CA2105306A1 - Receptors for bombesin-like peptides - Google Patents

Abstract

Receptors for bombesin-like peptides are solubilized and purified in active form. The amino acid sequence and DNA encoding various subtypes of the receptors are disclosed. Uses of the purified receptor gene and polypeptide are disclosed, including means for screening for agonists and antagonists of the receptor ligands, for producing diagnostic or therapeutic reagents, and for producing antibodies. Therapeutic or diagnostic reagents and kits are also provided.

Description

~ '0 92/16~3 2 1 0 ~ 3 0 6 PCT/~S9~/02091 ECEP~ORS ~OR BOM~3E~IN~ E PEPTIDES
This application is a continuation-in-part of U.S.
Patent Application Serial No. 07/670,603 filed on March 15, 1991; of U.S. Patent Application Serial No. 07/533,659 filed on June 5, 1990; an~ of U.S. Patent Application Serial No.
07/426,150 filed on October 24, 1989; each of which is incorporated herein by reference and benefit is claimed of the i respective filing dates.
;'. 10 Field of the Invention The present invention relates generally to nucleic acids and polypeptides characteristic of receptors for bombesin-like peptides, and more particularly to their uses in preparing new reagents useful for dia~nosing or treating various receptor related medical conditions.

; BACKGROUND O~ THE INVENTION
s Growth factors are involved in numerous physiological and pathological processes. An increasing number of small regulatory peptides have been discovered in the neural and neuroendocrine cells of mammalian tissues. More recent evidence has pointed to the role of neuropeptides in the regulation of animal cell growth, e.g., in the action of mitogenic peptides on the Swiss 3T3 cell system. One of the first neuropeptides studied was the tetra-decapeptide bombesin which was originally isolated from amphibian skin, Anastasi et al. (1971) Experientia 27:166-167. Over ten bombesin-related peptides have been subsequently isolated from various sources and c}assified into three sub~amilies according to their C-terminal sequences. These~subfamiIies are the bombesin, the ranatensin, and litorin subfamilies.
Several endogenous mammalian peptides are ~ struckurally related to bombesin-like peptides. The gastrin - 35 releasing peptide (GRP~) is~a member of the bombesin subfamily, and neuromedin B ~NMB)~is~a;;member of the ranatensin subPamily.
Gastrin releasing peptide (GRPJ is a 27 amino acid peptide having the following sequence in humans:
NH2-Val-Pro-Leu-P~b-~la-Gly-~G~-Gl.-Thr-Val-Leu-Thr-L--s-~let-T, W0~2/166~3 2 1 PCT/US92/~2091 0~306 ~ ~

-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-L~u-Met-(NH2). GRP is of significant interest because it functions as an autocrine - growth factor in the pathogenesis of cancer. In particular, ~- GRP has been found to promote the growth of human small cell lung carcinoma (SCLC). GRP binding to cell surface receptors ;~ is thought to stimulate cellular growth by promoting the hydrolysis of phosphatidyl inositides and by activating protein kinase C. A large number of biological responses to GRP have been observed including: stimulation of Na~/H~ antiport, mobilization of intracellular Ca2+, transient expression of c-fos and c-myc proto-oncogenes, induction of tyrosine kinase activity, elevation of DNA synthesis, and promotion of cell division.
Other bombesin-like peptides, including neuromedin B, mediate a variety of similar biological and pharmacological activities. These peptides appear to function as growth factors, and to be involved in regulation of homeostasis, thermoregulation, metabolism, and behavior.
` For example, the role of GRP in maintaining the growth of SCLC suggests that effective therapeutic agents could be developed to interrupt the autocrine growth cycle by inactivating GRP or inhibiting its receptor. The active site of GRP is the C-terminal region which binds high affinity receptors on SCLC membranes. Blocking this binding can inhibit SCLC growth. This has already been accomplished with ; monoclonal antibodies to bombesin which bind to the active site on GRP, thus inactivating the peptide, see Cuttitta et al.
~1985) Nature 316:823-826.
Another means to block GRP from binding to its rèceptor, and therefore useful in treating SCLC, is to inhibit the receptor itself. Unfortunately, means to find such , reagents have been severely hampered by the absence of purified :
GRP receptor in an active form. This problem can be overcome by~use of the recombinant~ receptor. ~ Along with providing an ~35 improved renewable source of the receptor from a specific source, using the recombinant GRP receptor in screening for GRP
receptor reactive drugs also has the following advantages: a ;`

WO9~/16623 ~ ) 3~ ~ PCT/U~92/02091 ` '' ' :

potentially gxeater number of receptors per cell giving greater yield of reagent and higher signal to noise ratio in assays;
and receptor subtype specificity (giving greater biological and disease specificity).
Cross-linking of the GRP receptor to bound radiolabeled GRP has been used to visualize the GRP
receptor-ligand conjugate on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and to deduce certain other characteristics of the receptor, see Rozengurt et al., PCT/GB88/00255. However, the technique used did not " involve isolation of the receptor, but rather involved characterization of a modified form of the receptor protein.
Unfortunately, in order to characterize the structural properties of the GRP receptor in greater detail and to understand the mechanism of action at the molecular level, the receptor needs to be purified. For many applications, the " receptor must be isolated in an active state retaining the binding activity of the receptor. These applications include the generation of antibodies against active receptor epitopes, . 20 structural studies of the ligand binding site, and the use of the purified receptor for screens for agonists and antagonists of GRP binding. Isolation of the receptor gene should provide an economical source of the receptor, allow expression of more ~ receptors on a cell leading to increased assay sensitivity, i 25 promote characterization of various receptor subtypes, and allow correlation of activity with receptor structures.
Similarities in other bombesin-like peptide functions exist. In particular, the NMB receptor shares many functions and characteristics with the GRP receptor, but also exhibits different structural and functional properties. To date, few receptors have been isolated and characterized in their active form. The amount of receptor present in most tissues is minute. Furthermore, the receptor must often be solubilized from membranes with detergents that can perturb or disrupt the structure of the receptor protein. Further compounding these -difficulties is the unpredictable nature of receptor isolation -~
in that the method for successfully solubilizlng one proteln ., ': ~

WO9~/16623 PCT/US92/02091 21~o~ ~' receptor type or subclass may not be successful ~or a different protein receptor type or subclass.
Thus, a need exists for the isolation and characterization of receptors for bombesin-like peptides, e.g., GRP, NMB, and other bombesin-like peptides. The present invention provides these and the means for preparing many other useful reagents.

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SU~MARY OF THE INVENTION
The present invention provides gene and protein sequences of various receptors for bombesin-like peptides (RBP), including subtypes RlBP and R2BP, which are receptors for GRP and NMB, respectively, as well as other similar receptor molecules, e.g., R3BP.
This invention provides recomblnant nucleic acids, and isolated or substantially pure nucleic acids, which are substantially homologous to a sequence encoding a receptor for a bombesin-like peptide, or a fragment thereof. Nucleic acids encoding fusion polypeptides are contemplated, as are vectors, cells, and organisms comprising such nucleic acids. Exemplary embodiments are different RBP subtypes, i.e., RlBP (GRP
receptor), R2sp (NMB receptor), and R3BP (a third related gene for a receptor-like protein whose ligand has ~ot yet been identified~.
Recombinant polypeptides, and isolated or substantially pure polypeptides derived from these RBP protein ` sequences are encompassed herein. Fusion polypeptides are provided, along with cells and organisms comprising the < polypeptides. Compositions comprising these polypeptides are embraced herein. Exemplary embodiments are, again, GRP
- receptor, NMB receptor, and R3BP.
The invention provides antibodies specific for epitopes unique to, or characteristic of, the receptors for bombesin-like peptides. ~These include antibodies which bind specifically to either epitopes which are shared by the genus j~ of receptors for bombesin-like peptides, or epitopes which distinguish between the different receptor subtypes.
Kits comprising any of these compositions are included herein. Thus, various nucleic acid molecules, polypeptides, and antibodies may provide the basis of various diagnostic or therapeutic kits.
The various compositions also provide bases for methods for treating hosts, particularly those suffering from abnormal receptor function, e.g., proliferative cell . ', :

W092/l~623 2`1 ~ 5 3 0 ~ PCT/US92/02091 ~' conditions, by administering effective amounts of these reagents, or contacting biological samples with them.
The compositions, e.g., ligand binding fragments, also provide the means to select and screen for additional agonists and antagonists for the respective receptor subtypes.
Selected compounds are made available, both ligands and molecules which interact at polypeptide regions separate from the ligand binding regions. Of particular utility are compounds ~ffecting multiple receptor subtypes, e.g., those exhibiting desired spectra of specificity for modulating biological activity.
The group of RBP subtypes is also very useful in providing a group of receptor polypeptides having both substantial similarities and critical differences. These RBP, :
as a group, allow dissecting of structure and function for the class in a manner impossible from characterization of a single subtype.
The following description specifically describes mostly the mouse RlBP (GRP receptor), but similar concepts could be applied to other related receptors for bombesin-like peptides, including human RlBP (GRP receptorj, rat R2BP (NMB
receptor~, human R2BP (NMB receptor), and human R3BP
(incompletely characterized homologous putative receptor). In :
particular, analogous uses and reagents derived from other similar receptors for bombesin-like peptides will be developed.
Identification of new bioactive ligands for new receptor subtypes will also result.
This invention pertains to expressing DNA encoding the GRP receptor in host cells, e.g., transcribing and i 30 translating, thereby enabling the synthesis of GRP receptor compositions having the amino acid sequence of the naturally-occurring GRP receptor which are entirely free of other proteins of the species of origin and further enabling the synthesis of novel mutant GRP receptors.
In addition, this invention relates to the use of DNA -encoding the GRP receptor or its fragments in hybridization , ~ diagnosis of defective GRP receptor DNA or mRNA, and for . : ' . .

W~ 92/16623 ~ G PCT/U592/02091 obtaining DNA encoding the GRP receptor from natural sources.
Similar uses of genes for other receptors for bombesin-like peptides (RBP) are likewise provided, e.g., R2BP (~B receptor subtype), R3BP, or other closely related receptors.
More specifically, this invention pertains to the use of the recombinant RlBP, R2BP, or R3BP, and related proteins;
to cell lines transfected with vectors directing the expression of RlBP, R2BP, R3BP, or related receptors; to membranes from such cell lines, e.g., in drug screening assays for compounds lo having suitable binding af~inity for the respective receptors, `~ individually or in combination; and to antibodies and other ;
reagents made available therefrom.
; Even more specifically, this invention p~rtains to recombinant RlBP (GRP-R), R2BP (NMB-R), or R3BP, along with protein fragments of the receptors, and antibodies directed thereto, that are useful in diagnostic assays to determine the levels of expression in a patient's tissues of the respective , receptor subtypes. Assays based on detection of antibodies to the receptors and/or detection of the receptors can also have ~; 20 prognostic value.
` Additionally, this invention pertains to using the recombinant receptors or fragments or derivatives thereof, e.g., to make reagents such as antibodies to the receptors or -fragments, or to isolate specific receptor agonists or antagonists defined in screening assays.
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BRIEF DESC:RIPT~ON OF THE DRAWINGS
Figure l is a graphic comparison of the ability of several detergents to solubilize RlBP (GRP receptor) and shows the effect of solubilization on binding activity.
Figure 2 is a graph of GRP-binding activity and RlBP
(GRP receptor) solubilization as a function of detergent -(CHAPS) concentration.
Figure 3 is a graph of RlBP solubilization and activity as a function of the soluble cholesteryl ester stabilizing agent (CHS) conc2ntration.
Figure 4 is a graph of GRP binding activity as a function of detergent (CHAPS) concentration.
Figure 5 is a gel display of SDS-PAGE analysis of - l25I-GRP cross-linked to RlBP (GRP-R) in a crude soluble extract.
Figure 6 is a silver stained gel display of SDS-PAGE
`~ analysis of the purified RlBP.
- Figure 7 shows the separation of tryptic fragments of ~lBP by reverse-phase HPLC.
Figure 8 shows a hydropathy analysis of the deduced amino acid sequence of the mouse RlBP (GRP-R). This was generated using the Pepplot (window = 20 amino acids) in the `~
~ Sequence Analysis Software Package of the University of :~
,~ Wisconsin Genetics Group, see Devereaux et al (1984) Nuc. Acids Res. 12:387-395, which is incorporated herein by reference. --Positive regions are relatively hydrophobic, and negative regions are hydrophilic. Putative transmembrane domains are n:mbered sequentially by numbers I through VII. Solid line:
,~ Kyte-Doolittle criterion, Dotted line: Goldman criterion.
, 30 Figure ~ shows Northern hybridization analysis of mRNA from Swiss 3T3 cells.
Figure l0 shows Northern hybridization analysis of mRNA from human fetal lung cells (HFL).
Figure ll shoWs GRP ligand-dependent induction of chloride current in a Xenopus oocyte expressing an in vitro tr~nscript from a RlBP (GRP-R) cDNA clone.

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~ 9~/16623 2 1 ~ S 3 0 r~ PCT~US92/020~1 1 Figure 12 shows a hydropathy analysis plot of t~e deduced amino acid sequence of a rat NMB receptor.
Figure 13 shows a hydropathy plot derived from a human GRP receptor.
Figure 14 shows biological response of receptors for two bombesin-like peptides. This figure shows the increase ln intracellular-Ca2~ of NCI-H345 cells in response to Tyr4-bombesin (BN) and neuromedin B (NMB). An increase in ~luorescence at 492 nm appears after addition of the indicated ligand~ The ligands were added to achieve a final concentration of lO0 nM. lO ~l 10% Triton-X was added as indicated at the termination of the experimental determination.
5 X lo6 cells were used per determination. The time scale is displayed at the lower right hand corner of the figure.
Figure 15 shows a concentration effect relationship -~
of NMB or BN in NCI-H345 cells. Data are shown as nM change from rasting baseline values. The values are the mean of 2-3 separate determinations. 5 X lo6 cells were used in each assay. ~ : NMB agonist response, ~:Bombesin response.
Figure 16 shows concentration effect relationship of the antagonist or inhibitor, [D-Phe6]BN(6-l3) ethyl ester, in ~ the presence of 50 nM NMB or BN in NCI-~345 cells. The -- inhibitor was incubated with the cells for 5 minutes prior to the addition of ligand. The percent change in [Ca2+]i was - -~, 25 ~ determined as described. The values are the mean of two ,~ separate determinations. 5 X 106 cells were used per determlnation. ~ :N~ response, :Bombesin response.
, Figure 17 shows a hydropathy plot derived from a ¢1 human R2BP (NM~-R).
Figure 18 shows functional expression of a human RlBP
~ (GRP-R) and human R2BP (NMB-R). The electrophysiological j response (chloride cUrrent versus time) is shown o~ Xenopus oocytes to~GRP or NMB application a~ter expression of injected human GRP-receptor m~NA or NMB-recptor transcribed from cDNA
clone templates ln vitro. Panel A shows GRP-R response to agonists tlO nM) and to [D-Phe6]BN(6~l3) (l ~M), plus agonists (10 nM). Panel B shows~ ~ B-R response to agonists (lO nM) and .

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W092/l6623 ~ 1 ~ 5 3 ~ 6 PCT/US92/~2091 - ~) , ''~ 10 to [ D-phe6 ] BN ( 6-l3 ), ( 1 ~M) plus agonists (lo nM). Uninjected oocytes did not respond to GRP or NMB.
Figure l9 shows RNase protection analysis ~f steady state RlBP (GRP-R) mRNA and R2BP (NMB-R) mRNA levels in various 5 lung cancer cell lines. 30 ~g of total RNA was hybridized to either a 32P-labeled GRP-R or NMB-R cRNA probe as described. A
portion of a resulting autoradiograph is shown;
: A) RlBP (GRP-R), 5 day exposure in the presence of an ~ intensifying screen;
; loB) R2BP (NMB-R), 12 day exposure in the presence of an intensifying screen.
The results from all cell lines examined are summarized in Table 10. The signal strength on the resultiny autoradiograph was assessed and assigned a relative value that is exemplified by the following in Figure 19:
: ~+ NCI-H345 + NCI-N592 tr NCI-H510 - NCI-H209 ~ To ascertain that equivalent amounts of intact RNA i~
was analyzed,~total RNA from each sample analyzed was also electrophoresed, blotted, and probed with human beta-actin.
Signals from all RNA samples were comparable, indicating that - the RNA analyzed is not degraded. RNA from the human glioblastoma cell line U118 was included as a positive control in the GRP-R experiments.
~i 25 Figure 20 shows results of a low stringency genomic blot of human DNA cut with Eco RI. A mouse RlBP (GRP-R) probe was used, revealing six new fragments, none of which corresponds to the receptors earlier characterized herein. The six novel hands are indicated by the arrows.

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W~2/166~3 11 . PCI/US92/0209 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
- CONTENTS
I. General II. Nucleic Acids III. Receptor Variants V. Making Receptor V. Receptor Isolation VI. Receptor Analogues VII. Antibodies 10 VIII. Other Uses of Receptors IX. Ligands: Agonists and Antagonists X. Kits XI. Therapeutic Applications XII. Receptor Subtypes I. General The present invention provides the amino acid sequence and DNA sequence of various receptors for bombesin-` like peptides, e.g., a mouse receptor subtype one for a~ 20 bombesin-like peptide, also designated RlBP, which corresponds to a gastrin-releasing peptide (GRP) receptor. These sequences were obtained after an RlBP, or GRP receptor (GRP-R), was ; purified and the amino acid sequence of tryptic fragments of the receptor was determined. Similar sequences for a human RlBP tGRP-R), a rat receptor subtype two for a bombesin-like peptide, i.e., a neuromedin B receptor (NMB-R), a human NMB-R, and a human third receptor subtype, designated R3BP, are provided. The descriptions below are often directed to a mouse RlBP (GRP-R) but are likewise applicable to other receptor subtypes. Human RlBPj rat R2BP, human R2BP, and human R3BP are exemplary emhodiments of the class of RBP.
~ Partial amino acid sequences obtained from a purified ; RlBP were used to deduce DNA probes which were then used to isolate an RlBP cDNA ~orm of the gene. Some of the standard 35 methods are described or referenced, e.g., in Maniatis et al.
(1982) Molecular Clonina A Laboratorv Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook et al.
(1989) Molecular Cloninq: A Laboratory ~anual, (2d ed.), vols 1-3, CSH Press, NY; Ausubel et al., Bioloqv, Greene Publishing Associates, Brooklyn, NY; or Ausubel et al. (1987 and Supplements) Current Protocols ln Molecular Bioloqy~

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; Greene/Wiley, New York, all of which are each incorporated herein by reference. Isolation of this RlBP gene allowed isolation of a gene for a homologous second subtype, R2BP, commonly referred to as a NMB receptor, which further led to the isolation of a third subtype, designated R3BP. These genes will allow isolation of other receptor genes for bombesin-like peptides, further extending the family beyond the herein ~-; described three subtypes, and five specific embodiments. The procedure is broadly set forth below.
A cDNA library, constructed in lambda gtlO
bacteriophage, was prepared from RNA isolated from Swiss 3T3 cells. Several modifications and unique techniques had to be utilized to overcome problems associated with isolating a cDNA
clone when probing the library with oligonucleotides. In ` particular, it was necessary to enrich the library for cDNA
species encoding the RlBP due to the under representation of ` such species in unenriched cDNA libraries. Oligonucleotide probes were designed having a nucleotide sequence based upon the most likely codon usage. The cDNA library was plated out, allowing the lambda phage containing cDNA inserts to lyse their E. coli hosts and form plaques, each containing individual cDNA
inserts. The plaques were screened for RlBP DNA sequences with labeled oligonucleotide probes. Subtype one RBP cDNA species were isolated, but these encoded an incomplete RlBP.
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Polymerase chain reaction technology was used to ~ isolate additional cDNA species encoding portions of an RlBP
-~ (GRP-R), and its 5' and 3' fl;anking regions. Gene-specific ~ primer-directed cDNA cloning was then used to obtain a single s cDNA clone encoding an entire receptor subtype one translation product. The actual cloning techniques utilized herein are set forth in detail in Examples 12 and 13 below. Using the ~ isolated subtype one receptor gene from mouse, a homologous :'! second subtype (R2BP, or NMB receptor) was isolated from rat.
Similarly, human RlBP and R2BP sequences have been isolated, along with a t~ird subtype, designated R3BP, which is as yet incompletely characterized.
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9~16623 l3 PCT/US92/02091 Once a cDNA for a receptor subtype one was isolated from mou~e, it was sequenced. The nucleotide sequence revealed the amino acid sequence of the primary translation product of a - GRP receptor, i.e., the amino acid sequence before any post-translational modification.
A complete mouse amino acid sequence is shown in Table l. This sequence corresponds to SEQ ID NO: l. Table 1 discloses both the nucleotide sequence of the receptor subtype ; one, which binds GRP, and its deduced amino acid sequence, also - lO published in Battey et al. tl99l) Proc. Nat'l Acad. Sci. USA
88:395-399, which is incorporated herein by reference. The experimentally determined amino acid sequence of the intact receptor subtype one protein and of isolated tryptic peptides to the receptor are indicated by underlining. Putative transmembrane se.quences are labeled I through VII. Consensus sequences for N-linked glycosylation are boxed.
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i"~ W092/16623 , PCT/US92/02091 ~1053~6 As used herein, tha terms "receptor subtype one for bombesin-like peptides," "RlBP," or "GRP receptor" shall be ~` defined as including a protein or peptide having the amino acid seguence shown in Table 1, or a fragment thereof. It also refers to a polypeptide which functionally and similarly binds , to a GRP protein with high affinity, e.g., at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and more preferably at better than about 3 nM.
The terms shall also be used herein to refer to a subtype one receptor gene, the alleles of the mouse subtype one receptor, or other subtype one receptors in a mouse, and the subtype one : receptors of species other than mouse, for example, humans and other mammals. The term does not encompass natural antibodies which bind the ligand, since the structural features o~ an antibody binding site are different from ligand binding sites.
A human subtype two receptor (R2BP) seguence is shown in Table

2. This sequence corresponds to SEQ ID NO: 3.
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The mouse subtype one receptor DNA was used as a probe to isolate a rat second subtype receptor gene sequence, see Table 3, and a human subtype one receptor gene sequence, see Table 2. The human subtype one receptor sequence was used as a probe to isolate a human sequence designated R3BP. This designation as a member of the RBP family results from its high homology to receptor subtypes one and two, see Table 12. The subtype one receptor (for bombesin-like peptides) is commonly referred to as a GRP receptor, whereas the subtype two receptor 10 (for bombesin-like peptides) is commonly referred to as a NMB
receptor. The sequence in Table 3 corresponds to SEQ ID NO: 5;
` and the sequence in Table 4 corresponds to SEQ ID NO: 7; and the sequence in Table 12 corresponds to SEQ ID NO: 9. The isolated ra, subtype two receptor gene sequence was then used to isolate a human subtype two receptor gene sequence, see Table 4. Similar procedures will be applicable to isolate homologous receptors from other species, or other receptors in the same species, e.q., a human subtype 3 receptor. In ; particular, receptors for other bombesin~like peptides will be isolated. See Example 29, below, Figure 20, and Table 12.

