comprises a full length antibody or fragment thereof that binds to an antigen and is conjugated to a cytotoxic or immunosuppressive agent, wherein the antibody drug conjugate exerts (a) a cytotoxic or cytostatic effect on antigen-expressing or antigen-targeting cell lines, or (b) a cytotoxic, cytostatic or immunosuppressive/immunoactivating effect on antigen-expressing immune cells, wherein conjugation occurs at unnatural amino acids in the antibody. In some embodiments, the antigen, antigen-expressing cell, antigen-targeting cell, or antigen-expressing immune cell is PD-1、PD-L1、PSMA、CD70、CD3、HER2、HER3、TROP2、GPC3、VEGFR、EGFR、c-Met(HGFR)、CD19、CD22、CD25(IL-2Rα)、CD30、CD33、CD37、CD46、CD48、CD56(NCAM-1)、CD71( transferrin R), CD74, CD79B, C-D123 (IL-3Rα), CD138 (syndecan-1), CD142, CD166 (ALCAM), CD203C (ENPP 3), CD205 (LY 75), CD221 (IGF-1R), CD262 (TRAIL R2), CD276 (B7-H3), mesothelin, epCAM, CEACAM5, CEACAM6, DLL3, ROR1, ROR2, GPNMB, GCC, GUCY C, naPi2B, flt-1, flt-3, folate receptor α, tissue factor (TF)、CA6、MUC1、MUC16(CA-125)、BCMA、SLAMF7(CS1)、TIM1、CanAg、Ckit(CD117)、EphA2、Nectin4、SLTRK6、FGFR2、LYPD3(C4.4a)、 cadherin 3, 5T4 (TPBG), STEAP1, PTK7, ephrin-A4, LIV-1 (SLC 39A6 or ZIP 6), SLC1A5, TENB2, ETBR, integrin v3, critto, AGS-5 (SLC 44A 6), E, AXL, LAMP, MN 15/TNF α, or TNF α is not limited thereto. In some embodiments, the antigen, antigen-expressing cell, or antigen-targeting cell, or antigen-expressing immune cell is a TROP2, or HER2 or CD70 antigen, or antigen-targeting cell, or antigen-expressing immune cell. In some embodiments of the disclosure, the antibody of the ADC comprises a full length antibody or fragment thereof that binds to CD70 and is conjugated to a cytotoxic or immunosuppressive agent, wherein the antibody drug conjugate exerts (a) a cytotoxic or cytostatic effect on a cancer cell line expressing CD70, or (b) a cytotoxic, cytostatic or immunosuppressive/immunoactivating effect on immune cells expressing CD70, wherein the conjugation occurs at an unnatural amino acid in the antibody.

In some embodiments, an antibody, variant, or composition of the disclosure may be an antibody, variant, or composition that binds to an antigen receptor. In other embodiments, the antibody, variant, or composition may be one that binds to the extracellular surface of an antigen receptor. In some embodiments, an antibody, variant, or composition of the disclosure may be an antibody, variant, or composition having CDRs grafted onto a framework region of a variable region. In other embodiments, the antibodies, variants, or compositions of the present disclosure may be antibodies, variants, or compositions having unnatural amino acids. In some embodiments, the antibody, variant, or composition may be an antibody, variant, or composition described by more than one embodiment elsewhere herein of the disclosure. In some embodiments, an antibody, antibody variant, or antibody composition disclosed herein may be fully humanized. In other embodiments, the antibodies, antibody variants, or antibody compositions disclosed herein may be chimeric. In some embodiments, the antibody may be an antibody that is a full length antibody (variable region+fc region), fab, bispecific, fab dimer, fab bispecific, fab trispecific, bispecific T cell conjugate, dual affinity re-targeting antibody, igG1/IgG3 bispecific antibody, diabody, bispecific diabody, scFv-Fc, minibody.

In some embodiments, the ADC comprises an antibody conjugated to a drug, wherein conjugation occurs via unnatural amino acids in the antibody. Antibodies comprise at least one unnatural amino acid, non-limiting examples of which are disclosed herein.

In some embodiments, the ADC comprises an antibody conjugated to a drug, wherein conjugation occurs via unnatural amino acids in the heavy chain of the antibody. In some embodiments, the ADC comprises an antibody conjugated to a drug, wherein conjugation occurs via unnatural amino acids in the light chain of the antibody. In some embodiments, the ADC comprises a full length antibody conjugated to the drug, wherein conjugation occurs via unnatural amino acids in the antibody. In some embodiments, the ADC comprises a full length antibody conjugated to the drug, wherein conjugation occurs via unnatural amino acids in the heavy chain of the antibody. In some embodiments, the ADC comprises a full length antibody conjugated to the drug, wherein conjugation occurs via unnatural amino acids in the light chain of the antibody.

In some embodiments, the ADC comprises a full length antibody conjugated to the drug, wherein the first conjugation occurs via an unnatural amino acid in the heavy chain of the antibody and the second conjugation occurs via an unnatural amino acid in the light chain of the antibody. In some embodiments, the full length antibody comprises two full length heavy chains and two full length light chains, wherein the first pair of conjugation occurs via unnatural amino acids in each heavy chain of the antibody and the second pair of conjugation occurs via unnatural amino acids in each light chain of the antibody.

In some embodiments, the drug of the ADC is a cytotoxic drug or agent. In some aspects of the invention, the cytotoxic drug is a bicubicin analog, such as a bicubicin analog of the present disclosure. In some embodiments, the drug is a drug generated as described in the examples herein. In some embodiments, the ADC comprises an antibody, antibody fragment, or variant thereof engineered to have one or more unnatural amino acids that are site-specifically incorporated into heavy and/or light chain amino acid sequences conjugated to a drug via a phosphate-based linker.

In some aspects, the present disclosure provides an ADC of formula (II):

Wherein:

Ab is an antibody, wherein Ab comprises one or more unnatural amino acids;

l is a linker;

E is a moiety linking Ab and L;

d is an integer of 1 to 100, and

Wherein:

Each X ³ is C;

Each X ⁸ is C, and

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3;

or a pharmaceutically acceptable salt thereof.

In some embodiments, a has the structure of formula (a):

In some embodiments, the corresponding amine of formula (a) has a ClogP value of at least about 1.

In some embodiments, a has the structure of formula (b):

In some embodiments, the corresponding amine of formula (b) has a ClogP value of at least about 1.

In some embodiments, a has the structure of formula (c):

In some embodiments, the corresponding amine of formula (c) has a ClogP value of at least about 1.

In some embodiments, a has the structure of formula (d):

in some embodiments, the corresponding amine of formula (d) has a ClogP value of at least about 1.

In some embodiments, the antibody Ab comprises one or more unnatural amino acids.

In some embodiments, d is an integer from 1 to 100. In some embodiments, d is an integer from 1 to 10. In some embodiments, d is 1,2,3, 4, 5,6, 7, 8, 9, or 10.

In some embodiments, d is 1,2,3, or 4. In some embodiments, d is 1. In some embodiments, d is 2. In some embodiments, d is 3. In some embodiments, d is 4.

In some further embodiments of formula (II):

X ³ is C;

X ⁸ is C, and

In some embodiments, each of the heteroalkyl groups is an alkoxy group. In some embodiments, each of the alkoxy groups is independently-OR ^k, wherein each R ^k is independently alkyl, wherein the alkyl is optionally substituted with-N (R ^d)(R^e) OR heterocyclyl, wherein each R ^d and R ^e is independently H, alkyl, alkenyl, OR alkynyl, and wherein each heterocyclyl contains at least one nitrogen atom.

In some embodiments, X ⁴ is N, X ⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is C (R ⁷). In some other embodiments, X ⁴ is C (R ⁴),X⁵ is N, X ⁶ is C (R ⁶) and X ⁷ is C (R ⁷). In some other embodiments, X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is N and X ⁷ is C (R ⁷). in some embodiments, X ⁷ is CH. In some other embodiments, at least one of X ⁴ and X ⁷ is CH.

In some embodiments, X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is C (R ⁷). In some embodiments, X ⁴ is CH, X ⁷ is CH, or both.

In some embodiments, d is an integer from 1 to 100. In some embodiments, d is an integer from 1 to 10. In some embodiments, d is 1, 2, 3, or 4. In some embodiments, d is 1. In some embodiments, d is 2. In some embodiments, d is 3. In some embodiments, d is 4.

In some embodiments, L is a phosphate-based linker. In some embodiments, the phosphate-based linker comprises a phosphate-based moiety selected from the group consisting of phosphate, pyrophosphate, triphosphate, tetraphosphate, phosphonate, bisphosphonate, phosphoramidate, jiao Anji phosphate, triamino phosphate, tetraphosphoric acid, phosphorothioate, and phosphorodithioate. In some embodiments, the phosphate-based moiety is a pyrophosphate. In some embodiments, the phosphate-based moiety is a bisphosphonate.

In some embodiments, L further comprises at least one additional moiety, wherein each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, substituted alkylene, - (alkylene-O) -, optionally substituted arylene, -O-, -C (O) -, -N (R _w)-、-S(O)_0-2 -, water-soluble polymer, and amino acid, wherein each R _w is independently H or C ₁-C₈ alkyl, and combinations thereof.

In some embodiments, each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, - (alkylene-O) -, -C (O) -, -N (R _w) -, a water-soluble polymer, and an amino acid, wherein each R _w is independently H or C ₁-C₈ alkyl, and combinations thereof.

In some embodiments, L comprises at least one alkylene group. In some embodiments, L comprises an amino acid. In some embodiments, the amino acid is selected from the group consisting of serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and N _ε -methyl-lysine. In some embodiments, the amino acid is lysine or N _ε -methyl-lysine. In some embodiments, the amino acid is lysine. In some embodiments, the amino acid is N _ε -methyl-lysine.

In some embodiments, L comprises a water-soluble polymer. In some embodiments, L comprises a water-soluble polymer and an amino acid, wherein the water-soluble polymer is conjugated to the amino acid. In some embodiments, the water-soluble polymer is conjugated to a side chain of an amino acid. In some embodiments, the water-soluble polymer is conjugated to the amino acid via a spacer element.

In some embodiments, L is selected from the group of linkers of table 6. In some embodiments, L is selected from the group of linkers of table 7. In some embodiments, L is selected from the group consisting of:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) - (alkylene-O) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene- (O-alkylene) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J- (alkylene-O) _n -alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +,

* -P (=O) (OH) -O-P (=O) (OH) - (O) -alkylene- (O-alkylene) _n -U-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U- (alkylene-O) _n -alkylene- +;

Wherein:

each U is independently selected from the group consisting of:

Each J is independently

Each alkylene is independently selected from the group consisting of:

Each n is independently an integer from 1 to 100;

* Represents a linkage to an-O-atom of formula (II), and

+ Represents a linkage to E;

In some embodiments, each n is independently an integer from 1 to 10. In some other embodiments, each n is independently 1,2, or 3.

In some embodiments, L is substituted with one or more water-soluble polymers.

In some embodiments, L is a divalent linker. In some embodiments, L is a divalent linker optionally substituted with one or more water soluble polymers. In some embodiments, L is a linker substituted with one or more water-soluble polymers. In some embodiments, L is a linker substituted with a water soluble polymer.

In some embodiments, L is:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- + wherein:

u is selected from the group consisting of:

Each alkylene is independently selected from the group consisting of:

* Represents a linkage to an-O-atom of formula (II), and

+ Represents a linkage to E;

Wherein L is optionally substituted with one or more water soluble polymers. In some embodiments, L is substituted with one or more water-soluble polymers. In some embodiments, a water-soluble polymer is conjugated to the amino acid side chain of group U. In some embodiments, a water-soluble polymer is conjugated to the amino acid side chain of group U via a spacer element. In some embodiments, the spacer element is a carbonyl group.

In some embodiments, U is:

In some embodiments, L has the following structure:

Wherein T is the water-soluble polymer, R ^t is H or methyl, represents a linkage to an-O-atom of formula (II), and +represents a linkage to E.

In some embodiments, the water-soluble polymer is a polysaccharide.

In some embodiments, the water-soluble polymer is a polyethylene glycol (PEG) moiety.

In some embodiments, the PEG moiety has a molecular weight in the range of about 100Da to about 100,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100Da to about 10,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100Da to about 5,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100Da to about 1,000 Da.

In some embodiments, PEG is linear. In some other embodiments, PEG is branched.

In some embodiments, the drug-linker compound comprising a reactive moiety is conjugated to an antibody or antibody fragment by reacting the drug-linker compound with an antibody, antibody fragment, or variant thereof (or simply "antibody") comprising one or more natural or unnatural amino acids. The conjugation reaction provides an ADC in which the drug linker is conjugated to the natural or unnatural amino acid of the antibody via a covalent bond. The covalent bond may be the product of a reactive moiety of the drug linker and an additional moiety present in the natural or unnatural amino acid, where the additional moiety can react with the reactive moiety to form a covalent bond. Methods of conjugating drug linkers to antibodies are known in the art (see, e.g., johann, k. Et al, polymer Chemistry,27 (11): 4396-4407 (2020); bioconjug chem.,27 (12): 2791-2807 (2016); northrop, b.h. et al, polymer Chemistry,18 (6): 3415-3430 (2015); axup, j.y. Et al, proc.Natl. Acad. Sci.,109 (40): 16101-16016 (2012); hartmuth, c. Et al, angew.Chem., int.Ed.,40 (11): 2004-2021 (2001); sletten, e.m. and Bertozzi, C.R., angew.Chem., int.Ed.,48 (38): 6974-6998 (2009); WO2006/050262A2; and WO2013/185177A1, the contents of each of which are hereby incorporated by reference in their entirety). Non-limiting examples of reactions and linkages formed between a drug linker compound and natural or unnatural amino acids incorporated into antibodies of the present disclosure include the following.

A. (i) Reacting a drug linker comprising-N ₃ with an unnatural amino comprising an alkynyl group, thereby providing a bond comprising a 1,2, 3-triazolyl moiety, or (ii) reacting a drug linker comprising an alkynyl group with an unnatural amino acid comprising-N ₃, thereby providing a bond comprising a 1,2, 3-triazolyl moiety. In some embodiments, the alkynyl group is a cyclooctynyl group. In some embodiments, the unnatural amino acid is para-azido-L-phenylalanine. In some embodiments, the bond comprising a 1,2, 3-triazolyl moiety has the following structure:

Wherein:

Each s is independently 0 or an integer from 1 to 50, optionally, each s is independently 0, 1,2, 3,4, 5, or 6;

Each t is independently 0 or an integer from 1 to 50, optionally, each t is independently 0, 1,2, 3,4, 5 or 6;

Each + represents a linker connection to a drug linker, and

Each wavy line represents a link to an antibody.

B. (i) Reacting a drug linker comprising a tetrazinyl group with an unnatural amino acid comprising an (E) -cyclooctenyl group, thereby providing a bond comprising a1, 4-dihydropyridazinyl moiety, or (ii) reacting a drug linker comprising a tetrazinyl group with an unnatural amino acid comprising an (E) -cyclooctenyl group, thereby providing a bond comprising a1, 4-dihydropyridazinyl moiety. In some embodiments, the bond comprising a1, 4-dihydropyridazinyl moiety has the structure:

Wherein:

Each R ^f is independently H or alkyl, optionally unsubstituted C ₁-C₆ alkyl;

Each + represents a linker connection to a drug linker, and

Each wavy line represents a link to an antibody.

C. (i) Reacting a drug linker comprising a-ONH ₂ group with an unnatural amino acid comprising a carbonyl or keto group, thereby providing a bond comprising an oxime moiety, or (ii) reacting a drug linker comprising a carbonyl or keto group with an unnatural amino acid comprising a-ONH ₂ group, thereby providing a bond comprising an oxime moiety. In some embodiments, the carbonyl or ketone group is-C (O) R ^q, where R ^q is unsubstituted C ₁-C₆ alkyl. In some embodiments, R ^q is methyl. In some embodiments, the bond comprising the oxime moiety has the structure:

Wherein:

Each R ^q is independently unsubstituted C ₁-C₆ alkyl, optionally, each R ^q is methyl;

Each + represents a linker connection to a drug linker, and

Each wavy line represents a link to an antibody.

D. (i) Reacting a drug linker comprising a maleimide group with a natural or unnatural amino acid comprising a thiol (-SH) to provide a bond comprising a pyrrolidine-2, 5-dione moiety, such as a 3- (λ1-sulfanyl) pyrrolidine-2, 5-dione moiety, or (ii) reacting a drug linker comprising a thiol (-SH) group with an unnatural amino acid comprising a maleimide group to provide a bond comprising a pyrrolidine-2, 5-dione moiety, such as a 3- (λ1-sulfanyl) pyrrolidine-2, 5-dione moiety. In some embodiments, the natural amino acid is cysteine. In some embodiments, the bond comprising a pyrrolidine-2, 5-dione moiety (such as a 3- (λ1-sulfanyl) pyrrolidine-2, 5-dione moiety) has the structure:

Wherein:

Each + represents a linker connection to a drug linker, and

Each wavy line represents a link to an antibody.

E. (i) Reacting a drug linker comprising a primary or secondary amine with a natural or unnatural amino acid comprising a carboxylic acid group, a protected carboxylic acid or an activated ester group, thereby providing a bond comprising an amide moiety, or (ii) reacting a drug linker comprising a carboxylic acid group, a protected carboxylic acid or an activated ester group with a natural or unnatural amino acid comprising a primary or secondary amine group, thereby providing a bond comprising an amide moiety. In some embodiments, the natural amino acid is aspartic acid or glutamic acid. In some other embodiments, the natural amino acid is lysine.

In some embodiments, the reaction is a peptide coupling reaction or other well known method for amide formation, each of which may be performed using methods readily understood by one of ordinary skill in the art. In some embodiments, the bond comprising the amide moiety has the structure:

Wherein:

Each R ^j is independently H or alkyl, optionally unsubstituted C ₁-C₆ alkyl;

Each + represents a linker connection to a drug linker, and

Each wavy line represents a link to an antibody.

F. (i) Reacting a drug linker comprising a hydroxyl group (-OH) with a natural or unnatural amino acid comprising a carboxylic acid group, a protected carboxylic acid or an activated ester group, thereby providing a bond comprising an ester moiety, or (ii) reacting a drug linker comprising a carboxylic acid group, a protected carboxylic acid or an activated ester group with a natural or unnatural amino acid comprising a hydroxyl group (-OH), thereby providing a bond comprising an ester moiety. In some embodiments, the natural amino acid is aspartic acid or glutamic acid. In some other embodiments, the natural amino acid is serine, threonine, or tyrosine. Methods of forming such ester linkages may be performed using methods readily understood by one of ordinary skill in the art. In some embodiments, the bond comprising the ester moiety has the structure:

Wherein:

Each + represents a linker connection to a drug linker, and

Each wavy line represents a link to an antibody.

G. (i) Reacting a drug linker comprising a thiol group (-SH) with a natural or unnatural amino acid comprising a carboxylic acid group, a protected carboxylic acid, or an activated ester group, thereby providing a linkage comprising a sulfate moiety, or (ii) reacting a drug linker comprising a carboxylic acid group, a protected carboxylic acid, or an activated ester group with a natural or unnatural amino acid comprising a thiol group (-SH), thereby providing a linkage comprising a sulfate moiety. In some embodiments, the natural amino acid is aspartic acid or glutamic acid. In some other embodiments, the natural amino acid is cysteine. Methods of forming such thioester linkages can be performed using methods readily understood by one of ordinary skill in the art. In some embodiments, the bond comprising the ester moiety has the structure:

Wherein:

Each + represents a linker connection to a drug linker, and

Each wavy line represents a link to an antibody.

H. Reacting a drug linker comprising a-C (O) CH ₂NH₂ group with a natural or unnatural amino acid comprising a carboxylic acid group, a protected carboxylic acid or an activated ester group, thereby providing a bond comprising a-C (O) CH ₂ NHC (O) -moiety, or (ii) reacting a drug linker comprising a carboxylic acid group, a protected carboxylic acid or an activated ester group with a unnatural amino acid comprising a-C (O) CH ₂NH₂ group, thereby providing a bond comprising a-C (O) CH ₂ NHC (O) -moiety. In some embodiments, the natural amino acid is aspartic acid or glutamic acid. Methods of forming such bonds may be performed using methods readily understood by one of ordinary skill in the art. In some embodiments, the bond has the following structure:

Wherein:

Each + represents a linker connection to a drug linker, and

Each wavy line represents a link to an antibody.

I. The drug linker comprising a thiol group (-SH) is reacted with a natural or unnatural amino acid comprising a thiol group, thereby providing a disulfide-containing linkage. In some embodiments, the natural amino acid is cysteine. The method of forming disulfide bonds may be performed using methods readily understood by one of ordinary skill in the art.

Thus, in some embodiments, E comprises an amide, an ester, a thioester, a pyrrolidine-2, 5-dione, an oxime, a 1,2, 3-triazole, or a 1, 4-dihydropyridazine, wherein each of the 1,2, 3-triazole and the 1, 4-dihydropyridazine is optionally fused to an 8 membered ring. In some embodiments, E is selected from the group consisting of:

Wherein:

Each R ^j is independently H or unsubstituted C ₁-C₆ alkyl;

Each R ^q is independently unsubstituted C ₁-C₆ alkyl;

Each R ^f is independently H or unsubstituted C ₁-C₆ alkyl;

each s is independently 0,1, 2, 3, 4, 5, or 6;

Each t is independently 0,1, 2, 3, 4, 5, or 6;

Each + represents a connection to L, and

Each wavy line represents a connection to Ab.

In some embodiments, E is:

Wherein R ^q is unsubstituted C ₁-C₆ alkyl. In some embodiments, R ^q is methyl.

In some embodiments, ab comprises 1 to 10 unnatural amino acids. In some embodiments, ab comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unnatural amino acids. In some embodiments, ab comprises 1, 2, 3, or 4 unnatural amino acids. In some embodiments, ab comprises 1 unnatural amino acid. In some embodiments, ab comprises 2 unnatural amino acids. In some embodiments, ab comprises 3 unnatural amino acids. In some embodiments, ab comprises 4 unnatural amino acids.

In some embodiments, ab is configured to bind to an antigen.

In some embodiments, the Ab is configured to bind to a tumor-associated antigen (TAA) or a cancer antigen.

In some embodiments, ab binds ：PD-1、PD-L1、PSMA、CD70、CD3、HER2、HER3、TROP2、GPC3、VEGFR、EGFR、c-Met(HGFR)、CD19、CD22、CD25(IL-2Rα)、CD30、CD33、CD37、CD46、CD48、CD56(NCAM-1)、CD71( transferrin R) to a Tumor Associated Antigen (TAA) selected from the group consisting of CD74, CD79B, CD123 (IL-3Rα), CD138 (multi-ligand glycan-1), CD142, CD166 (ALCAM), CD203c (ENPP 3), CD205 (LY 75), CD221 (IGF-1R), CD262 (TRAIL R2), CD276 (B7-H3), mesothelin, epCAM, CEACAM5, CEACAM6, DLL3, ROR1, ROR2, GPNMB, GCC, GUCY c, naPi2B, flt-1, flt-3, folate receptor α, tissue factor (TF)、CA6、MUC1、MUC16(CA-125)、BCMA、SLAMF7(CS1)、TIM1、CanAg、Ckit(CD117)、EphA2、Nectin4、SLTRK6、FGFR2、LYPD3(C4.4a)、 cadherin 3, 5T4 (TPTPP), STEAP1, PTK7, ephrin-A4, LIV-1 (SLC 39A6 or ZIP 6), SLC1A5, TENB2, ETBR, integrin v3, cripto, AGS-5 (SLC 44 RC 4), LR 6, MN 1/TNF 3, and TNF IX.