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Table 3: The nucleotide sequence and predicted amino acid sequence derived from two independent rat subtype two receptor (NMB-R) cDNA clones. Horizontal underlining between nucleotide and amino acid sequences indicate the location of seven predicted transmembrane domains (numbered sequentially) based on homology to other G-protein coupled receptor superfamily members. Heavy dots show the location of potential - sites of N-linked glycosylation. The sequence is also disclos~d in Wada et al. (1991) Neuron 6:421-430, which is incorporated herein by reference.

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, 24 -Table 4: The nucleotide sequence and predicted amino acid sequence derived from human. Both the human genomic receptor subtype one (GRP-R) clone and the human SCLC cell line NCI-H345 cDNA indicate the same protein sequence. Inverted triangles indicate intron positions as determined from the genomic clone.

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~ ~ g2/l~623 2 ~ ~ ~ 3 ~ ~ PCT/~S92/02~91 This invention also encompasses proteins or peptides having substantial amino acid sequence homology with the amino acid sequenc~s in Tables l, 2, 3, or 4, or SEQ ID NO: lO, but excluding any protein or peptide which exhibits substantially the same or lesser amino acid sequence homology than does the substance P or substance K receptors. The substance K receptor sequence is shown in Table 6, as compared with the mouse GRP
receptor.
A polypeptide "fragment"; or "segment", is a stretch of amino acid residues of at least about 8 amino acids, -generally at least lO amino acids, more generally at least 12 amino acids, often at least 14 amino acids, more often at least 16 amino acids, typically at least 18 amino acids, more typically at least 20 amino acids, usually at least 22 amino :
acids, more usually at least 24 amino acids, preferably at least 26 amino acids, more preferably at least 2~ amino acids, and, in particularly preferred embodiments, at least about 30 or more amino acids.
~ Amino acid sequence homology, or sequence identity, is determined by optimizing residue matches, if necessary, by introducing gaps as required. This changes when considering -~
~ conservative substitutions as matches. Conservative ji substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine;
serine, threonine; lysine, arginine; and phenylalanine, -tyrosine. Homologous amino acid sequences are intended to - includ~ natural allelic and interspecies variations in each respective receptor sequence. Typical homologous proteins or peptides will have from 25-100% homology (if gaps can be introduced), to 50-100% homology (if consarvative substitutions are included) with the amino acid sequence of Tables l, 2, 3, or 4, or SEQ ID N0: lO. Homology measures will be at least about 35%, generally at least 40%, more generally at least 45%, often at least 50%, more often at least 55%, typically at least , 60%, more typically at least 65%, usually at least 70%, more usually at least 75%, preferably at least 80%, and more .
.

';~ W092/16623 PCT/US92/02091 preferably ~t least 80%, and in particularly preferred embodiments, at least 85% or more. some homologous proteins or peptides, such as the various receptor subtypes, will share various biological activities with the receptors for bombesin-like peptides of Tables l, 2, 3, or 4, or SEQ ID NO: lO. As used herein, the term "biological activity" is defined as including, without limitation, bombesin-like protein ligand ` binding, cross-reactivity with antibodies raised against each respective receptor from natural sources, and coupling to guanyl nucleotide regulatory proteins (G-proteins). The G-protein linkage typically causes other functionally downstr~am ; biochemical effects including protein phosphorylation and release of sequestered Ca~+, both of which are often used to assay receptor function. It should be noted that various ~ 15 different bombesin-like peptides ~ffect different cellular - responses in the same or different cell types. A "ligand-related activity" refers either to ligand binding itself, or to biological activities which are mediated by ligand binding, including, e.g., G protein interaction, and protein phosphorylation or Ca~ sequestration effects.
The term "ligand" refers to molecules, usually members of the family of bombesin-like peptides, that bind the segments involved in peptide ligand binding. Also, a ligand is a molecule which serves either as a natural ligand to which the ; 25 receptor, or an analogue thereof, binds, or a molecule which is a functional analogue of a natural ligand. The functional ~, analogue may be a ligand with structural modifications, or may be a wholly unrelated molecule which has a molecular shape which interacts with the appropriate ligand binding determinants. The ligands may serve as agonists or antagonists, see, e.g., Goodman et al. (eds) (l990) Goodman &
Gilman's: The Pharmacoloqical Bases of Thera~eutics (8th ed), Pergamon Press.
Solubility of a polypeptide or fragment depends upon , 35 the environment and the polypeptide. Many parameters affect j polypeptide solubility, including temperature, electrolyte environment, size and molecular characteristics of the 1:

. . ~ - .
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~` ~ 92/16623 ~ PCT/VS92/02091 polypeptide, and nature of the solvent. Typically, the temperature at which the polypeptide is used ranges from about 4 C to about 65 C. Usually the temperature at use is greater than about 18 C and more usually greater than about 22O C.
For diagnostic purposes, the temperature will usually be about room temperature or warmer, but less than the denaturation temperature of components in the assay. For therapeutic purposes, the temperature will usually be body temperature, typically about 37 C for humans, though under certain situations the temperature may be raised or lowered in situ or in vitro.
The electrolytes will usually approximate in situ : physiological conditions, but may be modified to higher or lower ionic strength where advantageous. The actual ions may be modified to conform to standard buffers used in : physiological or analytical contexts.
The size and structure of the polypeptide should :i generally be in a substantially stable state, and usually not in a denatured state. The polypeptide may be associated with `l 20 other polypeptides in a quaternary structure, e.g., to confer solubility, or associated with lipids or detergents in a manner i which approximates its natural lipid bilayer interactions.
.~, The solvent will usually be a biologically compatible buffer, of a type used for preservation of biological ~- 25 activities, and will usually approximate a physiological solvent. Usually the solvent will have a neutral pH, typically ; ~ between about 5 and 10, and preferably about 7 5. On some occasions, a detergent will be added, typically a mild non-denaturing one, e.g., CHS or CHAPS.
i~ 30 Solubility is usually measured in Svedberg units, which are a measure of the sedimentation velocity of a molecule I under particu~lar conditions. The determination of the J sedimentation velocity was classically performed in an analytical ultracen~rifuge, but is typically now performed in a standard ultracentrifuge. See, Freifelder (1982) Phvsical Biochemistry (2d ed.), W.H. Freeman; and Cantor and Schimmel (1980) BioPhYsical Chemistry, parts 1-3, W.H. Freeman & Co., ~--: -:

~l~i W O 92/16623 :21 D:~3 ~ ~ PC~r/US92/02091 ~

~ .
~`; 30 San Francisco; each of which is hereby incorporated herein by reference. As a crude determination, a sample containing a putatively soluble polypeptide is spun in a standard full sized ultracentrifuge at about 50K rpm for about 10 minutes, and soluble molecules will remain in the supernatant. A soluble particle or polypeptide will typically be less than about 30S, more typically less than about 15S, usually less than about lOS, more usually less than about 6S, and, in particular embodiments, preferably less than about 4S, and more preferably less than about 3S.
;
II. Nucleic Acids This invention contemplates use of isolated DNA or fragments which encode these receptors for bombesin-like ` 15 peptides, e.g., each respective raceptor subtype, or any fragment thereof, to encode a biologically active corresponding receptor polypeptide. In addition, this invention covers isolated or recombinant DNA which encodes a biologically active ~1 protein or polypeptide having receptor activity and which is ; 20 capable of hybridizing under appropriate conditions with the DNA sequences shown in Tables 1, 2, 3, 4, or 12. Said ,",! biologically active protein or polypeptide can be a receptor itself, or fragment, and have an amino acid sequence shown in ~ Tables 1, 2, 3, or 4, or SEQ ID N0: 10. Further, this~ 25 invention covers the use of isolated or recombinant DNA, or ~ragments thereof, which encode proteins which are homologous s~ to each respective receptor subtype or which was isolated using cDNA encoding a receptor for a bombesin-like peptide as a probe. The isolated DNA can have the respective regulatory sequences in the 5' and 3' flanksj e.g., promoters, enhancers, poly-A addition signals, and others.
An "isolated" nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially , separated from other~components which naturally accompany a native sequence, e.g., ri~osomes, polymerases, and flanking genomic sequences from the originating species. The term embraces a nucleic acid sequence~which has been removed from 3 ~

~ 2/16623 2 ~ ~ ~ 3 ~ ~ PCT/US9~/020~

i~

its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A
substantially pure molecule includes isolated forms of the ~- 5 molecule.
An isolated nucleic acid will generally be a ; homogeneous composition of molecules, but will, in some embodiments, contain minor heterogeneity. This heterogeneity is typically found at the polymer ends or portions not critical to a desired biological ~unction or activity.
" A "recombinant" nucleic acid is defined either by its method of production or its structure. In reference to its ` method of production, e~g., a product made by a process, the process is use of recombinant nucleic acid techniques, e.g., involving human intervention in the nucleotide sequence.
Alternatively, it can be a nucleic acid made by generating a ~` sequance comprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, e.g., naturally occurring mutants. Thus, for example, products made by transforming cells with any ;~' unnaturally occurring vector is encompassed, as are nucleic acids comprising sequence derived using any synthetic oligonucleotide process. Such is often done to replace a codon with a redundant codon encoding the same or a conservative ~ 25 amino acid, while typically introducing or removing a sequence $; recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate '''! a single genetic entity comprising a desired combination of `, functions not found in the commonly available natural forms.
Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, J control sequences~, or other useful features may be incorporated by design. A similar concept;is intended for a recombinant, ~ 35 e.g., fusion, polypeptide. ~Specifically included are synthetic -', nucleic acids which, by genetic code redundancy, encode similar .!.

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21,0~.3~6 ~? WO 92/166~3 PCT/US92tO2091~;

.
?~ 32 polypeptides to fragments of these receptors, and fusions of sequences from various different subtypes.
A 'Ifragment'l in a nucleic acid context is a contiguous segment of at least about 17 nucleotides, generally at least 20 nucleotides, more generally at least 23 nucleotides, ordinarily at least 26 nucleotides, more ordinarily at least 29 nucleotides, often at least 32 nucleotides, more often at least 35 nucleotides, typically at least 38 nucleotides, more typically at least 41 nucleotides, usually at least 44 nucleotides, more usually at least 47 nucleotides, preferably at least 50 nucleotides, more preferably at least 53 nucleotides, and in particularly preferred embodiments will be at least 56 or more nucleotides.
A DNA which codes for a receptor for a bombesin-like -` 15 peptide will be particularly useful to identify genes, mRNA, and cDNA species which code for related or homologous J receptors, as well as DNAs which code for receptor sub-types and receptors from different species. There is at least one receptor sub-type described with a different selectivity - towards bombesin-like peptides from the subtype one which specifically binds GRP, e.g., a second subtype specific for ` binding NMB (subtype two), and there are likely others. In particular, a genetic sequence encoding another putative RBP
has been isolated and designated "subtype three" or l'R3BP", - -~ 25 though it has not been completely characterized. Various bombesin-like peptide receptor sub-types should be highly homologous and are encompassed herein. However, even receptor proteins that have a more distant evolutionary relationship to -' the RlBP and do not bind gastrin releasing peptide can readily be isolated using these bombesin-like peptide receptor ~ sequences if they are sufficiently homologous. Rat NMB
I receptors and human GRP and NMB receptors are examples of I related receptors, as is the~human R3BP. Mammalian receptors are of particular interest.
, 35 Preferred probes for such screens are those regions of the receptors which are conserved between different receptor subtypes. In particular, the third transmembrane segment, -~, : , :.

~ ~ ~ 9~tl~3 2 ~ ~ 5 3 ~ ~ PCT/US92/02091 c~rresponding approximately to nucleotides 345 to 410 of Table `~ 1, is expected to show high homology to corresponding regions of other receptor subtypes. Other conserved regions will be identified by comparisons to other similar receptors or receptor subtypes, e.g., the sixth, seventh, and second transmembrane segments.
`~ This invention further covers recombinant DNA
~-~ molecules and fragments havinq a DNA sequence identical to or ;~ highly homologous to the isolated DNAs set forth herein. In particular, the sequences will often be operably linked to DNA
segments which control transcription, translation, and DNA
replication.
Homologous nucleic acid sequences, when compared, exhibit significant similarity. The standards for homology in ~` ` 15 nucleic acids are either measures for homology generally used in the art by sequence comparison or based upon hybridization conditions. The hybridization conditions are described in ~; greater detail below, but are further limited by the homology to either of the substance P and substance K receptors.
;` 20 Homology measures will be limited, in addition to any stated parameters, to exceed any such similarity to the receptors for jl substance P or substance K.
-"sl! Substantial homology in the nucleic acid sequence ;;~ comparison context means either that the segments, or their ~ complementary strands, when compared, are identical when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 50% of the nucleotides, generally at least 56%, more generally at least 59%, ordinarily at least 62%, more ordinarily at least 65%, often at least 68%, more often at least 71%, typically at least 74%, more typically at least 77%, usually at least 80%, more usually at least about -~l 85~, preferably at least about 90%, more preferably at least ~-about 95 to 98% or more, and in particular embodiments, as high at about 99~ or more of the nucleotides. Alternatively, ~substantial homology eXlsts when the segments will hybridize ¦ under selective hybridization conditions, to a strand, or its complement, typically using a sequence derived from Tables 1, ~;3 ~

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?~ WO 92/16623 PCT/US92/0209l ~

2, 3, 4, or 12. Typically, selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 1.4 nucleotides, preferably at least about 65~, more preferably at least about 75%, and most preferably at least about ~o%. See, Kanehisa (1984) Nuc. Ac ds Res. 12:203-213, which is incorporated herein by reference. The length of homolo~y comparison, as described, may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 40 nucleotides, preferably at least about 50 nucleotides, and more preferably at least about 75 to 100 or mora nucleotides.
- 15 Stringent conditions, in referring to homology in the hybridization context, will be stringent combined conditions of salt, temperature, organic solvents, and other parameters typically controlled in hybridization reactions. Stringent temperature conditions will usually include temperatures in excess of about 30 C, more usually in excess of about 37 C, typically in excess of about 45 C, more typically in excess of about 55 C, preferably in excess of about 65~ C, and more preferably in excess of about 70 C. Stringent salt conditions will ordinarily be less than about 1000 mM, usually less than ~ -about 500 mM, more usually less than about 400 mM, typically less than about 300 mM, pre~erably less than about 200 mM, and more preferably less than about 150 mM. However, the - combination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370, which is here~y incorpoFated herein by reference.

III. RecePtor Variants The isolated receptor DNA can be readily modified by ~;
nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of nucleotide stretches. These modifications result in novel DNA sequences which encode these ' . .

``~;~;
~ 9~ 623 2 ~ ~ ~ 3 ~ ~ PCT~US92~02091 receptors, their derivatives, or proteins having GRP receptor activity. These modified sequences can be used to produce mutant receptors or to enhance the expression of receptor species. Enhanced expression may involve gene amplification, increased transcription, increased translation, and other mechanisms. Such mutant receptor derivatives include predetermined or site~specific mutations of the respective receptor or its fragments. "Mutant GRP receptor" is defined herein as encompassing a polypeptide otherwise falling within ~' 10 the homology definition of the GRP receptor as set forth above, but having an amino acid se~uence which differs from that of `~ GRP receptor as found in nature, whether by way of deletion, . substitution or insertion. In particular, "site specific mutant GRP receptor" is defined as having homology with a receptor of Tables 1, 2, 3, or 4, or SEQ ID NO: lo, and as sharing various biological activities with those receptors.
Similar concepts apply to each of the mouse and human RlBP (GRP
receptor), the rat and human R2BP (NMB receptor), the human R3BP, and other receptors for bombesin-like peptides, particularly those receptors found in warm blooded animals, i e.g., mammals and birds. As stated before, it is emphasized ,1 that descriptions are generally meant to encompass all receptors for bombesin-like peptides, not limited to the GRP
. receptor example specifically discussed.
Although site specific mutation sites are predetermined, mutants need not be site specific. GRP receptor ,- mutagenesis can be conducted by making amino acid insertions or deletions. Substitutions, deletions, insertions, or any combinations may be generated to arrive at a final construct.
Insertions include amino- or carboxy- terminal fusions. Random mutagenesis can be conducted at a target codon and the expressed GRP receptor mutants can then be screened for the i desired activity. Methods for making substitution mutations at predetermined sites in DNA having a known sequence are well known in the art, e.g., by M13 primer mutagenesis. See also Sambrook et al. (19891 and Ausubel et al. (1987 and ' ~ Supplements).
~: . ~: .
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W0~2/]6623 ~ 3 ~ ~ PCT/US92/02091 The mutations in the DNA normally should not place coding sequences out of reading frames and pre~erably will not create complementary regions that could hybridize to produce secondary mRNA structure such as loops or hairpins.
The present invention also provides recombinant proteins, e.g., heterologous fusion proteins using segments from these receptors. A heterologous fusion protein is a fusion of proteins or segments which are naturally not normally fused in the same manner. Thus, the fusion product of an immunoglobulin with a receptor polypeptide is a continuous protein molecule having sequences fused in a typ}cal peptide linkage, typically made as a single translation product and exhibiting properties derived from each source peptide. A
similar concept applies to heterologous nucleic acid sequences.
'~ 15 In addition, new constructs may be made from ~1 combining similar functional domains from other proteins. For example, ligand-binding or other segments may be "swapped"
` between di~ferent new fusion polypeptides or fragments. See, ; e.g., Cunningham et al. (1989) Science 243:1330-1336; and O'Dowd et al. (1988) J. siol. Chem. 263:15985-15992, each of which is incorporated herein by reference. Thus, new chimeric polypeptides exhibiting new combinations of specificities will ;--result from the functional linkage of ligand-binding ` specificities and intracellular regions. For example, the si 25 ligand binding domains from other related receptors may be added or substituted for other binding domains of these ~!: receptors. The resulting protein will often have hybrid function and properties.
~l The phosphoramidite method described by Beaucage and 'i 30 Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce `i suitable synthetic DNA ~ragments. A double stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA
:.~ 35 polymerase with an appropriate primer sequence.
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~ ~ 92/~6623 2 1 ~ 5 3 ~ ~ PCT/US92/02~91 IV. Makin~ Receptor DNA which encodes the GRP receptor or fragments thereof can be obtained by chemical synthesis, screening cDNA
libraries, or by screening genomic libraries prepared from a wide variety of cell lines or tissue samples.
This DNA can be expressed in a wide varlety of host cells for the synthesis of a full-len~th receptor or fragments of a receptor which can in turn, for example, be used to generate polyclonal or monoclonal antibodles; for binding studies; for construction and expression of modified receptor molecules; and for structure/function studies. Each receptor or its fragments can be expressed in host cells that are transformed or transfected with appropriate ~xpression vectors.
These molecules can be substantially free of protein or lS cellular contaminants, other than those derived from the recombinant host, and therefore are particularly useful in pharmaceutical compositions when combined with a pharmaceutically acceptable carrier and/or diluent. The receptor, or portions thereof, may be expressed as fusions with other proteins.
Expression vectors are typically self-replicating DNA
or RNA constructs containing the desired receptor gene or its ~- fragments, usually operably linked to suitable genetic control elements that are recognized in a suitable host cell. These control elements are capable of effecting expression within a suitable host. The specific type of control elements necessary to effect expression will depend upon the eventual host cell used. Generally, the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system, and typically include a transcriptional promoter, an optional operator to control the onset o~
transcription, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation. Expression vectors also usually contain an origin of xeplication that allows the vector to replicate independently of the host cell.

:

W092/166~3 2 ~ ~ PCT/US92/0209 The vectors of this invention contain DNA which encodes a rec~ptor for a bombesin-like peptide, or a fragment thereof encoding a biologically active receptor polypeptide.
The DNA can be under the control of a vlral promoter and can encode a selection marker. This invention further contemplates use of such expression vectors which are capable of expressing eukaryotic cDNA coding for a receptor in a prokaryotic or ` eukaryotic host, where the vector is compatible with the host - and where the eukaryotic cDNA coding for the receptor is inserted into the vector such that growth of the host containing the vector expresses the cDNA in question. Usually, ` expression vectors are designed ~or stable replication in their `~ host cells or for amplification to greatly increase the total , number of copies of the desirable gene per cell. It is not always necessary to require that an expression vector replicate in a host cell, e.g., it is possible to effect transient I expression of the GRP receptor or its fragments in various -~ hosts using vectors that do not contain a replication origin that is recognized by the host cell. It is also possible to use vectors that cause integration of GRP receptor or its fragments into the host DNA by recombination.
Vectors, as used herein, comprise plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles ~"1~ , .
;l which enable the integration of DNA fragments into the genome .,~J ~ 25 of the host. Expression vectors are specialized vectors which contain genetic control elements that effect expressian of 3 operably linked genes. Plasmids are the most commonly used form of vector but all other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels et al. (1985 and Supplements) Cloninq Vectors: A Laboratory Manual, Elsevier, N.Y., and Rodriquez et al. (eds) Vectors: A Survey of Molecular Cloninq Vectors and Their_Uses, Buttersworth, Boston, j' 1988, which~are incorporated herein by reference.
'~ 35 Transformed cells are cells, preferably mammalian, that have been transformed or transfected with receptor vectors , ~ constructed using recombinant DNA techniques. Transformed host 3, 1: : , W~92/16623 PCT/US92/02091 ~ 21053~5 ~ 39 cells usually express the receptor or its fragments, but for ` purposes of cloning, amplifying, and manipulating its DNA, do not need to express the receptor. This invention further contemplates culturing transformed cells in a nutrient medium, thus permitting the receptor to accumulate in the culture. The receptor can be recovered, either from the culture or from the culture medium.
For purposes of this invention, DNA sequences are operably linked when they are functionally related to each lO- other. For exampl2, DNA for a presequence or secretory leader is operably linked to a polypeptide if it is expressed as a preprotein or participates in directing the polypeptide to the cell membrane or in secretion of the polypeptide. A promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide; a ribosome binding site is -operably linked to a coding sequence if it is positioned to permit translation. Usually, operably linked means contiguous and in reading frame, however, certain genetic elements such as repressor genes are not contiguously linked but still bind to operator sequences that in turn control expression.
Suitable host cells include prokaryotes, lower eukaryotes, and higher eukaryotes. Prokaryotes include both gram negatiue and gram positive organisms, e.g., E. coli and B.
subtilis. Lower eukaryotes include yeasts, e.g., S. cerevisiae ~'i 25 and Pichia, and species of the genus Dictvostelium. Higher eukaryotes include established tissue culture cell lines from animal cells, both of non-mammalian origin, e.g., insect cells, and birds, and of mammalian origin, e.g., human, primates, and rodents.
Prokaryotic host-vector systems include a wide variety of vectors for many different species. As used herein, ~1 E. coli and its vectors will be used generically to include l equivalent vectors used in other prokaryotes. A representative J vector for amplifying DNA is pBR322 or many of its derivatives. -¦ 35 Vectors that can be used to express the receptor or its fragments include, but are~not limited to, such vectors as :i~ those containing the Iac promoter (pUC-series); trp promoter ]~ :
,~.: -S ~ ~

W092/16623 ~ 3:~ 5 PCT/US92/02091 :- 40 (pBR322-trp); Ipp promoter (the pIN-series); lambda-pP or pR
promoters (pOTS); or hybrid promoters such as ptac (pDR540).
See Brosius et al. (1988) "Expression Vectors Employing Lambda-, trp-, lac-, and Ipp~derived Promoters", in Vectors: A
Survey of Molecular Cloninq Vectors and Their Uses, (eds.
- Rodriguez and Denhardt), Buttersworth, Boston, Chapter l0, pp.
205-236, which is incorporated herein by reference.
Lower eukaryotes, e.g., yeasts and Dictyostelium, may be transformed with GRP receptor sequence containing vectors.
For purposes of this invention, the most common lower eukaryotic host is the baker's yeast, Saccharomyces cerevisiae.
It will be used to generically represent lower eukaryotes although a number of other strains and species are also available. Yeast vectors typically consist of a replication origin (unless of the integrating type), a selection gene, a promoter, DNA encoding the receptor or its fragments, and sequences for translation termination, polyadenylation, and transcription termination. Suitable expression vectors for ~ yeast include such constitutive promoters as 3-phosphoglycerate `~ 20 kinase and various other glycolytic enzyme gene promoters or . such inducible promoters as the alcohol dehydrogenase 2 promoter or metallothionine promoter. Suitable vectors include derivatives of the following types: self-replicating low copy number (such as the YRp-series), self-replicating high copy number (such as the YEp-series); integrating types (such as the YIp-series), or mini-chromosomes (such as the YCp-series).
Higher eukaryotic tissue culture cells are the preferred host cells for expression of the functionally active GRP receptor protein. In principle, any higher eukaryotic tissue culture cell line is workable, e.g., insect baculovirus expression systems, whether from an invertebrate or vertebrate source. However, mammalian cells are preferred.
Transformation or transfection and propagation of such cells has become a routine procedure. Examples of useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insect cell lines, bird cell lines, and monkey (CQS) cell lines. Expression vectors for : . . .
', .:

~ ~ 92~]6fi23 2~305 PCT/US92/02091 ~1 such cell lines usually include an origin of replication, a promoter, a translation initiation site, RNA splice sites (if genomic DNA is used), a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection gene or amplification gene. Suitable expression vectors may be plasmids, viruses, or retroviruses -- carrying promoters derived, e.g., from such sources as from adenovirus, SV40, parvoviruses, vaccinia virus, or cytomegalovirus. Representative examples of suitable expresision vectors include pCDNAl; pCD, see Okayama et al.
(1985) Mol. Cell Biol. 5:1136-1142: pMClneo PolyA, see Thomas et al. ~1987) Cell 51:503~512; and a baculovlrus vector such as pAC 373 or pAC 610.
It will often be desired to express a receptor polypeptide in a system which provides a specific or defined glycosylation pattern. In this case, the usual pattern will be that provided naturally by the expression system. However, the patt~rn will be modifiable by exposing the polypeptide, e.g., an unglycosylated form, to appropriate glycosylating proteins ~-~ 20 introduced into a heterologous expression system. For example, -~; the receptor gene may be co-transformed with one or more genes ,~`r encoding mammalian or other glycosylatlng enzymes. Using this I approach, certain mammalian glycosylation patterns will be achievable in prokaryote or other cells.
~-i 25 `7 V. Receptor Isolation The GRP receptor can be solubilized from mêmbranes in an active form, and purified without loss of activity by the ; methods outlined below. Again, although the methods are applied to GRP receptor, other receptors for bombesin-like ,.,! peptides will behave similarly and should be isolatable using ;~! analogous methods.
i~ The source of GRP receptor can be a eukaryotic or prokaryotic host expressing recombinant GRP receptor DNA, such as is described above. The source can also be a cell line such as mouse Swiss 3T3 fibroblasts, but other mammalian cell lines .!

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:~ , : ' '~ WO9~/~66~3 ~105~6 PCT/US9t/02~91 ~

are also contemplated by this invention, with the preferred cell line being from the human species.
The active GRP receptor was solubilized from membranes containing the GRP receptor using a stabilizing agent and a detergent. The stabilizing agent is preferably a soluble cholesteryl ester. Particularly good results have been obtained using cholesteryl hemisuccinate (c~S). The detergent can be non~ionic, zwitter-ionic, or the like. Particularly good results have been obtained using the zwitter-ionic . 10 detergent 3-[(3-cholamidopropyl) dimethylammonio]-l-propane sulfonate (CHAPS).
i .
Cellular membranes containing the GRP receptor are prepared by lysis of a cultured GRP receptor containing cell line, e.g., Swiss 3T3 fibroblasts, followed by centrifugation.
The resulting pellets are washed by resuspension and `~ centrifuged again.
Once the membranes are obtained from a suitable cell line, as described above and in Example 1, the final concentration of protein is adjusted. A suitable final protein ; 20 concentration is about 15 mg/ml.
The membranes are then salt washed prior to solubilization of the GRP receptor. The membranes are washed twice with buffer and sodium chloride (NaCl), then washed with '`! a solubilization buffer and finally suspended in the , 25 solubilization~buffer at an adjusted protein concentration. A
~'ii suitable buffer composition for the first two washings comprises a medium such as 50 mM 4-(2-hydroxyethyl)-piperazine ethane sulfonic acid (HEPES), pH 7.5, a chelator such as 2 mM ethylenediamine-tetraacetic acid (EDTA), and J 30 protease inhibitors. A suitable NaCl concentration is 1.0 M.
The solubilization buffer, both for the washing and suspension, can be typically comprised of 50 mM HEPES, pH 7.5, 2 mM EDTA, another chelator~such as 1 mM
` [ethylenebis-(oxyethylenenitrilo)]tetraacetic acid (EGTA), lO0 mM NaCl, and protease inhibitors. The protein concentration is adjusted to about 7 mg/ml, ~for example. This salt washing step provides a two-fold purification. Similar results can be ~92/16623 ~1 ~ r ~ ~ PCT/US92/0209l b achieved by washing the membranes with 2 M urea, high pH
b~ffers (pH 10), or chaotropic salts, e.g., potassium iodide (KI). This procedure also increases the stability of the GRP
receptor in the extract. Other constituents of the buffers may include, e.g., sucrose, and suitable protease inhibitors include, without limitation, aprotinin, leupeptin, pepstatin, bacitrin, and phenylmethylsulfonyl fluoride ~PMSF).
A mixture of detergent (CHAPS) and soluble chol steryl ester stabilizing agent (CHS) is then slowly added to the membrane suspension to give a set final detergent concentration. The weiqht ratio of detergent to soluble cholesteryl ester can be within the range of about 200:1 to

5:2, preferably about 10:1. Alternatively, the detergent can be added to the men~rane suspension, followed by the addition of the soluble cholesteryl ester. In that instance, initially there will be 100% detergent and the soluble cholesteryl ester is added until the weight ratio of detergent to ester is within the range of about 200:1 to 5:2, preferably about 10:1. For solubilization of the GRP receptor, the concentration of detergent should be 0.4 to 3.0% weight per volume ~w/v), and is optimally set at about 0.75% (w/v) for a membrane concentration - (prior to the membrane washing steps) of around 15 mg/ml.
Similarly, the concentration of soluble cholesteryl ester is within the range of about 0.0015 to 1.2% (w/v). Likewise, for a membrane concentration of around 15 mg/ml, the concentration of soluble cholesteryl ester is preferably about n . 075% (w/v).
The extract is then incubated at a temperature within the range of about 0 to 37 C, typically at room temperature such as 21 C, and then cooled to 0 to 21 C, typically 4 C.
The insoluble material is then centrifuged at high speeds, preferably about 100,000 times gravity, in a standard centrifuge for a suitable period of time, depending upon the volume involved, to obtain an extract containing the solubilized receptor (i.e., soluble extract).
At high detergent concentration (0.4 to 3.0%), the receptor loses biologica~l activity. However, upon dilution with a buffer solution, the receptor is reactivated. The ~: ' W O 92/1~623 ~ ~ ~ ` PC~r/US92/02091 presence of the soluble cholesteryl ester, which acts as a stabilizing agent, is necessary for the receptor to be reactivated at the low detergent concentration. For assays using the active solubilized GRP receptor to exhibit binding - -activity, the final concentration of detergent in the suspension should be diluted to within the range of about 0.025 to 0.2~ (w/v). The weight ratio of detergent to soluble cholesteryl ester is still maintained within the range of about 200:1 to 5:2, preferably about 10:1. Therefore, a suitable range for the soluble cholesteryl ester is about 0.000125 to 0.08% (w/v). The preferable assay concentrations are 0.075%
(w/v) detergent and about 0.0075~ (w/v) soluble cholesteryl ester.
The solubilized receptor in its active form is then purified and freed of contaminating proteins. Purification of the GRP receptor involves a multistep procedure which includes ~`~ the following steps, which follow the solubilization procedure ~: as set forth above.
(1) Polyethylene glycol precipitation. The GRP
; 20 receptor is precipitated from the soluble extract by addition ; of polyethylene glycol (PEG). Addition of PEG is preferably ~ done to obtain a final concentration of 20% (w/v). The '~ precipitate is then collected by centrifugation and resuspended in a buffer solution. The buffer solution can typically be comprised of 25 mM HEPES, pH 7.5, 25 mM TRIS/Cl, 2 mM EDT~, ~! ~ 075~ (w/v) detergent, 0.0075~ (w/v) soluble cholesteryl - ester, and protease inhibitors. The final volume of the suspension is preferably 25% that of the original soluble extract. Proteins remaining insoluble in the suspension are removed by centrifugation. This step provides a two-fold purification, and enhances the stability of the receptor.
(2) Wheat germ agglutinin chromatography. The ~ soluble extract is applied to a wheat germ agglutinin affinity J column equilibrated with a buffer solution typi~ally comprised of 50 mM HEPES, pH 7.5, 2 mM EDTA, 0.25% (w/v) detergent, 0.025% (w/v) cholesteryl ester, and protease inhibitors. The column is eluted with column buffer solution and 5 mM ~ -~': ' . , ',i: ', ';

~ ~ 92/16623 2 ~ ~ ~ 3 ~ ~ P~T/US92/02091 ; 45 - N-N'-N"-triacetyl-chitotriose. Fractions containing the GRP
receptor are then identified by l25I-GRP binding assays. This ~; step provides a five-fold purification by removing proteins that do not contain carbohydrate. To obtain a good yield, it is necessary to elut~ the column with chitotriose or chitobiose. The yield may also be enhanced by maintaining the detergent concentration above about 0.2% detergent and 0.02%
soluble cholesteryl ester.
(3) GRP-affinity chromatography. The wheat germ agglutinin c~lumn eluate is further fractionated on a GRP
affinity column. In the preferred embodiment, the column contains a beaded matrix with the peptide human ~Nlel4,27]GRPl3-27 ~the C~terminal portion of GRP) coupled to it at 2 mg peptide/ml packed gel. The column is equilibrated with a solution typically comprised of 25 mM TRIS, 25 mM HEPES, pH 7.5, 2 mM EDTA, 0.075% (w/v) CHAPS, 0.0075~ (w/v) CHS, and ; protease inhibitors. The concentration of detergent in the wheat germ agglutinin column eluate is preferably adjusted to 0.075% (w/v) by dilution with a solution typically comprised of 25 mM HEPES, 25 mM TRIS, pH 7.5, 2 mM EDTA, and protease inhibitors. After application of the sample and extensive washing of the column, bound protein is eluted with a salt at a ~` concentration above 0.2 M. Particularly suitable is 0.5 M
-~ NaCl. Fractions containing the GRP receptor are then identified by l25I-GRP binding assays. The GRP peptide used ([Nlel4,27]GRPl3-27) is an ana1Og made by Triton Biosciences ~- Inc. (Alameda, CAj which is resistant to oxidation. Other GRP
peptides and matrixes that will also work include, without limitation, GRPl-27, GRPl4-27, and [Lys3]Bombesin, though the ;
optimum yield and elution conditions may involve adjustment.
Elution of the bound protein with salt is important because receptor binding activity is preserved and a good yield is ~-achieved. The concentration of detergent in the sample loaded onto the column ~is carefully~ optimized. The suitable range of detergent is a~out 0.025 to 0.2% (w/v). The ratio of detergent to stabilizing agent is also the same, being 200:1 to 5:2, ~- -preferably l0~
.

. .

,,,,."~ .. ....,.,"`,",, ,:, :
wo g2,l6623 ~ 1 ~ 5 ~ ~ ~ PCT/US92/~2091~

(4) Second a~finity column. Fractions containing the GRP receptor eluted from the affinity column are desalted and the sample is applied to a second GRP affinity column, and eluted as described in step (3). Fractions containing the receptor are then identified by binding assays. Use of two consecutive affinity columns in this step is preferred to give a high degree of purity.
t5) Gel filtration. This is an optional step that yields a purer product. The gel filtration step is also useful to remove protease inhibitors, salt, and residual detergent from the receptor.
In general, the solubilized, unpurified and solubilized, purified GRP receptor of this invention binds gastrin releasing peptide with an affinity of at least KD=10 nM. The GRP receptor from a mouse swiss 3T3 fibroblast cell line, according to this invention was found to have the following characteristics: runs as a broad band on SDS-PAGE
with an apparent molecular weight of a~out 70 to 100 kilodaltons; binds selectively with polypeptides of the bombesin type; has a KD value of about 10-100 pM; is free of coupled G proteins; contains N-linked carbohydrates; when -deglycosylated, has an apparent molecular weight of 36+5 Xilodaltons on SDS-PAGE; and has a partial amino acid sequence near the N-tèrminus of:
; 25 -Leu-Asn-Leu-Asp-Val-Asp-Pro-Phe-Leu-Ser-.
'.!1 Now that the entire sequence is known, the GRP
receptor, fragments or derivatives thereof can be prepared by - conventional processes for synthesizing peptides. These include processes such as are described in Stewart and Young ;~,30 (1984) Solid Phase Peptide_Synthesis, Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of ~ Peptide Synthesis, Springer-Verlag, New York; and Bodanszky j3 (1984) The Principles of Peptide Synthesis, Springer-Verlag, j New York; all of each which are incorporated herein by reference. For example, an azide process, an acid chloride process, an acid anhydride process, a mixed anhydride process, an active ester process~(~for example, p-nitrophenyl ester, ~ 92/l6623 2 ~ ~ ~ 3 ~ ~ PCT/US9~/02091 ~7 N-hydroxysuccinimide ester, or cyanomethyl ester), a carbodiimidazole process, an oxidative-reductive process, or a dicyclohexylcarbodiimide (DCCD)/additive process can be used.
Solid phase and solution phase syntheses are both applicable to the foregoing processes.
The GRP receptor, fragments or derivatives are suitably prepared in accordance with the above processes as typically employed in peptide synthesis, generally either by a so-called stepwise process which comprises condensing an amino acid to the terminal amino acid, one by one in sequence, or by coupling peptide fragments to the terminal amino acid. Amino ; groups that are not being used in the coupling reaction must be protected to prevent coupling at an incorr~ct location.
If a solid phase synthesis is adopted, the C-terminal amino acid is bound to an insoluble carrier or support through its carboxyl group. The insoluble carrier is not particularly limited as long as it has a binding capability to a reactive carboxyl group. Examples of such insoluble carriers include `
` halomethyl resins, such as chloromethyl resin or bromomethyl ., 20 resin, hydroxymethyl resins, phenol resins, tert-alkyloxycarbonylhydrazidated resins, and the like.
An amino group-protected amino acid is bound in sequence through condensation of its activated carboxyl group and the reactive amino group of the previously~formed peptide or chain, to synthesize the peptide step by step. After synthesizing the complete sequence, the peptide is split off from the insoluble carrier to produce the peptide. This solid-phase approach is generally described by Merrifield et al. (1963) in J. Am. Chem. Soc. 85:2149-2156, which is incorporated herein by reference.
The prepared receptor and fragments thereof can be isolated and purified from the reaction mixture by means of . peptide separation, for~example, by extraction, precipitation, electrophoresis and various forms of chromatography, and the like. The receptor of this invention can be obtained in varying degrees of purity depending upon its desired use.
Purification can be accomplished by use of the protein '`

" ~ ;
,l :
, .