In some embodiments, ab binds to an antigen selected from the group consisting of TROP2, CD70, HER2, CD3, PSMA, HER3, and GPC 3. In some embodiments, ab binds to CD 70. In some embodiments, ab binds to GPC 3.

In some embodiments, the one or more unnatural amino acids are selected from the group consisting of p-acetylphenylalanine, 4-acetyl-L-phenylalanine (p-acetyl-L-phenylalanine (pAF)), 3-O- (N-acetyl-beta-D-glucosamine) -L-threonine, N4- (beta-N-acetyl-D-glucosamine) -L-asparagine, O-allyl-L-tyrosine, alpha-N-acetylgalactosamine-O-L-serine, alpha-N-acetylgalactosamine-O-L-threonine, 2-aminocaprylic acid, 2-amino-L-phenylalanine, 3-amino-L-phenylalanine, 4-amino-L-phenylalanine, 2-amino-L-tyrosine, 3-amino-L-tyrosine, 4-azido-L-phenylalanine, 4-benzoyl-L-phenylalanine, (2, 2-bipyridyl-5) -L-alanine, 3-boron-L-phenylalanine, 4-bromo-L-phenylalanine, p-cyano-phenylalanine, p-bromo-L-phenylalanine, p-amino-phenylalanine, 3-amino-L-phenylalanine, 3-azido-phenylalanine, and combinations thereof, 3, 4-dihydroxy-L-phenylalanine (L-DOPA), 4-ethynyl-L-phenylalanine, 2-fluoro-L-phenylalanine, 3-fluoro-L-phenylalanine, 4-fluoro-L-phenylalanine, O- (3-O-D-galactosyl-N-acetyl-beta-D-galactosamine) -L-serine, L-homoglutamine, (8-hydroxyquinoline-3-yl) -L-alanine, 4-iodo-L-phenylalanine, 4-isopropyl-L-phenylalanine, O-isopropyl-L-tyrosine, 3-isopropyl-L-tyrosine, O-mannopyranosyl-L-serine, 2-methoxy-L-phenylalanine, 3-methoxy-L-phenylalanine, 4-L-phenylalanine, 3-methyl-L-phenylalanine, 3- (2-naphtyl) -L-alanine, 5-nitro-L-histidine, 4-nitro-L-leucine, 2-nitro-L-phenylalanine, 4-nitro-L-phenylalanine, 4-nitro-L-tryptophan, 5-nitro-L-tryptophan, 6-nitro-L-tryptophan, 7-nitro-L-tryptophan, 2-nitro-L-tyrosine, 3-nitro-L-tyrosine, O-phospho-L-serine, O-phospho-L-tyrosine, 4-propargyloxy-L-phenylalanine, O-2-propargyl-1-yl-L-tyrosine, 4-sulfo-L-phenylalanine and O-sulfo-L-tyrosine.

In some embodiments, at least one of the one or more unnatural amino acids is a p-acetylphenylalanine. In some more specific embodiments, at least one unnatural amino acid of the one or more unnatural amino acids is 4-acetyl-L-phenylalanine (p-acetyl-L-phenylalanine (pAF)). In some embodiments, each of the one or more unnatural amino acids is a p-acetylphenylalanine. In some more specific embodiments, each unnatural amino acid of the one or more unnatural amino acids is 4-acetyl-L-phenylalanine (p-acetyl-L-phenylalanine (pAF)).

In some embodiments, the Ab comprises a heavy chain having a heavy chain sequence, a light chain having a light chain sequence, or both.

In some embodiments, the heavy chain sequence comprises at least one unnatural amino acid of the one or more unnatural amino acids.

In some embodiments, the light chain sequence comprises at least one unnatural amino acid of the one or more unnatural amino acids.

In some embodiments, the Ab comprises two heavy chains, each heavy chain having a heavy chain sequence, wherein each heavy chain sequence comprises at least one unnatural amino acid of the one or more unnatural amino acids.

In some embodiments, the Ab comprises two light chains, each light chain having a light chain sequence, wherein each light chain sequence comprises at least one unnatural amino acid of the one or more unnatural amino acids.

In some embodiments, the Ab comprises two heavy chains and two light chains, wherein each heavy chain sequence and each light chain sequence comprises at least one unnatural amino acid of the one or more unnatural amino acids.

In some embodiments, one or more unnatural amino acids are solvent accessible.

In some embodiments, each unnatural amino acid of the one or more unnatural amino acids is the same. In some embodiments, each of the one or more unnatural amino acids is a p-acetylphenylalanine. In some more specific embodiments, each unnatural amino acid of the one or more unnatural amino acids is 4-acetyl-L-phenylalanine (p-acetyl-L-phenylalanine (pAF)).

In some embodiments, ab comprises 1,2, 3, or 4 unnatural amino acids.

In some embodiments, ab comprises two heavy chains and two light chains, each of the heavy chains having a heavy chain sequence and each of the light chains having a light chain sequence, wherein each of the heavy chain sequences and each of the light chain sequences comprises one unnatural amino acid.

In some other embodiments, the Ab is an anti-CD 70 antibody (i.e., ab is an anti-CD 70 Ab), an antibody fragment, or a variant thereof. In some embodiments, the anti-CD 70 Ab comprises at least one of the sequences listed in table 2.

In some embodiments, the anti-CD 70 Ab comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises an amino acid sequence selected from the group consisting of the heavy chain sequences listed in table 2, and the light chain sequence comprises an amino acid sequence selected from the group consisting of the light chain sequences listed in table 2, wherein at least one of the heavy chain sequence and the light chain sequence comprises one or more unnatural amino acids.

In some embodiments, the anti-CD 70 Ab heavy chain sequence comprises at least one unnatural amino acid of the one or more unnatural amino acids, and the position in the heavy chain sequence occupied by the at least one unnatural amino acid is selected from the group consisting of Kabat positions 114, 115, 129, 136, 159, and 160. In some embodiments, the position in the heavy chain sequence occupied by the at least one unnatural amino acid is Kabat position 114.

In some embodiments, the anti-CD 70 Ab light chain sequence comprises at least one unnatural amino acid of the one or more unnatural amino acids, and the position in the light chain sequence that is occupied by the at least one unnatural amino acid is selected from the group consisting of Kabat positions 110, 112, 114, and 121.

In some embodiments, the anti-CD 70 Ab comprises two heavy chain sequences, wherein each heavy chain sequence comprises at least one unnatural amino acid of the one or more unnatural amino acids, and the position in each heavy chain sequence occupied by each at least one unnatural amino acid is independently selected from the group consisting of Kabat positions 114, 115, 129, 136, 159, and 160. In some embodiments, each heavy chain sequence comprises a single unnatural amino acid.

In some embodiments, the position in each heavy chain sequence occupied by each at least one unnatural amino acid is Kabat position 114. In some embodiments, each anti-CD 70 Ab heavy chain sequence comprises a single unnatural amino acid.

In some embodiments, the anti-CD 70 Ab comprises two light chain sequences, wherein each light chain sequence comprises at least one unnatural amino acid of the one or more unnatural amino acids, and the position in each light chain sequence occupied by each at least one unnatural amino acid is independently selected from the group consisting of Kabat positions 110, 112, 114, and 121.

In some embodiments, the anti-CD 70 Ab comprises two heavy chain sequences and two light chain sequences, wherein (i) each heavy chain sequence comprises one unnatural amino acid and the position occupied by each unnatural amino acid is Kabat position 114, (ii) each light chain sequence comprises one unnatural amino acid and the position occupied by each unnatural amino acid is selected from the group consisting of Kabat positions 110, 112, 114, and 121. In some embodiments, each unnatural amino acid is a p-acetylphenylalanine. In some more specific embodiments, each unnatural amino acid is 4-acetyl-L-phenylalanine (para-acetyl-L-phenylalanine (pAF)).

In some embodiments, the anti-CD 70 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO. 26.

In some embodiments, the anti-CD 70 antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO. 27.

In some embodiments, the anti-CD 70 antibody comprises the heavy chain of the amino acid sequence of the particular SEQ ID NO. 25.

In some embodiments, the anti-CD 70 antibody comprises a heavy chain of the amino acid sequence of a particular SEQ ID NO. 20.

In some embodiments, the anti-CD 70 antibody comprises a light chain having the amino acid sequence of SEQ ID NO. 19.

In some embodiments, an anti-CD 70 antibody comprises two heavy chains each having the amino acid sequence of SEQ ID NO. 20 and two light chains each having the amino acid sequence of SEQ ID NO. 19.

In some embodiments, an anti-CD 70 antibody comprises two heavy chains each having the amino acid sequence of SEQ ID NO. 25 and two light chains each having the amino acid sequence of SEQ ID NO. 19.

In some other embodiments, the Ab is an anti-TROP 2 antibody (i.e., the Ab is an anti-TROP 2 Ab), an antibody fragment, or a variant thereof. In some embodiments, the anti-TROP 2Ab comprises at least one of the sequences listed in table 1. In some other embodiments, the anti-TROP 2 antibody comprises a heavy chain of the amino acid sequence of the particular SEQ ID NO. 5. In some other embodiments, the anti-TROP 2 antibody comprises a light chain having the amino acid sequence of SEQ ID NO. 4.

In some other embodiments, the Ab is an anti-HER 2 antibody (i.e., ab is an anti-HER 2 Ab), an antibody fragment, or a variant thereof. In some embodiments, the anti-TROP 2 Ab comprises at least one of the sequences listed in table 3. In some other embodiments, the anti-HER 2 antibody comprises a heavy chain of the amino acid sequence of particular SEQ ID NO. 29. In some other embodiments, the anti-HER 2 antibody comprises a light chain having the amino acid sequence of SEQ ID NO. 30.

In some other embodiments, the Ab is an anti-PSMA antibody (i.e., the Ab is an anti-PSMA Ab), an antibody fragment, or variant thereof. In some embodiments, the anti-PSMA Ab comprises at least one sequence listed in table 4. In some embodiments, the anti-PSMA antibody comprises the heavy chain of the amino acid sequence of a particular SEQ ID NO. 39. In some embodiments, the anti-PSMA antibody comprises a light chain having the amino acid sequence of SEQ ID NO. 40.

In some other embodiments, the Ab is an anti-HER 3 antibody (i.e., the Ab is an anti-HER 3 Ab), an antibody fragment, or variant thereof. In some embodiments, the anti-HER 3 Ab comprises at least one of the sequences listed in table 5. In some embodiments, an anti-HER 3 antibody comprises the heavy chain of the amino acid sequence of the particular SEQ ID NO. 58. In some embodiments, the anti-HER 3 antibody comprises a light chain having the amino acid sequence of SEQ ID NO. 47. In some other embodiments, the anti-HER 3 antibody comprises a light chain having the amino acid sequence of SEQ ID NO. 51.

It will be appreciated that ADCs are typically produced as a composition containing a population of ADCs, i.e., a mixture of ADCs that are substantially identical except for drug loading. As disclosed herein, an ADC composition may be characterized by a drug to antibody ratio (DAR) that reports the average number of drugs conjugated to an antibody in the ADC composition.

Thus, in some aspects, the present disclosure provides an ADC composition comprising a mixture of ADCs, wherein each ADC in the mixture is identical except that the number of drugs or drug linkers conjugated to each ADC may vary. In one non-limiting example, the ADC of the present disclosure includes a first ADC, a second ADC, a third ADC, and a fourth ADC, wherein the first ADC, the second ADC, the third ADC, and the fourth ADC are identical, except that the first ADC comprises one drug or drug linker, the second ADC comprises two drugs or drug linkers, the third ADC comprises three drugs or drug linkers, and the fourth ADC comprises four drugs or drug linkers.

In some other embodiments, an ADC composition is provided, the ADC composition comprising:

(a) An ADC of formula (II), wherein d is 1;

(b) An ADC of formula (II), wherein ADC is the same as (a) except that d is 2;

(c) An ADC of formula (II), wherein ADC is the same as (a) except that d is 3;

(d) An ADC of formula (II), wherein ADC is the same as (a) except that d is 4;

(e) An ADC of formula (II), wherein ADC is the same as (a) except that d is 5;

(f) An ADC of formula (II), wherein ADC is the same as (a) except that d is 6;

(g) An ADC of formula (II), wherein ADC is the same as (a) except that d is 7;

(h) An ADC of formula (II), wherein ADC is the same as (a) except that d is 8;

(i) An ADC of formula (II), wherein ADC is the same as (a) except that d is 9, or

(J) An ADC of formula (II), wherein ADC is the same as (a) except that d is 10;

or a combination of any two or more of the foregoing, wherein the composition is characterized as having a DAR of at least about 1 and at most about 10.

In some embodiments, the ADC composition is characterized as having a DAR of at least about 1 and at most about 8. In some embodiments, the ADC composition is characterized as having a DAR of at least about 2 and at most about 8, at least about 2 and at most about 6, or at least about 2 and at most about 4. In some embodiments, the ADC composition is characterized as having a DAR of at least about 3 and at most about 4. In some embodiments, the ADC composition is characterized as having a DAR of at least about 1 and at most about 2. In some embodiments, the ADC composition is characterized as having a DAR of at least about 1 and at most about 3. In some embodiments, the ADC composition is characterized as having a DAR of at least about 2 and at most about 4. In some embodiments, the ADC composition is characterized as having a DAR of at least about 3 and at most about 4. In some embodiments, the ADC composition is characterized as having a DAR of about 2. In some other embodiments, the ADC composition is characterized as having a DAR of about 4.

In some aspects of the disclosure, it is desirable to have antibodies, antibody fragments, variants, or drug conjugates that have increased serum half-life, water solubility, bioavailability, therapeutic half-life, or circulation time, or that have modulated immunogenicity, or that have modulated biological activity. One way to achieve such desirable characteristics of the compositions disclosed herein is through covalent attachment of polymeric polyethylene glycol (PEG). In order to maximize the desired properties of PEG, the total molecular weight and hydration state of the one or more polymers attached to the bioactive molecule must be high enough to impart the favorable properties typically associated with such polymer attachment, such as increased water solubility and circulation half-life, while not adversely affecting the bioactivity of the PEG-attached molecule. PEG derivatives are typically linked to the bioactive molecule through reactive chemical functional groups such as amino acid residues, N-terminal and/or carbohydrate moieties. In some aspects of the invention, PEG derivatives are linked to bioactive molecules through reactive chemical functionalities to improve the biophysical properties of the resulting ADCs. WO99/67291 discloses a method for conjugating a protein to PEG wherein at least one amino acid residue on the protein is substituted with a synthetic amino acid and the protein is contacted with PEG under conditions sufficient to effect conjugation to the protein.

In some aspects of the disclosure, it is desirable for an antibody, antibody fragment, variant, or drug conjugate to have increased serum half-life, water solubility, bioavailability, therapeutic half-life, or circulation time, or to modulate immunogenicity or biological activity. One way to achieve such desirable characteristics of the compositions disclosed herein is through covalent attachment of polymeric polyethylene glycol (PEG). In order to maximize the desired properties of PEG, the total molecular weight and hydration state of the one or more polymers attached to the bioactive molecule must be high enough to impart the favorable properties typically associated with such polymer attachment, such as increased water solubility and circulation half-life, while not adversely affecting the bioactivity of the PEG-attached molecule. PEG derivatives are typically linked to the bioactive molecule through reactive chemical functional groups such as amino acid residues, N-terminal and/or carbohydrate moieties. In some aspects of the invention, PEG derivatives are linked to bioactive molecules through reactive chemical functionalities to improve the biophysical properties of the resulting ADCs. WO99/67291 discloses a method for conjugating a protein to PEG wherein at least one amino acid residue on the protein is substituted with a synthetic amino acid and the protein is contacted with PEG under conditions sufficient to effect conjugation to the protein.

Proteins and other molecules typically have a limited number of reactive sites available for polymer attachment. Sites most suitable for modification by polymer attachment may play an important role in receptor binding, and such sites may be necessary to preserve the biological activity of the molecule, thus making them unsuitable for polymer attachment. Thus, the indiscriminate attachment of the polymer chains to such reactive sites on the bioactive molecule often results in a significant reduction or even complete loss of the bioactivity of the polymer modified molecule, and PEG attachment can be directed to specific locations within the protein such that the PEG moiety does not interfere with the function of the protein. One method of directing PEG attachment is to introduce synthetic amino acids into the protein sequence. The protein biosynthesis mechanism of the prokaryotes E.coli (ESCHERICHIA COLI/E.coli) can be altered to incorporate synthetic amino acids into proteins efficiently and with high fidelity in response to the amber codon UAG. See, e.g., J.W. Chin et al ,J.Amer.Chem.,Soc.124:9026-9027,2002;J.W.Chin,&P.G.Schultz,ChemBioChem 3(11):1135-1137,2002;J.W.Chin et al, PNAS USA 99:11020-11024,2002, and L.Wang, & P.G.Schultz, chem.Comm.,1:1-11,2002. A similar process can be accomplished with eukaryotic Saccharomyces cerevisiae (Saccharomyces cerevisiae/S.cerevisiae) (e.g., J.Chin et al Science 301:964-7,2003). Using this method, unnatural amino acids can be incorporated into antibodies, variants, or drug conjugates of the disclosure, thereby providing attachment sites for PEG. See, for example, WO 2010/01735 and WO2005/074650.

Methodology and techniques

The present disclosure encompasses methods and techniques well known in the art. These methods include mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacological conventional methods within the skill of the art. The compounds of the present disclosure may be synthesized using several methods or protocols employed in the art. See, for example Dubowchik et al, bioconjugate chem.13:855-869,2002; doronina et al, nature Biotechnology (7): 778-784,2003; wo2012/166560; wo2013/185117, each of which is incorporated herein by reference. Many methodologies and techniques for synthesizing pharmaceutical, diagnostic or therapeutic compounds are well known to those of ordinary skill in the art.

Unless otherwise indicated, the present disclosure also includes conventional techniques of molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, all of which are within the skill of the art. Such techniques are well explained in the literature, such as Molecular Cloning:ALaboratory Manual,Third Edition,Cold Spring Harbor Press,Cold Spring Harbor,NY(Sambrook et al edit ,2001);Oligonucleotide Synthesis:Methods And Applications(Methods in Molecular Biology),Herdewijn,P., humana Press, totowa, NJ, oligonucleotide Synthesis (Gait, M.J. edit ,1984);Methods In Molecular Biology,Humana Press,Totowa,NJ;Cell Biology:A Laboratory Notebook,Academic Press,New York,NY(Cellis,J.E., 1998), ANIMAL CELL Culture (Freshney, R.I. edit, 1987), introduction To Cell And Tissue Culture Plenum Press, new York, NY, (Mather, J.P. and Roberts, P.E. edit, 1998), cell And Tissue Culture: laboratory Procedures John Wiley and Sons, hoboken, NJ, (Doyle, A. Et al edit ,1993-8);Methods In Enzymology(Academic Press,Inc.)New York,NY;Weir's Handbook Of Experimental Immunology Wiley-Blackwell Publishers,New York,NY,(Herzenberg,L.A., 1987), current Protocols In Molecular Biology, greene Pub. Associates, new York, NY, (Ausubel, F.M. et al edit 1987), PCR The Polymerase Chain Reaction, birkhauser, boston, MA, (Mullis, K. Et al edit, 1994), current Protocols In Immunology, john Wiley and Sons, hoboken, NJ, (Colan, J. E. Et al edit ,1997);Gene Transfer Vectors For Mammalian Cells Cold Spring Harbor Press,Cold Spring Harbor,NY,(Miller,J.M., 1987), greene Pub. Associates, new York, NY, (Ausubel, F.M. et al edit 1987), ZK.3695, 1998, zdie, J.E. edit 9743, J. edit.

Therapeutic use of ADC

The antibodies or ADCs of the present disclosure may be used to treat a variety of diseases, disorders, conditions, or cancers. The compositions disclosed herein are useful for modulating immune responses. Modulation of an immune response may include stimulating, activating, increasing, enhancing, or upregulating the immune response. Modulation of an immune response may include suppressing, inhibiting, preventing, reducing, or downregulating the immune response. In some embodiments, the ADCs of the invention may be used to reduce or inhibit tumor growth or progression in cancers or cancer cells that contain an effective amount of an antigen expressing ADC.

Disclosed herein are methods of treating a condition in a subject with an ADC or pharmaceutical composition of the disclosure. In some cancers, overexpression of specific cell surface receptors may allow selective targeting of cancer cells with small molecules or drugs while minimizing the impact on healthy cells. The present invention provides a method of treating cancer by administering to a patient a therapeutically effective amount of an ADC of the invention comprising an antibody or antibody fragment conjugated to a drug linker as disclosed herein. The cancer to be treated by the ADC of the invention may be breast cancer including Triple Negative Breast Cancer (TNBC), brain cancer, pancreatic cancer, skin cancer, lung cancer, liver cancer, gall bladder cancer, colon cancer, ovarian cancer, prostate cancer, uterine cancer, bone cancer, blood cancer or a cancer or disease or condition associated with any of these cancers. In some embodiments, the cancer is a leukemia. In some embodiments, the blood cancer is leukemia, lymphoma, or myeloma. In some embodiments, the leukemia is leukemia, wherein the leukemia is Acute Myelogenous Leukemia (AML). In some embodiments, AML is responsive to treatment with an ADC of the disclosure comprising an anti-CD 70 antibody. In some other embodiments, the cancer is liver cancer. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC). In some embodiments, liver cancer is responsive to treatment with an ADC of the present disclosure comprising an anti-GPC 3 antibody.