WO9~/16623 2~ ~3 ~ $ PcTIus92/n209l~

~8 purification techniques disclosed herein or by the use of the antibodies herein described in immunoabsorbant affinity chromatography. This immunoabsorbant affinity chromatography is carried out by first linking the antibodies to a solid support and then contacting the linked antibodies with solubilized lysates of small cell lung cancer cells, lysates of other cells expressing the GRP receptor, or lysates or ; supernatants of cells producing the GRP receptor as a result of ~ DNA techniques, see below.
;" 1 0 VI. ReceE~or Analo~ues ~ "Derivativ~s" of the GRP receptor include amino acid `~ sequence mutants, glycosylation variants, and covalent or ; aggregative conjugates with other chemical moieties. Covalent derivatives can be prepared by linkage of functionalities to groups wh}ch are found in the GRP receptor amino acid side chains or at the N- or C- termini, by means which are well ~' known in the art. These derivatives can include, without i;~ limitation, aliphatic esters or amides of the carboxyl terminus, or of residues containing carboxyl side chains, ~`~ O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino terminal amino acid or '! amino-group containing residues, e.g., lysine or arginine.
Acyl groups are selected from the group of alkyl-moieties i 25 including C3 to Cl8 normal alkyl, thereby forming alkanoyl aroyl species. ~
In particularj glycosylation alterations are included, e.g., made by modifying the glycosylation patterns of ~,! a polypeptide during its synthesis and processing, or in further processing steps. Particularly preferred means for accomplishing this are by exposing the polypeptide to glycosylating enzymes derived from cells which normally provide such processing, e.g., mammalian glycosylation enzymes.
Deglycosylation enzymes are also contemplated. Also embraced are versions of the same primary amino acid sequence which have other minor modificat1ons, 1nc1uding phosphorylated amino acid :

:

~ 92/166~3 2 ~ ~ 5 3 ~ 6 PCT/US92/02091 residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
A major group of derivatives are covalent conjugates of the GRP receptor or fragments thereof with other proteins of polypeptides. These derivatives can be synthesized in recombinant culture such as N- or C-terminal fusions or by the use of agents known in the art for their usefulness in cross-linking proteins through reactive side groups. Preferred GRP derivatization sites with cross-linking agents are at free amino groups, carbohydrate moieties, and cyste.ine residues.
Fusion polypeptides between the receptors and other homologous or heterologous proteins are also provided.
Homologous polypeptides may be fusions between different growth factor receptors, resulting in, for instance, a hybrid protein exhibiting ligand specificity of one receptor and the intracellular region of another, or a receptor which may have broadened or weakened specificity of binding. Likewise, heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins. Typical examples are fusions of a reporter polypeptide, e.g., luciferase, with a segment or domain of a receptor, e.g., a ligand-binding segment, so that the presence or location of a desired ligand may be easily determined. See, e.g., Dull et al., U.S. Patent No. 4,859,609, which is hereby ^ 25 incorporated herein by reference. Other gene fusion partners ~; include bacterial ~-galactosidase, trpE, Protein A, ~-lactamase, alpha amylase, alcohol dehydrogenase, and yeast ` alpha mating factor. See, e.g,., Godowski et al. (1988) .~, Science 241:812-816.
The phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNA fragments. A double stranded fragment ;~ will often be obtained either by synthesizing the complementary i strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA
polymerase with an appropriate primer sequence.

, . .

!

w092/l66~3 ~ 1 0 ~ 3 0 6 ~CT/US9~/02~

Such polypeptides may also have amino acid residues - which have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties, particularly those which have molecular shapes similar to phosphate groups. In some embodiments, the modifications will be useful labeling reagents, or serve as purification targets, e.g., affinity ligands.
Fusion proteins will typically be made by either recombinant nucleic acid methods or by synthetic polypeptide-~ 10 methods. Techniques for nucleic acid manipulation and expression are described generally, for example, in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.), ~; Vols. 1-3, Cold Spring Harbor Laboratory, which are incorporated herein by reference. Techni~ues for synthesis of polypeptides are described, for example, in Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2155; Merrifield (1~86) Science 232: 341-347; and Atherton et al. (1989) Solid Phase Peptide ~ Synthesis: A Practical Approach, IRL Press, Oxford; each of -~ which is incorporated herein by reference.
'; 20 This invention also contemplates the use of ~, derivatives of the GRP receptor other than variations in amino 1~ .
i acid sequence or glycosylation. Such derivatives may involve ~ji covalent or aggregative association with chemical moieties.
These derivatives generally fall into three classes: (1) salts, i3 25 (2) side chain and terminal residue covalent modifications, and ~ii (3) adsorption complexes, for example with cell membranes.
Such covalent or aggregative derivatives are useful as immunogens, as reagents in immunoassays or in purification methods such as for affinity purification of gastrin releasing peptide or other binding ligands. For example, the GRP
receptor can be immobilized by covalent bonding to a solid support such as cyanogen bromide-activated Sepharose, by methods which are we`ll~known in the art,~or adsorbed onto polyolefin surfaces,`~with or without glutaraldehyde cross-linking, for use in the assay or purification of anti-GRP
receptor antibodies or gastrin releasing peptide. The GRP
receptor can also be labeled with a detectable group, for , '` : .

y~ 92~16623 PCT/US92/02091 "`~i 21~306 example radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays.
The solubilized GRP receptor of this invention can be used as an immunogen for the production of antisera or antibodies specific for the receptor or any fragments thereof.
The purified receptor can be used to screen monoclonal antibodies or antigen-binding fragments prepared by ` immunizaticn with various forms o~ impure preparations containing the GRP receptor. In particular, the term "antibodies" also encompasses antigen binding fragments of natural antibodies. The purified receptor can also be used as ~ a reagent to detect any antibodies generated in response to the ; presence of elevated levels of gastrin releasing peptide; 15 receptor or cell fragments containing the GRP receptor.
Additionally, GRP receptor fragments may also serve as immunogens to produce the antibodies of the present invention, `~
as described immediately below. For example, this invention `~ contemplates antibodies having binding affinity to or being raised against the amino acid sequence shown in Tables 1, 2, 3, or 4, or SEQ ID N0: 10, or fragments thereof. In particular, this invention contemplates antibodies having binding affinity ~, to or being raised against specific fragments which are predicted to lie~outside of the lipid bilayer. These fragments~ 25 include the following ten amino acid sequence (residues 9-18, inclusive) near the N-terminus:
~, 9 18 Leu-Asn-Leu-Asp-Val-Asp-Pro-Phe-Leu-Ser-~ In addition, this invention covers fragments of the GRP
¦ 30 receptor which are predicted to reside on the extracellular ;~ side of the membrane: residues 1-39, inclusive; residues 98-115, inclusive; residues 176-209, inclusive; and residues 288-300, inclusive; and to the following portions of the `
~receptor which are predicted to reside on the intracellular side of the membrane: residues 64-77, inclusive; residues ! 138-157, inclusive; residues 236-266, inclusive; and residues ' ~ .

.~
: `

:

W0~2/16623 2 1 0 ~ 3 0 ~ PCT/US92/02091~

330-385, inclusive. Analogous regions of other receptors for bombesin-like peptides will also be used.

VII. Antibodles Antibodies can be raised to the various subtypes of RBP, e.g., GRP and related receptors, and fragments thereof, ` both in their naturally occurring forms and in their recombinant forms. Additionally, antibodies can be raised to GRP receptors in either their active forms or in their inactive forms, the diffierence being that antibodies to the active receptor are more likely to recognize epitopes which are only present in the active receptor. Anti idiotypic antibodies are also contemplated.
Antibodies, including binding fragments and single chain versions, against predetermined fragments of the GRP
receptor can be raised by immunization of animals with conjugates of the fragments with immunogenic proteins.
.~. Monoclonal antibodies are prepared from cells secreting the . desired antibody. These antibodies can be screened for binding ~ 20 to normal or defecti~le GRP receptors, or screened for agonistic ''`J or antagonistic GRP receptor activity. These monoclonal antibodies will usually bind with at least a KD of about 1 mM, more usually at least about 300 ~M, typically at least about lO
~M, more typically at least about 30 ~M, preferably at least about lO ~M, and more preferably at least about 3 ~M or better.
Although the foregoing addresses GRP receptors, similar ~, antibodies will be raised against other receptors, or receptor subtypes, for bombesin-like peptides.
; The antibodies, including antigen binding fragments, of this invention can have significant diagnostic or therapeutic value. They can be potent antagonists that bind to ~ the GRP receptor and inhibit ligand binding to the receptor or 'l~ inhibit the ability~of gastrin releasing peptide to elicit a i biological response. They also can be useful as non-neutralizing antibodies and can be coupled to toxins or x radionuclides so that when the~antibody binds to the receptor, the cell itself is killed. Further, these antibodies can be ~`:

~,:

~ 92/1~623 2 ~ ~ ~ 3 ~ ~ PCT/US92/~2091 conjugated to drugs or other therapeutic agents, either directly or indirectly by means of a linker.
The antibodies of this invention can also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can bind to the GRP receptor without inhibiting ligand binding. As neutralizinf~ antibodies, they ~; can be useful in competitive binding assays. They will also be useful in detecting or quantifying GRP or GRP receptors.
Receptor fragments may be joined to other materials, particularly polypeptides, as fused or covalently joined - polypeptides to be used as immunogens. The GRP receptor and its fragm~ents may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbiology, Hoeber ~edical Division, Harper and Row,~1969; Landsteiner (1962) Specificit~
of Seroloaical Reactions, Dover Publications, New York, and Williams et al. (1967) Methods in Immunoloqy and ImmunochemistrY, Vol. 1, Academic Press, New York, each of which are incorporated herein by reference, for descriptions of ; 20 methods of preparing polyclonal antisera. A typical method involves hyperimmunization of an animal with an antigen. The ~`
i blood of the animal is then collected shortl~J after the i-~3 repeated immunizations and the gamma globulin is isolated.
In some instances, it is desirable to prepare ~ 25 monoclonal antibodies from various mammalian hosts, such as f.~'~ mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites et al. (eds) Basic and Clinical I~munoloqy (4th ed.), Lange Medical Publications, hos Altos, i 30 CA, and references cited therein; Harlow and Lane (1988) I Antibodies: A Laboratory Manual, CSH Press; Goding tl986) 'f : Monoclonal Antibodies: PrinciPles and Practice (2d ed) Academic Press, New York;~ and particularly in Kohler and `
I Milstein (1975)~ in Nature 256: 495-497, which discusses one ¦ 35 method of generating monoclonaI antibodies. Each of these ~-references is incorporated~herein by reference. Summarized br1efly, thls method involves injecting an animal w1th an f ,`: ~ ' .' ` .

W092/~6~23 2 ~ ~ ~-3 ~ ~ Pi~T/US92/020g~

immunogen. The animal is then sacrificed and cells taken from its spleen, which are then fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing ln vitro. The population of hybridomas is then - 5 screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
Other suitable techniques involve ln vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors. See, Huse et al. (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281; and Ward et al. (1989) Nature 341:544-546, each of which is hereby incorporated herein by reference. The polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable `; signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent r. moieties, chemiluminescent moieties, magnetic particles, and the like. Patents, teaching-the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
'i 30 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see Cabilly, U.S. Patent No.
4,816,567. These patents are incorporated herein by reference.
~-j The antibodies of this invention can also be used for , affinity chromatography in isolating the receptor. Columns can jl 35 be prepared where the antibodies are linked to a solid support, ! e.g., particles, such as agarose, Sephadex, or the like, where a cell lysate may be passed through the column, the column ;

, , - W~2~16623 2 ~ 0 5 3 0 ~ PCr/US9Z/020gl washed, followed by increasing concentrations of a mild denaturant, whereby the purified receptor protein will be released.
The antibodies may also be used to screen expression libraries for particular expression products. Usually the antibodies used in such a procedure will be labeled with a moiety allowing easy detection of presence of antigen by antibody binding.
~ Antibodies raised against each receptor will also be~ 10 used to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various immunological conditions related to expression of the respective receptors.
., .
VIII. Other Uses of Receptors ~ .
`~ 15 Both the naturally occurring and the recombinant form ~ -of the receptors for bombesin-like peptides of this invention are particularly useful in kits and assay methods which are capable of screening compounds for binding activity to the receptors. Several methods of automating assays have been developed in recent years so as to permit screening of tens of ' thousands of compounds per year. See, e.g, Fodor et al. (1991) `i Science 251:767-773, which is incorporated herein by reference and which describes means for testing of binding affinity by a plurality of defined polymers synthesized on a solid substrate.
25 The development of suitable assays can be greatly facilitated by the availability of large amounts of purified, soluble receptor in an~active state such as is provided by this ~-nventlon .
i For example, antagonists can normally be found once the receptor has been pharmacologically defined, as is the case now with the GRP and NMB receptors. Testing of potential ` receptor antagonists is now possible upon the development of ;~j highly automated assay methods using a purified receptor. In ~ particular, new agonists and antagonists will be discovered by `~ 35 using screening techniques made available herein. Of ~3 particular importance are compounds found to have a combined binding affinity for multiple receptor subtypes, e.g., .qi ~ : :

-:, '.
.~ ~ . . .
1:

6~23 2 1 0 ~ ~ O ~ PCT/US92/~20~1~

compounds which can serve as antagonists for both a GRP
receptor and a NMB receptor. Such compounds provide methods for simultaneously affecting multiple receptor subtypes.
This invention is particularly useful for screening compounds by using the recombinant receptors in any of a variety of drug screening techniques. The advantages of using a recombinant receptor in screening for receptor reactive drugs include: (a) improved renewable source of the receptor from a specific source; (b) potentially greater number of receptors per cell giving better signal to noise ratio in assays; and (c) receptor subtype specificity (theoretically givlng greater biological and disease specificity).
One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing the a receptor. Cells may be isolated which express a single receptor subtype insolation from any others. Such cells, either in viable or fixed form, can be used for standard receptor/ligand binding assays. See also, Parce et al. (1989) Science 246:243-247; and Owicki et al. (1990) Proc. Nat'l Acad. Sci. USA 87:4007--4011, which are incorporated herein by reference and describe sensitive methods to detect cellular responses. Competitive assays are particularly useful, where the ceLls (source of RBP) are contacted and incubated with a labeled ligand having known binding affinity to the receptor, such as 125I-GRP, and a test compound whose binding affinity to the GRP receptor is being measured. The bound ligand and free ligand are then separated to assess the degree of ligand binding. The amount of test compound bound is inversely proportional to the amount of labeled ligand binding measured. Any one of numerous techniques can be used to separate bound from free ligand to assess the degree of ligand binding. This separation step could typically~involve a procedure such as adhesion to filters followed by washing, adhesion to plastic followed by washing, -or centrifugation of the cell membranes. Viable cells could also be used to screen for the effects~of drugs on GRP receptor ; mediated functions, e.g.! second messenger levels, i.e., Ca++;
- : .
.

~ , . .

~Q9~ 6~3 PCT/US~2/02091 2:L0~3~6 cell proliferation; inositol phosphate pool changes; and others. Some detection methods allow for elimination o~ a separation step, e.g., a proximity sensitive detection system.
~- Calcium sensitive dyes will be useful for detecting Ca++
levels, with a fluorimeter or a fluorescence cell sorting apparatus.
Another method utilizes membranes from transformed eukaryotic or prokaryotic host cells as the source of the GRP
receptor. These cells are stably transformed with DNA vectors directing the expression of the GRP receptor. Essentially, the membranes would be prepared from the cells and used in any ~` receptor/ligand binding assay such as the competitive assay set forth above.
Still another approach is to use solubilized, unpurified or solubilized, purified receptors from transformed eukaryotic or prokaryotic host cells. This allows for a ~ "molecular" binding assay with the advantages of increased : specificity, the ability to automate, and high drug test throughput.
Another technique for drug screening involves an ~, approach which provides high throughput screening for compounds having suitable binding affinity to the igastrin- releasing peptide receptor and is described in detail in Geysen, European -Patent Application 84/03564, published on September 13, 1984, , 25 which is incorporated herein by reference. First, large i numbers of different small peptide test compounds~are ;i synthesized on a solid substrate, e.g., plastic pins or some other appropriate surface, see Fodor et al. (1991). Then all '! the pins are reacted with solubilized, unpurified or solubilized, purified GRP receptor, and washed. The next step involves detecting bound GRP receptor.
~ Rational drug design may also be based upon `'! structural studies of the molecular shapes of the receptor and other effectors or ligands. Effectors may be other proteins which mediate other functions in response to ligand binding, or ~¦ other proteins which normally interact with the receptor. One means for determining~which sites interact with specific other ~' !

W0~2/16623 2 ~ ~ ~ 3 ~ ~ PCT/US92/02091~

proteins is a physical structure determination, e.g., x-ray crystallography or 2 dimensional NMR techniques. These will provide guidance as to which amino acid residues form the molecular contact regions. For a detailed description of protein structural determination, see, e.g., Blundell and Johnson (1976) Protein Crystalloq-raphy~ Academic Press, New York, which is hereby incorporated herein by reference.
Purified receptor can be coated directly onto plates for use in the aforementioned drug screening techni~ues.
lo However, non-neutralizing antibodies to these receptors can be used as capture antibodies to immobilize the respective receptor on the solid phase.

IX. Liqands: Aq~nists and Antaqonists The blocking of physiological response to bombesin-like peptides may result from the inhibition of binding of the - ligand to the receptor, likely through competitive inhibition.
Thus, in vitro assays of the present invention will often use --isolated membranes from cells expressing a recombinant receptor, soluble fragments comprising the ligand binding segments of these receptors, or fragments attached to solid phase substrates. These assays will also allow for the I diagnostic determination of the effects of either binding - segment mutations and modifications, ox ligand mutations and modifications, e.g., ligand analogues.
This invention also contemplates the use of competitive drug screening assays, e.g., where neutralizing antibodies to the receptor or receptor fragments compete with a test compound for binding to the receptor. In this manner, the antibodies can be used to detect the presence of any polypeptide which shares one or more binding sites o~ the receptor and can also be used to occupy binding sites on the receptor that might otherwise be occupied by a bombesin-like peptide.
Additionally, neutralizing antibodies against the receptor and soluble fragments of the receptor which contain the high affinlty ligand binding site, can be used to inhibit ` ~ 92/16623 2 1 ~ 5 ~ ~ ~ PCT/US92/02091 gastrin releasing peptide receptor function in cancerous tissues, e.g., tissues experiencing proliferative abnormalities.