In some embodiments, the invention provides a method of treating cancer by administering to a patient a therapeutically effective amount of an ADC of the invention. The cancer may be an antigen-expressing cancer. The cancer may be ovarian cancer including, but not limited to, epithelial, mesenchymal and germ cell tumors. Ovarian cancer may include fallopian tube cancer or primary peritoneal cancer. Cancers may be characterized by high expression of antigen receptors. Cancers can be treated by recruiting cytotoxic T cells to tumor cells that express antigen receptors. In some embodiments, the present disclosure provides methods of treating any cancer, disease, or condition associated with high expression of an antigen receptor by administering to a patient a therapeutically effective amount of an antibody or ADC of the present disclosure. In some embodiments, the invention provides a method of treating a disorder, or condition, or disease, or cancer by administering to a patient a therapeutically effective amount of an antibody or ADC of the invention. in some embodiments, the antibody, antibody fragment, or variant thereof binds to a tumor-associated antigen (TAA) selected from the group consisting of ：PD-1、PD-L1、PSMA、CD70、CD3、HER2、HER3、TROP2、GPC3、VEGFR、EGFR、c-Met(HGFR)、CD33、CD19、CD22、CD25(IL-2Rα)、CD30、CD37、CD46、CD48、CD56(NCAM-1)、CD71( transferrin R), CD74, CD79b, C-D123 (IL-3 ra), CD138 (multi-ligand glycan-1), CD142, CD166 (ALCAM), CD203C (ENPP 3), CD205 (LY 75), CD221 (IGF-1R), CD262 (TRAIL R2), CD276 (B7-H3), mesothelin, epCAM, CEACAM5, CEACAM6, DLL3, ROR1, ROR2, GPNMB, GCC, GUCY c, naPi2B, flt-1, flt-3, folate receptor alpha, tissue factor (TF)、CA6、MUC1、MUC16(CA-125)、BCMA、SLAMF7(CS1)、TIM1、CanAg、Ckit(CD117)、EphA2、Nectin4、SLTRK6、FGFR2、LYPD3(C4.4a)、 cadherin 3, 5T4 (TPBG), STEAP1, PTK7, hepatin-A4, LIV-1 (SLC 39A6 or ZIP 6), flt-3, folic acid receptor alpha, tissue factor (TF)、CA6、MUC1、MUC16(CA-125)、BCMA、SLAMF7(CS1)、TIM1、CanAg、Ckit(CD117)、EphA2、Nectin4、SLTRK6、FGFR2、LYPD3(C4.4a)、 cadherin 3, 5T4 (TPBG), STEAP1, PTK7, hepatin-A4, LIV-1 (SLC 39A6 or ZIP 6), SLC1A5, TENB2, ETBR, integrin v3, cripto, AGS-5 (SLC 44A 4), LY6E, AXL, LAMP1, LRRC15, TNF- α, MN/CA IX, but are not limited thereto. In some embodiments, the invention provides a method of treating a disorder, or condition, or disease, or cancer by administering to a patient a therapeutically effective amount of an anti-TROP 2 antibody or ADC of the invention. In some embodiments, the invention provides a method of treating a disorder, or condition, or disease, or cancer by administering to a patient a therapeutically effective amount of an anti-HER 2 antibody or ADC of the invention. In some embodiments, the invention provides a method of treating a disorder, or condition, or disease, or cancer by administering to a patient a therapeutically effective amount of an anti-CD 3 antibody or ADC of the invention. In some embodiments, the invention provides a method of treating a disorder, or condition, or disease, or cancer by administering to a patient a therapeutically effective amount of an anti-PSMA antibody or ADC of the invention. in some embodiments, the invention provides a method of treating a disorder, or condition, or disease, or cancer by administering to a patient a therapeutically effective amount of an anti-CD 70 antibody or ADC of the invention.

In some aspects, the disclosure provides ADCs for use in treating diseases or conditions in cells expressing high TROP2 receptor numbers. In some aspects, the disclosure provides ADCs for use in treating diseases or conditions in cells that express high numbers of HER2 receptors. In some aspects, the disclosure provides ADCs for use in the treatment of diseases or conditions in cells expressing high numbers of CD3 receptors. In some aspects, the disclosure provides ADCs for use in treating diseases or conditions in cells expressing high PSMA receptor numbers. In some aspects, the disclosure provides ADCs for use in the treatment of diseases or conditions in cells expressing high numbers of CD70 receptors. Antibodies and ADCs of the present disclosure are useful for the treatment of cancers, including but not limited to ovarian cancers, including but not limited to epithelial, mesenchymal and germ cell tumors. Ovarian cancer may include fallopian tube cancer or primary peritoneal cancer. Cancers may be characterized by high expression of antigen receptors, such as ovarian cancer. Cancers can be treated by recruiting cytotoxic T cells to tumor cells that highly express antigen receptors. The antibodies of the present disclosure are useful for treating genetic diseases, AIDS, or diabetes, but are not limited thereto. The antibodies, compounds, or compositions or conjugates of the present disclosure can be used to manufacture a medicament for treating a disease or condition in a cell expressing a high number of receptors. The antibodies, compounds, or compositions or conjugates of the present disclosure can be used to manufacture a medicament for the treatment of cancers including, but not limited to, breast cancer (including triple negative breast cancer), ovarian cancer (including, but not limited to, epithelial, mesenchymal, and germ cell tumors). The antibodies of the invention are useful in the manufacture of a medicament for the treatment of a disease, condition, or cancer associated with or associated with the expression of an antigen receptor (e.g., TROP2, HER2, CD3, PSMA, CD70, HER3, or GPC3 or antigen receptor). The anti-TROP 2 antibodies of the invention are useful for the manufacture of a medicament for the treatment of diseases, conditions or cancers associated with or associated with high TROP2 receptor numbers. In other embodiments, the anti-HER 2 antibodies of the present disclosure may be used in the manufacture of a medicament for treating a disease, condition, or cancer associated with or associated with HER2 expression. In other embodiments, the anti-CD 3 antibodies of the present disclosure may be used in the manufacture of a medicament for treating a disease, condition, or cancer associated with or associated with CD3 expression. In other embodiments, the anti-PSMA antibodies of the invention may be used in the manufacture of a medicament for the treatment of a disease, condition, or cancer associated with or associated with PSMA expression. In other embodiments, the anti-CD 70 antibodies of the present disclosure may be used in the manufacture of a medicament for the treatment of a disease, condition, or cancer associated with or associated with CD70 expression. In other embodiments, the anti-CD 70 antibodies of the present disclosure may be used in the manufacture of a medicament for treating a disease, condition, or cancer associated with or associated with HER3 expression. In other embodiments, the anti-CD 70 antibodies of the present disclosure may be used in the manufacture of a medicament for treating a disease, condition, or cancer associated with or associated with GPC3 expression.

In some embodiments, the condition to be treated is cancer. The cancer may be, but is not limited to, breast cancer (including Triple Negative Breast Cancer (TNBC)), brain cancer, pancreatic cancer, skin cancer, lung cancer, liver cancer, gall bladder cancer, colon cancer, ovarian cancer, prostate cancer, uterine cancer, bone cancer, and blood cancer, or a cancer or disease or condition associated with any of these cancers. The blood cancer may be leukemia, lymphoma or myeloma. In some embodiments, the cancer is leukemia and the leukemia is AML. Cancer is a cancer that begins with epithelial cells, which are cells that cover the body surface, produce hormones, and constitute glands. As non-limiting examples, cancers include breast cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, bladder cancer, stomach cancer, prostate cancer, liver cancer, ovarian cancer, brain cancer, vaginal cancer, vulvar cancer, uterine cancer, oral cancer, penile cancer, testicular cancer, esophageal cancer, skin cancer, fallopian tube cancer, head and neck cancer, gastrointestinal stromal cancer, adenocarcinoma, skin or intraocular melanoma, anal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urinary tract cancer, renal pelvis cancer, ureter cancer, endometrial cancer, cervical cancer, pituitary cancer, central Nervous System (CNS) tumors, primary CNS lymphoma, Brain stem gliomas and spinal axis tumors. In some cases, the cancer is a skin cancer, such as basal cell carcinoma, squamous cell carcinoma, melanoma, non-melanoma, or actinic (solar) keratosis. In some embodiments, the cancer is any cancer having a highly expressed number of antigen receptors, such as a TROP2 antigen receptor number, HER2 antigen receptor number, CD3 antigen receptor number, PSMA antigen receptor number, CD70 antigen receptor number, HER3 antigen receptor number, or GPC3 antigen receptor number. In some embodiments, the condition to be treated is a disease or condition associated with or having a high number of antigen receptors, such as, for example, TROP2 antigen receptor number, HER2 antigen receptor number, CD3 antigen receptor number, PSMA antigen receptor number, CD70 antigen receptor number, HER3 antigen receptor number, or GPC3 receptor number. The disease or condition may be a pathogenic infection. The pathogenic infection may be a bacterial infection. The pathogenic infection may be a viral infection. The disease or condition may be an inflammatory disease. The disease or condition may be an autoimmune disease. The autoimmune disease may be diabetes. The disease or condition may be cancer. In some embodiments, the disease or condition is any disease or condition having a highly expressed number of antigen receptors (e.g., a TROP2 antigen receptor number). The disease or condition may be a pathogenic infection. The bioactive molecule can interact with a cell surface molecule on the infected cell. The bioactive molecule can interact with a molecule on a bacterium, virus, or parasite. Pathogenic infections may be caused by one or more pathogens. In some cases, the pathogen is a bacterium, fungus, virus, or protozoan. Exemplary pathogens include, but are not limited to, bacillus (Bordetella), leptospira borrelii (Borrelia), brucella (Brucella), campylobacter (Campylobacter), chlamydia, chlamydophila thermophila (Chlamydophila), clostridium, corynebacterium, enterococcus (Enterobacter), escherichia, francisella (FRANCISELLA), clostridium, and the like, Haemophilus (Haemophilus), helicobacter (Helicobacter), legionella (Legionella), leptospira, listeria (Listeria), mycobacterium (Mycobacterium), mycoplasma, neisseria (Neisseria), pseudomonas (Pseudomonas), rickettsia (Rickettsia), salmonella (Salmonella), shigella (Shigella), staphylococcus, streptococcus, treponema (Treponema), Vibrio or Yersinia (Yersinia). The pathogen may be a virus. Examples of viruses include, but are not limited to, adenovirus, coxsackie virus (coxsackievirus), epstein-Barr virus, hepatitis virus (e.g., hepatitis a, hepatitis b, and hepatitis c), herpes simplex virus (types 1 and 2), cytomegalovirus, herpes virus, HIV, influenza virus, measles virus, mumps virus, papilloma virus, parainfluenza virus, polio virus, respiratory syncytial virus, rubella virus, and varicella zoster virus. Examples of diseases or conditions caused by viruses include, but are not limited to, cold, influenza, hepatitis, AIDS, varicella, rubella, mumps, measles, warts, and poliomyelitis. The disease or condition may be an autoimmune disease or an autoimmune related disease. Autoimmune diseases may be dysfunctions of the human immune system that cause the human body to attack its own tissues. Examples of autoimmune diseases and autoimmune related diseases include, but are not limited to, addison's disease, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome (APS), autoimmune aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, behcet's disease, celiac disease, crohn's disease, dermatomyositis, eosinophilic fasciitis, erythema nodosum, giant cell arteritis (temporal arteritis), goodpasture ' ssyndrome, goodpasture's disease, Graves 'disease, hashimoto's disease, idiopathic Thrombocytopenic Purpura (ITP), igA nephropathy, juvenile arthritis, diabetes mellitus, juvenile diabetes, kawasaki syndrome (Kawasaki syndrome), lambert-Eton syndrome (Lambert-Eaton syndrome), lupus (SLE), mixed Connective Tissue Disease (MCTD), multiple sclerosis, myasthenia gravis, pemphigus, polyarteritis nodosa, autoimmune polyadenylic syndrome type I, type II and type III, Rheumatic polymyalgia, polymyositis, psoriasis, psoriatic arthritis, reiter's syndrome, recurrent polychondritis, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren's syndrome, sperm and testis autoimmune, stiff person syndrome, polyarteritis (Takayasu 'sarteritis), temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo and Wegener's granulomatosis.

The disease or condition may be an inflammatory disease. Examples of inflammatory diseases include, but are not limited to, alveolitis, amyloidosis, vasculitis, ankylosing spondylitis, ischemic necrosis, baretoposi's Disease (Basedow's Disease), bell's palsy, bursitis, carpal tunnel Syndrome, celiac Disease, cholangitis, patellar chondromalacia, chronic active hepatitis, chronic fatigue Syndrome, kegen's Syndrome (Cogan's Syndrome), congenital hip dysplasia, costal chondritis, crohn's Disease, cystic fibrosis, de Quervain 'S TENDINITIS, diabetes-related arthritis, diffuse idiopathic hyperosteogeny, lupus, ehlers-Danlos Syndrome (Ehlers-Dannlos Syndrome), familial mediterranean fever, fasciitis, fibrositis/fibromyalgia frozen shoulder, ganglion cyst, giant cell arteritis, gout, graves' Disease, HIV-related rheumatic Disease Syndrome, hyperparathyroidism-related arthritis, infectious arthritis, inflammatory bowel Syndrome/irritable bowel Syndrome, juvenile rheumatoid arthritis, lyme Disease (LYME DISEASE), marfan's Syndrome (Marfan's Syndrome), milkova Disease (Mikulicz's Disease), mixed Betty tissue Disease, multiple sclerosis, myofascial pain Syndrome, osteoarthritis, osteomalacia, osteoporosis and corticosteroid-induced osteoporosis, paget's Disease (Paget's Disease), recurrent rheumatism, parkinson's Disease (Parkinson's Disease), pu Lu Moshi Disease (Plumer's Disease), polymyalgia, polymyositis, pseudogout, psoriatic arthritis, raynaud's phenomenon/syndrome, litt's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, sciatica (lumbar radiculopathy), scleroderma, scurvy, sickle cell arthritis, sjogren's syndrome, spinal stenosis, lumbar spondylolisthesis, stilll's Disease, systemic lupus erythematosus, gaultheria (Takayasu's Disease) (pulseless), tendinitis, tennis elbow/golf elbow, thyroid-related arthritis, trigger finger, ulcerative colitis, wegener's granulomatosis, and Whipple's Disease.

Pharmaceutical compositions containing the antibodies or ADCs of the invention may be formulated at an effective intensity for administration by various means to human patients suffering from conditions that may be affected by the antibody agonist or antagonist, such as, but not limited to, antiproliferative agents, anti-inflammatory agents, or antiviral agents, alone or as part of a condition or disease. The average amount of antibody or ADC may vary and in particular should be based on the recommendations and prescriptions of the qualified physician. The precise amount of antibody or ADC is a preferred problem, depending on factors such as the exact type of condition being treated, the condition of the patient being treated, and other ingredients in the composition. The present disclosure also provides for the administration of a therapeutically effective amount of another active agent, such as an anti-cancer chemotherapeutic or immunotherapeutic agent, but is not limited thereto. The amount administered can be readily determined by one skilled in the art based on the treatment with the antibodies or ADCs of the invention.

Pharmaceutical composition

In other aspects of the invention, the antibody, antibody fragment, variant or ADC further comprises a pharmaceutical composition or formulation. Such pharmaceutical compositions can employ various pharmaceutically acceptable excipients, stabilizers, buffers, and other components for administration to animals. See, e.g., remington, THE SCIENCE AND PRACTICE of Pharmacy, 19 th edition, gennaro editions, mack Publishing co., easton, PA,1995. Since a variety of components (e.g., purified, stabilized components) need to be considered, the stability, administration to a subject, and activity of a suitable composition or formulation is determined as a function of each compound. Suitable salts for inclusion in the composition or formulation may include, but are not limited to, sodium chloride, potassium chloride, or calcium chloride. Buffers and/or stabilizers, such as sodium acetate, may be used. Suitable buffers may include phosphate-citrate buffer, phosphate buffer, citrate buffer, L-histidine, L-arginine hydrochloride, bicarbonate buffer, succinate buffer, citrate buffer, and TRIS buffer, alone or in combination. Surfactants may also be used, including polysorbates (e.g., polysorbate 80), dodecyl sulfate (SDS), lecithin, alone or in combination.

In some aspects of the invention, the pharmaceutical composition or formulation may be in the form of an aqueous composition or in the form of a reconstituted liquid composition or as a powder. When the formulation is in liquid form, the composition or formulation may have a pH ranging from about 4.0 to about 7.0 or from about 4.5 to about 6.5. However, the skilled practitioner may adjust the pH to provide acceptable stability and administration.

The composition may be stored in a vial or cartridge, pen delivery device, syringe, intravenous administration tube, or intravenous administration bag, but is not limited thereto. In other embodiments, the pharmaceutical compositions of the invention may be administered as a single dose, or one or more subsequent doses may be administered minutes, days or weeks after the first administration. Further administration is contemplated as necessary to treat, alleviate or prevent a cancer, condition, disorder or disease.

In some cases, the antibodies, antibody fragments, variants, or ADCs disclosed herein may be used in combination with additional therapies or therapies including, but not limited to, surgery, radiation, cryosurgery, thermotherapy, hormonal therapy, chemotherapy, vaccines, and other immunotherapies. In some embodiments, such additional treatments may include therapeutic agents, such as chemotherapeutic agents, hormonal agents, anti-tumor agents, immunostimulants, immunomodulators, corticosteroids, or combinations thereof.

In other embodiments, the antibodies, antibody fragments, variants, or ADCs of the invention may be administered with one or more immunostimulants to induce or enhance an immune response. Immunostimulants that can stimulate specific branches of the immune system, such as Natural Killer (NK) cells that mediate antibody-dependent cellular cytotoxicity (ADCC). Such immunostimulants include, but are not limited to, IL-2, immunostimulatory oligonucleotides (e.g., cpG motifs), alpha-interferon, gamma-interferon, tumor necrosis factor alpha (TNF alpha). In other embodiments, the ADC of the invention may be administered with one or more immunomodulatory agents including, but not limited to, cytokines, chemokines (including, but not limited to, SLC5 ELC, MIP3 a, MIP3 β, IP-IO, MIG, and combinations thereof). The other therapeutic agent may be a vaccine that immunizes the subject against an antigen (e.g., TROP2, HER2, CD3, PSMA, CD70, HER3, or GPC 3). In some embodiments, such vaccines include an antigen, and optionally one or more adjuvants to induce or enhance an immune response. Many classes of adjuvants are known in the art.

Chemotherapeutic agents or any agent directed to treating, alleviating or preventing a disease, condition or cancer in a subject in need thereof may also be administered in combination with the ADCs disclosed herein. Chemotherapeutic agents may include, but are not limited to, erlotinibGenentech/oscharm.) bortezomib (bortezomib) ("aMillennium pharm, fulvestrant (fulvestrant)AstraZeneca), sunitinib (sutent) (SU 11248, pfizer), letrozole (letrozole)Novartis), imatinib mesylate (imatinib mesylate)Novartis), PTK787/ZK 222584 (Novartis), oxaliplatin (oxaliplatin) ("ASanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Rapamycin) (Sirolimus),Wyeth), lapattinib (lapatinib)GSK572016, glaxoSmithKline), lonafanil (lonafarnib) (SCH 66336), sorafenib (BAY 43-9006, bayer Labs.) and gefitinib (gefitinib)AstraZeneca), AG1478, AG1571 (SU 5271; sugen), alkylating agents such as thiotepa andCyclophosphamide, alkyl sulfonates such as busulfan, endosulfan and triamcinolone, antifolate antineoplastic agents such as pemetrexed (pemetrexed)Eli Lilly), aziridines such as benzotepa (benzodopa), carboquinone (carboquone), methoprene (meturedopa) and urapide (uredopa), ethyleneimine and methyl melamine including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide and trimethylol melamine, annonaceous lactones (especially bullatacin and bullatacin ketone (bullatacinone)), camptothecins (including the synthetic analogue topotecan), bryostatin, calostatin (callystatin), CC-1065 (including adonew (adozelesin) thereof), Carzelesin and Bizelesin synthetic analogues, nostalgins (cryptophycins) (particularly nostalgin 1 and nostalgin 8), dolastaxins, bicaryoxins (including synthetic analogues, KW-2189 and CB1-TM 1), eleutherobin (eleutherobin), podocarpine (pancratistatin), sarcandol (sarcodictyin), sponge chalone (spongistatin), nitrogen mustards (nitrogen mustards) such as chlorambucil, Nafipronil (chlornaphazine), cholesteryl phosphoramide (cholophosphamide), estramustine (estramustine), ifosfamide, mechlorethamine, oxazamine hydrochloride (mechlorethamine oxide hydrochloride), melphalan, mechlorethamine (novembichin), mechlorethamine cholesterol (PHENESTERINE), prednimustine (prednimustine), trefosfolamine (trofosfamide), uracil mustard (uracil mustard); nitrosoureas (nitrosoureas), such as carmustine, Chlorourea, fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranimnustine), antibiotics such as enediyne antibiotics, calicheamicin (calicheamicin), calicheamicin gamma and calicheamicin omega, danamycins (dynemicin) including danamycin A, bisphosphonates such as chlorophosphonate, esperamicin (esperamicin), and new carcinomycin chromophores and related chromoprotein enediyne antibiotic chromophores, Aclacinomycin (aclacinomysins), actinomycin (actinomycin), aflatoxin (anthramycin), diazoserine, bleomycin (bleomycins), actinomycin C (cactinomycin), cartrubicin (carabicin), carminomycin (caminomycin), carcinophilin (carzinophilin), chromomycin (chromomycinis), dactinomycin (dactinomycin), daunorubicin (daunorubicin), and pharmaceutical compositions, Ditropinix (detorubicin), 6-diazo-5-oxo-L-norleucine,Doxorubicin (doxorubicin) (including morpholino doxorubicin, cyanomorpholino doxorubicin, 2-pyrrolidodoxorubicin, and deoxydoxorubicin), epirubicin (epirubicin, esorubicin (esorubicin), idarubicin (idarubicin), doxycycline (marcellomycin), mitomycin (mitomycins) (such as mitomycin C), mycophenolic acid, norgamycin (nogalamycin), olivomycin (olivomycins), pelomycin (peplomycin), methylmitomycin (potfiromycin), puromycin (puromycin), doxorubicin (quelamycin), rodobutynin (rodorubicin), streptozotocin (streptonigrin), streptozocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), jingstadine (zinostatin), zorubicin (zorubicin), antimetabolites such as methotrexate and 5-fluorouracil (5-FU), folic acid analogs such as dimethyl folic acid (denopterin), ubenimex (ubenimex), and combinations thereof, Methotrexate, pterin (pteropterin), trimellitate, purine analogs such as fludarabine (fludarabine), 6-mercaptopurine, thioguanine (thiamiprine), thioguanine, pyrimidine analogs such as ancitabine (ancitabine), azacytidine (azacitidine), 6-azauridine (azauridine), carmofur (carmofur), cytarabine, dideoxyuridine (dideoxyuridine), deoxyfluorouridine (doxifluridine), Enocitabine (enocitabine), fluorouridine, androgens such as carbo Lu Gaotong (calusterone), drotasone propionate (dromostanolone propionate), cyclothioandrostanol (epitiostanol), mestane (mepitiostane), testosterone (testolactone), antiandrogen or androgen deprivation therapy, antiandrogens such as aminoglutethimide, mitotane, and the like, Trolesteine (trilostane), folic acid supplements such as folinic acid (frolinic acid), acetoglucurolactone (aceglatone), aldehyde phosphoramide glycoside (aldophosphamide glycoside), aminolevulinic acid (aminolevulinic acid), eniluracil (eniluracil), amsacrine (bestrabucil), bicalutamide (bisantrene), idazoxan, dimelamide (defofamide), dimetacin (demecolcine), deaquinone, difluoornithine (elformithine), irinotecan (elliptinium acetate), epothilone (epothilone), etodol (etoglucid), gallium nitrate, hydroxyurea, lentinan (lenan), lonidamine (lonidainine), maytansinoids such as maytansine (maytansine) and ansamitocin (ansamitocins), mitoguazone (mitoguazone), mitoxantrone (Mo Pai) alcohol (mopidanmol), diamine nitroacridine (nitraerine), penstatin (phenamet), pyrimorph (pirarubicin), sorangine (podophyllinic acid), carbazide 2-ethyl propizine; Polysaccharide complexes (JHS Natural Products, eugene, OR), lezocine (razoxane), risperidin, xician (sizofuran), spiral germanium (spirogermanium), tenasconic acid (tenuazonic acid), triamine quinone (triaziquone), 2' -trichlorotriethylamine, trichothecenes (trichothecenes) (especially T-2 toxin, warts (verracurin) A, cephalosporins (roridin) A and serpentine (anguidine)), uratein (urethan), vindesine, dacarbazine, mannatide (mannomustine), dibromomannitol (mitobronitol), dibromodulcitol (mitolactol), pipobroman (pipobroman), gacytosine, arabinoside ("Ara-C")), cyclophosphamide, thiotepa, taxoids (taxoids), such as paclitaxel (paclitaxel) Bristol-Myers Squibb Oncology, prencton, N.J.), ABRAXANE ^TM, nanoparticle formulations free of Cremophor (Cremophor), albumin, paclitaxel (American Pharmaceutical Partners, schaumberg, ill.) andDocetaxel (doxetaxel) (Rhone-Poulenc Rorer, antony, france); Gemcitabine (gemcitabine), 6-thioguanine, mercaptopurine, methotrexate, platinum analogues such as cisplatin and carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine; Vinorelbine, noose An Tuo (novantrone), teniposide, idatroxas, daunomycin, aminopterin (aminopterin), hilded, ibandronate, CPT-11, topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO), retinoids such as retinoic acid, capecitabine, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

Examples

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

EXAMPLE 1 general Experimental procedure

All commercially available anhydrous solvents were used without further purification and stored under nitrogen atmosphere. TLC was performed on Merck silica gel 60F254 plates, stained with UV light and/or with KMnO ₄ aqueous solution for observation. Chromatographic purification was performed on CombiFlash Rf from Teledyne ISCO using the conditions detailed in the experimental procedure. Analytical HPLC was performed on a Shimadzu system using Phenomenex Gemini-NX C18 μm 50X 4.6mm column eluting with a linear gradient of acetonitrile/aqueous solution containing 0.05% TFA at 1 ml/min. (mobile phase A:0.05% TFA/Water; mobile phase B:0.05% TFA/90% Acetonitrile (ACN) in Water) or Water BEH 1.7. Mu. M v 2.1.1X 50mmmm column. Analytical methods 1:1% B, 0-50% B in 11 min, 50-100% B in 0.5 min, 100% B in 1.5 min, 100-0% B in 1min, 0% B in 2 min, 10-20% B in 1min, 20-70% B in 11 min, 70-100% B in 0.5 min, 100% B in 1.5 min, 100-10% B in 1min, 10% B in 2 min, method 3: 0-40% B in 2:1 min, 40-90% B in 11 min, 90-100% B in 0.5 min, 100% B in 1.5 min, 100-10% B in 1min, 10% B in 2 min. Preparative HPLC was performed on Shimadzu systems using Gemini-NX C18 5 μm 100X 30mm, 150X 30mm or 250X 50mm columns, depending on scale. Mass Spectra (MS) were recorded on Shimadzu LCMS-2020 system and processed using Shimadzu LabSolutions software. Agilent 1260Infinity Binary LC coupled to the 6230Accurate-Mass TOFMS system was used for HR-ESI-TOF analysis. NMR spectroscopic data were collected on a 500MHz Bruker NMR spectrometer. Chemical shift (δ) is reported in ppm and referenced to deuterium solvent signal. Coupling constants (J) are reported in hertz (Hz). Spin multiplexing is described as s (singlet), br (broad), d (doublet), dd (doublet), t (triplet), q (quartet), or m (multiplet).

Chemical names of compounds were deduced from chemical structures using ChemDraw 20.1.1 (cambridge soft).

Abbreviations used in the examples herein are ACN, acetonitrile, CDI, 1' -carbonyldiimidazole, DCM, dichloromethane, DIPEA, N-diisopropylethylamine, DIAD, diisopropyl azodicarboxylate, DMF, dimethylformamide, EDC, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide HCl, etOAc, ethyl acetate, etOH, HATU, 1- [ bis (dimethylamino) methylene ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-oxide hexafluorophosphate, HPLC, high performance liquid chromatography, meOH, methanol, MS, TFA, trifluoroacetic acid, THF, TLC, thin layer chromatography.

EXAMPLE 2 Synthesis of Compound 4

The general synthetic scheme for compound 4 is shown below.

(S) -4- (benzyloxy) -8- (chloromethyl) -6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indole-2-carboxylic acid methyl ester (compound 2). (S) -4- (benzyloxy) -8- (chloromethyl) -6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indole-2-carboxylic acid methyl ester (compound 1, obtained by benzylation of Ring opening-biscarmycin SA, CAS No. 152785-82-5,MedChemExpress,Monmouth Junction,NJ,USA); 1.25g,2.07 mmol) was dissolved in THF/MeOH (20 mL/10 mL) and treated with LiOH solution (1M, 2 mL). The reaction was allowed to proceed at room temperature for 1 hour, then additional LiOH (2 mL) was added, and after 1 hour more, additional LiOH (2 mL) was added. The solution was then kept at-20 ℃ overnight. On the next day, volatiles were removed and water (20 mL) was added when cooled with an ice water bath. HCl (0.5N) solution was added until ph=1, and the precipitate was collected by filtration, and the filter cake was washed with water (5 ml×3) and dried under high vacuum to give compound 2 (1.2 g, 98%) as a pale green solid. HPLC (method 2) 7.0min, MSm/z 590 (M+H) ⁺.

(S) - (5- (benzyloxy) -1- (chloromethyl) -7- (indoline-1-carbonyl) -1, 2-dihydropyrrolo [3,2-e ] indol-3 (6H) -yl) (5, 6, 7-trimethoxy-1H-indol-2-yl) methanone (compound 3). Acid 2 (140 mg,0.237 mmol) and indoline (CAS number 496-15-1, 30. Mu.L, 0.264mmol;Sigma Aldrich) were dissolved in DMF (2 mL) and treated with HATU (110 mg,0.289 mmol) followed by DIPEA (62.1. Mu.L, 0.36 mmol). The reaction was kept at room temperature for 1 hour. Water (18 mL) was added to the solution, and the precipitate was collected by filtration. The filter cake was washed with water (3 ml×3) and dried under high vacuum to give a pale yellow solid (180 mg, > 100%) which was used directly in the next step. HPLC (method 2) 12.4min, MSm/z 713 (M+Na) ⁺.

(S) - (8- (chloromethyl) -4-hydroxy-6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indol-2-yl) (indolin-1-yl) methanone (compound 4). Substrate 3 (180 mg,0.237 mmol) was dissolved in THF (30 ml, clear solution) and degassed and purged with N ₂. Pd/C (120 mg) was added and again degassed and purged twice with H ₂. The reaction was stirred at room temperature for 4 hours. The reaction was quenched with celite and filtered, and concentrated to give compound (4) as a brown solid (130 mg, 91%). HPLC (method 2) 10.8min, MSm/z 601 (M+H) ⁺.

EXAMPLE 3 Synthesis of Compound 8

The general synthetic scheme for compound 8 is shown below.

(S) -6- (tert-Butoxycarbonylamino) -2- (2- (1, 3-dioxoisoindolin-2-yloxy) acetylamino) hexanoic acid (Compound 6). A suspension of 2, 5-dioxopyrrolidin-1-yl 2- (1, 3-dioxoisoindolin-2-yloxy) acetate (Compound 5 (CAS No. 1333377-78-8), 1g,4.17mmol; combi-Blocks, catalog No. QD-9979), (S) -2-amino-6- (tert-butoxycarbonylamino) hexanoic acid (1.15 g,4.67 mmol) and DIPEA (800.9. Mu.L, 4.59 mmol) in DCM (40 ml) was stirred at room temperature for 1 hour (becoming an almost clear solution). HPLC showed most of the starting material was consumed. The solvent was removed via rotary evaporator and the residue was dissolved in ACN/H ₂ O/TFA (adjusted to pH 1-2) and purified by HPLC and freeze dried to give a white powder as compound 6 (460 mg, 25%). HPLC (method 2) 6.6min, MS M/z 450 (M+H) ⁺.

(S) -6-amino-2- (2- (1, 3-dioxoisoindolin-2-yloxy) acetamido) hexanoic acid (Compound 7). Boc protected compound (6) was dissolved in DCM (6 mL) and treated with TFA (2 mL) at room temperature for 15min, and the volatiles were removed in vacuo and co-evaporated with DCM (3 ml×2) to give residue 7 (480 mg, > 100%) which was used directly in the next step. HPLC (method 1) 5.1min.

(S) -44- (2- (1, 3-dioxoisoindolin-2-yloxy) acetamido) -38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-aza-tetra-pentadecane-45-acid (Compound 8). Acid (7) (480 mg, calculated 1.02 mmol), m-dPEG12-O succinimide (780 mg,1.14 mmol) and DIPEA (800.9. Mu.L, 4.59 mmol) were dissolved in DCM (20 mL) and stirred at room temperature for 3 hours. The solvent was removed via rotary evaporator and the residue was dissolved in ACN/H ₂ O/TFA (adjusted to pH 1-2) and purified by HPLC and freeze dried to give a white powder as compound 8 (575 mg,3 step 61%). HPLC (method 1) 8.9min, MSm/z 920 (M+H) ⁺.

EXAMPLE 4 Synthesis of Compound 12

The general synthetic scheme for compound 12 is shown below.

(S) -8- (chloromethyl) -2- (indoline-1-carbonyl) -6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indol-4-yl dihydrogen phosphate (9). A solution of Compound 4 (130 mg,0.216 mmol) in THF/ACN (30 mL/20mL; containing some suspension) was cooled to-20 ℃ (ACN-dry ice bath) and slowly treated with POCl ₃ (205. Mu.L, 2.2 mmol) followed by DIPEA (227. Mu.L, 1.3 mmol). The reaction mixture was kept at-20 ℃ for 30 minutes, then at 4 ℃ (ice-water bath) for 30 minutes. HPLC showed starting material still present. The reaction mixture was cooled again to-20 ℃ and additional POCl ₃ (205 μl,2.2 mmol) and DIPEA (227 μl,1.3 mmol) were added and the reaction mixture was warmed to 4 ℃ for 30 min. HPLC showed no starting material and the reaction was quenched with 1M NaH ₂ PO4 and left overnight at 4 ℃. On the next day, intermediates are still present. The reaction mixture was allowed to warm to room temperature for 3 hours. After all volatiles were removed, the residue was suspended in water (20 mL) and filtered. The filter cake was washed with water (10 ml×3) to give a pale brown solid which was dried under high vacuum to crude compound 9 (160 mg, > 100%) and used directly in the next step. HPLC (method 3) 5.7min, MS M/z 679 (M-H) ⁺.

(S) -4- (((2-aminoethoxy) (hydroxy) phosphoryloxy) -8- (chloromethyl) -2- (indoline-1-carbonyl) -6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indole (10). Tert-butyl 2- (phosphonooxy) ethylcarbamate (147 mg,0.43 mmol) was dissolved in DMF (1 mL) and treated with CDI (215 mg,1.33 mmol) for 30min at room temperature. After quenching with MeOH (3 drops) for 15 minutes, all volatiles were removed to give a slurry. To a solution of the above slurry in DMF (2 mL) was added compound 9 (160 mg, calculated amount 0.216 mmol). The reaction was kept at room temperature overnight. After dilution with DCM (4 mL) and cooling to 4℃on an ice bath, TFA (4 mL) was added over 30 minutes. DCM was removed and the residue was purified by HPLC and the desired fractions were collected and concentrated using a rotary evaporator to give the TFA salt of the target molecule (10) (67 mg, 34%). HPLC (method 2) 4.9min, MSm/z 802 (M-H) ⁺.

(8S) -4- (((44- (2- (aminooxy) acetamido) -38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39, 46-diazatetraoctadeca-48-yl) hydroxyphosphoryloxy) (hydroxy) phosphoryloxy) -8- (chloromethyl) -2- (indoline-1-carbonyl) -6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indole (11). Acid (8) (95 mg,0.103 mmol) was dissolved in DCM (3 mL) and treated with N-hydroxysuccinimide (16.8 mg,0.146 mmol) and EDC (32.2 mg,0.168 mmol). The reaction was kept at room temperature for 6 hours and added to a suspension of 10 (67 mg,0.073 mmol) and DIPEA (63.7 μl,0.365 mmol) in DCM (3 mL). The mixture was kept at room temperature for 2 hours and then at 4 ℃ overnight (16 hours). After some DCM (about 2mL remaining) was removed, NH ₂NH₂ (36.6 μl,0.73 mmol) was added to the crude product 11 at room temperature for 10 min. The volatiles were removed completely and redissolved in ACN/H ₂ O (1/1, 5 mL) and the pH was adjusted to about 2 with 10% citric acid solution. The clear solution was then injected into HPLC and purified. The desired fraction was collected and freeze-dried to give the target compound (11) (42 mg, 34%) as a white powder. HPLC (method 1) 8.9min, MSm/z 1575 (M+H) ⁺.

EXAMPLE 5 Synthesis of Compound 14

The general synthetic scheme for compound 14 is shown below.

(8S) -4- (((44- (2- (aminooxy) acetamido) -38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39, 46-diazatetraoctadecan-48-yl) hydroxyphosphoryloxy) (hydroxy) phosphoryloxy) -8- (chloromethyl) -2- (5, 6-dimethoxyindoline-1-carbonyl) -6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indole (14) compound 13 (see example 2) was prepared using a similar procedure as described for compound 4. Compound 14 (see example 4) was then prepared via compound 13 using a method similar to that described for compound 12, yielding the target compound 14 (15 mg,6.9%, from 1). HPLC (method 3) 4.1min, MSm/z 1633 (M-H) ⁺.

EXAMPLE 6 Synthesis of Compound 16

The general synthetic scheme for compound 16 is shown below.

(8S) -4- (((44- (2- (aminooxy) acetamido) -38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39, 46-diazatetraoctadeca-48-yl) hydroxyphosphoryloxy) (hydroxy) phosphoryloxy) -8- (chloromethyl) -2- (2, 3-dihydro-1H-pyrrolo [2,3-c ] pyridine-1-carbonyl) -6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indole (Compound 16). Compound 15 was prepared using a similar method as described for compound 4 (see example 2). Compound 16 (see example 4) was then prepared via compound 15 using a method similar to that described for compound 12, yielding the target compound 16 (16 mg,0.64%, from 1). HPLC (method 2) 5.6min, MSm/z 1577 (M+H) ⁺,1575(M-H)⁺.

EXAMPLE 7 Synthesis of Compound 18

The general synthetic scheme for compound 18 is shown below.

(8S) -4- (((44- (2- (aminooxy) acetamido) -38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39, 46-diazatetraoctadeca-48-yl) hydroxyhydroxyphosphoryloxy) (hydroxy) phosphoryloxy) -8- (chloromethyl) -2- (5- (2-morpholinoethoxy) indoline-1-carbonyl) -6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indole (18). Compound 17 was prepared using a similar method as described for compound 4 (see example 2). Compound 18 (see example 4) was then prepared via compound 17 using a method similar to that described for compound 12, yielding the target compound 18 (15 mg,7.8%, from 1). HPLC (method 2) 6.0min, MSm/z 1705 (M+H) ⁺,1703(M-H)⁺.

EXAMPLE 8 Synthesis of Compound 19

The general synthetic scheme for compound 19 is shown below.

(S) -4- (((2- (2- (aminooxy) acetamido) ethyl) hydroxyhydroxyphosphoryloxy) (hydroxy) phosphoryloxy) -8- (chloromethyl) -2- (indoline-1-carbonyl) -6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indole (19). Compound 19 (see example 4) was prepared using a similar procedure to that described for compound 12, using 2, 5-dioxopyrrolidin-1-yl 2- (((tert-butoxycarbonyl) amino) oxy) acetate instead of compound 8, to give the title compound 19 (10 mg,7.5%, from 1). HPLC (method 3) 4.0min, MSm/z 877 (M-H) ⁺.

EXAMPLE 9 Synthesis of Compound 20

The general synthetic scheme for compound 20 is shown below.

(S) -4- (((2- (2- (aminooxy) acetamido) ethyl) hydroxyhydroxyphosphoryloxy) (hydroxy) phosphoryloxy) -8- (chloromethyl) -2- (5, 6-dimethoxyindoline-1-carbonyl) -6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indole (20). Compound 20 (see example 4) was prepared using a similar procedure to that described for compound 12, using 2, 5-dioxopyrrolidin-1-yl 2- (((tert-butoxycarbonyl) amino) oxy) acetate instead of compound 8, to give the title compound 20 (14 mg,4.0%, from 1). HPLC (method 3) 3.7min, MSm/z 935 (M-H) ⁺.

EXAMPLE 10 Synthesis of Compound 21

The general synthetic scheme for compound 21 is shown below.

(S) -4- (((1- (aminooxy) -2-oxo-6, 9, 12-trioxa-3-azatetradecan-14-yl) hydroxyphosphoryloxy) (hydroxy) phosphoryloxy) -8- (chloromethyl) -2- (5, 6-dimethoxyindoline-1-carbonyl) -6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indole (21). Compound 21 (see example 4) was prepared using a similar method to that described for compound 12, yielding the target compound 21 (10 mg,2.3%, from 1). HPLC (method 3) 4.2min, MS M/z 1067 (M-H) ⁺.

EXAMPLE 11 Synthesis of Compound 22

The general synthetic scheme for compound 22 is shown below.

(8S) -4- (((32- (2- (aminooxy) acetamido) -26, 33-dioxo-2,5,8,11,14,17,20,23-octaoxa-27,34-diazatricetyl-36-yl) hydroxyphosphoryloxy) (hydroxy) phosphoryloxy) -8- (chloromethyl) -2- (5, 6-dimethoxyindoline-1-carbonyl) -6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indole (22). Compound 22 (see example 4) was prepared using a similar method to that described for compound 12, yielding the target compound 22 (0.9 mg,2.2%, from 1). HPLC (method 3) 4.0min, MSm/z 1459 (M+H) ⁺,1457(M-H)⁺.

EXAMPLE 12 Synthesis of Compound 23

The general synthetic scheme for compound 23 is shown below.

(S) -4- (((2- (2- (aminooxy) acetamido) ethyl) hydroxyhydroxyphosphoryloxy) (hydroxy) phosphoryloxy) -8- (chloromethyl) -2- (2, 3-dihydro-1H-pyrrolo [2,3-c ] pyridine-1-carbonyl) -6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indole (23). Compound 23 (see example 4) was prepared using a similar method to that described for compound 12, affording the title compound 23.HPLC (method 2) 5.0min, MSm/z 876 (M-H) ⁺.

EXAMPLE 13 Synthesis of Compound 24

The general synthetic scheme for compound 24 is shown below.

(8S-4- (((2- (2- (aminooxy) acetamido) ethyl)) hydroxyphosphoryloxy) (hydroxy) phosphoryloxy) -8- (chloromethyl) -2- (5- (2-morpholinoethoxy) indoline-1-carbonyl) -6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indole (24). Compound 24 (see example 4) was prepared using a similar method to that described for compound 12, yielding the target compound 24 (10 mg,8.1%, from 1). HPLC (method 2) 5.7min, MSm/z 1006 (M+H) ⁺,1004(M-H)⁺.

EXAMPLE 14 Synthesis of Compound 25

The general synthetic scheme for compound 25 is shown below.

(S) - (8- (chloromethyl) -4-hydroxy-6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indol-2-yl) (6- (2- (dimethylamino) ethoxy) indolin-1-yl) methanone (25) compound 25 (see example 2) was prepared using a similar method to that described for compound 4 to give the title compound 25 (4 mg,11%, from 1). HPLC (method 2) 6.9min, MSm/z688 (M+H) ⁺.

EXAMPLE 15 Synthesis of Compound 26

The general synthetic scheme for compound 26 is shown below.

(S) - (8- (chloromethyl) -4-hydroxy-6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indol-2-yl) (5- (2- (dimethylamino) ethoxy) indolin-1-yl) methanone (26) compound 26 (see example 2) was prepared using a similar method to that described for compound 4 to give the title compound 26 (2 mg,10%, from 1). HPLC (method 2) 6.4min, MSm/z688 (M+H) ⁺.

EXAMPLE 16 Synthesis of Compound 27

The general synthetic scheme for compound 27 is shown below.

(S) - (8- (chloromethyl) -4-hydroxy-6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indol-2-yl) (6- (2-morpholinoethoxy) indol-1-yl) methanone (27). Compound 27 (see example 2) was prepared using a similar method to that described for compound 4, affording the target compound 27 (2 mg,10%, from 1). HPLC (method 2) 7.0min, MSm/z 730 (M+H) ⁺.

EXAMPLE 17 Synthesis of Compound 28

The general synthetic scheme for compound 28 is shown below.

(S) - (8- (chloromethyl) -4-hydroxy-6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indol-2-yl) (6- (2- (pyrrolidin-1-yl) ethoxy) indolin-1-yl) methanone (28). Compound 28 (see example 2) was prepared using a similar method to that described for compound 4, yielding the target compound 28 (2 mg,10%, from 1). HPLC (method 2) 7.1min, MS M/z 714 (M+H) ⁺.

EXAMPLE 18 Synthesis of Compound 29

The general synthetic scheme for compound 29 is shown below.

(S) - (8- (chloromethyl) -4-hydroxy-6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indol-2-yl) (5- (2- (pyrrolidin-1-yl) ethoxy) indolin-1-yl) methanone (29). Compound 28 (see example 2) was prepared using a similar method to that described for compound 4, affording the title compound 29 (4 mg,17%, from 1). HPLC (method 2) 7.0min, MS M/z 714 (M+H) ⁺.

EXAMPLE 19 Synthesis of Compound 30

The general synthetic scheme for compound 30 is shown below.

(S) - (8- (chloromethyl) -4-hydroxy-6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indol-2-yl) (6- (2- (4-methylpiperazin-1-yl) ethoxy) indol-1-yl) methanone (30). Compound 30 was prepared using a similar method to that described for compound 4, affording the title compound 30 (6 mg,30%, from 1). HPLC (method 2) 6.0min, MSm/z 743 (M+H) ⁺.

EXAMPLE 20 Synthesis of Compound 31

The general synthetic scheme for compound 31 is shown below.

(S) - (8- (chloromethyl) -4-hydroxy-6- (5, 6, 7-trimethoxy-1H-indole-2-carbonyl) -3,6,7, 8-tetrahydropyrrolo [3,2-e ] indol-2-yl) (5- (2- (4-methylpiperazin-1-yl) ethoxy) indol-1-yl) methanone (31). Compound 31 (see example 2) was prepared using a similar method to that described for compound 4, affording the title compound 31 (7.8 mg,34%, from 1). HPLC (method 2) 5.7min, MS M/z 743 (M+H) ⁺.

EXAMPLE 21 Synthesis of Compound 35

The general synthetic scheme for compound 35 is shown below.

Compound 35 was prepared using a similar method as described for compound 4 (see example 2). MSm/z 602 (M+H) ⁺.

EXAMPLE 22 Synthesis of Compound 36

The general synthetic scheme for compound 36 is shown below.

Compound 36 was prepared using a similar method as described for compound 4 (see example 2).

EXAMPLE 23 Compounds 37, 38 and 39

Compound 37 was prepared as described in WO 2015/153401 A1, the entire contents of which are hereby incorporated by reference in their entirety.

Compound 38 (CAS accession No. 157922-77-5) and compound 39 (CAS accession No. 152785-82-5, also known as (S) -ring-opening-bicubicin SA), each of which is commercially available (MedChemExpress, monmouth Junction, NJ, USA), have the following structures:

example 24 site-specific conjugation of drug linker to anti-CD 70 antibody containing unnatural amino acid pAF.