X. Kits -This invention also contemplates use of the GRP
receptor, fragments thereof, peptides, and their fusion products in a variety of diagnostic kits and methods for detecting the presence of the gastrin releasing peptide lo receptor. Typically the kit will have a compartment containing either a defined receptor peptide or gene segment or a raagent which recognizes one or the other.
A kit for determining the binding affinity of a test compound to the gastrin releasing peptide receptor would typically comprise a test compound; a labeled compaund, for example a ligand or antibody having known binding affinity for the gastrin releasing peptide receptori a source of gastrin ` releasing peptide receptor (naturally occurring or recombinant); and a means for separating bound from free labeled compound, such as a solid phase for immobilizing the gastrin releasing peptide receptor. Once compounds are screened, those having suitable binding affinity to the GRP
j receptor can be evaluated in suitable biological assays, as are ~, well known in the artj to determine whether they act as i 25 agonists or antagonists. The availability of recombinant ~!, receptor polypeptides also provide well defined standards for calibrating such assays.
A preferred kit for determining the concentration of, i for example, gastrin releasing peptide receptor in a sample would typically comprise a labeled compound, e.g., ligand or antibody, having known binding affinity for the gastrin releasing peptide receptor, a source of gastrin releasing peptide receptor (naturally occurring or recombinant) and a means for separating the bound from free labeled compound, for example a solid phase for immobilizing the gastrin releasing peptide receptor. Compartments containing reagents, and instructions, will normally be provided.
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one method for determining the concentration of gastrin-releasing peptide receptor in a sample would typically comprise the steps of: (l) preparing membranes from a sample comprised of a GRP receptor source; (2) washing the membranes and suspending them in a buffer; (3) solubili2ing the GRP
receptor by incubating the membranes in a culture medium to which a detergent and a soluble cholesteryl ester has been added; (4) adjusting the detergent concentration of the solubilized receptor; (5) contacting and incubating said dilution with radiolabeled GRP to form GRP:GRP receptor complexes; ~6) recovering the complexes such as by filtration through polyethyleneimine treated filters; and (7) measuring the radioactivity of the recovered complexes. Similar methods should be applicable to other members of the family of RBP.
Antibodies, including antigen binding fragments, -~ specific for ths receptor or receptor fragments are useful in diagnostic applications to detect the presence of elevated ;i levels of the receptor and/or its ~ragments. Such diagnostic assays can employ lysates, live cells, fixed cells, immunofluorescence, cell cultures, body fluids, and further can involve the detection of antigens related to the GRP receptor in serum, or the like. Diagnostic assays may be homogeneous (without a separation step between free reagent and receptor-ligand complex) or heterogeneous (with a separation step). Various commercial assays exist, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay ; (ELISA), enzyme immunoassay (EIA), enzyme-multiplied - immunoassay technique (EMIT), substrate-labeled fluorescent ; immunoassay tS~FIA) and the like. For example, unlabeled antibodies can be employed by using a second antibody which is labeled and which recognizes the antibody to the GRP receptor or to a particular fragment thereof. These assays have also been extensively discussed in the literature. See, e.g., 3 Harlow and Lane (1988) Antibodies:_A Laboratory Manual, CSH.
Anti-idiotypic antibodies may have similar use to diagnose presence of antibodies against a receptor, as such may be diagnostic of various abnormal states. For example, , ,': -92/1~623 2 1 0 ~ PCT/US92/02091 overproduction of RBP may result in production of various immunological reactions which may be diagnostic of abnormal receptor expression, particularly in proliferative cell conditions such as cancer.
Frequently, the reagents for diagnostic assays are supplied in kits, so as to optimize the sensitivity of the assay. For the subject invention, depending upon the nature of the assay, the protocol, and the label, either labeled or - unlabeled antibody, or labe1ed receptor is provided. ~his is usually in conjunction with other additives, such as buffers, stabilizers, materials necessary for signal production such as substrates for enzymes, and the like. Preferably, the kit will - also contain instructions for proper use and disposal of the ~ .
contents after use. Typically the kit has compartments for each useful reagent. Desirably, the reagents are provided as a dry lyophilized powder, where the reagents may be reconstituted in an aqueous medium having appropriate concentrations for performing the assay.
Any of the aforementioned constituents of the drug ' 20 screening and the diagnostic assays may be used without ' modification or may be modified in a variety of ways. For -- example, labeling may be achieved by covalently or .;! non-covalently joining a moiety which directly or indirectly i provides a detectable signal. In any of these assays, the ligand, test compound, GRP receptor, or antibodies thereto can ~--i be labeled either directly or indirectly. Possibilities for `l direct labeling include label groups: radiolabels such as 125I, ~1 enzymes (U.S. Pat. No. 3,645,090) such as peroxidase and `~ alkaline phosphatase, and fluorescent labels (U.S. Pat. No.
3,940,~75) capable of monitoring the change in fluorescence intensity, wavelength shift, or fluorescence polarization.
I Both of the patents are incorporated herein by reference.
j Possibilities for indirect label ng include biotinylation of ~; one constituent~followed by binding to avidin co~pled to one of the above label groups.
~; There are also numerous methods of separating the bound from the free ligand, or alternatively the bound from the 'I : .

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WO92/16623 2 i O ~ 3 a G PCT/US92/02091 free tast compound. The receptor can be immobilized on various matrixes followed by washing. Suitable matrixes include plastic such as an ELISA plate, filters, and beads. Methods of immobilizing the receptor to a matrix include, without limitation, direct adhesion to plastic, use of a capture antibody, chemical coupling, and biotin-avidin. The last step in this approach involves the precipitation of receptor/ligand complex by any of several methods including those utilizing, e.g., an organic solvent such as polyethylene glycol or a salt such as ammonium sulfate. Other suitable separation techniques - include, without limitation, the fluorescein antibody magnetizable particle method described in Rattle et al. (1984) Clin. Chem. 30(9):1457-1461, and the double antibody magnetic particle separation as described in U.s. Pat. No. 4, 659, 678, each of which is incorporated herein by reference.
' The methods ~or linking protein receptors or their fragments to the various labels have been extensively reported in the literature and do not require detailed discussion here.
Many of the techniques involve the use of activated carboxyl groups either through the use of carbodiimide or active esters ~ to form peptide bonds, the formation of thioethers by reaction `1 of a mercapto group with an activated halogen such as chloroacetyl, or an activated olefin such as maleimide, for linkage, or the like. Fusion proteins will also find use in f 25 these applications.
`~f Another diagnostic aspect of this invention involves 1 use of oligonucleotide or polynucleotide seguences taken from `f the sequence of a receptor for GRP or other bombesin-like peptide. These sequences can be used as probes for detecting levels of the receptor in patients suspected of having a proliferative cell conditions, e.g.`, cancer. The preparation ~! of both RNA and DNA nucleotide sequences, the labeling of the ! sequences, and the preferred size of the sequences has received ! ample description and discussion~in the literature. Normally 1 35 an oligonucleotide probe should have at l~ast about 14 .~ .
nucleotides, usually at least about 18 nucleotides, and the polynucleotide probes may be up to several kilobases. Various !

~92/16623 PCT/US92/0~091 ~ 21053~6 labels may be employed, most commonly radionuclides, particularly 32p. However, other techniques may also be employed, such as using biotin modified nucleotides for introduction into a polynucleotide. The biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, or the like. Alternatively, antibodies may be employed which can recognize specific duplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes, or DNA-protein duplexes. The antibodies in turn may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, `~ the presence of antibody bound to the duplex can be detected.
The use of probes to the novel anti-sense RNA may be carried ~-~ 15 out in any conventional techniques such as nucleic acid hybridization, plus and minus screening, recombinational probing, hybrid released translation ~HRT), and hybrid arrested translation (HART). This also includes amplification techniques such as polymerase chain reaction (PCR).
Diagnostic kits which also test for the qualitative or quantitative presence of other markers are also contemplated. Diagnosis or prognosis may depend on the ~- combination of multiple indications used as markers. Thus, kits may test for combinations of markers. See, e.g., Viallet : 25 et al. (1989~ Proqress in Growth Factor Res. 1:89-97.
Similar reagents are made available for application of these concepts to receptors for other bombesin-like ~l peptides.
..
XI. Therapeutic APPlications This invention provides reagents with significant therapeutic value. The GRP receptor (naturally occurring or recombinant), fragments thereof and antibodies thereto, along with compounds identified as having binding affinity to the GRP
receptor, should be useful in~the treatment of conditions exhibiting proliferative growth, e.g., cancerous tissues, such as prostatic and pancreatic tumors, and particularly in the ,; : , :, $ ;; ~ : :

2~ ~3~5 treatment of small cell lung cancer. Additionally, this invention should have therapeutic value in any disease or disorder associated with abnormal expression or abnormal triggering of receptors for GRP or other bombesin-like peptides. For example, it is believed that the GRP receptor likely plays a role in neurologic function, and can affect gastrointestinal, pulmonary, and brain tissue. As before, the basic principles underlying the descriptions here directed towards G~P receptors will also be applicable to other receptors for bombesin-like peptides. -Recombinant GRP receptor or GRP receptor antibodies can be purified and then administered to a patient. These reagents can be combined for therapeutic use with additional active ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, along with physiologically innocuous stabilizers and excipients. These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations. This invention also contemplates use of antibodies or binding fragments thereof which are not complement binding. ~ -Drug screening using the GRP receptor or fragments ! thereof can be performed to identify compounds having binding affinity to the GRP receptor. Subsequent biological assays can then be utilized to determine if the compound has intrinsic stimulating activity and is therefore a blocker or antagonist in that it blocks the activity of gastrin releasing peptide.
Likewise, a compound having intrinsic stimulating activity can activate the receptor and is thus an agonist in that it simulates the activity of gastrin releasing peptide. This invention further contemplates the therapeutic use of antibodies to the GRP receptor as antagonists. This approach should be particularly useful with other receptors for bombesin-like peptides. For example effective antagonists for the NMB receptor have not been found, and identification of a ligand ~or the R3BP has noe yet been done. ~' -"
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~ 92/l6623 2 ~ O ~ ~ ~ 6 PCT/US92/02091 The quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy.
Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these `~ reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication `- lO of human dosage. Various considerations are described, e.g., in Gilman et al. (eds) (1990) Goodman and Gilman's: The Pharmacological Bases of TheraPeutics, 8th Ed., Pergamon Press;
;~ and Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Penn.; each of which is hereby -` 15 incorporated herein by reference. Methods for administration are discussed therein and below, e.g., for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal ', diffusion, and others. Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index, Merck & Co., Rahway, New ! Jersey. Because of the high affinity binding between a bombesin-like peptide and its receptors, low dosages of these , . .
reagents would be initially expected to be effective. Thus, dosage ranges would ordinarily be expected to be in amounts ~, 25 lower than 1 mM concentrations, typically less than about lO ~M
concentrations, usually less than about 100 nM, preferably less than about 10 pM (picomolar), and most preferably less than ;~ about 1 fM (femtomolar), with an appropriate carrier. Slow , release formulations, or slow release apparatus will often be utilized for continuous administration. The intracellular segments of the receptors,- both the GRP receptor and related receptors will find additional uses as described in detail below ~ ~
i - The GRP receptor, fragments thereof, and antibodies :; 35 to the receptor or its fragments, antagonists, and agonists, .i may be administered directly to the host to be treated or, ; depending on the size of the compounds, it may be desirable to : ' ' ', ':
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W092/l66~3 2 1 0 5 3 0 6 PCI/US92/02091~

conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration. Therapeutic formulations may be administered in any conventional dosage formulation. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. Formulations comprise at least one - -active ingredient, as defined above, together with one or more acceptable carriers thPreof. Each carrier must be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Formulations include those suitable for oral, -- rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art o~ pharmacy. See, e.g., Gilman et al. (eds) (199O~ Goodman and Gilman's: The Pharmacoloqical Bases of Therapeutics, 8th Ed., Pergamon Press; and Reminqton's Pharmaceutical Sciences, 17th `~ ed. (199O), Mack Publishing Co., Easton, Penn.; each of which is hereby incorporated herein by reference. The therapy of -` this invention may be combined with or used in association with . .
other chemotherapeutic or chemopreventive agents.

XII. Receptor Subtypes The present invention contemplates the isolation of ~, additional closely related receptors for other bombesin-like ' peptides. As described above, these are various types of boS~besin-like peptides having different functions. See, e.g., LeBacq-Verheyden et al. (1990), which is incorporated herein by ; 30 reference. Various of these peptides have been functionally classified as digestive hormones, central modulators of metabolism, growth factors, or~neuropeptides. A wide variety of pharmacological effects are mediated by these peptides.
' The present invention provides direct means to isolate a group of related receptors displaying both distinctness and similarities in structure, expression, and function. Elucidation of many of the physiological effects of .
,s ' ., , ~ 92/l6~23 2 1 ~ ~ 3 ~ ~ PCT/US92/02091 the bombesin-like peptides will be greatly accelerated by the isolation and characterization of distinct members of the receptor family. In particular, the present invention provides useful probes for identifying additional homologous proteins, as described in Example 29. The human R3BP is one such - example. These additional proteins are candidates for receptors which bind other bombesin-like peptides, e.g., phyllolitorin or litorin.
The isolated genes will allow transformation of cells lac~ing expression of related receptors, e.g., either specie types or cells which lack corresponding receptors and exhibit negative background activity. Expression of trans~ormed genes will allow isolation of pharmacologically pure cell lines, with defined or single receptor subtypes. This approach will allow - 15 for more sensitive detec~ion and discrimination of the physiological effects of each receptor subtype in isolation from others. Subcellular fragments, e.g., cytoplasts or membrane fragments, can be isolated and used.
Although the various receptors often have unrelated functions, they share significant structural similarities.
Dissection of its structural elements which ef~ect the various physiological functions provided by the receptors is possible using standard techniques of modern molecular biology, particularly in comparing membérs of a related class. See, e.g., the homolog-scanning mutagenesis technique described in Cunningham et al. (1989) Science 243:1339-1336; and approaches used in O'Dowd et al. tl988) J. Biol. Chem. 263:15985-15992;
and Lechleiter et al. (1990) EMBO J. 9:4381-4390; each of which is incorporated herein by reference.
In particular, ligand binding segments can be substituted between receptors to determine what structural features are important in both ligand binding affinity and specificity. The segments of receptor accessib1e to an extracellular ligand would be primary targets of such analysis.
An array of different receptors will be used to screen for ligands exhibiting combined properties of interaction with different receptor subtypes. Particularly interesting segments .

W~92/1~623 ~ PC~/US92/02091 of those receptors include, without limitation, the third transmembrane segment, the amino end of the cytoplasmic segment, the second cytoplasmic loop, and the cysteine residues in the cytoplasmic COOH-tail.
Intracellular functions would probably involve segments of the receptor which are normally accessible to the cytosol. However, receptor internalization may occur under - certain circumstances, and interaction between intracellular components and the designated "extracellular" segments may occur. These intracellular functions usually involve signal transduction from ligand binding; and G-protein interaction has been reported. The specific segments of interaction of ; receptor with G-protein may be identified by mutagenesis or direct biochemical means, e.g., cross-linking or affinity methods. Structural analysis by crystallographic or other physical methods will also be applicable. Identification of the similarities and differences between receptor subtypes exhibiting distinct fùnctions will lead to new diagnostic and therapeutic reagents or treatments.
~` 20 Further study of the expression and control of these .~7 receptor subtypes will be useful. The controlling elements associated with the receptors exhibit differential developmental tissue specific, or other expression patterns.
Upstream or downstream genetic regions, e.g. ! control elements, are of interest.
~' Structural studies of the re~eptor subtypes will lead ~,~ to design of new ligands, particularly analogues exhibiting agonist or antagonist properties. This can be combined with previously described screening methods to isolate ligands exhibiting desired spectra of activities.
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Expression in other cell types will often result in `:'! glycosylation differences in a particular receptor. Various receptor subtypes may exhibit distinct functions based upon ~ -structural differences~other than amino acid sequence.
~ 35 Dif~erential modifications may be responsible for differential -1 function, and elucidation of the effects are now made possible.

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Although the foregoing description has focused primarily upon the GRP receptor, those of skill in the art will immediately recognize that the invention encompasses receptors for other ~; bombesin-like peptldes, e.g. a NMB receptor and an R3BP.
: The broad scope of this invention is best understood . with reference to the following examples, which are not - intended to limit the inventions in any manner.

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-~ EXPERIMENTAL
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EX~MPLE 1 Preparation of Mouse 3T3 Fibroblast Membranes Mouse Swiss 3T3 fibroblasts were grown to confluence in ; Dulbecco's modified Eagles medium supplemented with 10%
(vol/vol) fetal calf serum in T-850 roller bottles (lots of 100) at 37 C in a 10% CO2/90~ air environment. Upon harvest, the medium was poured off and each bottle was rinsed twice with 50 ml calcium/magnesium free phosphate buffered saline (PBS-CMF). Cells were incubated with 25-30 ml 0.04~ (wt/vol) ~` EDTA in PBS-CMF (warmed to 37~ C) for 15 minutes at room temperature. The cells were then removed with firm knocks and ` pipetted into conical 250 ml centrifuge tubes on ice. Cells -from six roller bottles were combined into each centrifuge tube. Roller bottles were rinsed a final time wi~h 25 ml PBS-CMF. Cells were pelleted at 1800 rpm for 10 minutes at 4 C in a Sorvall RC-3B centrifuge. Each pellet was resuspended in 50 ml fresh PBS-CMF at 4 C. Cells from 2-3 centrifuge tubes were combined, pelleted and washed with an additional 120 ml 'f PBS-CMF. The final cell pellets were resuspended in 200 ml -i lysis buffer (50 mM HEPES, pH 7.5, 2 mM MgC12! 1 mM EGTA, 50 g/ml leupeptin, 2.5 ~g/ml pepstatin, 10 ~g~ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)). Cells were lysed ~ 25 by N2 cavitation. Briefly, 100 ml of the cell suspension was '`t placed in ice in a sealed stainless steel container which was pressurized to 900 psi Qf N2. The suspension was slowly released from the chamber through a small orifice into a collection tube, causing rapid decompression and lysis of the ~; 30 cells. Cell lysis appeared complete by microscopic visualization. Membranes were pelleted at 39,000 x g for 30 minutes at 4 C, resuspended in lysis buffer and pelleted again. The pellet was suspended at a concentration of 15 mg ~ ~ membrane protein/ml~in a storage buffer (50 mM HEPES, pH 7.5, 1 `i~ 35 mM EGTA, 0.25 M sucrose, 50 ~g/ml leupeptin, 2.5 ~g/ml ' ~pepstatin, 10 ~g/ml aprotinin,~and 0.5 mM PMSF). Membranes .~ : .: , . ~ :
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~ ~ 92tl6623 2 ~ ~ ~ 3 ~ ~ PCT/US92/0~091 :i 71 were aliquoted in volumes of l and 5 ml, flash-frozen in liquid N2, and stored at -80~ c.

Comparison of Deterqents for - Solubilization of the GRP RecePtor Several detergents employed for receptor extraction in other systems were tested to measure their ability to solubilize GRP receptor from Swiss 3T3 fibroblast membranes.
Digitonin, Triton X-100, CHAPS, and CHAPS with CHS were all used to extract membranes at a detergent concentration of 0.50%
and all were effective in solubilizing receptor that had been radio-labeled by cross-linking to 125I-GRP. The binding of 125I-GRP (0.02 nM), measured as counts/minute (CPM) bound, was assayed in the presence of the detergent (0.1%) used in the extraction and several concentrations of the unlabeled 14-27 C-terminal amino acids of GRP (GRP14-27), as is shown in Figure 1. Only extraction with CHAPS plus CHS yielded detectable binding activity. Since all detergents were effective in -~ 20 solubiliæing the GRP receptor, the failure to observe binding activity in extracts prepared individually with digitonin, Triton X-100, or CHAPS was a result of receptor inactivation during the solubilization process. It was noted however, that partial reactivation of the receptor extracted with CHAPS
(without CHS) could be achieved by subsequent addition of CHS.
;l This established that CHS acts as a stabilizer in promoting the active GRP~receptor.
Com~arison_o~ Deterqent Concentration for Solubilization of the GRP Receptor Swiss 3T3 fibroblast membranes, prepared as in Example ~ 1, were incubated with various concentrations of the detergent `~! CHAPS. After separation o~ lnsoluble material by i centrifugation, ~soluble GRP binding activiJcy was measured in `~
the supernatant. When 0.75% (w/v) CHAPS was used to solubilize 35 the GRP receptor, maximal receptor binding was observed, as is shown in Figure 2. However, to obtain maximal solubilization of protein a CHAPS concentration of l.0% (w/v~ or greater was used. The GRP receptor binding declined steadily at higher 1, ; :

~ W092/1~23 2 1 ~ 5 3 0 6 PCT/US92/0209 ~

detergent concentrations. In order to observe specific GRP
binding to receptors solubilized by CHAPS, it was useful to include the stabilizing agent CHS. The ratio of CHAPS:CHS was maintained at 10:1 under both extraction and assay conditions.
Comparison of Stabilizing Agent Concentration for Solubilization of the GRP Receptor Swiss 3T3 fibroblast membranes, prepared as in Example 1, were solubilized with 0.75% (w/v) CHAPS in the presence of various amounts of cholesteryl hemisuccinate (CHS).
After the removal of insoluble material by centrifugation, soluble GRP receptor binding activity was measured in the supernatant at a 0.075~ (w/v) CHAPS concentration and a CHS
concentration 10 fold less than that used in the solubilization step. As shown in Figure 3, the optimal ratio of CHAPS to CHS
was about 10~
Comparison of peterqent Concentration for Bindinq Activity of the Solubilized GRP Receptor The dependency of binding activity on the concentration of detergent was studied. As is shown in Figure 4, GRP binding to `20 the receptor has a narrow optimum between 0.075 and 0.1~ CHAPS, and specific binding falls dramatically at CHAPS concentrations greater than 0.4%. Detergent levels above a concentration of 0.4% also cause a large increase in the nonspecific background in the assay which~is possibly due to the formation of detergent aggregates. While the GRP receptor is maximally extracted from membranes with detergent levels that are highly ~ inhibitory (0.75%), the inactivation of receptor molecules by ;' CHAPS appeared to be reversible. Complete binding activity of the receptor incubated in 0.75% CHAPS and 0.15% CHS could be recovered upon reducing the concentration of detergent by dialysis. Optimum pH for GRP Bindinq 5I-GRP binding was determined in 500 ~1 of 20 mM
MES, 20 mM CHES, 20 mM HEPES, 2 mM EDTA, 10 mg/ml BSA, 30 ~g/ml bacitracin, 0.02 nM 125I-GRP, and 5 ~g CHAPS extracted membrane protein at several pH values, ranging from pH 5-10. After incubation at 15 C for 30 minutes, samples were cooled on ice.
This was followed by the addition of 5.0 ml of 50 mM HEPES, pH

7.5, to neutralize the pH before the separation of bound and . ~ . .
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' ~ 92/16623 2 ~ O ~ 3 ~ S PCT/US92/0~091 free ligand. Receptor binding was found to be optimal at a pH
of 7.5. However, the receptor was able to tolerate incubation at a pH of lO for at least 24 hours at 4 C without loss of activity. In contrast, incubation of the receptor with a pH 5 buffer at 4 C caused a rapid loss of binding activity.