Anti-CD 70 antibody buffer with para-acetylphenylalanine (pAF) incorporated into each of the two heavy chains at Kabat position 114 was exchanged to 50mM sodium acetate, 2.5% trehalose, 0-20% DMSO (pH 4.0-4.3) and concentrated to 1mg/mL-20mg/mL. Acetylhydrazine (50 mM-100 mM) and amino-oxygen drug linker payload compounds 12, 14, 16 and 18 (10 molar equivalents-15 molar equivalents) prepared as described above were added and reacted at 30℃for 16 hours-72 hours. Antibody Drug Conjugate (ADC) was purified via Capto SPImpres column (Cytiva) to remove excess reagents. ADC buffer was exchanged to 50mM histidine, 100mM NaCl, 2.5% trehalose, pH 6.0, 0.22 μm filtered and stored at 4 ℃.

Specifically, an anti-CD 70 ADC was prepared by conjugating each of compounds 12, 14, 16 and 18 with an anti-CD 70 mAb as described above, wherein each of the two heavy chains has the amino acid sequence of SEQ ID NO:20, pAF at Kabat position 114, and each of the two light chains has the amino acid sequence of SEQ ID NO: 19. The cytotoxicity of the corresponding ADCs (hereinafter anti-CD 70-12, anti-CD 70-14, anti-CD 70-16 and anti-CD 70-18, respectively) was tested (see example 25).

As a benchmark, an anti-CD 70 ADC containing the drug (S) -ring-opened-bicubicin SA (compound 39) was prepared by conjugating the drug linker compound 37 with an anti-CD 70 mAb as described above, wherein each of the two heavy chains has the amino acid sequence of SEQ ID NO:25, pAF at Kabat position 114, and each of the two light chains has the amino acid sequence of SEQ ID NO:19 (hereinafter anti-CD 70-37). SEQ ID NO. 20 and 25 share the same heavy chain variable region amino acid sequence (i.e., SEQ ID NO: 26). As shown in Table 2, SEQ ID NO. 25 contains a tripeptide amino acid sequence "DEL" in the Fc constant region that is replaced with "EEM" in the Fc constant region of SEQ ID NO. 20, and SEQ ID NO. 20 and 25 are otherwise identical.

Example 25 in vitro cytotoxicity against CD70 ADC

The cytotoxicity of small molecule bicubicin analogs (compounds 4, 13, 15, 17 and 38) and anti-CD 70ADC (anti-CD 70-12, anti-CD 70-14, anti-CD 70-16 and anti-CD 70-18) was evaluated. Briefly, 786-O (CD 70 positive) cells were seeded into 96-well clear bottom whiteboards at 1,000 cells/well and incubated overnight in a 37 ℃ and 5% CO ₂ incubator. NCI-H929 (CD 70 negative) cells were seeded at 30,000 cells/well immediately prior to treatment of the test article. Serial dilutions of small molecule drugs or anti-CD 70 ADCs were added to the wells and the plates were incubated in a 37 ℃ and 5% CO ₂ incubator for 4 days. At the end of the incubation, cellTiter-Glo2.0 reagent (Promega, madison, wis.) was added to the room temperature equilibrated plate and luminescence was measured in a SpectraMax M5E plate reader. Relative viability was calculated as the percentage of untreated cells. Half maximal inhibitory concentration (IC ₅₀) was determined by nonlinear 4-parameter dose-response curve fitting using GRAPHPAD PRISM (GraphPad Software, san Diego, CA). Maximum killing (Emax) was determined by subtracting% viability at the indicated dose from 100%. The results are shown in fig. 1A and 1B and summarized in table 10.

Table 10 cytotoxicity data for the bicubicin analog and anti-CD 70 ADC.

As described above, the cytotoxicity of (S) -ring-opened-bicubicin SA (Compound 39) and anti-CD 70-37ADC on 786-O (CD 70 positive) cells was also tested, with IC ₅₀ values of 0.065nM and 0.321nM for free drug and ADC, respectively. anti-CD 70-37ADC had an IC ₅₀ value of 0.55nM when tested repeatedly under the same assay conditions. Thus, anti-CD 70-37ADC has an average IC ₅₀ value of 0.436 nM.

In summary, in the 786-O (CD 70 positive) cytotoxicity assay, the activity of anti-CD 70-12, anti-CD 70-14, anti-CD 70-16 and anti-CD 70-18 ADC was about 10-fold higher than the activity of anti-CD 70-37 ADC.

EXAMPLE 26 anti-GPC 3 ADC

Antibodies that specifically bind glypican-3 (GPC 3) are known in the art, including wild-type GPC3 and recombinant monoclonal Anti-GPC 3 antibodies, and can be prepared substantially as described in Pilia G et al (1996) Nature Genetics 12:241-247; kiyotaka N et al (2010) Anti-Cancer Drugs,21 (10:907-916), terrette J.A. et al (US 2010/0209432 A1), and Feng M et al (2013) Proc.Natl. Acad. Sci. USA,110 (12): E1083-91, the entire contents of each of which are hereby incorporated by reference in their entirety. Pilia g. et al (1996) Nature Genetics 12:241-247 (see, e.g., fig. 2, which is incorporated by reference in its entirety) and any other anti-GPC 3 antibodies, including those disclosed in the aforementioned references (i.e., kiyotaka n. Et al (2010); terrette j. A. Et al (US 2010/0209432 A1); and Feng m. Et al (2013)), can be modified using the methods described herein to incorporate one or more unnatural amino acids at any desired position.

Anti-GPC 3 ADCs were prepared by conjugating GPC3 clone 1mAb containing the unnatural amino acid pAF to each of compounds 12, 14, 16, and 18. Cytotoxicity of the corresponding ADCs (hereinafter, anti-GPC 3-12, anti-GPC 3-14, anti-GPC 3-16, and anti-GPC 3-18, respectively) and small molecule bicubicin analogs (compounds 15 and 17) was evaluated. Briefly, hepG2 and SNU449 cells were seeded at 3,000 cells/well into 96-well white opaque plates and incubated overnight in a 37 ℃ and 5% co ₂ incubator. Serial dilutions of small molecule drugs or anti-GPC 3 ADC were added to the wells and the plates were incubated in a 37 ℃ and 5% CO ₂ incubator for 3 days. At the end of the incubation, cellTiter-Glo2.0 reagent (Promega, madison, wis.) was added to the room temperature equilibrated plate and luminescence was measured in a SpectraMax M5E plate reader. Relative viability was calculated as the percentage of untreated cells. Half maximal inhibitory concentration (IC ₅₀) was determined by nonlinear 4-parameter dose-response curve fitting using GRAPHPAD PRISM (GraphPad Software, san Diego, CA). Maximum killing (Emax) was determined by subtracting% viability at the indicated dose from 100%. The results are shown in fig. 2A and 2B and summarized in table 11.

Table 11. Cytotoxicity data for the bicubicin analog and anti-GPC 3 ADC.

EXAMPLE 27 antitumor Activity of anti-GPC 3 ADC

Anti-GPC 3 ADC (anti-GPC 3-12, anti-GPC 3-14, anti-GPC 3-16 and anti-GPC 3-18) was evaluated at a low dose of 1mg/kg in a HepG2 xenograft mouse model of human hepatocellular carcinoma (HCC) in order to detect small changes in tumor growth rate and determine relative efficacy between ADCs in the model. The study design is summarized in table 12. Tumor growth inhibition was observed for all ADCs tested relative to vehicle control, with anti-GPC 3-14 exhibiting 66% tumor growth inhibition and 30 days tumor growth delay (data not shown).

TABLE 12 study design of in vivo test against GPC3 ADS in HepG2 xenograft model

Group of	Treatment of	Dosage of	Pathway	Time schedule	N
						1	Vehicle body	NA	IV	QWx3	10
2	GPC3-12 against	1mg/kg	IV	QWx3	9
						3	GPC3-14 against	1mg/kg	IV	QWx3	10
4	GPC3-16 against	1mg/kg	IV	QWx3	9
						5	GPC3-18 against	1mg/kg	IV	QWx3	9

Example 28 cloning of molecules for expression of anti-CD 70, anti-TROP 2 and anti-HER 3 antibodies

CHO cell codon optimized antibody heavy and light chain cDNA sequences were obtained from commercial DNA synthesis service (INTEGRATED DNA Technologies (IDT), san Diego, CA) for each of the CD70, TROP2 and HER3 antibodies. The synthesized DNA fragment was digested with HindIII and EcoRI (both from NEW ENGLAND BioLabs, (NEB), ipswich, mass.) and purified using the PCR purification kit (Qiagen, valencia, calif.). The digested antibody gene fragments were then ligated into expression vectors via a quick connect kit (NEB) to generate constructs for expression of wild-type antibody heavy and light chains. The resulting plasmid was propagated in E.coli and verified by DNA sequencing services (Eton Biosciences, san Diego, calif.).

Generation of amber codon containing mutants-heavy chain amino acid A114 (Kabat numbering) was selected to genetically incorporate the unnatural amino acid pAF based on the crystal structure of the immunoglobulin G1[ IgG1] mAb. For HER3, additional sites within the light chain sequence were also selected for pAF incorporation (see table 5). The genetic codon at the selected site is then mutated to an amber codon (TAG) via site-directed mutagenesis to generate an expression plasmid for the antibody mutant. Primers were purchased from IDT. All site-directed mutagenesis experiments were performed using the Q5 site-directed mutagenesis kit according to the instructions for use (NEB). Expression plasmids of the mutants were propagated in E.coli and verified by DNA sequencing services (Eton Biosciences).

Example 29 protocol for the production of antibodies containing pAF at heavy chain amino acid sequence position 114 (Kabat numbering; anti-CD 70, anti-TROP 2, and anti-HER 3 heavy chains) and light chain amino acid sequence position 121 (Kabat numbering; anti-HER 3 light chains).

Transient expression-the anti-CD 70, anti-TROP 2 and anti-HER 3 plateau CELL lines were maintained in EX-CELL 302 (Sigma) supplemented with 3mM L-glutamine (Gibco) and 3mM Glutamax (Gibco). Cells were passaged every 3 to 4 days and seeded at a density of 40 ten thousand cells per milliliter. Cells were seeded at 60 ten thousand cells per ml one day prior to transfection. On day 0, cells were transfected with antibody expression plasmids encoding the light and heavy chains using MaxCyte electroporation platform according to the instructions for use. After transfection, the cells were placed in an empty 125ml shake flask and incubated in a static incubator at 37 ℃ for 30 minutes. Basal expression medium (50% Dynamis: 50% ExCELL supplemented with 3mM L-glutamine and 3mM Glutamax) was added to transfected cells in shake flasks at a final density of 3X 10 ⁶ cells/ml. Transfected cells were incubated at 37℃with 5% CO ₂ on an orbital shaker set at 140rpm or 155 rpm. pAF (final concentration in culture: 1 mM), long R3 IGF-1 (Sigma; final concentration in culture: 120. Mu.g/L), glutaMAX (final concentration in culture: 2 mM) and (a) Cell Boost 5 (GE HEALTHCARE; final concentration in culture: 7 g/L), or (b) Cell Boost 4 (GE HEALTHCARE; final concentration in culture: 3.75 g/L) and Cell Boost7b (GE HEALTHCARE; final concentration in culture: 0.2 g/L) were added to the culture on day 1. The incubator temperature was changed from 37 ℃ to 32 ℃. Additional Glutamax (final concentration: 2 mM) and (a) Cell Boost 5 (final concentration: 7 g/L) were added on day 3 and supernatants were collected on day 5, or (b) Cell Boost 4 (GE HEALTHCARE; final concentration in culture: 2 g/L) and CellBoost b (final concentration in culture: 0.1 g/L) were added on day 3 and 5 and supernatants were collected on day 7. The medium glucose level was monitored using a glucose meter and additional glucose was added to the culture when the glucose level was below 2 g/L. Viable Cell count and viability were measured by Vi-Cell instrument. Antibody production was determined by Octet using a protein G sensor.

Stable bulk pool (bulk pool) generation-expression plasmids were linearized using Pvu I (NEB) digestion for four hours. After linearization, the DNA was purified using phenol/chloroform/isoamyl alcohol extraction and dissolved in endotoxin-free water at a concentration of 2.5. Mu.g/. Mu.l. The plateau CELL line was maintained in EX-CELL 302 supplemented with 3mM L-glutamine and 3mM Glutamax. Cells were passaged every 3 to 4 days and seeded at a density of 0.3X10 ⁶/ml. Cells were seeded at 0.6X10 ⁶/ml the day before transfection. On day 0, 15X 10 ⁶ cells were transfected with 25. Mu.g of linearized antibody expression plasmid using MaxCyte electroporation (OC-100) platform, according to the instructions for use. After transfection, the cells were placed in an empty 125ml shake flask and incubated in a static incubator at 37 ℃ for 30 minutes. 30ml of recovery medium (50% Ex-302:50% CD-CHO supplemented with 3mM L-glutamine and 3mM Glutamax) was then added to the flask and shaken overnight. On the first day, transfected cells were counted, centrifuged, washed and resuspended in selection medium (50% ExCELL 302:50% CD-CHO containing 50. Mu.M MSX) to create a stable batch pool. Viable cell number and viability were monitored and medium was changed every 3 to 4 days until the viability of the stable batch pool was restored to 90%. When the selection is completed, a frozen cell bank is prepared and the resulting stable batch cell is used to produce material for fed-batch expression.

Fed-batch expression-A batch pool of previously generated antibody stabilized at a density of 0.5X10 ⁶/ml was inoculated on day 0 into basal expression medium (50% Dynamis:50% ExCELL 302 supplemented with 50. Mu.M MSX, or 50% ExCELL 302 supplemented with 1 XGS, 2. Mu.g/ml insulin, 0.5mM ornithine, 2g/L glucose and 1 Xanti-caking agent, and 50% Dynamis:50% ExCELL 302 of 25. Mu.M MSX) in shake flasks. Transfected cells were incubated at 37℃with 5% CO ₂ on an orbital shaker set at 150 rpm. pAF (final concentration in culture: 0.5 mM), cell Boost 4 (GE HEALTHCARE; final concentration in culture: 10 g/L) and Cell Boost 7b (GE HEALTHCARE; final concentration in culture: 0.52 g/L) were added to the culture on day 3. Long R3 IGF-1 (final concentration in culture: 120. Mu.g/L) was added to the medium on day 5. The medium glucose level was monitored using a glucose meter and additional glucose was added to the culture when the glucose level was below 2 g/L. Viable Cell count and viability were measured by Vi-Cell instrument. Supernatants were collected on day 7 or 10 for purification. Antibody production was determined by Octet using a protein G sensor.

EXAMPLE 30 purification of antibodies from EuCODE expression System

Clarified cell culture medium containing target antibody containing unnatural amino acid pAF was loaded onto protein A ProSep Ultra column (EMD Millipore) equilibrated in 20mM sodium phosphate, 100mM sodium chloride (pH 7.5). After loading, the column was washed with buffer a (20 mM sodium phosphate, 100mM sodium chloride, pH 7.5) followed by washing with wash buffer B (5 mM succinic acid, pH 5.8) to remove host cell contaminants. The target antibody was eluted from the column with elution buffer C (50 mM glycine, 10mM succinic acid, pH 3.2). The target antibodies were pooled and the pH was adjusted to pH5.0 with 2.0M tris base. The target antibody was further purified by loading the conditioned protein a pool onto Capto SPImpres column (GE HEALTHCARE) equilibrated in 30mM sodium acetate (pH 5.0). The target antibody was eluted from the column with a linear gradient to 100% buffer B (30 mM sodium acetate, 0.5M sodium chloride, pH 5.0) and fractions containing monomeric antibody were pooled, filtered at 0.22. Mu.M and stored at < 65℃until further use.

Example 31 anti HER2 and anti PSMA antibodies

Anti-HER 2 and anti-PSMA antibodies of the present disclosure and variants thereof, as well as ADCs of the present disclosure containing the antibodies and variants thereof, may be substantially as described in WO2022/212899A1 and WO2019/191728A1 and/or prepared by adaptation methods explicitly disclosed herein, the entire contents of each of these patents being incorporated herein by reference in their entirety.

Additional non-limiting embodiments of the invention are set forth below.

A1. A compound of formula (I):

Wherein:

R is H or L-W, wherein L is a linker and W is a reactive moiety, and

Wherein:

Each X ³ is C;

Each X ⁸ is C, and

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3;

Or a salt thereof.

A2. the compound of embodiment A1, wherein the compound is a compound of formula (Ia) having the structure:

Wherein:

R is H or L-W, wherein L is a linker and W is a reactive moiety;

X ³ is C;

X ⁸ is C;

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3.

A3. The compound of embodiment A1, wherein the compound is a compound of formula (Ib) having the structure:

Wherein:

R is H or L-W, wherein L is a linker and W is a reactive moiety;

X ³ is C;

X ⁸ is C;

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3.

A4. the compound of embodiment A1, wherein the compound is of formula (Ic) having the structure:

Wherein:

R is H or L-W, wherein L is a linker and W is a reactive moiety;

X ³ is C;

X ⁸ is C, and

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3.

A5. the compound of embodiment A1, wherein the compound is of formula (Id) having the structure:

Wherein:

R is H or L-W, wherein L is a linker and W is a reactive moiety;

X ³ is C;

X ⁸ is C, and

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3.

A6. The compound according to any one of embodiments A1 to A5, wherein:

X ³ is C;

X ⁸ is C, and

A7. The compound of any one of embodiments A1 through A6, wherein each of X ¹ and X ² is CH ₂, and X ⁹, when present, is CH ₂.

A8. the compound according to any one of embodiments A1 to A7, wherein:

X ⁴ is C (R ⁴) or N, wherein R ⁴ is H, halogen or heteroalkyl;

X ⁵ is C (R ⁵) or N, wherein R ⁵ is H, halogen or heteroalkyl;

x ⁶ is C (R ⁶) or N, wherein R ⁶ is H, halogen or heteroalkyl, and

X ⁷ is C (R ⁷) or N, wherein R ⁷ is H, halogen or heteroalkyl.

A9. The compound of any one of embodiments A1-A8, wherein each of the heteroalkyl is an alkoxy.

A10. The compound of embodiment A9 wherein each of the alkoxy groups is independently-OR ^k, wherein each R ^k is independently alkyl, wherein the alkyl is optionally substituted with-N (R ^d)(R^e) OR heterocyclyl, wherein each R ^d and R ^e is independently H, alkyl, alkenyl, OR alkynyl, and wherein each heterocyclyl contains at least one nitrogen atom.

A11. the compound of any one of embodiments A1-a 10, wherein X ⁴ is N, X ⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is C (R ⁷).

A12. the compound of any one of embodiments A1-a 10, wherein X ⁴ is C (R ⁴),X⁵ is N, X ⁶ is C (R ⁶) and X ⁷ is C (R ⁷).

A13. The compound of any one of embodiments A1-a 10, wherein X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is N and X ⁷ is C (R ⁷).

A14. the compound of any one of embodiments a11, a12, and a13, wherein X ⁷ is CH.

A15. the compound of embodiment a12 or a13, wherein at least one of X ⁴ and X ⁷ is CH.

A16. The compound of any one of embodiments A1-a 10, wherein X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is N.

A17. a compound of embodiment a16 wherein X ⁴ is CH.

A18. The compound of any one of embodiments A1-a 10, wherein X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is C (R ⁷).

A19. A compound of embodiment a18 wherein X ⁴ is CH, X ⁷ is CH, or both.

A20. The compound according to any one of embodiments A1 to a19, wherein R is H.

A21. The compound of any one of embodiments A1 to a19, wherein R is L-W.

A22. The compound of embodiment A1 or a21, wherein R is L-W and L is a phosphate-based linker comprising a phosphate-based moiety selected from the group consisting of phosphate, pyrophosphate, triphosphate, tetraphosphate, phosphonate, bisphosphonate, phosphoramidate, jiao Anji phosphate, triamino phosphate, tetraphosphoric acid ester, phosphorothioate, and phosphorodithioate.

A23. the compound of any one of embodiments A1-a 22, wherein the corresponding amine of moiety a has a ClogP value of at least about 1.

A24. the compound of embodiment a22, wherein the phosphate-based moiety is a bisphosphonate.

A25. The compound of any one of embodiments a22 through a24, wherein L further comprises at least one additional moiety, wherein each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, substituted alkylene, - (alkylene-O) -, optionally substituted arylene, -O-, -C (O) -, -N (R _w)-、-S(O)_0-2 -, water-soluble polymer, and amino acid, wherein each R _w is independently H or C ₁-C₈ alkyl, and combinations thereof.

A26. The compound of embodiment A25 wherein each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, - (alkylene-O) -, -C (O) -, -N (R _w) -, a water-soluble polymer, and an amino acid, wherein each R _w is independently H or C ₁-C₈ alkyl, and combinations thereof.

A27. The compound of any one of embodiments a 22-a 26, wherein the phosphate-based moiety is covalently bonded to a-O-atom of formula (I), or formula (Ia), or formula (Ib), or formula (Ic), or formula I (d) via a phosphorus atom of the phosphate-based moiety.

A28. The compound of embodiment a25, a26 or a27, wherein L comprises at least one alkylene group.

A29. the compound of any one of embodiments a25 to a28, wherein L comprises an amino acid.

A30. the compound of any one of embodiments a 25-a 29, wherein L comprises a water-soluble polymer.

A31. The compound of any one of embodiments a 25-a 30, wherein L comprises a water-soluble polymer and an amino acid, wherein the water-soluble polymer is conjugated to the amino acid.

A32. the compound of embodiment a31, wherein the water-soluble polymer is conjugated to a side chain of the amino acid.

A33. The compound of embodiment a31 or a32, wherein the water-soluble polymer is conjugated to the amino acid via a spacer element.

A34. the compound according to any one of embodiments a25 to a33, wherein the amino acid is selected from the group consisting of serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and N _ε -methyl-lysine.

A35. The compound of any one of embodiments a25 to a34, wherein the amino acid is lysine or N _ε -methyl-lysine.

A36. The compound of embodiment a21, wherein L is selected from the group of linkers of table 6.

A37. the compound of embodiment a21, wherein L is selected from the group of linkers of table 7.

A38. the compound of embodiment a21, wherein L is selected from the group consisting of:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) - (alkylene-O) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene- (O-alkylene) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J- (alkylene-O) _n -alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +,

* -P (=O) (OH) -O-P (=O) (OH) - (O) -alkylene- (O-alkylene) _n -U-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U- (alkylene-O) _n -alkylene- +;

Wherein:

each U is independently selected from the group consisting of:

Each J is independently

Each alkylene is independently selected from the group consisting of:

Each n is independently an integer from 1 to 100;

* Represents a linkage to an-O-atom of formula (I), or formula (Ia), or formula (Ib), or formula (Ic) or formula I (d), and

+ Represents a connection to W;

A39. The compound of any one of embodiments a 36-a 38, wherein each n is independently an integer from 1 to 10, or wherein each n is independently 1,2, or 3.

A40. the compound of any one of embodiments a 36-a 39, wherein L is substituted with the one or more water-soluble polymers.

A41. the compound of any one of embodiments a36 to a40, wherein L comprises the group U, and one water-soluble polymer is conjugated to the amino acid side chain of the group U.

A42. The compound of embodiment a41, wherein the water-soluble polymer is conjugated to the amino acid side chain of group U via a spacer element.

A43. the compound of any one of embodiments A1-A5 and a 21-a 42, wherein R is L-W and L is a divalent linker.

A44. the compound according to any one of embodiments A1 to A5 and a36 to a43, wherein R is L-W, and L is:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +, wherein:

u is selected from the group consisting of:

Each alkylene is independently selected from the group consisting of:

+ Represents a connection to W;

wherein L is optionally substituted with one or more water soluble polymers.

A45. the compound of embodiment a44, wherein L is substituted with the one or more water-soluble polymers.

A46. the compound of a45, wherein one water-soluble polymer is conjugated to the amino acid side chain of group U.

A47. The compound of embodiment a46, wherein the one water-soluble polymer is conjugated to the amino acid side chain of group U via a spacer element.

A48. the compound of any one of embodiments a 44-a 47, wherein U is:

A49. The compound of embodiment a47 or a48, wherein the spacer element is a carbonyl group.

A50. the compound of any one of embodiments a47 to a49, wherein L has the structure:

Wherein T is the water-soluble polymer, R ^t is H or methyl, R is the linkage to an-O-atom of formula (I), or formula (Ia), or formula (Ib), or formula (Ic) or formula (d), and +is the linkage to W.

A51. The compound of any one of embodiments a 25-a 50, wherein the water-soluble polymer is a polyethylene glycol (PEG) moiety.

A52. The compound of embodiment a51, wherein the PEG moiety has a molecular weight in the range of about 100Da to about 100,000 Da.

A53. The compound of embodiment a52, wherein the PEG moiety has a molecular weight in the range of about 100Da to about 10,000Da, about 100Da to about 5,000Da, or about 100Da to about 1,000 Da.

A54. The compound of any of embodiments A1-A5 and a 21-a 53, wherein R is L-W, and the reactive moiety W comprises-N ₃、-OH、-SH、-NH(R^j)、-C(O)R^q、-C(O)OR^x、-C(O)CH₂NH₂, an activated ester, -O-NH ₂, maleimide, tetrazine, alkyne, cyclooctyne, or (E) -cyclooctene, wherein R ^j is H or unsubstituted alkyl, R ^q is unsubstituted alkyl, and R ^x is H, unsubstituted alkyl, or a carboxylic acid protecting group.

A55. The compound of embodiment a54, wherein the reactive moiety W is selected from the group consisting of:

Wherein:

r ^j is H or unsubstituted C ₁-C₆ alkyl,

R ^q is unsubstituted C ₁-C₆ alkyl,

R ^f is H or unsubstituted C ₁-C₆ alkyl,

S is 0,1, 2, 3,4, 5 or 6, and

T is 0,1, 2, 3, 4, 5 or 6.

A56. The compound of embodiment a55, wherein the optionally substituted mono-or polycyclic group comprising the cyclooctyne is selected from the group consisting of:

A57. a compound according to any one of embodiments A1 to A5 and a21 to a56, wherein R is L-W, and W is-ONH ₂.

A58. A compound of embodiment A2 wherein R is H.

A59. The compound of embodiment A58 wherein the compound is selected from the group consisting of compounds 4, 13, 15, 17, 25, 26, 27, 28, 29, 30, 31, 35 and 36, each having a structure as disclosed in the examples herein, and salts thereof.

A60. A compound according to embodiment A2 wherein R is L-W and the compound is selected from the compounds listed in table 9 and salts thereof.

A61. An Antibody Drug Conjugate (ADC) of formula (II):

Wherein:

Ab is an antibody, wherein Ab comprises one or more unnatural amino acids;

l is a linker;

E is a moiety linking Ab and L;

d is an integer of 1 to 100, and

Wherein:

Each X ³ is C;

Each X ⁸ is C, and

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3;

or a pharmaceutically acceptable salt thereof.

A62. the ADC of embodiment a61, wherein a has the structure: Wherein the remaining variables are as defined in embodiment 61.

A63. the ADC of embodiment a61, wherein a has the structure: Wherein the remaining variables are as defined in embodiment 61.

A64. The ADC of embodiment a61, wherein a has the structure: Wherein the remaining variables are as defined in embodiment 61.

A65. the ADC of embodiment a61, wherein a has the structure: Wherein the remaining variables are as defined in embodiment 61.

A66. the ADC of any one of embodiments a 61-a 65, wherein:

X ³ is C;

X ⁸ is C, and

A67. The ADC of any of embodiments A61 to A66 wherein each of X ¹ and X ² is CH ₂ and X ⁹, when present, is CH ₂.

A68. The ADC of any one of embodiments a 61-a 67, wherein:

X ⁴ is C (R ⁴) or N, wherein R ⁴ is H, halogen or heteroalkyl;

X ⁵ is C (R ⁵) or N, wherein R ⁵ is H, halogen or heteroalkyl;

x ⁶ is C (R ⁶) or N, wherein R ⁶ is H, halogen or heteroalkyl, and

X ⁷ is C (R ⁷) or N, wherein R ⁷ is H, halogen or heteroalkyl.

A69. The ADC of any one of embodiments a 61-a 68, wherein each of the heteroalkyl groups is an alkoxy group.

A70. The ADC of embodiment A69 wherein each of the alkoxy groups is independently-OR ^k, wherein each R ^k is independently alkyl, wherein the alkyl is optionally substituted with-N (R ^d)(R^e) OR heterocyclyl, wherein each R ^d and R ^e is independently H, alkyl, alkenyl OR alkynyl, and wherein each heterocyclyl contains at least one nitrogen atom.

A71. The ADC of any of embodiments a 61-a 70, wherein X ⁴ is N, X ⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is C (R ⁷).

A72. The ADC of any of embodiments a 61-a 70, wherein X ⁴ is C (R ⁴),X⁵ is N, X ⁶ is C (R ⁶) and X ⁷ is C (R ⁷).

A73. The ADC of any of embodiments a 61-a 70, wherein X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is N and X ⁷ is C (R ⁷).

A74. The ADC according to any one of embodiments a71, a72 and a73, wherein X ⁷ is CH.

A75. The ADC of embodiment a72 or a73, wherein at least one of X ⁴ and X ⁷ is CH.

A76. The ADC of any of embodiments a 61-a 70, wherein X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is N.

A77. The ADC of embodiment a76, wherein X ⁴ is CH.

A78. The ADC of any of embodiments a 61-a 70, wherein X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is C (R ⁷).

A79. The ADC of embodiment a78, wherein X ⁴ is CH, X ⁷ is CH, or both.

A80. The ADC of any one of embodiments a 61-a 79, wherein d is an integer from 1 to 10, or is 1,2, 3, or 4.

A81. The ADC according to any one of embodiments a61 to a80, wherein L is a phosphate-based linker.

A82. The ADC of any one of embodiments a 61-a 81, wherein R is and L is a phosphate-based linker comprising a phosphate-based moiety selected from the group consisting of phosphate, pyrophosphate, triphosphate, tetraphosphate, phosphonate, bisphosphonate, phosphoramidate, jiao Anji phosphate, triamino phosphate, tetraphosphoric acid ester, phosphorothioate, and phosphorodithioate.

A83. The ADC according to any of embodiments a 61-a 82, wherein the corresponding amine of part a has a ClogP value of at least about 1.

A84. The ADC of embodiment a82, wherein the phosphate-based moiety is a bisphosphonate.

A85. The ADC according to any one of embodiments a82 to a84, wherein L further comprises at least one additional moiety, wherein each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, substituted alkylene, - (alkylene-O) -, optionally substituted arylene, -O-, -C (O) -, -N (R _w)-、-S(O)_0-2 -, water-soluble polymer, and amino acid, wherein each R _w is independently H or C ₁-C₈ alkyl, and combinations thereof.

A86. The ADC of embodiment a85, wherein each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, - (alkylene-O) -, -C (O) -, -N (R _w) -, a water-soluble polymer, and an amino acid, wherein each R _w is independently H or C ₁-C₈ alkyl, and combinations thereof.

A87. The ADC of any one of embodiments a 82-a 86, wherein the phosphate-based moiety is covalently bonded to the-O-atom of formula (II) via a phosphorus atom of the phosphate-based moiety.

A88. The ADC of embodiment a85, a86, or a87, wherein L comprises at least one alkylene group.

A89. The ADC according to any one of embodiments a85 to a88, wherein L comprises an amino acid.

A90. The ADC of any one of embodiments a 85-a 89, wherein L comprises a water-soluble polymer.

A91. the ADC according to any one of embodiments a85 to a90, wherein L comprises a water-soluble polymer and an amino acid, wherein the water-soluble polymer is conjugated to the amino acid.

A92. the ADC of embodiment a91, wherein the water-soluble polymer is conjugated to a side chain of the amino acid.

A93. The ADC of embodiment a91 or a92, wherein the water-soluble polymer is conjugated to the amino acid via a spacer element.

A94. The ADC according to any one of embodiments a85 to a93, wherein the amino acid is selected from the group consisting of serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and N _ε -methyl-lysine.

A95. The ADC according to any one of embodiments a85 to a94, wherein the amino acid is lysine or N _ε -methyl-lysine.

A96. The ADC of embodiment a81, wherein L is selected from the group of linkers of table 6.

A97. the ADC of embodiment a81, wherein L is selected from the group of linkers of table 7.

A98. the ADC of embodiment a81, wherein L is selected from the group consisting of:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) - (alkylene-O) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene- (O-alkylene) _n -J-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-J- (alkylene-O) _n -alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene- (O-alkylene) _n -U-alkylene- +,

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U- (alkylene-O) _n -alkylene- + wherein:

each U is independently selected from the group consisting of:

Each J is independently

Each alkylene is independently selected from the group consisting of:

Each n is independently an integer from 1 to 100;

* Represents a linkage to an-O-atom of formula (II), and

+ Represents a linkage to E;

A99. The ADC of any one of embodiments a 96-a 98, wherein each n is independently an integer from 1 to 10, or wherein each n is independently 1,2, or 3.

A100. The ADC of any one of embodiments a 96-a 99, wherein L is substituted with one or more water-soluble polymers.

A101. The ADC according to any one of embodiments a96 to a100, wherein L comprises a group U, and one water-soluble polymer is conjugated to the amino acid side chain of the group U.

A102. the ADC of embodiment a101, wherein the water-soluble polymer is conjugated to the amino acid side chain of group U via a spacer element.

A103. The ADC of any one of embodiments a 61-a 102, wherein L is a divalent linker optionally substituted with a water soluble polymer.

A104. The ADC of any one of embodiments a 96-a 103, wherein L is:

* -P (=o) (OH) -O-P (=o) (OH) - (O) -alkylene-U-alkylene- +, wherein:

u is selected from the group consisting of:

Each alkylene is independently selected from the group consisting of:

* Represents a linkage to an-O-atom of formula (II), and

+ Represents a linkage to E;

wherein L is optionally substituted with one or more water soluble polymers.

A105. The ADC of embodiment a104, wherein L is substituted with one or more water-soluble polymers.

A106. the ADC of a105 wherein one water soluble polymer is conjugated to the amino acid side chain of group U.

A107. The ADC of embodiment a106, wherein the one water-soluble polymer is conjugated to the amino acid side chain of group U via a spacer element.

A108. the ADC according to any one of embodiments a104 to a107, wherein U is:

A109. the ADC of embodiment a107 or a108, wherein the spacer element is a carbonyl group.

A110. The ADC of any one of embodiments a 107-a 109, wherein L has the structure:

A111. The ADC according to any one of embodiments a85 to a110, wherein the water-soluble polymer is a polyethylene glycol (PEG) moiety.

A112. the ADC of embodiment a111, wherein the PEG moiety has a molecular weight in the range of about 100Da to about 100,000 Da.

A113. The ADC of embodiment a112, wherein the PEG moiety has a molecular weight in the range of about 100Da to about 10,000Da, about 100Da to about 5,000Da, or about 100Da to about 1,000 Da.

A114. The ADC according to any of embodiments a61 to a113, wherein E comprises an amide, an ester, a thioester, a pyrrolidine-2, 5-dione, an oxime, A1, 2, 3-triazole or A1, 4-dihydropyridazine, wherein the 1,2, 3-triazole and the 1, 4-dihydropyridazine are each optionally fused to an 8 membered ring.

A115. The ADC of embodiment a114, wherein E is selected from the group consisting of:

Wherein:

Each R ^j is independently H or unsubstituted C ₁-C₆ alkyl;

Each R ^q is independently unsubstituted C ₁-C₆ alkyl;

Each R ^f is independently H or unsubstituted C ₁-C₆ alkyl;

each s is independently 0,1, 2, 3, 4, 5, or 6;

Each t is independently 0,1, 2, 3, 4, 5, or 6;

Each + represents a connection to L, and

Each wavy line represents a connection to Ab.

A116. the ADC of any one of embodiments a 61-a 115, wherein E is: Wherein R ^q is unsubstituted C ₁-C₆ alkyl.

A117. The ADC of embodiment a116, wherein R ^q is methyl.

A118. the ADC according to any one of embodiments A1 to a117, wherein E connects L to an unnatural amino acid of an Ab.

A119. The ADC of any one of embodiments a 61-a 118, wherein Ab is configured to bind to an antigen.

A120. The ADC of embodiment a119, wherein Ab is configured to bind to a Tumor Associated Antigen (TAA) or a cancer antigen.

A121. The ADC of embodiment a119 or a120, wherein the antigen is selected from the group ：PD-1、PD-L1、PSMA、CD70、CD3、HER2、HER3、TROP2、GPC3、VEGFR、EGFR、c-Met(HGFR)、CD19、CD22、CD25(IL-2Rα)、CD30、CD33、CD37、CD46、CD48、CD56(NCAM-1)、CD71( transferrin R consisting of CD74, CD79B, CD123 (IL-3 ra), CD138 (multi-ligand glycan-1), CD142, CD166 (ALCAM), CD203c (ENPP 3), CD205 (LY 75), CD221 (IGF-1R), CD262 (TRAIL R2), CD276 (B7-H3), mesothelin, epCAM, CEACAM5, CEACAM6, DLL3, ROR1, ROR2, GPNMB, GCC, GUCY c, naPi2B, flt-1, flt-3, folate receptor alpha, tissue factor (TF)、CA6、MUC1、MUC16(CA-125)、BCMA、SLAMF7(CS1)、TIM1、CanAg、Ckit(CD117)、EphA2、Nectin4、SLTRK6、FGFR2、LYPD3(C4.4a)、 cadherin 3, 5T4 (tpt), STEAP1, PTK7, ephrin-A4, LIV-1 (SLC 39A6 or ZIP 6), SLC1A5, TENB2, ETBR, integrin v3, cripto, aglc 5 (rc 4), SLC E, AXL, LAMP, MN-15, TNF-IX and lrbg 4.

A122. the ADC of embodiment a121, wherein the antigen is selected from the group consisting of TROP2, CD70, HER2, CD3, PSMA, and GPC 3.

A123. The ADC of embodiment a122, wherein the antigen is GPC3.

A124. the ADC of embodiment a122, wherein the antigen is CD70.

A125. The ADC of embodiment a124, wherein Ab is an anti-CD 70 antibody comprising at least one sequence listed in table 2.

A126. The ADC of embodiment a125, wherein the anti-CD 70 Ab comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises an amino acid sequence selected from the group consisting of the heavy chain sequences listed in table 2, and the light chain sequence comprises an amino acid sequence selected from the group consisting of the light chain sequences listed in table 2, wherein the heavy chain sequence comprises the one or more unnatural amino acids.

A127. The ADC of embodiment a126, wherein the anti-CD 70 Ab comprises two heavy chain sequences selected from the group consisting of the heavy chain sequences listed in table 2, wherein each heavy chain sequence comprises one unnatural amino acid.

A128. The ADC of any of embodiments A61 to A127 wherein the one or more unnatural amino acids are selected from the group consisting of p-acetylphenylalanine, 4-acetyl-L-phenylalanine (p-acetyl-L-phenylalanine (pAF)), 3-O- (N-acetyl-beta-D-glucosamine) -L-threonine, N4- (beta-N-acetyl-D-glucosamine) -L-asparagine, O-allyl-L-tyrosine, alpha-N-acetylgalactosamine-O-L-serine, alpha-N-acetylgalactosamine-O-L-threonine, 2-aminocaprylic acid, 2-amino-L-phenylalanine, 3-amino-L-phenylalanine, 4-amino-L-phenylalanine, 2-amino-L-tyrosine, 3-amino-L-tyrosine, 4-azido-L-phenylalanine, 4-benzoyl-L-phenylalanine, (2, 2-bipyridyl-5) -L-alanine, 3-boron-L-phenylalanine, 4-methyl-phenylalanine, 4-carboxyl-L-phenylalanine, 4-bromo-L-phenylalanine, 4-methyl-L-phenylalanine, 4-bromo-L-phenylalanine, 4-phenylalanine, and combinations thereof, P-cyano-L-phenylalanine, 3, 4-dihydroxy-L-phenylalanine (L-DOPA), 4-ethynyl-L-phenylalanine, 2-fluoro-L-phenylalanine, 3-fluoro-L-phenylalanine, 4-fluoro-L-phenylalanine, O- (3-O-D-galactosyl-N-acetyl-beta-D-galactosamine) -L-serine, L-homoglutamine, (8-hydroxyquinolin-3-yl) -L-alanine, 4-iodo-L-phenylalanine, 4-isopropyl-L-phenylalanine, O-isopropyl-L-tyrosine, 3-isopropyl-L-tyrosine, O-mannopyranosyl-L-serine, 2-methoxy-L-phenylalanine, 3-methoxy-L-phenylalanine, 4-methoxy-L-phenylalanine, 3-methyl-L-phenylalanine, O-methyl-L-tyrosine, 3- (2-naphtyl) -L-alanine, 5-nitro-L-histidine, 4-nitro-L-phenylalanine, 2-nitro-L-phenylalanine, 3-nitro-L-phenylalanine, L-nitro-L-phenylalanine, 4-nitro-L-phenylalanine, 4-nitro-L-tryptophan, 5-nitro-L-tryptophan, 6-nitro-L-tryptophan, 7-nitro-L-tryptophan, 2-nitro-L-tyrosine, 3-nitro-L-tyrosine, O-phospho-L-serine, O-phospho-L-tyrosine, 4-propargyloxy-L-phenylalanine, O-2-propargyl-1-yl-L-tyrosine, 4-sulfo-L-phenylalanine and O-sulfo-L-tyrosine.

A129. The ADC of any one of embodiments a 61-a 128, wherein each of the one or more unnatural amino acids is a p-acetylphenylalanine.

A130. The ADC according to any one of embodiments a61 to a129, wherein Ab comprises 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 unnatural amino acids.

A131. A pharmaceutical composition comprising a compound according to any one of embodiments A1 to a60 or an ADC according to any one of embodiments a61 to a130, and at least one pharmaceutically acceptable adjuvant, binder, buffer, carrier, diluent or excipient.

A132. The pharmaceutical composition of embodiment a131, further comprising a chemotherapeutic agent, hormonal agent, anti-tumor agent, immunostimulant, immunomodulator, corticosteroid, or combination thereof.

A133. a method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound according to any one of embodiments A1 to a60, an ADC according to any one of embodiments a61 to a130, or a pharmaceutical composition according to embodiments a131 or a 132.

A134. the method of embodiment a133, wherein the disease or condition is cancer.

A135. The method of embodiment a134, wherein the cancer is a leukemia.

A136. the method of embodiment a135, wherein the blood cancer is leukemia, lymphoma, or myeloma.

A137. The method of any one of embodiments a 133-a 136, wherein the treatment comprises administering the ADC of any one of embodiments a 61-a 130, or a pharmaceutical composition comprising the ADC of any one of embodiments a 61-a 130.

A138. the method of embodiment a137, wherein Ab is an anti-CD 70 antibody.

A139. The method of embodiment a134, wherein the cancer is liver cancer.

A140. The method of embodiment a139, wherein the liver cancer is hepatocellular carcinoma.

A141. The method of embodiment a139 or a140, wherein the treatment comprises administration of the ADC of any one of embodiments a 61-a 130, or a pharmaceutical composition comprising the ADC of any one of embodiments a 61-a 130, wherein Ab is an anti-GPC 3 antibody.

Additional non-limiting embodiments of the invention are set forth below.

B1. A compound of formula (I) having the structure:

Wherein:

R is H or L-W, wherein L is a linker and W is a reactive moiety, and

Wherein:

Each X ³ is C;

Each X ⁸ is C, and

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3;

Or a salt thereof.

B2. The compound according to embodiment B1, wherein a has the structure of formula (a), and the compound is a compound of formula (Ia) having the structure:

Wherein:

R is H or L-W, wherein L is a linker and W is a reactive moiety;

X ³ is C;

X ⁸ is C;

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3.

B3. The compound of embodiment B1 or B2, wherein:

X ³ is C;

X ⁸ is C, and

B4. the compound of embodiment B1, B2 or B3, wherein:

X ¹ is C (R ^1a)(R^1b), wherein each of R ^1a and R ^1b is H;

X ² is C (R ^2a)(R^2b), wherein each of R ^2a and R ^2b is H;

X ³ is C;

x ⁴ is C (R ⁴) or N, wherein R ⁴ is H;

X ⁷ is C (R ⁷) or N, wherein R ⁷ is H;

X ⁸ is C, and

X ⁹, when present, is CH ₂.

B5. the compound of any one of embodiments B1-B4, wherein each of the heteroalkyl is an alkoxy group.

B6. the compound according to any one of embodiments B1 to B5, wherein:

X ¹ is C (R ^1a)(R^1b), wherein each of R ^1a and R ^1b is H;

X ² is C (R ^2a)(R^2b), wherein each of R ^2a and R ^2b is H;

X ³ is C;

x ⁴ is C (R ⁴) or N, wherein R ⁴ is H;

X ⁵ is C (R ⁵) or N, wherein R ⁵ is H, halogen or alkoxy;

X ⁶ is C (R ⁶) or N, wherein R ⁶ is H, halogen or alkoxy;

X ⁷ is C (R ⁷) or N, wherein R ⁷ is H;

X ⁸ is C, and

X ⁹, when present, is CH ₂.

B7. A compound according to embodiment B6 wherein X ⁵ is C (R ⁵) or N, wherein R ⁵ is H or alkoxy, and X ⁶ is C (R ⁶) or N, wherein R ⁶ is H or alkoxy.

B8. The compound of any one of embodiments B1 to B7, wherein X ⁴ is N, X ⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is C (R ⁷).

B9. The compound of any one of embodiments B1 to B7, wherein X ⁴ is C (R ⁴),X⁵ is N, X ⁶ is C (R ⁶) and X ⁷ is C (R ⁷).

B10. the compound of any one of embodiments B1 to B7, wherein X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is N and X ⁷ is C (R ⁷).

B11. The compound of any one of embodiments B1-B7, wherein X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is C (R ⁷).

B12. The compound of any one of embodiments B1-B7 and B9-B11, wherein at least one of X ⁴ and X ⁷ is CH.

B13. The compound of any one of embodiments B1-B12, wherein each of X ⁴ and X ⁷ is CH.

B14. the compound of any one of embodiments B1-B13, wherein at least one of R ⁵ and R ⁶ is alkoxy.

B15. The compound of any one of embodiments B5 through B14 wherein each of the alkoxy groups is independently-OR ^k, wherein each R ^k is independently alkyl optionally substituted with heterocyclyl OR-N (R ^d)(R^e), wherein the heterocyclyl contains at least one nitrogen atom, and each R ^d and R ^e is independently H, alkyl, alkenyl, OR alkynyl.

B16. The compound of embodiment B15 wherein each of said alkoxy groups is selected from the group consisting of -OCH₃、-OCH₂CH₃、-OCH₂CH₂CH₃、-OCH(CH₃)₂、-OCH₂CH₂N(CH₃)₂、

A group of groups.