Solubilization of the GRP Receptor for Assavs Swiss 3T3 ibroblast membranes, prepared in Example 1, were suspended at 15 mg protein/ml in 50 mM HEPES, p~ 7.5, ~1 1.0 mM EGTA, lO0 mM NaCl, 0.25 M sucrose, 50 ~g/ml leupeptin, 5 ~g/ml pepstatin, lO ~g/ml aprotinin, 30 ~g/ml bacitracin, and 0.5 mM phenylmethylsulfonyl fluoride. A mixture of 3-[(3-cholamidopropyl) dimethylammonio]-l-propane sulfonate (CHAPS) and cholesteryl hemisuccinate (CHS) in a ratio of lO:l ~`~ was added slowly to yield a final concentration of 0.75% CHAPS.
The extract was incubated at 2l C for 30 minutes, cooled to 4 ;i C and the insoluble materia} was removed by centrifugation at lO0,000 x gravity for 60 minutes. The clear supernatant was frozen in liquid N2 and stored at -80 C without loss of activity.

Liqand Bindinq Assavs ~ specific l25I-GRP (3-(l25Iodotyrosyl-l5~ gastrin releasing peptide, 1900-2000 Ci/mmol) binding to intact or .i~ detergent s~lubilized membranes (20-50 ~gj prepared as in ~; Example 3) was assayed in 50 mM HEPES, pH 7.5, 2 mM EDTA, lO
mg/ml bovine serum albumin (BSA), 30 ~g/ml bacitracin, and 0.02 nM 125I-GRP. For assays of detergent solubilized membrane extracts, the final CHAPS detergent concentration was adjusted to between 0.050% and 0.20%. The concentration of CHS was ~, maintained at l/5 to l/lO the concentration of CHAPS. Samples `
, were also prepared omitting the BSA. After incubation at 15 C
for 30 minutes, samples were cooled to 0 C. Bound ligand 5I-GRP:GRP receptor complex) was recovered by rapid ~ filtration through polyethyleneimine treated Whatman GF/B

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filters, followed by four washes with 4 ml of ice cold TRIS
buffer (50 mM TRIS/Cl, pH 7.5). The filters were counted in an ~ Isodata 500 gamma counter. Nonspecific backgrounds were : determined by inclusion of 100 nM unlabeled GRP in the assay to compete for specific binding sites and typically reprèsented 1.5-2% of the specific radioactivity bound. The nonspecific binding could be attributed to a small degree of binding of the 125I-GRP to the filters. It was found that binding activity of the solubilized receptor is highly dependent on the ; 10 concentration of the detergent. As shown in Figure 4, GRP
binding to the receptor has a narrow optimum between 0.075%
~ CHAPS/0.015% CHS and 0.10% CHAPS/0.02% CHS, and specific '~ binding falls dramatically at CHAPS/CHS concentrations greater than 0.4%/0.08%. Detergent levels above about 0.4% CHAPS with 0.08% CHS present also cause a large increase in the I nonspecific background possibly due to the formation of -~ detergent aggregates. Since the receptor is maximally extracted from membranes with detergent levels that are highly inhibitory (0.75% CHAPS), inactivation of the receptor by CHAPS
appeared to be reversible. Indeed, complete binding activity of receptor incubated in 0.75% CHAPS plus 0.15% CHS could be , recovered upon reducing the concentration of detergent by :~ dialysis. ;

~ 25 EXAMPLE 5 -¦ Receptor Kinetics i~ Assays were performed for various times of incubation ,~ ~ and BSA (10 mg/ml) was either included in the assay or omitted.
~! 125I-GRP binding to the soluble receptor at 15 C leveled off J 30 by 20 minutes and remained constant for up to 2 hours.
Omission of the BSA that had been added to prevent proteolysis ~-~ of the ligand had no significant effect on the binding !i kinetics.; ~
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o 5 ~ 3 ~ PCT/US92/02091 2/166~3 G-Protein Complex The GRP receptor in Swiss 3T3 fibroblast membranes was found to be G-protein coupled. Therefore, the effect of guanylnucleotides on 1~5I-GRP binding to soluble receptors was studied. The final detergent concentration was 0.075~ CHAP
and 0.015% CHS was present. The G-protein coupling of the GRP
receptor in intact Swiss 3T3 fibroblast membranes was inferred from the observation that the ligand affinity of the receptor was reduced about ten fold in the presence of the nucleotides GDP and GTP and the non-hydrolyzable GTP analogue GMPPNP n the presence of Mg+2, guanylnucleotides are presumed to ln uce the dissociation of G-proteins from the high affinity ligand/receptor/G-protein ternary complex, resultlng ln formation of the ligand/receptor complex that displays lower affinity. The GRP receptor extracted from membranes by CH~PS
showed no change in their ligand binding propertles ln t e presence of Mg+2 and GTP or GMPPNP at levels that reduce GRP
` binding to membranes by about 80%. The lack of an effect of`- 20 GTP on GRP binding in the presence of Mg+ indicates that interaction of the receptor with its G-protein lS not , maintained in the detergent extract. The control ln Table 5, contains MgCl2.

' , ...

.:

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X WOg2~16623 PCT/US92/02~91 ~` TABLE 5: GRP binding in presence o~ guanylnucleotide - Solubilized Membranes Counts/mlnute Bound l25I-GRP
Measured as % of Total Added control 28 control + lO mM AMPPNP 27.8 control + lO mM GTP 27.5 control + lO mM GMPPNP 26.5 - control + lO mM GMPPNP
+ lO0 nM GRPl-27 2.0 Intact Membranes ~i~ Counts/minute Bound l25I-GRP
Neasured as % of Total Added control 28.9 . control + 5 mM ATP 29.7 :
: control + 5 mM AMPPNP 33.4 control ~ 5 mM GTP lO.7 :
; control + 5 mM GMPPNP lO.5 control + 5 ~M GMPPNP . ..... ... :
+ lO0 nM GRPl-27 l.4 ~ .

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~ 92/16623 2 1 0 ~ 3 ~ ~ PCT/US92/02091 EXAMiPLE 7 Scatchard Anal~sis of the Soluble GRP RecePtors Scatchard analysis of l25I-GRP binding to intact and solubilized swiss 3T3 membranes was done. one particular experiment is discussed below, where the binding parameters of the solubilized and the membrane bound form of the receptor are determined under similar conditions. Assays were determined at 15 C. For assays of solubilized or intact membranes, the binding reactions were terminated at 30 and 180 minutes, respectively. The following are the binding parameters, where KD is the dissociation constant and Bm is the maximum binding capacity:
KD (intact membranes) = 37 pM
KD (solubilized membranes) = lO pM
Bm (intact membranes) = 0.79 pmol/mg proteln Bm (solubilized membranes) = l.0 pmol/mg protein Scatchard analysis revealed the presence of a hlgh affinity binding site. Some non-linearity and scatter in the data was ~'20 observed at low values of bound/free ligand where determination of precise binding data is most difficult. The dissociation , constant of the ligand binding to the soluble receptors (lO pM) was less than that exhibited by the receptors in intact memibranes (37 pM) despite the lack of G-protein coupling to the soluble receptors that was observed. As'noted above, such G- -protein coupling boosts the affinity of the membrane receptors by an order of magnitude. However, the assay was performed ' under conditions that had been optimized for GRP binding to the soluble receptor which may~have compensated for the affinity lost by G-protein interactions. In other experiments, the dissociation' constant of the solubilized receptor was calculated to range from lO to 30 pM, The data demonstrated that the functional conformation of the receptor binding site was maintained in 'detergent solution.
, The Scatchard data from this experiment also indicated that there were 0.79 pmol receptors/mg protein in crude Swiss 3~3 cell membranes and about 50% of the receptor - ~

W092/16623 PCT/U~92/02091 ? ~ ` ` 78 binding site~ were solubilized by extracting the membranes with detergent. some of the factors that were found to be necessary for the most efficient solubilization of receptor activity were inclusion of NaCl (>loO mM), elimination of divalen* cations, 5 and the extraction of membranes at room temperature. Although NaCl was necessary for the optimal solubilization of the receptors, the salt inhibited GRP binding to both the Swiss 3T3 : fibroblast membranes and detergent solubilized receptor (IC50 =
approx. 50 mM). However, the inhibitlon of the receptors by 10 NaCl at concentrations up to 1.0 M was found to be completely ; reversible.
:
:' .` W~ ..
Lig~n~_~}~5l~lcity of GRP Bindinq Sites in Soluble Membrane Extracts The binding of 125I-GRP to solubilized 3T3 membranes was assayed in the presence of various unlabeled competitor peptides. The C-terminal eight amino acids of GRP (GRP20-27) were found to be essential for high affinity binding to the GRP
receptors in whole cells. The complete GRP sequence (GRPl-27), the N-terminal portion of GRP ~GRPl-16), substance P, substance j P antagonist, physalemin (all of which were from Peninsula 3 Laboratories, Belmont CA), and the C-terminal portion of GRP -with norleucine substituted for methionine referred to as [Nlel4,27]GRP13-27.~(i.e. Lys-Nle-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Nle-NH2), were tested for their ability to , compete for 125I-GRP binding to soluble 3T3 fibroblast membrane extracts. The concentration of [Nlel4,27]GRP13-27 required to '! cause 50% inhibition of 125I-GRP binding to the soluble '~ 30 receptor (IC50 = 0-3 nM) was slightly higher than that of ; GRPl-27 (IC50 = 0.1 nM). In contrast, the N-terminal portion (GRPl-16) was unable to compete with 125I-GRP for binding to ' the soluble receptor. Additionally, substance P, substance :l antagonist, and physalemin had no inhibitory effect at the ~ 35 concentrations tested (up to 1000 nM). These results parallel ¦ closely that which was found in similar studies in whole cells~j and isolated membranes.

'~;
`: ~ ' ' ' ' L.' . , , .' ' . . , ; , ~ f~,O92/16623 PCT/US92/~2091 ,~ ~, ~` 79 Cross-l inkinq of 12 5 -GRP ~Receptors The molecular weight of the GRP receptor in solubilized swiss 3T3 membranes was estimated by covalently `
cross-linking it to bound 125I-GRP via the homobifunctional cross-linking reagent bis~sulfosuccinimidyl)suberate (BS3) and analyzing the affinity of labeled receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This ;~- cross-linker is specific for primary aminv groUps. Soluble 3T3 lo fibroblast membrane protein (40 ~g) was incubated ~or 30 minutes at 15 C in a final volume of 500 ~1 of 50 mM HEPES, 2 -mM EDTA, 0.075% CHAPS, 0.015% CHS, 30 ~g/ml bacitracin, and 0.2 nM 125I-GRP. The binding reaction was cooled to 0~ C and BS3 was added to yield a final concentration of 3 mM.~ 15 Cross-linking was quenched by addition of 0.10 ml of TRIS
buffer (l.o M TRIS/Cl, pH 7.5). After another 10 minute incubation, 0.1 ml TCA (100%) was added and the solution was -, further incubated at 0 C for 30 minutes. Precipitated material was collected by centrifugation, washed with ice cold acetone, and heated at 95 C for 3 minutes in SDS-PAGE sample , buffer. The samples were subjected to SDS-PAGE on a 7.5% gel ~ and the gel was fluorographed. A detailed description of the s~i- SDS-PAGE technique is found in ~aemmli et al. (1970) Nature - 2270680, which is incorporated herein by reference. Figure 5 iIlustrates the gel display.
, Lane Composition `r,,.'~ A no addition B 0.1 nM unlabeled~ GRP
C O.5 nM unlabeled GRP
.~! 30 D 1.0 nM unlabeled GRP
, E 100 nM unlabeled GRP

A strongly~labeled species migrated in a diffuse band with an apparent~Mr of about 75-100 kDa. Low levels of unlabeled GRP
inhibited the labeling of this species, indicating that the ~ labeling is highly specific. The broadness of the labeled band -,~ is consistent with the fact that the GRP receptor has been ~: :

~ WO92/166~3 ~ ~ 0~ 3 ~ 6 PCT/US92!0209l ~

;~ 80 ~ound to contain carbohydrate. The labeled product is very -similar to that derived from whole cell or membrane cross-linking experiment. N-Glycanase treatment of samples derived from cross-linked whole cells indicated that the labeled protein contained N-linked carbohydrates. The deglycosylated protein displayed an apparent Mr of 38 kDa on ~-SDS-PAGE.

Purification of the GRP RecePtor `, Solubilization of the GRP Receptor Swiss 3T3 fibroblast membranes (2-3 g of protein) were prepared as described in Example l and suspended in 200 ml storage buffer (see Example l). The membranes were mixed with 50 ml of NaCl (5.0 M), bringing the NaCl concentration to about -, l M, pelleted by centrifugation at 40,000 x g for 30 minutes, and washed twice at 4 C with 200 ml of high salt buffer (50 mM
HEPES, pH 7.5, 2 mM EDTA, l.o M NaCll 25 ~g/ml leupeptin, lO
' ~g/ml aprotinin, 2.5 ~g/ml pepstatin, and 0.5 mM PMSF). The membranes were then washed with low salt buffer (50 mM HEPES, pH 7.5, 2 mM EDTA, 25 ~g/ml leupeptin, lO ~g/ml aprotinin, 2.5 ~g/ml pepstatin, and ~0.5 mM PMSF) and resuspended in 200 ml 50 mM HEPES, pH 7.5, 2 mM EDTA, 1 mM EGTA, lO0 mM NaCl, 0.03 l~g/ml bacitracin, 25 ~g/ml leupeptin, lO ~g/ml aprotinin, 2.5 ~g/ml pepstatin, and 0.5 mM PMSF. ~A skock solution containing a ~ mixture of CHAPS and CHS was added slowly to~the membranes to 3 give a final concentration of 0.75% CHAPS and 0.075% CHS. The .
mixture was incubated for 30 minutes at 21 C, cooled to 4 C
and centrifuged at lO0,000 x g or 60 minutes at 4 C. The supernatant contained the solubilized GRP receptor.
Preci~itation bv Polyethvlene G1YCO1 ~o the solubilized extract (l90 ml), 126 ml of ice cold polyethylene glycol (PEG)~ 8,000;~50 w/v% in H2O) was added~ After thorough~m1xing, the precip1tate tha~formed was collected~by c~ntrifugation~at lO0,000 x g for lO minutes. The pellet was suspended in 25 mM~HEPES, 25 mM TRIS,~ pH 7.5, 2 mM
TA, ~0,5 C ~ ~5, 0,0~ C~6~ g/~ n, and 10 WjQ9~/16623 2 1 0 ~ 3 ~ ~ PCT/US92/02091 ` ~g/ml bacitracin in a total volume of 50 ml with the aid of a Potter-Elvehjem homogenizer. The suspension, which contained some insoluble protein, was centrifuged at 69,000 x g for lo minutes, and the pellet was discarded.
Wheat Germ Aqqlutlnin Chromatoqraphy Following precipitation by PEG, the GRP receptor was further purified by lectin affinity chromatography. A column (1.6 x 9 cm~ containing wheat germ agglutinin-agarose resin (3-5 mg lectinfmg of wet gel) (E-Y Laboratories, San Mateo, CA) was equilibrated with 50 mM HEPES, pH 7.5, 2 ~ EDTA, 0.~5%
CHAPS, 0.025% CHS, 5 ~g leupeptin, and 10 ~g/ml bacitracin at C. The soluble extract was diluted with one volume of column buffer, and the final detergent concentration was ; adjusted to 0.25% CHAPS and 0.025% CHS. The sample was applied to the lectin column at a flow rate of 1.5 ml/min. The column was then washed with about 10 column volumes of buffer, and eluted with column buffer plus 5 mM
N,N',N " -triacetyl-chitotriose. Fractions containing the GRP
receptor binding activity were pooled and diluted with 2.3 volumes of 25 mM HEPES, 25 mM TRIS, pH 7.5, 2.0 mM EDTA, 5 ~g/ml leupeptin, and 10 ~g/ml bacitracin.
GRP Affinity Chromatoaraphy - Actigel superflow resin (10 ml)tSterogene, San . Gabriel, CA) was washed with 5 volumes of 100 mM KPO4, pH 7Ø
~; 25 The resin was incubated with 10 ml of 100 mM KPO4, 100 mM
~' NaCNBH3, pH 7.0 containing 2 mg/ml [Nlel4,27]GRP13-27 for 2 - hours with gentle agitation. The resin was washed with 100 mM
KPO4, pH 7.0, followed by alternating washes with 100 mM KAc, pH 4.0, 0.5 M NaCl; and 100 mM TRIS pH 8.0, 0.5 M NaCl. A
column of the resin (1.6 x 5 cm) was equilibrated with 25 mM
. .
TRIS, 25 mM HEPES, pH 7.5, 2.0 mM EDTA, 0.075% CHAPS, 0.0075%
CHS, 5 ~g/ml leupeptin, and 10 ~g/ml bacitracin at 4 C. The crude GRP receptor eluted from the lectin column was loaded i onto the GRP affinity column at 0.1 ml/min. The column was ;J~ 35 then washed with about 20 volumes of the equilibration buffer. -The bound receptor was eluted from the column with ~1 equili~ration buffer plus 0.5 M NaCl at a flow rate of 0.2 :; . :
~'i ~ ~ ' '. ~
.j ~ .~.
f ~

" ' -W~92/16623 2 ~ PCT/US92/0209 ml/min. Fractions containing the receptor were identified by assays of 125I-GRP binding activity and were pooled (10-13 ml).
- The elution pool was concentrated to about 1 ml by ultrafiltration using a Centriprep-10 device (~micon, Danvers, MA). The sample was then desalted by dilution of the sample ~-- with 15 volumes of affinity column equilibration buffer and -- re-concentration of the sample to 1 ml. This desalting step was repeated and the resulting 1 ml sample was diluted to 5 ml with affinity column equilibration buffer. PAGE analysis of the purified GRP receptor revealed the presence of a - significant level of contamination.
~ This solution of semi-pure receptor was loadied onto a -~ second [Nlel4,27]GRP13-27-actigel superflow column (1.0 x 3 cm), prepared as described above, at 1.8 ml/h. The column was washed with 20 column volumes of equilibration buffer, and the bound receptor was eluted with equilibration buffer plus 0.5 M
NaCl at a flow rate of 0.1 ml/min. Fractions containing GRP
receptor binding activity were pooled and concentrated to 0.3 ml by ultrafiltration.
Gel Filtration The purified receptor was desalted by chromatography on a Superose-6 HR 10/30 column (Pharmacia LKB, Piscataway, NJ). The column was equilibrated with 20 m~ HEPES, pH 7.5, 2 - mM EDTA, 0.075% CHAPS, 0.0075~ CHS, and 100 mM NaCl. The receptor was chromatographed at 0.4 ml/min. The receptor was eluted from the column in about 2 ml.
Characterization o~ the Purified 5RP Rcceptor The overall yield of the pure GRP receptor from the crude solubilized extract ranged from 10-20%, based on recovery of high affinity 125I-GRP binding activity. Scatchard analysis of binding data obtained with the purified receptor indicated that its affinity for GRP (KD = 10-30 pM) was essentially the same as the receptor in the crude detergent solubilized extract. The data show that 30-50 pmoles of receptor sites are typically obtained in the final purified fractions of the receptor, as outlined in this example. This corresponds to about 1-2 ~g of receptor protein, taking into account that the . . .
( ::
...

~ wo g2/16623 2 ~ ~ 5 3 ~ ~ PCT/~S92102091 ~ 83 .:
deglycosylated receptor exhibits an apparent molecular weight ~- of 36+5 kilodaltons on SDS-PAGE gels.
A silver stained SDS-PAGE gel of the receptor preparation showed a single intensely staining diffuse band with an apparent molecular weight of 70-lO0 kD. The receptor preparation was essentially free of contaminants. Figure 6 illustrates the silver stained gel display of the purified GRP
receptor. The relative level of silver staining of the GRP
receptor band was compared with known amounts of protein to determine the approximate amount of receptor protein loaded on the gel. The rough value obtained was in the range of that estimated to be present by Scatchard analysis of l25I-GRP
binding data, which confirmed that the intensely staining band on the gel was the GRP receptor. Furthermore, the apparent molecular weight of the purified GRP receptor corresponded to ~ that obtained with affinity labeled receptor. This was 3 obtained by binding l25I-GRP to the receptor in whole cells, intact membranes, or crude soluble extracts, and cross-linking the receptor-ligand complex with a homobifunctional .~ 20 cross-linking reagent.
~, The diffuse nature of the GRP receptor band on SDS
~3 PAGE is characteristic of proteins containing carbohydrate. A
small portion of the purified receptor was radiolabeled by ~ iodination~with l25I-NaI in the presence of Iodogen (Pierce, ^~ 25 Rockford, IL) to enhance the detection of the receptor on gels.
Treatment of the radiolabeled receptor with N-glycanase resulted in loss of the 70-lO0 kDa band, and the generation of ~; a new band at about 36+5 kilodaltons, representing the deglycosylated receptor.
Determination of Partial Amino Acid ~1 Seauence of the GRP RecePtor ;1 A partial sequence near the N-terminus of the , purified GRP receptor was determined by sequential Edman ~ degradation. The~sequence~obtained for residues 8-17 was:
`~ ~` 35 -Leu-Asn-Leu-Asp-Val-Asp-Pro-Phe-Leu-Ser-.