B17. the compound according to any one of embodiments B1 to B16, wherein R is L-W.

B18. a compound of embodiment B17 wherein L is a phosphate-based linker.

B19. The compound of embodiment B18, wherein the phosphate-based linker comprises a phosphate-based moiety having the structure:

Wherein represents a linkage to an-O-atom at position R of formula (I) or formula (Ia), wherein L further comprises at least one additional moiety and the wavy line of the phosphate-based moiety represents a linkage to one of the at least one additional moiety, wherein the at least one additional moiety is selected from the group consisting of unsubstituted alkylene, substituted alkylene, - (alkylene-O) -, optionally substituted arylene, -O-, -C (O) -, -N (R _w)-、-S(O)_0-2 -, a water-soluble polymer, and an amino acid, wherein each R _w is independently H or C ₁-C₈ alkyl, and combinations thereof.

B20. The compound of embodiment B19 wherein each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, - (alkylene-O) -, -C (O) -, -N (R _w) -, a water-soluble polymer, and an amino acid, wherein each R _w is independently H or C ₁-C₈ alkyl, and combinations thereof.

B21. The compound of any one of embodiments B1 through B17, wherein R is L-W, and L is selected from the group of linkers of table 6.

B22. the compound of any one of embodiments B1 through B17, wherein R is L-W, and L is selected from the group of linkers of table 7.

B23. The compound of any one of embodiments B1-B17, wherein R is L-W, and L is selected from the group of linkers of table 8.

B24. the compound of any one of embodiments B1 to B17, wherein R is L-W, and L has the structure:

wherein represents a linkage to an-O-atom at position R of formula (I) or formula (Ia), and +represents a linkage to W.

B25. the compound of any one of embodiments B1 to B17, wherein R is L-W, and L has the structure:

wherein T is a water-soluble polymer, R ^t is H or methyl, represents a linkage to the-O-atom at position R of formula (I) or formula (Ia), and +represents a linkage to W.

B26. The compound of embodiment B25, wherein the water-soluble polymer is a polyethylene glycol (PEG) moiety.

B27. The compound of embodiment B26, wherein the PEG moiety has a molecular weight in the range of about 100Da to about 100,000Da, about 100Da to about 10,000Da, about 100Da to about 5,000Da, or about 100Da to about 1,000 Da.

B28. The compound of embodiment B26, wherein the PEG moiety is- (CH ₂CH₂O)_nCH₃, wherein n is an integer from 1 to 24.

B29. The compound of embodiment B26, wherein the PEG moiety is- (CH ₂CH₂O)_nCH₃, wherein n is 8, 9, 10, 11, or 12.

B30. The compound of any one of embodiments B1 through B29 wherein R is L-W and the reactive moiety W comprises-N ₃、-OH、-SH、-NH(R^j)、-C(O)R^q、-C(O)OR^x、-C(O)CH₂NH₂, an activated ester, -O-NH ₂, maleimide, tetrazine, alkyne, cyclooctyne, or (E) -cyclooctene, wherein R ^j is H or unsubstituted alkyl, R ^q is unsubstituted alkyl, and R ^x is H, unsubstituted alkyl, or a carboxylic acid protecting group.

B31. the compound of embodiment B30, wherein the reactive moiety W is selected from the group consisting of:

Wherein:

r ^j is H or unsubstituted C ₁-C₆ alkyl,

R ^q is unsubstituted C ₁-C₆ alkyl,

R ^f is H or unsubstituted C ₁-C₆ alkyl,

S is 0,1, 2, 3,4, 5 or 6, and

T is 0,1, 2, 3, 4, 5 or 6.

B32. A compound according to any one of embodiments B1 to B31, wherein W is-ONH ₂.

B33. The compound of embodiment B1 or B2, wherein the compound is selected from the group consisting of:

And salts thereof.

B34. a compound according to embodiment B1 or B2 having the structure:

Or a salt thereof.

B35. A compound according to embodiment B1 or B2 having the structure:

Or a salt thereof.

B36. A compound according to embodiment B1 or B2 having the structure:

Or a salt thereof.

B37. a compound according to embodiment B1 or B2 having the structure:

Or a salt thereof.

B38. The compound according to any one of embodiments B1 to B16, wherein R is H.

B39. the compound of embodiment B1 or B2, wherein the compound is selected from the group consisting of:

And salts thereof.

B40. the compound of embodiment B1 or B2, wherein the compound is selected from the group consisting of:

And salts thereof.

B41. an Antibody Drug Conjugate (ADC) of formula (II):

Wherein:

Ab is an antibody, wherein Ab comprises one or more unnatural amino acids;

l is a linker;

E is a moiety linking Ab and L;

d is an integer of 1 to 10, and

Wherein:

Each X ³ is C;

Each X ⁸ is C, and

Wherein:

Each R ^a and R ^b is independently H, alkyl, alkenyl, or alkynyl;

Each m is independently 0,1, 2 or 3;

or a pharmaceutically acceptable salt thereof.

B42. the ADC of embodiment B41, wherein a has the structure: And wherein the remaining variables are as defined in embodiment 41.

B43. the ADC of embodiment B41 or B42, wherein:

X ³ is C;

X ⁸ is C, and

B44. the ADC of embodiment B41, B42 or B43, wherein:

X ¹ is C (R ^1a)(R^1b), wherein each of R ^1a and R ^1b is H;

X ² is C (R ^2a)(R^2b), wherein each of R ^2a and R ^2b is H;

X ³ is C;

x ⁴ is C (R ⁴) or N, wherein R ⁴ is H;

X ⁷ is C (R ⁷) or N, wherein R ⁷ is H;

X ⁸ is C, and

X ⁹, when present, is CH ₂.

B45. the ADC according to any one of embodiments B41 to B44, wherein each of the heteroalkyl groups is an alkoxy group.

B46. the ADC of any one of embodiments B41-B45, wherein:

X ¹ is C (R ^1a)(R^1b), wherein each of R ^1a and R ^1b is H;

X ² is C (R ^2a)(R^2b), wherein each of R ^2a and R ^2b is H;

X ³ is C;

x ⁴ is C (R ⁴) or N, wherein R ⁴ is H;

X ⁵ is C (R ⁵) or N, wherein R ⁵ is H, halogen or alkoxy;

X ⁶ is C (R ⁶) or N, wherein R ⁶ is H, halogen or alkoxy;

X ⁷ is C (R ⁷) or N, wherein R ⁷ is H;

X ⁸ is C, and

X ⁹, when present, is CH ₂.

B47. The ADC of embodiment B46, wherein X ⁵ is C (R ⁵) or N, wherein R ⁵ is H or alkoxy, and X ⁶ is C (R ⁶) or N, wherein R ⁶ is H or alkoxy.

B48. The ADC of any of embodiments B41-B47, wherein X ⁴ is N, X ⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is C (R ⁷).

B49. The ADC of any of embodiments B41-B47, wherein X ⁴ is C (R ⁴),X⁵ is N, X ⁶ is C (R ⁶) and X ⁷ is C (R ⁷).

B50. The ADC of any of embodiments B41-B47, wherein X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is N and X ⁷ is C (R ⁷).

B51. The ADC of any of embodiments B41-B47, wherein X ⁴ is C (R ⁴),X⁵ is C (R ⁵),X⁶ is C (R ⁶) and X ⁷ is C (R ⁷).

B52. The ADC of any of embodiments B41-B51, wherein at least one of X ⁴ and X ⁷ is CH.

B53. The ADC of any one of embodiments B41-B47 and B49-B52, wherein each of X ⁴ and X ⁷ is CH.

B54. The ADC of any of embodiments B41-B53, wherein at least one of R ⁵ and R ⁶ is alkoxy.

B55. The ADC of any of embodiments B45 to B54 wherein each of the alkoxy groups is independently-OR ^k, wherein each R ^k is independently alkyl optionally substituted with a heterocyclyl OR-N (R ^d)(R^e), wherein the heterocyclyl contains at least one nitrogen atom, and each R ^d and R ^e is independently H, alkyl, alkenyl, OR alkynyl.

B56. the ADC of embodiment B55, wherein each of the alkoxy groups is selected from the group consisting of -OCH₃、-OCH₂CH₃、-OCH₂CH₂CH₃、-OCH(CH₃)₂、-OCH₂CH₂N(CH₃)₂、

A group of groups.

B57. the ADC of any of embodiments B41-B56, wherein d is 1,2, 3, or 4.

B58. The ADC according to any one of embodiments B41 to B57, wherein L is a phosphate-based linker.

B59. the ADC of embodiment B58, wherein the phosphate-based linker comprises a phosphate-based moiety having the structure:

Wherein each of the at least one additional moieties is independently selected from the group consisting of unsubstituted alkylene, - (alkylene-O) -, -C (O) -, -N (R _w) -, a water-soluble polymer, and an amino acid, wherein each R _w is independently H or C ₁-C₈ alkyl, and combinations thereof.

B60. the ADC according to any one of embodiments B41 to B57, wherein L is selected from the group of linkers of table 6.

B61. the ADC according to any one of embodiments B41 to B57, wherein L is selected from the group of linkers of table 7.

B62. The ADC according to any one of embodiments B41 to B57, wherein L is selected from the group of linkers of table 8.

B63. The ADC of any one of embodiments B41-B57, wherein L has the structure:

Wherein represents a linkage to an-O-atom at position L of formula (II), and +represents a linkage to E.

B64. the ADC of any one of embodiments B41-B57, wherein L has the structure:

Wherein T is a water-soluble polymer, R ^t is H or methyl, represents a linkage to the-O-atom at position L of formula (II), and +represents a linkage to E.

B65. The ADC of embodiment B64, wherein the water-soluble polymer is a polyethylene glycol (PEG) moiety.

B66. the ADC of embodiment B65, wherein the PEG moiety has a molecular weight in the range of about 100Da to about 100,000Da, about 100Da to about 10,000Da, about 100Da to about 5,000Da, or about 100Da to about 1,000 Da.

B67. The ADC of embodiment B65, wherein the PEG moiety is- (CH ₂CH₂O)_nCH₃, wherein n is an integer from 1 to 24.

B68. The ADC of embodiment B65, wherein the PEG moiety is- (CH ₂CH₂O)_nCH₃, wherein n is 8, 9, 10, 11, or 12.

B69. The ADC according to any of embodiments B41 to B68, wherein E comprises an amide, an ester, a thioester, a pyrrolidine-2, 5-dione, an oxime, a1, 2, 3-triazole or a1, 4-dihydropyridazine, wherein the 1,2, 3-triazole and the 1, 4-dihydropyridazine are each optionally fused to an 8 membered ring.

B70. the ADC of embodiment B69, wherein E is selected from the group consisting of:

Wherein each R ^j is independently H or unsubstituted C ₁-C₆ alkyl, each R ^q is independently unsubstituted C ₁-C₆ alkyl, each R ^f is independently H or unsubstituted C ₁-C₆ alkyl, each s is independently 0, 1,2,3, 4,5, or 6, each t is independently 0, 1,2,3, 4,5, or 6, each +represents a linkage to L, and each wavy line represents a linkage to Ab.

B71. The ADC according to any one of embodiments B41 to B70, wherein E is: Wherein R ^q is unsubstituted C ₁-C₆ alkyl.

B72. The ADC of embodiment B71, wherein R ^q is methyl.

B73. The ADC according to any one of embodiments B41-B72, wherein E connects L to an unnatural amino acid of Ab.

B74. The ADC of any one of embodiments B41-B73, wherein Ab is configured to bind to an antigen.

B75. The ADC of embodiment B74, wherein the antigen is selected from the group consisting of ：PD-1、PD-L1、PSMA、CD70、CD3、HER2、HER3、TROP2、GPC3、VEGFR、EGFR、c-Met(HGFR)、CD19、CD22、CD25(IL-2Rα)、CD30、CD33、CD37、CD46、CD48、CD56(NCAM-1)、CD71( transferrin R), CD74, CD79B, CD123 (IL-3 ra), CD138 (syndecan-1), CD142, CD166 (ALCAM), CD203c (ENPP 3), CD205 (LY 75), CD221 (IGF-1R), CD262 (TRAIL R2), CD276 (B7-H3), mesothelin, epCAM, CEACAM5, CEACAM6, DLL3, ROR1, ROR2, GPNMB, GCC, GUCY c, naPi2B, flt-1, flt-3, folate receptor alpha, tissue factor (TF)、CA6、MUC1、MUC16(CA-125)、BCMA、SLAMF7(CS1)、TIM1、CanAg、Ckit(CD117)、EphA2、Nectin4、SLTRK6、FGFR2、LYPD3(C4.4a)、 cadherin 3, 5T4 (TPBG), STEAP1, PTK7, ephrin-A4, LIV-1 (SLC 39A6 or ZIP 6), SLC1A5, TENB2, ETBR, integrin v3, cripto, AGS-5 (SLC 44 A4), lrix 6, MN-E, AXL, LAMP, and TNF-a.

B76. The ADC of embodiment B75, wherein the antigen is TROP2, CD70, HER2, PSMA, HER3, or GPC3.

B77. the ADC according to any one of embodiments B41 to B76, wherein Ab is an anti-CD 70 antibody comprising a sequence set forth in table 2.

B78. The ADC of embodiment B77, wherein the anti-CD 70 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID No. 26.

B79. The ADC according to embodiment B77 or B78, wherein the anti-CD 70 antibody comprises a light chain variable region having the amino acid sequence of SEQ ID No. 27.

B80. The ADC according to embodiment B77, B78 or B79, wherein the anti-CD 70 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 25.

B81. the ADC according to embodiment B77, B78 or B79, wherein the anti-CD 70 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 20.

B82. The ADC according to any of embodiments B77 to B81, wherein the anti-CD 70 antibody comprises a light chain having the amino acid sequence of SEQ ID No. 19.

B83. The ADC of embodiment B77, wherein the anti-CD 70 antibody comprises two heavy chains each having the amino acid sequence of SEQ ID No. 20 and two light chains each having the amino acid sequence of SEQ ID No. 19.

B84. The ADC according to any one of embodiments a61 to a122 and B41 to B76, wherein Ab is an anti-TROP 2 antibody comprising a sequence set forth in table 1.

B85. the ADC of embodiment B84, wherein the anti-TROP 2 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 5.

B86. The ADC of embodiment B84 or B85, wherein the anti-TROP 2 antibody comprises a light chain having the amino acid sequence of SEQ ID No. 4.

B87. The ADC of any one of embodiments a 61-a 122 and B41-B76, wherein Ab is an anti-HER 2 antibody comprising a sequence set forth in table 3.

B88. The ADC of embodiment B87, wherein the anti-HER 2 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 29.

B89. the ADC of embodiment B87 or B88, wherein the anti-HER 2 antibody comprises a light chain having the amino acid sequence of SEQ ID No. 30.

B90. The ADC of any one of embodiments a 61-a 122 and B41-B76, wherein Ab is an anti-PSMA antibody comprising a sequence listed in table 4.

B91. The ADC of embodiment B90, wherein the anti-PSMA antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 39.

B92. the ADC of embodiment B90 or B91, wherein the anti-PSMA antibody comprises a light chain having the amino acid sequence of SEQ ID No. 40.

B93. The ADC of any one of embodiments a 61-a 122 and B41-B76, wherein Ab is an anti-HER 3 antibody comprising a sequence set forth in table 5.

B94. the ADC of embodiment B93, wherein the anti-HER 3 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 58.

B95. the ADC of embodiment B93 or B94, wherein the anti-HER 3 antibody comprises a light chain having the amino acid sequence of SEQ ID No. 47.

B96. The ADC according to any one of embodiments B41 to B95, wherein the antibody comprises two heavy chains and one unnatural amino acid is incorporated into each of the heavy chains.

B97. The ADC according to any one of embodiments B41 to B96, wherein the unnatural amino acid is p-acetyl-L-phenylalanine.

B98. the compound of any one of embodiments B1 to B4, or the ADC of any one of embodiments B41 to B97, wherein the corresponding amine of moiety a has a ClogP value of at least about 1.

B99. A pharmaceutical composition comprising a compound according to any one of embodiments B1 to B40 and B98 or an ADC according to any one of embodiments B41 to B98, and at least one pharmaceutically acceptable adjuvant, binder, buffer, carrier, diluent or excipient.

B100. A method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound according to any one of embodiments A1 to a60, B1 to B40, and B98, an ADC according to any one of embodiments a61 to a130, B41 to B98, or a pharmaceutical composition according to embodiments a131, a132, or B99.

B101. The method of embodiment B100, wherein the disease or condition is cancer.

B102. the method of embodiment B101, wherein the cancer is a CD70 expressing cancer.

B103. The method of embodiment B101 or B102, wherein the cancer is renal cell carcinoma.

B104. the method of embodiment B101 or B102, wherein the cancer is a blood cancer.

B105. the method of embodiment B104, wherein the blood cancer is leukemia, lymphoma, or myeloma.

While certain embodiments of the present invention have been described, these embodiments are presented by way of example only and are not intended to limit the scope of the present disclosure. Indeed, the novel compositions, methods, and systems described herein may be embodied in a variety of other forms. Further, various omissions, substitutions, and changes in the compositions, systems, and methods described herein may be made without departing from the spirit of the disclosure. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the disclosure. Accordingly, the scope of the invention is to be limited only by reference to the following claims.

Features, materials, characteristics or groups described in connection with a particular aspect, embodiment or example are to be understood as applicable to any other aspect, embodiment or example described in this section or elsewhere in this specification unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the compositions so disclosed, any method or process step, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The present protection is not limited to the details of any of the foregoing embodiments. This protection extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Furthermore, certain features that are described in this disclosure in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations separately or in any suitable subcombination. Although features may be described above as acting in certain combinations, one or more features from a claimed combination can in some cases be excised from the combination, and the combination may be directed to a subcombination or variation of a subcombination.

The features and attributes of the specific embodiments disclosed above may be combined in different ways to form additional embodiments, all of which fall within the scope of the present disclosure. Moreover, the separation of various system components in the implementations described above should not be understood as requiring such separation in all implementations, and it should be understood that the described components and systems can generally be integrated together in a single product or packaged into multiple products.

For the purposes of this disclosure, certain aspects, advantages and novel features are described. Not all such advantages may be realized in accordance with any particular embodiment. Thus, for example, those skilled in the art will recognize that the present disclosure may be embodied or carried out in a manner that achieves one advantage or a set of advantages as taught herein without necessarily achieving other advantages as taught or suggested herein.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Conditional language such as "capable," "may," or "may" are generally intended to convey that certain embodiments include, but other embodiments do not include, certain features, elements, and/or steps unless specifically stated otherwise or otherwise understood within the context as used. Thus, such conditional language is not generally intended to imply that features, elements and/or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without user input or prompting, whether these features, elements and/or steps are included in or are to be performed in any particular embodiment.

A connective language such as the phrase "at least one of X, Y and Z" is understood in the general context of use to convey that an item, term, etc. may be X, Y or Z, unless specifically stated otherwise. Thus, such connection language is not generally intended to imply that certain embodiments require the presence of at least one of X, at least one of Y, and at least one of Z.

The terms "about," "generally," "substantially" and "essentially" as used herein mean a value, quantity or characteristic that is close to the value, quantity or characteristic that still performs the desired function or achieves the desired result. For example, the terms "about," "generally," "substantially," and "substantially" may refer to amounts within less than 10%, less than 5%, less than 1%, less than 0.1%, and less than 0.01% of the stated amount.

As used herein and in the appended claims, the term "comprising" is open ended and applies to the broadest reasonable interpretation of that term. The present disclosure contemplates alternative embodiments, wherein the term "consisting of" and "consisting of" may be used in place of each statement of the term "comprising" herein.

The scope of the present disclosure is not intended to be limited by the specific disclosure of the preferred embodiments in this section or elsewhere in this specification, and may be defined by the claims presented in this section or elsewhere in this specification or presented in the future. The language of the claims should be construed broadly based on the language employed in the claims and not limited to examples described in the present specification or during the prosecution of the present application, which examples should be construed as non-exclusive.

Claims

1. A compound of formula (I) having the following structure:

in:

R is H or L-W, wherein L is a linker and W is a reactive moiety; and

A is selected from the group consisting of formula (a), (b), (c) and (d) having the following structures:

in:

Each X ¹ is C(R ^1a )(R ^1b ); wherein each R ^1a and R ^1b are independently H, halogen, alkyl, alkenyl or alkynyl;

Each X ² is C(R ^2a )(R ^2b ); wherein each R ^2a and R ^2b are independently H, halogen, alkyl, alkenyl or alkynyl;

Each X ³ is C;

Each X ⁴ is C(R ⁴ ) or N, wherein R ⁴ is H, halogen, -OH, -SH, -NO ₂ ,

-CN, -N ₃ , -N(R ^a )(R ^b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ^c , -C(O)OR ^c , -C(O)N(R ^a )(R ^b ),

-C(S)R ^c , -C(S)OR ^c , -C(S)N(R ^a )(R ^b ), -C(O)SR ^c or -S(O) _m (R ^s );

Each X ⁵ is C(R ⁵ ) or N, wherein R ⁵ is H, halogen, -OH, -SH, -NO ₂ ,

-C(S)R ^c , -C(S)OR ^c , -C(S)N(R ^a )(R ^b ), -C(O)SR ^c or -S(O) _m (R ^s );

Each X ⁶ is C(R ⁶ ) or N, wherein R ⁶ is H, halogen, -OH, -SH, -NO ₂ ,

-C(S)R ^c , -C(S)OR ^c , -C(S)N(R ^a )(R ^b ), -C(O)SR ^c or -S(O) _m (R ^s );

Each X ⁷ is C(R ⁷ ) or N, wherein R ⁷ is H, halogen, -OH, -SH, -NO ₂ ,

-C(S)R ^c , -C(S)OR ^c , -C(S)N(R ^a )(R ^b ), -C(O)SR ^c or -S(O) _m (R ^s );

Each X ⁸ is C; and

each X ⁹ , when present, is C(R ^9a )(R ^9b ); wherein each R ^9a and R ^9b is independently H, halogen, alkyl, alkenyl or alkynyl;

in:

Each ^Ra and ^Rb is independently H, alkyl, alkenyl or alkynyl;

each R ^c is independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl;

each ^Rs is independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl; and

Each m is independently 0, 1, 2 or 3;

or a salt thereof.