~` W092/]~623 2 1 ~ 5 3 ~ 6 PCT/US9~/02091 .~`.:~ ~.
`.` 84 Trypsinization of the Purified GRP Receptor and the Isolation of Tryptic fraqments Purified GRP receptor was prepared as described in - 5 Example 10. After Superose-6 chromatography, 40 picomoles of receptor were obtained based on Scatchard analysis of 125I-GRP
binding data. This corresponded to about 1.6 ~g of protein.
The sample (3 ml) was concentrated to about 100 ~1 by ultrafiltration using a Centricon 10 device (Amicon). The - 10 sample was then diluted with 2 ml of H2O, and concentrated to ~ 100 ~1~ Once again, the sample was diluted with 2 ml H2O, and - concentrated to 100 ~1, and was finally diluted with 1 ml of H2O, and concentratad to 138 ~1. To digest the receptor with trypsin, 0.1 ~g of trypsin was added, and the sample was incubated at 37 C. After 2 hours, an additional 0.1 ~g of trypsin was added, followed by another 0.2 ~g of trypsin after - 5 hours of incubation. After 22 hours at 37 C, the sample was rapidly frozen in liquid N2 and stored at -80 C.
Trypsin digested GRP receptor was thawed to room temperature and reduced with dithiothreitol (DTT) at a final concentration of 10 mM for 30 minutes at 37 C. The entire DTT
treated tryptic digest was then fractionated by reverse phase high pressure liquid chromatography (HPLC) using a 2.1 mm X 3 cm C4 column ~Brownlee, Aquapore Butyl, 300 angstrom pore ~' 25 size), and a linear gradient of 0.05% trifluoroacetic acid -~ (TFA) in water (solvent A) to 0.05% TFA in 100% acetonitrile (solvent B), see Figure 7. The conditions for the HPLC
gradient were 0% solvent B to 100% solvent B in 60 minutes at a -` flow rate of 0.2 milliliters per minute. Effluent fractions were detected at 215 nm, collected at one minute intervals, and " stored at 4a C.
For peptide sequence analysis, consecutive fractions were pooled and concentrated on a Speed Vac (Savant, Farmingdale, NY) to a~final volume of approximately 50 ~1. The sample was loaded in entirety onto a glass fiber filter which ! had been treated and precycled with Biobrene (Applied ~i Biosystems (ABI~, Foster City, CA). Automated amino acid sequence analysis was performed on an ABI model 475A gas phase .i , .
, ~

, . . .

~ WO~ 6fi23 2 ~ ~ 5 3 ~ ~ PCT/US92/02091 ~5 sequencer according to Hewick et al. (1981) J. Biol. Chem.
256:7990-7997, equipped with an ABI model 120A on-line detection HPLC system for identification of phenylthiohydantoin - (PTH-) amino acids. Quantitation of PTH-amino acids was performed by an ABI model 900 data system using 60 picomoles of a set of known PTH-amino acid standards (ABI). In this manner, the combined tryptic HPLC fractions 56 through 59 gave the amino acid sequence MASFLVFYVIPLAII (designated ~56/59); the tryptic HPLC fraction 44 yielded the amino acid sequence QLTSVGVSV (designated T44), and the tryptic HPLC fraction 50 gave the amino acid sequence PNLFISXLALG (designated T50), where X denotes a residue that could not be identified.
NH2-terminal sequence analysis was performed on the ;; intact purified GRP receptor following washing of the sample - 15 with H?O and concentration of the sample on a Centricon 10 ultrafiltration device (Amicon, Danvers, MA). The sample (95%
~ or approximately 95 ~l was loaded onto a Biobrene (ABI) -~ precycled glass filter and NH2-terminal sequence analysis was .~ performed through 30 cycles of automated Edman degradation on .~ 20 an ABI 475A gas phase sequencer ~Hewick et al.(1981)).
;~ PTH-amino acid identification and quantitation were performed using an ABI 120A PTH-amino acid analyzer and ABI 900 data - system. Following two separate NH2-terminal sequence runs on two purified preparations of the GRP receptor, the following consensus NH2-terminal amino acid sequence was obtained for 17 residues, where X denotes a residue for which an accurate assignment of a specific amino acid was not made:

A!P N X X S X L N L D V D P F L S.
; 30 ;~ Identification of cDNA Clone Encodina the Swiss 3T3 GRP Receptor Preliminary studies established that a murine embryonal fibroblast cell line (Balb 3T3) expressed a repertoire of mRNAs very similar in abundance and distribution to the GRP receptor-expressing Swiss 3T3 murine fibroblast cell line, but did not have any cell surface GRP receptors ., .

2 ~ 0 ~ U ~:~

detectable in standard binding assays see Kris et al. (1987) J. Biol. Chem. 262:11215-11220; and Zachary et al. (1985) Proc.
Natl. Acad. sci. USA 82:7616-7620, each of which is incorporated herein by reference. These observations suggested that the GRP receptor mRNA would be one of a limited number of transcripts present in swiss 3T3, but absent from salb 3T3 mRNA. Polyadenylated mRNA was isolated from both Swiss 3T3 and Balb 3T3 cell lines and was used to generate a Swiss 3T3 subtracted cDNA library enriched for cDNAs derived from Swiss 3T3 mRNA but not represented in Balb 3T3 mRNA using published methodology, e.g., Timlin et al. (1990) Nuc. Acids. Res.
18:1587-1593, which is incorporated herein by reference. The cDNA inserts whose length exceeded 300 base pairs were ligated into the lambda gtlO bacteriophage cDNA cloning`vector and the ,~ 15 library amplified using the established methods, e.g., Davis et al. (1986) Basic Methods in Molecular BioloqY, Elsevier Science Publishing Company, New York.
The library was screened with'an oligonucleotide probe whose sequence was based on the amino acid sequence of an `
', 20 internal tryptic fragment (T 56/59) purified by HPLC from a -~ tryptic digest of the purified GRP receptor protein. The amino acid sequence (MASFLVFYVIPLAII) of the internal peptide was ~,' used to design a long non-degenerate antisense oligonucleotide whose sequence was based on optimal codon usage frequency as ' described in the literature by Lathe (1985) Mol.Biol. 183:1-12, resulting in a 44~base long probe referred to as I3:
(5'ATGATGGCCAGGGGGATCACATAGAAGACCAGGAAGGAGGCCAT 3'). The I3 ~, probe was labeled by phosphorylation of the 5' end using gamma ' 32P-ATP and polynucleotide kinase employing the established techniques of Davis et al. (1986). The labeled probe was used to screen lOO,OOO member clones from the subtracted library ~ using hybridization and wash conditions as described. See Wood ~' ; tl987~ Chapter 48 in Methods in Enzvmolo~Y 152:443-447, which is incorporated herein by reference. Duplicate screening identified five' positive clones, which were plaque purified.
The EcoRI inserts from the five clones were subcloned into the plasmid vector pGEN 4 (Promega), and the nucl'eotide sequence of `' ;, :
., .
~ .
., , W092/1~623 21 D 5 3 ~ ~ PCT/US92/02091 the hybridizing inserts was determined using the Sequenase 2.0 double stranded sequencing klt tUs Biochemical). Two of the five clones (Tl and T2) had an identical region of overlapping DNA sequence which encoded the internal peptide used to design the oligonucleotide probe. The fragment was removed from the plasmid vector by EcoRI digestion and purified by gel electrophoresis and electroeIution as described by Davis et al.
- (1986). The purified insert fragments were labeled by random primer extension using a commercially available kit and the lo supplier's recommendations (Bethesda Research Laboratories) to generate a probe to identify other overlapping cDNA clones from the subtracted library in a second screening of the 100,000 library members. Nucleotide sequence analysis of the nine additional clones identified revealed a single long open reading frame whose predicted translation product included the internal tryptic fragment amino acid sequence, which ended in a termination codon within the composite sequence. The amino terminal end of the open reading frame was not present in any of the clones isolated from the subtracted library.
To obtain the 5' end of the c~NA and the sequence at the amino terminal end of the open reading frame, an in vitro polymerase chain reaction amplification (PCR) cDNA cloning procedure (5' RACE) was perf~rmed essentially as described in Frohman et al. (1988) Proc Natl. Acad. Sci. USA 85:8998-9002, - 25 using two nested gene-specific oligonucleotides (EXT 3: 5' GGGGAGCCAGCAGAAGGC 3'; EXT 2: 5' CCATGGAATGGATTTTA) derived from the known nucleotide sequence of the cDNA clones previously analyzed. EXT 3 was used as a gene-specific primer for reverse transcription of Swiss 3T3 mRNA, and EXT 4 was used ;~ 30 as a gene specific primer for Taq DNA polymerase catalyzed PCR.
Nineteen 5' RACE cDNAs were isolated and characterized, and five of the clones that extended the longest distance were sequenced as described;previously. Nucleotide sequence analysis revealed an extension of the long open reading frame encoding the internal tryptic peptide amino acid sequence, beginning with an initiator methionine codon. The predicted amino acid sequence of the open reading frame was compared with - :
- :

. . .

W092~]6623 ~ PCT/US92~02091 .,` ~

amino terminal sequence derived from the purified GRP receptor (See Example 11). The experimentally determined amino acid - sequence did not contain the methionine at position 1 of the deduced sequence, but corresponded well to residues 2-18.
Deduced amino acids 2-4 and 8-18 (Table 1) were identical. The amino acids that did not match ~amino acids 5-7, Table 1) were ambiguous in the original amino acid sequence, probably because they are located at an N-linked glycosylation site (Asn-Cys-Ser). In addition, the amino acid sequence from internal tryptic peptides T44 (QLTSVGVSV) and T50 (PNLFISXLALG), derived from the purified Swi55 3T3 GRP receptor (Example 11), matched segments within the long open reading frame of the composite GRP receptor cDNA.
Gene-specific primer-directed cDNA cloning was used - 15 to obtain a single cDNA clone which encodes the entire uninterrupted open reading frame. In this procedure, a gene-specific oligonucleotide (EXT7: 5' TACTTTGAGATACAATGG 3') complementary to an 18 nucleotide segment of the 3' -.` untranslated region of the GRP receptor mRNA was used to prime the synthesis of first-strand cDNA by MuLV reverse ~; transcriptase. Double-stranded cDNA was generated, and cloned into lambda gtlO using standard methodology of Davis et al.
~, (1986). Five hundred thousand clones were screened with a cDNA
; fragment probe derived from one of the 5' RACE cDNA clones which extended into the 5' untranslated region of the cDNA.
Over twenty clones were identified, and ten were plaque purified and subcloned into plasmid vectors by standard methods ~;~
of Davis et al. (1986). Nucleotide sequence analysis confirmed that the clones contained the entire uninterrupted open reading frame of the GRP receptor protein. The DNA sequence of the GRP
receptor from mouse and its deduced amino acid sequence is shown in Table 1.
Analysis of the nucleotide sequence of the open reading frame revealed several interesting features of the predicted protein. The predicted molecular weight of the protein is about 43,100 daltons, in good agreement with that reported for the N-glycanase treated GRP binding protein from i~ -~` WO92/16623 2 1 0 ~ PCTJUS92/02091 Swiss 3T3 cells, described in Example 10. Hydrophobicity analysis is presented in Figure 8 and predicts the presence of seven putative transmembrane domains, consistent with earlier observations that the GRP receptor is coupled to a guanine-nucleotide binding protein (G-protein), see Fischer et al. (1988) J. Biol. Chem. 26~:2808-2816. The superfamily of -~
G-protein coupled receptor genes typically share certain conserved residues within or adjacent to the seven transmembrane domains, see Masu et al. (1987) Nature .
329:836-838. These conserved amino acids are found in the ; predicted locations within the open reading frame of the mouse GRP receptor sequence (Table 1). Five potential sites for N-linked glycosylation (Asn-X-Ser/Thr) are noted (Table 1), ; consistent with the observation that the GRP receptor is heavily glycosylated, and that N-glycanase treatment of the GRP
receptor glycoprotein reduces the apparent molecular weight of the protein in SDS- polyacrylamide gels from about 70-lOO
kilodaltons to about 38~5 kilodaltons (Example 10). Table 6 -shows a comparison between the GRP receptor and the~ substance K
receptor.
.:. . ' .

,. .
i~ ..
-"'1 :''-:;.
:~ ,. . :
' : .
.. j , .

.. . .
`1~
"
. j , .

,i' , : ~ - ~ ' 1 :
.~:
1 ~ .

~ W092~16623 2~ 3,~,~ PCr/US92/02091 .. ~` ~, Table 6: A comparison of the amino acid sequences of the GRP receptor and the Substance K receptor.
- . . .

-. 1 MAPNNCSHLNLDVDPFLSCNDTFNQSLSPPKMDNWFHPGFIYVIPAVYGL 50 D
I I I I I I
1 .MGACW MTDINISSGLDSNATGITAFSMPGWQ...... ~L,ALWT~AYLA 42-~ 51 IIVIGLIGNITLIKIFCTV~SMRNVPNLFISSLALGDLLL~VTCAPVDAS 100 , I I I I I I I I I I I I I I
.. 43 LvLvAvMGNATyIwTILAHQRMRTvTNyFIvNLALAD~cMAA~NAAFNFv 92 ; 101 ~YLADRWLFG~IGCXLIPFIQLTSVGVSVFTLTALSADRYKAIVRPMDIQ 150 :~ I 111 1, l 11 11 111.1 111 1 .
`~ 93 YASHNIWYFG~AFCYFQN~FPITAMFVSIYSMTAIAADRYMAIVHPFQPR 142 ..`
... 151 ASHAL~KICLKAALIWIVSML~AIPEA~FSD~HPFHVKDTNQTFISCAPi 200 I I I I I I I I I i I I . .
143 LSAPGTRAVI..AGIWLVALALAFPQCFYSTI....T~DEGATXC W A~P 186 ;,,~-, :
`.- 201 PHSNEL~PKIHSMASFLVFYVIPLAIISVYYYFIARN'LIQSAYNLPVEGN 250 ".- 187 FDSGGKMLLLYHLIVIALIYFLPLVVMFVAYSVIGLTL~R~SVPGHQA~G 236 .`.. ,.. ~ ~
:`` 251 IHVKKQIESRKRLAKTVLVFVGLFAFCWLPN~VIYLYRSY~YSE~DTSML 300 ~;. 1: - II I I I I III I I
"~ 237 ANL.RH~QA~RRFVKTMVLVVVTFAIC~LPYHLYFI~GTF... QEDIYC~ 282 ~ 301 HFVTSI~AHLLAFTNSCV. NPFALYLLSKSFRXQ~NTQ~LCCQ,....... 342 1 : ! I ~ 11 1 11 1 11 ,........ ~ 283 XFIQQVYLALFWLAMSSTMYNPIIYCCLNHRFRSGFRLAFRCCPWVTPTE 332 .-,, .-343 ......... PGLMNRSHSTGRSTTCMTSFKSTN~SATFs~INRNICHEGi 38l .,.: I I I I I I .
333 EDKMELTYTPSL...STRVNRCHTKEIFFMSGDVAPSEAVNGQAESPQAG 379 384 V*.... 385 ~
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v~ lEGEN0: ~ TABLE 6 GRPRC~EP~OR:
SUBS~AN~E K RElE P~OR

` WO92/1~623 2 1 ~ 5 3 0 6 PCT/US92/02091 Northern blot analysis was undertaken to identify thenature of the transcripts encoding the swiss 3T3 GRP receptor.
~he results are shown in Figure 9. One microgram of ` polyadenylated mRNA derived from Swiss 3T3 and Balb 3T3 cells was purified and resolved by electrophoresis on a formaldehyde-containing one percent agarose gel, which was subsequently transferred to a nitrocellulose filter. The filter was hybridized with a 450-base pair cDNA fragment probe encoding the carboxy terminal transmembrane domains 5, 6, and 7 as well as a portion of the 3' untranslated sequences. The probe was labeled with 32p to a specific activity 500 cpm/picogram using a commercially available random primer extension kit (Bethesda Research Laboratories). Two mRNAs specifically hybridized to the probe, whose sizes were estimated to be 7.2 kb and 3.0 kb by comparison to mouse 28S
(5.0 kb) and 18S (2.0 kb) markers (Figure 9). As expected, the two mRNA forms were only detected in mRNA from Swiss 3T3, with no GRP receptor transcripts observed in mRNA from Balb 3T3 cells.

Human mRNA Species Homoloqous to Mouse GRP Rece~tor cDNA
Northern blot analysis was performed to determine the !~ 25 degree of homology between the GRP receptor expressed in human 3! fetal lung cells, see Kris et al. (1987) J. Biol. Chem. -262:11215-11220; and the Swiss 3T3 cell receptor.
Polyadenylated mRNA was isolated from human fetal lung ce~ls, and subjected to Northern analysis as described in Example 12, using the same 450-base pair cDNA fragment of the Swiss 3T3 cell GRP receptor as a probe, except that the stringency of the hybridization filter washing steps was reduced. Two mRNA
species of approximately 7.2 and 3.0 kb were detected in the human cell line, corresponding to those observed in mouse Swiss 3T3 cell mRNA. See Figure 10. Based on the conditions used for the blot, the mRNA species identified were at least 8C%
homologous to the Swiss~3T3 GRP receptor probe. The results ' indicate that the mouse GRP receptor cDNA, described in Example 1' -.. :
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W09~166~3 2 ~ ~ 3 3 ~ 6 PCT/US92tO2091 ~ 92 `` 12, can be used to readily isolate cDNAs or genomic DNA
fragments encoding the GRP receptor in other mammalian species, including humans.
These homologous receptors will be available to isolate other homologous receptors by using similar techniques.

Expression of the Mouse GRP Receptor Derived from the cDNA Clone in Xenopus Oocytes to Demonstrate lo Rece~tor Function A sense in vitro transcript was prepared from the mouse GRP receptor cDN~ protein coding region (Table 1) cloned in the transcription vect~r pGEM 4 (Promega) using sp6 RNA
polymerase and established methods of Davis et al. (1986). The synthesized transcript (about 20 nanograms) was injected-into ` XenoPus oocytes. Sixteen hours later, the oocytes were voltage clamped and bathed in a solution containing 10 9 M GRP. As ' shown in Figure 11, a GRP ligand dependent chloride current `~ (magnitude of about 160 nanoamperes) was coincident with addition of the ligand. These results demonstrate the expression of an in vitro transcript-dependent GRP receptor on the Xenopus oocyte cell surface, which is coupled through G-proteins to a Ca++ dependent chloride channel. The ligand dependent chloride current was not observed in control oocytes injected with an antisense in vitro transcript, thus demonstrating speci~icity of the response.

Isolation of Candidate NMB-R cDNA Clones A hexamer-prlmed cDNA library was constructed from rat esophagus, and screened by hybridization at low stringency with the Swiss 3T3 GRP-R cDNA probe. Several candidate clones ~ ~ were isolated, two of which contained the entire coding region j~ of a Iong open reading frame. Several criteria were used to establish that the cDNA clones~encode a NMB-preferring bombesin ~, receptor protein distinct from the GRP-R initially isolated.
;f ~ The properties~distinguishing these two bombesin receptor subtypes include protein structure, sensitivity of receptor `f ` ~ W092~16623 2 i ~ ~ 3 ~ ~ PCT/US92/02091 ` 93 function to specific antagonists, relative binding affinity for bombesin peptide ligands, and tissue distribution of - expression. These properties were studied using the cDNA
clones isolated at low stringency from the esophageal cDNA
library.