2. The compound according to claim 1, wherein A has a structure of formula (a), and the compound is a compound of formula (Ia) having the following structure:

in:

R is H or L-W, wherein L is a linker and W is a reactive moiety;

X ¹ is C(R ^1a )(R ^1b ); wherein each R ^1a and R ^1b is independently H, halogen, alkyl, alkenyl or alkynyl;

^X2 is C( ^R2a )( ^R2b ); wherein each ^R2a and ^R2b are independently H, halogen, alkyl, alkenyl or alkynyl;

X ³ is C;

^X4 is C( ^R4 ) or N, wherein ^R4 is H, halogen, -OH, -SH, _-NO2 , -CN, _-N3 , -N(R ^a )(R ^b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c, -C(O)OR ^c , -C(O)N(R ^a )(R ^b ), -C(S)R ^c , -C(S)OR ^c , ^-C (S)N(R ^a )(R ^b ), -C(O)SR ^c , or -S(O) _m (R ^s );

^X5 is C( ^R5 ) or N, wherein ^R5 is H, halogen, -OH, -SH, _-NO2 , -CN, _-N3 , -N( ^Ra )( ^Rb ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O) ^Rc , -C(O)ORc, -C(O)N( ^Ra )(Rb), -C(S) ^Rc , -C(S) ^ORc , ^-C ( ^S )N( ^Ra )( ^Rb ), -C(O) ^SRc , or -S(O) _m ( ^Rs );

^X6 is C( ^R6 ) or N, wherein ^R6 is H, halogen, -OH, -SH, _-NO2 , -CN, _-N3 , -N(R ^a )( ^Rb ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)Rc, -C(O) ^ORc , -C(O)N(R ^a )(Rb), -C(S) ^Rc , -C(S) ^ORc , ^-C ( ^S )N(R ^a )(Rb), -C(O) ^SRc , or -S( ^O ) _m ( ^Rs );

^X is C( ^R ) or N, wherein ^R is H, halogen, -OH, -SH, -NO, _-CN , -N, _-N , -N( ^R )( ^R ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O) ^{R, -C(O)OR, -C(O)N(R)(R} ⁾ , -C(S) ^R , ^-C (S)OR, ^-C ( ^S )N( ^R )( ^R ), -C(O) ^SR , or -S(O) _m ( ^R ); and

X ⁸ is C;

in:

Each ^Ra and ^Rb is independently H, alkyl, alkenyl or alkynyl;

Each m is independently 0, 1, 2 or 3.

3. The compound according to claim 1 or 2, wherein:

^X1 is C( ^R1a )( ^R1b ); wherein each ^R1a and ^R1b is independently H, halogen or unsubstituted alkyl;

^X2 is C( ^R2a )( ^R2b ); wherein each ^R2a and ^R2b are independently H, halogen or unsubstituted alkyl;

X ³ is C;

X ⁴ is C(R ⁴ ) or N, wherein R ⁴ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl;

^X5 is C( ^R5 ) or N, wherein ^R5 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl;

^X6 is C( ^R6 ) or N, wherein ^R6 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl;

X ⁷ is C(R ⁷ ) or N, wherein R ⁷ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl;

X ⁸ is C; and

^X9 , when present, is C( ^R9a )( ^R9b ); wherein each ^R9a and ^R9b is independently H, halogen or unsubstituted alkyl.

4. A compound according to claim 1, 2 or 3, wherein:

^X1 is C( ^R1a )( ^R1b ); wherein each of ^R1a and ^R1b is H;

^X2 is C( ^R2a )( ^R2b ); wherein each of ^R2a and ^R2b is H;

X ³ is C;

^X4 is C( ^R4 ) or N, wherein ^R4 is H;

^X7 is C( ^R7 ) or N, wherein ^R7 is H;

X ⁸ is C; and

^X9 , when present, is _CH2 .

5. The compound of any one of claims 1 to 4, wherein each of the heteroalkyl groups is an alkoxy group.

6. A compound according to any one of claims 1 to 5, wherein:

^X1 is C( ^R1a )( ^R1b ); wherein each of ^R1a and ^R1b is H;

^X2 is C( ^R2a )( ^R2b ); wherein each of ^R2a and ^R2b is H;

X ³ is C;

^X4 is C( ^R4 ) or N, wherein ^R4 is H;

^X5 is C( ^R5 ) or N, wherein ^R5 is H, halogen or alkoxy;

X ⁶ is C(R ⁶ ) or N, wherein R ⁶ is H, halogen or alkoxy;

^X7 is C( ^R7 ) or N, wherein ^R7 is H;

X ⁸ is C; and

^X9 , when present, is _CH2 .

7. The compound of claim 6, wherein ^X5 is C( ^R5 ) or N, wherein ^R5 is H or alkoxy; and ^X6 is C( ^R6 ) or N, wherein ^R6 is H or alkoxy.

8. A compound according to any one of claims 1 to 7, wherein ^X4 is N, ^X5 is C( ^R5 ), ^X6 is C( ^R6 ) and ^X7 is C( ^R7 ).

9. A compound according to any one of claims 1 to 7, wherein ^X4 is C( ^R4 ), ^X5 is N, ^X6 is C( ^R6 ) and ^X7 is C( ^R7 ).

10. A compound according to any one of claims 1 to 7, wherein ^X4 is C( ^R4 ), ^X5 is C( ^R5 ), ^X6 is N and ^X7 is C( ^R7 ).

11. A compound according to any one of claims 1 to 7, wherein ^X4 is C( ^R4 ), ^X5 is C( ^R5 ), ^X6 is C( ^R6 ) and ^X7 is C( ^R7 ).

12. The compound according to any one of claims 1 to 7 and 9 to 11, wherein at least one of ^X4 and ^X7 is CH.

13. The compound according to any one of claims 1 to 12, wherein each of ^X4 and ^X7 is CH.

14. The compound according to any one of claims 1 to 13, wherein at least one of R ⁵ and R ⁶ is alkoxy.

15. A compound according to any one of claims 5 to 14, wherein each of the alkoxy groups is independently ^-ORk , wherein each ^Rk is independently alkyl optionally substituted with a heterocyclyl or -N( ^Rd )( ^Re ); wherein the heterocyclyl contains at least one nitrogen atom, and each ^Rd and ^Re is independently H, alkyl, alkenyl or alkynyl.

16. _The compound of claim 15, wherein each of the alkoxy groups is selected from the group consisting of _-OCH3 , _-OCH2CH3 , _-OCH2CH2CH3 , _-OCH ₍ CH3 ₎ ₂ _, _-OCH2CH2N ( _CH3 ) ₂ , Composed of groups.

17. A compound according to any one of claims 1 to 16, wherein R is L-W.

18. The compound of claim 17, wherein L is a phosphate-based linker.

19. The compound of claim 18, wherein the phosphate-based linker comprises a phosphate-based portion having the following structure:

wherein * represents a connection to the -O- atom at position R of formula (I) or formula (Ia); wherein L further comprises at least one additional moiety, and the wavy line of the phosphate-based moiety represents a connection to one of the at least one additional moiety; wherein the at least one additional moiety is selected from the group consisting of unsubstituted alkylene, substituted alkylene, -(alkylene-O)-, optionally substituted arylene, -O-, -C(O)-, -N( _Rw )-, -S(O) _0-2- , a water-soluble polymer and an amino acid; wherein each _Rw is independently H or _C1 - _C8 alkyl; and combinations thereof.

20. The compound of claim 19, wherein each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, -(alkylene-O)-, -C(O)-, -N( _Rw )-, a water soluble polymer, and an amino acid; wherein each _Rw is independently H or _C1 - _C8 alkyl; and combinations thereof.

21. A compound according to any one of claims 1 to 17, wherein R is L-W, and L is selected from the group of linkers of Table 6.

22. A compound according to any one of claims 1 to 17, wherein R is L-W, and L is selected from the group of linkers of Table 7.

23. A compound according to any one of claims 1 to 17, wherein R is L-W, and L is selected from the group of linkers of Table 8.

24. A compound according to any one of claims 1 to 17, wherein R is L-W, and L has the following structure:

wherein * represents a connection to the —O— atom at position R of formula (I) or formula (Ia); and + represents a connection to W.

25. A compound according to any one of claims 1 to 17, wherein R is L-W, and L has the following structure:

wherein T is a water-soluble polymer; R ^t is H or methyl; * indicates a connection to the —O— atom at position R of formula (I) or formula (Ia); and + indicates a connection to W.

26. The compound of claim 25, wherein the water soluble polymer is a polyethylene glycol (PEG) moiety.

27. The compound of claim 26, wherein the PEG moiety has a molecular weight ranging from about 100 Da to about 100,000 Da, from about 100 Da to about 10,000 Da, from about 100 Da to about 5,000 Da, or from about 100 Da to about 1,000 Da.

28. _{The compound of claim 26, wherein the PEG moiety is -(CH2CH2O)nCH3} _, _wherein n is _an integer from 1 to 24.

29. The compound _of claim 26, wherein the PEG moiety is -( _CH2CH2O ) _nCH3 , wherein n is 8, 9, 10 _, 11, or 12.

30. A compound according to any one of claims 1 to 29, wherein R is LW and the reactive moiety W comprises _-N3 , -OH, -SH, -NH( ^Rj ), -C(O) ^Rq , -C(O) ^ORx , -C(O) _CH2NH2 , _an activated ester, -O- _NH2 , maleimide, tetrazine, alkyne, cyclooctyne or (E)-cyclooctene; wherein ^Rj is H or unsubstituted alkyl, ^Rq is unsubstituted alkyl, and ^Rx is H, unsubstituted alkyl or a carboxylic acid protecting group.

31. The compound of claim 30, wherein the reactive moiety W is selected from the group consisting of:

_-N3 , -OH, -SH, -NH( ^Rj ), -C(O) ^Rq , -C(O) ^ORx , activated ester, -O- _NH2, and an optionally substituted monocyclic or polycyclic group comprising the cyclooctyne;

in:

R ^j is H or unsubstituted C ₁ -C ₆ alkyl,

^Rq is an unsubstituted C ₁ -C ₆ alkyl group,

^Rx is H, unsubstituted _C1 - _C6 alkyl or a carboxylic acid protecting group,

^Rf is H or unsubstituted C ₁ -C ₆ alkyl,

s is 0, 1, 2, 3, 4, 5, or 6, and

t is 0, 1, 2, 3, 4, 5, or 6.

32. The compound of any one of claims 1 to 31, wherein W is _-ONH2 .

33. The compound according to claim 1 or 2, wherein the compound is selected from the group consisting of:

and their salts.

34. The compound according to claim 1 or 2, which has the following structure:

or a salt thereof.

35. The compound according to claim 1 or 2, which has the following structure:

or a salt thereof.

36. The compound according to claim 1 or 2, which has the following structure:

or a salt thereof.

37. The compound according to claim 1 or 2, which has the following structure:

or a salt thereof.

38. A compound according to any one of claims 1 to 16, wherein R is H.

39. The compound according to claim 1 or 2, wherein the compound is selected from the group consisting of:

as well as

and their salts.

40. The compound according to claim 1 or 2, wherein the compound is selected from the group consisting of:

and their salts.

41. An antibody drug conjugate (ADC) of formula (II):

in:

Ab is an antibody, wherein Ab comprises one or more unnatural amino acids;

L is the connector;

E is the part that connects Ab and L;

d is an integer from 1 to 10; and

in:

Each X ³ is C;

each X ⁴ is C(R ⁴ ) or N, wherein R ⁴ is H, halogen, -OH, -SH, -NO ₂ , -CN, -N ₃ , -N(R ^a )(R ^b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ^{c , -C(O)OR c} , -C(O) ^N (R ^a )(R ^b ), -C(S)R ^c , -C(S)OR ^c , -C(S)N(R ^a )(R ^b ), -C(O)SR ^c , or -S(O) _m (R ^s );

each X ⁵ is C(R ⁵ ) or N, wherein R ⁵ is H, halogen, -OH, -SH, -NO ₂ , -CN, -N ₃ , -N(R ^a )(R ^b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ^{c , -C(O)OR c} , -C(O) ^N (R ^a )(R ^b ), -C(S)R ^c , -C(S)OR ^c , -C(S)N(R ^a )(R ^b ), -C(O)SR ^c , or -S(O) _m (R ^s );

each ^X6 is C( ^R6 ) or N, wherein ^R6 is H, halogen, -OH, _-SH , -NO2, -CN, _-N3 , -N( ^Ra )( ^Rb ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O) ^Rc , -C(O)ORc, -C(O)N( ^Ra )( ^Rb ), -C(S) ^Rc , -C(S) ^ORc , ^-C (S)N( ^Ra )( ^Rb ), -C(O) ^SRc , or -S(O) _m ( ^Rs );

each X ⁷ is C(R ⁷ ) or N, wherein R ⁷ is H, halogen, -OH, -SH, -NO ₂ , -CN, -N ₃ , -N(R ^a )(R ^b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ^{c , -C(O)OR c} , -C(O) ^N (R ^a )(R ^b ), -C(S)R ^c , -C(S)OR ^c , -C(S)N(R ^a )(R ^b ), -C(O)SR ^c , or -S(O) _m (R ^s );

Each X ⁸ is C; and

in:

Each ^Ra and ^Rb is independently H, alkyl, alkenyl or alkynyl;

Each m is independently 0, 1, 2 or 3;

or a pharmaceutically acceptable salt thereof.

42. The ADC of claim 41, wherein A has the following structure:

And wherein the remaining variables are as defined in claim 41.

43. An ADC according to claim 41 or 42, wherein:

X ³ is C;

X ⁸ is C; and

44. The ADC of claim 41, 42 or 43,

in:

^X1 is C( ^R1a )( ^R1b ); wherein each of ^R1a and ^R1b is H;

^X2 is C( ^R2a )( ^R2b ); wherein each of ^R2a and ^R2b is H;

X ³ is C;

^X4 is C( ^R4 ) or N, wherein ^R4 is H;

^X7 is C( ^R7 ) or N, wherein ^R7 is H;

X ⁸ is C; and

^X9 , when present, is _CH2 .

45. The ADC of any one of claims 41 to 44, wherein each of the heteroalkyl groups is an alkoxy group.

46. The ADC of any one of claims 41 to 45, wherein:

^X1 is C( ^R1a )( ^R1b ); wherein each of ^R1a and ^R1b is H;

^X2 is C( ^R2a )( ^R2b ); wherein each of ^R2a and ^R2b is H;

X ³ is C;

^X4 is C( ^R4 ) or N, wherein ^R4 is H;

^X5 is C( ^R5 ) or N, wherein ^R5 is H, halogen or alkoxy;

X ⁶ is C(R ⁶ ) or N, wherein R ⁶ is H, halogen or alkoxy;

^X7 is C( ^R7 ) or N, wherein ^R7 is H;

X ⁸ is C; and

^X9 , when present, is _CH2 .

47. The ADC of claim 46, wherein ^X5 is C( ^R5 ) or N, wherein ^R5 is H or alkoxy; and ^X6 is C( ^R6 ) or N, wherein ^R6 is H or alkoxy.

48. The ADC of any one of claims 41 to 47, wherein ^X4 is N, ^X5 is C( ^R5 ), ^X6 is C( ^R6 ) and ^X7 is C( ^R7 ).

49. The ADC of any one of claims 41 to 47, wherein ^X4 is C( ^R4 ), ^X5 is N, ^X6 is C( ^R6 ) and ^X7 is C( ^R7 ).

50. The ADC of any one of claims 41 to 47, wherein ^X4 is C( ^R4 ), ^X5 is C( ^R5 ), ^X6 is N and ^X7 is C( ^R7 ).

51. The ADC of any one of claims 41 to 47, wherein ^X4 is C( ^R4 ), ^X5 is C( ^R5 ), ^X6 is C( ^R6 ) and ^X7 is C( ^R7 ).

52. The ADC of any one of claims 41 to 51, wherein at least one of ^X4 and ^X7 is CH.

53. The ADC of any one of claims 41 to 47 and 49 to 52, wherein each of ^X4 and ^X7 is CH.

54. The ADC according to any one of claims 41 to 53, wherein at least one of R ⁵ and R ⁶ is alkoxy.

55. The ADC according to any one of claims 45 to 54, wherein each of the alkoxy groups is independently ^-ORk ;

wherein each ^Rk is independently alkyl optionally substituted with heterocyclyl or -N( ^Rd )( ^Re ); wherein the heterocyclyl contains at least one nitrogen atom, and each ^Rd and ^Re is independently H, alkyl, alkenyl or alkynyl.

56. _The ADC of claim 55, wherein each of the alkoxy groups is selected from the group consisting of _-OCH3 , _-OCH2CH3 , _-OCH2CH2CH3 , _-OCH ₍ CH3 ₎ ₂ _, _-OCH2CH2N ( _CH3 ) ₂ ,

Composed of groups.

57. The ADC of any one of claims 41 to 56, wherein d is 1, 2, 3 or 4.

58. The ADC of any one of claims 41 to 57, wherein L is a phosphate-based linker.

59. The ADC of claim 58, wherein the phosphate-based linker comprises a phosphate-based portion having the following structure:

wherein * represents a connection to the -O- atom at position L of formula (II); wherein L further comprises at least one additional moiety, and the wavy line of the phosphate-based moiety represents a connection to one of the at least one additional moiety;

wherein each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, -(alkylene-O)-, -C(O)-, -N( _Rw )-, a water soluble polymer, and an amino acid; wherein each _Rw is independently H or _C1 - _C8 alkyl; and combinations thereof.

60. The ADC of any one of claims 41 to 57, wherein L is selected from the group of linkers of Table 6.

61. The ADC of any one of claims 41 to 57, wherein L is selected from the group of linkers of Table 7.

62. The ADC of any one of claims 41 to 57, wherein L is selected from the group of linkers of Table 8.

63. The ADC of any one of claims 41 to 57, wherein L has the following structure:

wherein * represents a connection to the -O- atom at position L of formula (II); and + represents a connection to E.

64. The ADC of any one of claims 41 to 57, wherein L has the following structure:

wherein T is a water-soluble polymer; R ^t is H or a methyl group; * indicates a connection to the —O— atom at position L of formula (II); and + indicates a connection to E.

65. The ADC of claim 64, wherein the water soluble polymer is a polyethylene glycol (PEG) moiety.

66. The ADC of claim 65, wherein the PEG moiety has a molecular weight ranging from about 100 Da to about 100,000 Da, from about 100 Da to about 10,000 Da, from about 100 Da to about 5,000 Da, or from about 100 Da to about 1,000 Da.

The ADC of claim 65 , wherein the PEG moiety is —(CH ₂ CH ₂ O) _n CH ₃ , wherein n is an integer from 1 to 24.

The ADC of claim 65 , wherein the PEG moiety is —(CH ₂ CH ₂ O) _n CH ₃ , wherein n is 8, 9, 10, 11, or 12.

69. The ADC of any one of claims 41 to 68, wherein E comprises an amide, an ester, a thioester, a pyrrolidine-2,5-dione, an oxime, a 1,2,3-triazole, or a 1,4-dihydropyridazine, wherein each of the 1,2,3-triazole and the 1,4-dihydropyridazine is optionally fused to an 8-membered ring.

70. The ADC of claim 69, wherein E is selected from the group consisting of:

as well as

wherein each ^Rj is independently H or unsubstituted _C1 - _C6 alkyl; each ^Rq is independently unsubstituted _C1 - _C6 alkyl; each ^Rf is independently H or unsubstituted _C1 - _C6 alkyl; each s is independently 0, 1, 2, 3, 4, 5 or 6; each t is independently 0, 1, 2, 3, 4, 5 or 6; each + represents a connection to L; and each wavy line represents a connection to Ab.

71. The ADC of any one of claims 41 to 70, wherein E is:

wherein R ^q is an unsubstituted C ₁ -C ₆ alkyl group.

72. The ADC of claim 71, wherein ^Rq is methyl.

73. The ADC of any one of claims 41 to 72, wherein E connects L to the unnatural amino acid of Ab.

74. The ADC of any one of claims 41 to 73, wherein the Ab is configured to bind to an antigen.

75. The ADC of claim 74, wherein the antigen is selected from the group consisting of PD-1, PD-L1, PSMA, CD70, CD3, HER2, HER3, TROP2, GPC3, VEGFR, EGFR, c-Met (HGFR), CD19, CD22, CD25 (IL-2Rα), CD30, CD33, CD37, CD46, CD48, CD56 (NCAM-1), CD71 (Transferrin R), CD74, CD79b, CD123 (IL-3Rα), CD138 (Syndecan-1), CD142, CD166 (ALCAM), CD203c (ENPP3), CD205 (LY75), CD221 (IGF-1R), CD262 (TRAILR2), CD276 (B7-H3), Mesothelin, EpCAM, CEA CAM5, CEACAM6, DLL3, ROR1, ROR2, GPNMB, GCC, GUCY2c, NaPi2b, Flt-1, Flt-3, folate receptor α, tissue factor (TF), CA6, MUC1, MUC16 (CA-125), BCMA, SLAMF7 (CS1), TIM1, CanAg, Ckit (CD117), EphA2, Nectin4, SLTRK6, FGFR2, LYPD3 (C4.4a), cadherin 3, 5T4 (TPBG), STEAP1, PTK7, ephrin-A4, LIV-1 (SLC39A6 or ZIP6), SLC1A5, TENB2, ETBR, integrin v3, Cripto, AGS-5 (SLC44A4), LY6E, AXL, LAMP1, LRRC15, TNF-α, and MN/CA IX.

76. The ADC of claim 75, wherein the antigen is TROP2, CD70, HER2, PSMA, HER3, or GPC3.

77. The ADC of any one of claims 41 to 76, wherein Ab is an anti-CD70 antibody comprising a sequence listed in Table 2.

78. The ADC of claim 77, wherein the anti-CD70 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26.

79. The ADC of claim 77 or 78, wherein the anti-CD70 antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 27.

80. The ADC of claim 77, 78 or 79, wherein the anti-CD70 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 25.

81. The ADC of claim 77, 78 or 79, wherein the anti-CD70 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 20.

82. The ADC of any one of claims 77 to 81, wherein the anti-CD70 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 19.

83. The ADC of claim 77, wherein the anti-CD70 antibody comprises two heavy chains each having the amino acid sequence of SEQ ID NO: 20 and two light chains each having the amino acid sequence of SEQ ID NO: 19.

84. The ADC of any one of claims 41 to 76, wherein Ab is an anti-TROP2 antibody comprising a sequence listed in Table 1.

85. The ADC of claim 84, wherein the anti-TROP2 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO:5.

86. The ADC of claim 84 or 85, wherein the anti-TROP2 antibody comprises a light chain having the amino acid sequence of SEQ ID NO:4.

87. The ADC of any one of claims 41 to 76, wherein Ab is an anti-HER2 antibody comprising a sequence listed in Table 3.

88. The ADC of claim 87, wherein the anti-HER2 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 29.

89. The ADC of claim 87 or 88, wherein the anti-HER2 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 30.

90. The ADC of any one of claims 41 to 76, wherein Ab is an anti-PSMA antibody comprising a sequence listed in Table 4.

91. The ADC of claim 90, wherein the anti-PSMA antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 39.

92. The ADC of claim 90 or 91, wherein the anti-PSMA antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 40.

93. The ADC of any one of claims 41 to 76, wherein Ab is an anti-HER3 antibody comprising a sequence listed in Table 5.

94. The ADC of claim 93, wherein the anti-HER3 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 58.

95. The ADC of claim 93 or 94, wherein the anti-HER3 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 47.

96. The ADC of any one of claims 41 to 95, wherein the antibody comprises two heavy chains and an unnatural amino acid is incorporated into each of the heavy chains.

97. The ADC of any one of claims 41 to 96, wherein the unnatural amino acid is p-acetyl-L-phenylalanine.

98. A pharmaceutical composition comprising a compound according to any one of claims 1 to 40 or an ADC according to any one of claims 41 to 97, and at least one pharmaceutically acceptable adjuvant, binder, buffer, carrier, diluent or excipient.

99. A method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 40, an ADC according to any one of claims 41 to 97, or a pharmaceutical composition according to claim 98.

100. The method of claim 99, wherein the disease or condition is cancer.

101. The method of claim 100, wherein the cancer is a CD70 expressing cancer.

102. The method of claim 100 or 101, wherein the cancer is renal cell carcinoma.

103. The method of claim 100 or 101, wherein the cancer is a blood cancer.

104. The method of claim 103, wherein the blood cancer is leukemia, lymphoma, or myeloma.