The Nucleotide Sequence _nd Amino ~-id Se~uence of NMB-R cDNA
` 10 The nucleotide sequence and predicted amino acid sequence of a single long open reading frame present in two independent clon~s encoding the putative NMB-R is shown in Table 3. These cDNAs derive from mRNAs that encode a protein 390 amino acid in length, with a calculated molecular weight of 43 kDa. A hydropathy analysis of the predicted NMB-R protein reveals seven stretches of hydrophobic amino acids, consistent with a seven transmembrane-structure typical of G-pro~ein coupled receptors. See Figure 12. There are three potential ~- sites for N-linked glycosylation (Asnl, Asn71, Asnl92), consistent with the prediction that the NMB-R protein, like the GRP-R, may be a glycoprotein. See Table 3.
In Table 7, the predicted amino acid sequences of the mouse Swiss 3T3 GRP receptor and the rat NMB-R protein are compared. The NMB-R amino acid sequence has higher similarity ` 25 to the GRP-R than any other sequence reported to date (54%
`j identity). A previously reported comparison of the rat substance P and substance K receptors shows comparable amino ~;~ acid sequence identity between these two tachykinin receptor `~ subtypes (48% identity), see Yokota et al. (1989) J. Biol.
i~ 30 Chem. 264, 17649-17652. In contrast, the sequence identity between the putative rat NMB-R and the mouse GRP-R is considerably lower than observed when the substance K
, receptors are compared (85%) from bovine, see Masu et al.
(1987) Nature 329, 836-838, and rat, see Yokota et al. (1989) J. Biol. Chem. 264, 17649-17652.

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: ~` 94 Table 7: A comparison of the predicted amino acid sequences of a rat NMB-R with a mouse GRP-R. The predicted amlno acld sequence of a rat NMB~R (I'able 3) and mouse Swiss 3T3 GRP-R (Table 1) are aligned to maximize homology using the GAP Program ln the Software Package of the University of Wlsconsin Genetics Computer Group. See Devereaux et al. (1984) Nuc. Aclds Res. 12:387-395. Solid lines between amino acid residues which are typically conserved in many other known G-protein coupled receptor superfamily members are enclosed in b~xes.

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~i WV92/l6623 2 ~ 3 t .~ ~ P fCT/ US92/02091 A compari~on between the amino acid sequence predicted for the NMB-R and other members of the G-protein coupled receptor superfamily shows that many amino acid residues conserved in this family are present at corresponding positions in the NMB-R sequence. Two cysteine residues that may for~t a disulfide linkage situated in the first and second extracellular loop are conserved ln the predicted NMB-R
sefquence at positions 116 and 198. Another well conserved cysteine residue which is thought to be important in anchoring o the beta-adrenergic receptor to the plasma membrane is also present in the predicted sequence of NMB-R, 14 amino acid residues downstream from the end of the seventh transmembrane domain. In addition, numerous other amino acid residues which are typically conserved in members of the G-protein coupled receptor superfamily are also found in the predicted amino acid sequence of the NMB-R (Table 7 boxed residues). These ~-similarities indicate that, like the GRP-R, the NMB-R is a member of the G-protein coupled receptor superfamily.

Jf20 EXAMPLE 17 Analysis of the Functional Properties of the NMB-R
, To confirm the functional identity of the NMB-R cDNA, Xenopus oocytes were injected with RNA transcribed in vitro from cDNA clones containing the entire NMB-R protein coding domain. RN'A was transcribed and capped in vitro from either f the NMB-R or GRP-R cDNA clones using T7 RNA polymerase as ff recommended by the manufacturer (Promega). Defolliculated oocytes were microinjected with about 10 nanograms of mRNA per oocyte, and kept at 20 C in ND solution of Lupu-Meiri et al.
3Cff (I989) Pflugers Arch. 413:498-504. After 24 to 48 hours, oocytes were placed in a perfusion chambfer and voltage clamped at a holding potential of -60 mV. Ligands were added directly i to the chamber and ligand-dependant Cl currents were measured.
The GRP1-27 and NMB peptide were purchased from Peninsula f 35 tBurlingame, CA), and the ED-Phe6]BN(6-13) ethyl ester 'l antagonist was synthesized as described by Wang et al. (1990) i J. Biol. Chem. 265:15695-15703.

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. W092/16623 2 1 ~ ~ 3 ~ ~ PCT/U~92/020~1 Either NMB (lO 6 M) or GRP ~10 6 M) causes a depolarizing current which is typical for IP3- and Ca~2-mediated chloride channel opening. At lower agonist concentrations (10 9 M), only NMB could elicit a detectable response. These data establish that the cDNA clones isolated from the esophagus library encode a functional NMB-R that, in contrast to the GRP-R, responds to lower concentrations of NMB
than GRP.
The effect of a specific antagonist for the GRP-R on the function of the NMB-R expressed in oocytes was tested. The des-Met bombesin analog ([D-Phe6]BN(~-13) ethyl ester) functions as a specific antagonist for the pancreatic GRP-R but not the esophageal NMB-R. This antagonist completely blocks the electrophysiologic response of oocytes expressing the cloned Swiss 3T3 GRP-R when it is applied at a 10:1 molar ratio with micromolar concentrations of either GRP or NMB agonists.
In contrast, addition of the antagonist along with either NMB
or GRP agonist (10:1 molar ratio) did not diminish the response 1~ of the cloned NMB-R expressed in Xenopus oocytes.
;j 20 To establish that the differences in physiological response of the receptor to NMB and GRP were due to relative ~3~ binding affinities, the ligand binding properties of the cloned receptor expressed in Balb 3T3 fibroblasts were examined.
Preliminary ~inding studies showed that Balb 3T3 cells would be an appropriate host for expressing the cloned NMB-R, since they have very low levels of endogenous displaceable bombesin ~-~, binding.
An Eco RI fragment from the longest NMB-R cDNA clone encoding the entire open-reading frame was subcloned into a modified version of the pCD2 plasmid from Wada et al. (1989) Nature 342:684-689. Balb 3T3 cells were transfected with 40 micrograms of the NMB-R expression plasmid construct using the calcium phosphate precipitation method of Graham et al. (1973) Viroloqy 52:456-467, with a few modifications, see Davis et al. 35 (1986). Stably transfected cells were selected for resistance to the aminoglycoside G418 (800 ~g/ml). After a three week selection period, lO clones were screened for high affinity ., , ~ : . .
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`` 98 bin~ing. One cell line showing high levels of specific binding was selected for more detailed analysis.
Binding and displacement studies on the transfected Balb 3T3 cells were performed as described previously bv Kris et al. (1987) J. Biol. Chem. 262:11215-11220, in 24 well tissue culture dishes using 25 pM l25I-labeled bombesin purified after labeling by reverse phase high pressure liquid chromatography (von Schrenck et al. (l990) Amer. J. PhYsiol. 259:G468 G473).
Each point on the displacement curve was determined four times, ; lO and the average value plotted. The bombesin displacement studies performed to determine the KD values for NMB, GRP, and ` the ethyl ester antagonist on pancreatic and esophagus tissue sections were performed as described by von Schrenck et al.
` (1990).
The relative ligand affinity of the transfected NMB-R
was assessed by quantitative displacement of 125I-labeled bombesin (BN) binding by unlabeled NMB or GRP. NMB was more '~ potent than GRP in displacing labeled BN (XD for NMB = 2 nM; KD
for GRP = 43 nM). Ligand displacement properties determined for the transfected cells are compared in Table B to those obtained from esophageal tissue sections, known to express an NMB-R as well as the pancreatic acinar cell line AR42J, and .l pancreatic tissue sections known to express a GRP-R with properties similar to the Swiss 3T3 GRP-R. NMB was more potent than GRP in displacing 125I-BN bound to transfected Balb 3T3 J~ cells sxpressing the NMB-R, as was observed in esophagus tissue sections. In contrast, GRP is more potent than NMB in displacing l25I-bombesin binding to pancreatic acinar cells, 1 AR42J, or Swiss 3T3 cells. These result~ show that the cDNA
,1 30 under study encodes a functional NMB-preferring bombesin receptor, with binding properties resembling the esophageal NMB-preferring bombesin receptor reported previously. As expected, the ~specific GRP-R anta~onist [D-Phe6]BN(6-13) ethyl ester binds GRP-preferring receptors (pancreas, ~R42J, Swiss -3T3) at high affinity (KD = 1.6 to 5.3 nM), but has much lower affinity for NMB-preferring receptors on either esophagus or :., .J

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~ WO92/16~3 2 l 0 53 a 5 PC~/VS92/02091 ' --' 100 TABLE 8: Displacement of I-BN binding by GRP, NMB
and [D-Phe ~BN(6-13 ethyl ester antagonist ln dlfferent BN receptor subtypes*
Ki (nM) Cell Type NMB GRP
antagonist ~ salb 3T3/NMB-R 2 43 >1000 : 10 esophagus 0,3 30 >1000 pancreas 351 15 5. 3 15 AR42J 287 2 2.1 Swiss 3T3 62 2 1. 6 -_____ .
* Displacement of 125I-BN binding GRPl-27,6NMB, and a GRP-R antagonist [D-Phe ]BN(6-13) ethyl ester was analyzed in ~- 25 tissues and cultured cells expressing different bombesin receptor subtypes.
Whole cell binding studies on cell lines (Balb 3T3/NMB-R transfectants, Swiss 3T3) were performed essentially as descrihed by Kris et al. (1987) J. Biol. Chem. 2i52, 11215-11220. Binding displacement analysis ~,~ of tissue sections and AR42J cells was .-~
performed in a very similar manner, with a few modifications, to the method of von Schrenck et al. (1990) Amer. J. Physiol. ~-259:G468-G473. Binding properties of the NMB-R expressed on transfected Balb 3T3 fibroblast most closely resembIe the ~! esophagus NMB preferring receptor/ and are !' 40 clearly diffexent from GRP preferring BN
- receptor subtypes found on pancreatic acinar cells, Swiss 3T3 cells, and AR42J
ells.

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sombesin receptors have been described in both neural and non-neural tissues, as well as various cell lines. To determine which cells express the NMB preferring bombesin receptor subtype encoded in the cDNA clone, mRNA was examined in various tissues and cell lines using Northern blo~
hybridization analysis. Poly ~A)+ RNA isolated ~rom the rat brain, olfactory region, esophagus, and C6 glioma cell line each contain two hybridizing mRNA species present after a high stringency wash, with estimated sizes Qf approximately 3.2 kb and 2.7 kb. Both bands were observed together in all .~ expressing tissues and were still present after high-stringency ~ 15 washing, suggesting that they are transcripts from the same gene. In contrast, no NMB-R mRNA was detected in poly (A)+
~i mRNA samples isolated from pancreas, the AR42J rat pancreatic acinar cell line, and Swiss 3T3 cells, each shown previously to ~; express GRP-R mRNA. No hybridizing mRNA species were detected by either the GRP-R or the NMB-R probe in mRNA samples from lung, thymus, and Balb 3T3 cells. These results show that the , cloned NMB-R mRNA reported in this study is expressed in the `1 brain as well as in the esophagus. NMB-R mRNA within the brain was localized to the olfactory bulb, a brain region reported to -~ 25 express relatively high levels of binding sites for NMB-~3' preferring bombesin receptor.

; EXAMPLE 19 NMB-R and GRP-R mRNA in Different Brain Reqions RNA blot hybridization studies on rat brain mRNA
using both the NMB-R probe and the Swiss 3T3 GRP-R probe , indicated that both bombesin receptor subtypes are expressed in the brain. NMB-R and GRP-R mRNA expression in the rat CNS was examined in more detail using in situ hybridization histochemistry to determine the correlation between regions expressing the specific cloned NMB-R and GRP-R genes, and regions shown in prevlous ligand binding autoradiographic studies to express brain bombesin binding sites. The method of , - ' : ~ : ~ ' `': ~ .. . .

:
WO92/16623 ~ PCr/US92/0209l Wada at al. (1990) J. Neurosci. 10:2917-2930 was used for situ hybridization. Briefly, adult male rats were fixed by perfusion with 4~ paraformaldehyde, 0.05% glutaraldehyde.
After perfusion, the brain was removed and placed in post-fix solution (4% paraformaldehyde plus 10% sucrose) overnight at 4 C. Sections (25 micron thick) were mounted on polylysine-coated slides and then treated with proteinase K (10 g/ml, 37 C, 30 min), acetic anhydride, and dehydrated by successive ` imme.rsion in 50~, 70%, 95%, and 100% ethanol. 35S-label~d sense or antisense cRNA probes (specific activity about 2 X 109 ; cpm per microgram) were synthesized from a pGEM-4 plasmid vector (Promega) containing a 2.0 kb cDNA fragment encoding - either the rat NMB~R or rat GRP-R subcloned in the polylinker region between the SP6 and T7RNA polymerase promoters.
Hybridizations were performed in 50% formamide, 0.3 M NaCl, 10%
dextran sulfate, 10 mM DTT at 55 C overnight, with a probe concentration of 5 X 106 cpm per ml of hybridization buffer.
Sections were then washed in a solution containing 4 X SSC (1 x - SSC = 150 mM NaCl, 15 mM NaCitrate pH 7.0) and l mM DTT at room `
;20 temperature, incubated with RNAse A (20 ~g/ml at 37 C for 30 min), and washed at room temperature with solutions containing progressively lower concentrations of SSC and 1 m~1 DTT, beginning with 2 X SSC and ending with 0.5 X SSC. A final high ~- stringency wash was performed in a solution containing 0.1 X
SSC and 1 mM DTT at 55 C for 30 min. Slides were dehydrated in 50~, 70%, 95%, and 100% ethanol and exposed to ~max film (Amersham) at room temperature for 3-7 days.
Probes were hybridized to coronal rat brain sections from the olfactory regions as well as thalamic and hypothalamic 1 30 regions where labeled bombesin and NMB binding were prominent ; in previous studies. Overall, NMB-R expression was most striking in the olfactory and central thalamic regions, while GRP-R expression was most prominent in the hypothalamus. More . `
detailed analysis of the sections showed the NMB-R mRNA
expression was highest in the anterior olfactory nucleus, tenia ~ ~
tecta, and piriform cortex. In addition, many other regions, `
including the accessory olfactory bulb, frontal cortex, ~ WO9~/16623 2 1 ~ ~ 3 ~ 6 PCT/US92/U2~91 thalamic nuclei (paraventricular, antero dorsal, centromedial, centrolateral, and rhomboid), dentate gyrus, amygdalopiriform nucleus, and dorsal raphe also expressed NMB-R. GRP-R mRNA
expression was highest in the suprachiasmatic nucleus, paraventricular nucleus, nucleus of the lateral olfactory - tract, magnocellular preoptic nucleus, and lateral mammillary nucleus. Moderate expression was seen in the bed nucleus of ~- the accessory olfactory tract, lateral hypothalamic area, supraoptic nucleus, dentate gyrus, field CA3 of Ammon's horn, isocortex, medial amygdaloid nucleus, and nucleus ambiguous.
These results show that NMB-R and GRP-R mRNAs are selectively expressed in different rat brain regions. Similar selective expression should be found in other species.

Isolation and Characterlzatlon of Human Genomic and cDNA GRP-receptor Clones ~`~ To determine the germline sequence of the human GRP-R, a placental genomic library was screened using the coding region of the Swiss 3T3 GRP-R cDNA as a probe.
Approximately 1 x 106 recombinants from a human-placenta genomic library (Stratagene, La ~olla, CA) were screened with a 32P-labeled Swiss 3T3 GRP-R probe containing `
` the coding region. Filter hybridization was at 37 C usin~ -previously described methods o~ (Davis et al. (1986). Filters were washed twice at room temperature for 15 minutes in 300 mM
`;~ NaCl, 30 mM NaCitrate, 0.1% sodium dodecyl sulfate (SDS), and at 50~ C twice for 15 minutes in 15 mM NaCl, 1.5 mM NaCitrate, :
~! 0.1% SDS. Positive clones were plaque purified and smallerii 30 hybridizing fragments subcloned into pGEM4 (Promega, Madison, WI) and sequenced.
; After identifying the 3'-untranslated region of the l genomic human GRP-R clone, a primer was synthesized from this iJ region and used to prime~ first strand cDNA synthesis from NCI-H345 oligo-dT cellulose s~lected mRNA by methods previously described in Davis et al. (1986). The NCI-H345 cell line is a GRP-responsive SCLC cell line, see Cuttitta et al. (198-5) . :' W092/16623 2 ~ ~ ~ 3 ~ ~ PCT/US92/020gl Nature 316:823-825. From this lihrary four positive c~ones were plaque purified and sequenced. The 1152 nucleotides determining the protein coding region sequence of these clones from SCLC were found to be identical to those of the exons found in the genomic human GRP-R sequence. This result indicates that the GRP-R protein coding sequence is unaltered in this SCLC cell line.
' The sequence of the human GRP-R coding region is illustrated in Table 2. The human GRP-R is contained in three exons, and the predicted amino acid sequence encodes a 384-amino acid protein which is identical in length to that which has been determined for the Swiss 3T3 mouse GRP-R. Comparison ` of the amino acid sequence derived from the human clone to that of the mouse Swiss 3T3 sequence demonstrated a 90% amino acid identity (vertical lines in Table 9). There is far less conservation at the amino terminus of the GRP-R protein between mouse and human ~Table 9). Hydropathy analysis of the ~ predicted human GRP-R protein, see Figure 13, reveals seven "! regions of hydrophobic amino acids, consistent with a seven ,, 20 transmembrane structure typical of G-protein coupled receptors (see Dohlman et al. (1987) Biochemistry 26:2657-2663). There are also four conserved consensus sites of potential protein kinase C phosphorylation (see Xishimoto et al. (1985) J. Biol.
~; Chem. 260:12492-12499; Woodgett et al. (1986) Eur. J. BiochemO
161:177-184) (asterisks over potential phosphorylation sites in `i Table 9~.
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WO92/16623 2 ~ O ~ 3 ~ ~ PCT/US92/02091 Table 9: Comparison of the derived amino acid sequencPs for the mouse Swiss 3T3 (upper sequence) and the human GRP-R (lower sequence). Overall amino acid identity was 90%, indicated by vertical lines. Numbered bold lines above amino acids show the location of seven predicted hydrophobic :transmembrane domains. Asterisks indicate conserved sites for protein kinase C phosphorylation.
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W0~2/i66~3 ~ 3 ~ 6 PCT/~S92/02091 EXAMPLE 2l Functional_Evaluation of GRP-receptor cDNA
To evaluate the function and pharmacology of the cloned NCI H345 human GRP-R cDNA, Xenopus oocytes were injected with an in vitro transcript encompassing the coding region of the NCI-H345 GRP-R cDNA.
Functional Expression of Human GRP-R in Xenopus Oocytes RNA was transcribed and capped in vitro from the GRP-R cDNA clone usiny T7 RNA polymerase as recommended by the manufacturer (Promega). Defolliculated oocytes were - microinjected with approximately l0 nanograms of mRNA per oocyte, and kept at 20 in ND solution of Lupu-Meiri et al.
(l9~39) Pfluqers Arch. 413:498-504. After 24 to 48 hours, oocytes were placed in a perfusion chamber and voltage clamped ` at a holding potential of -60 mV. Ligands were added directly ; to the chamber, and ligand dependent Cl- currents were measured.
GRP applied at nanomolar concentrations was shown to elicit a depolarizing response in oocytes injected with the transcript. This response was shown to be blocked by an antagonist specific for the GRP-R, ([D-Phe6]BN(6-l3) ethyl ester) at a l0:l molar ratio of antagonist:agonist. Taken ~; 25 together, these data indicate that the cDNA isolated from NCI-s ~ H345 does encode a functional GRP-R that is functionally and ;1 pharmacologically indistinguishable to that isolated from Swiss 1 3T3 cells.
, .1 .
~ EXAMPLE 22 Analysis of the Expression of GRP-receptor mRNA
by Northern blot and RNase protection analysis Expression of GRP-R mRNA was examined in the SCLC
;;~ 35 cell line, NCI-H345, by Northern blot analysis. The predominant hybridizing mRNA species in this cell line had an 1 estimated size of 3.l kb. The human GRP-R probe also `~ hybridized to two sizes of mRNA from Swiss 3T3 cells ~- (approximately 7.2 kb and 3.l kb). The level of GRP-R mRNA `

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W092/~6623 2 3 ~ 5 3 ~ ~ PCT/US92/02091 ~i observed in NCI-H345 was low, near the threshold of detection.
Since RNA bl~t analysis might fail to detect low but significant levels of GRP-R m~NA, a more sensitive RNase protection assay was used to detect GRP-R mRNA in a panel of SCLC and non-SCLC lung cancer cell lines.
Lung cancer cell lines were obtained ~rom Dr. J.
Minna and Dr. A. Gazda. These cells were established and typed histologically as described, e.g., in Carney et al. (1985) Cancer Res. 45:2913-2919; Brower e,t al. ~1986) Cancer Res 46:798~806; Carmichael et al. (1988) Br. J. Cancer 58:437-440;
Harbour et al. (1988) Science 241:353-357; and Takahashi et al.
(1989) Science 246:491-493. Total ~NA was isolated from cells using guanidine thiocyanate homogenization and CsCl gradient '` purification as described by Davis et al. (1986). The probe ' ' 15 for this assay was transcribed with T7 polymerase from a Bql ', II-Hind III 600 bp genomic fragment cloned into pGEM4 according ,-', to the manufacturers directions (Promega). DNA template was - removed by digestion with 5 units RQl DNase (Promega).
Unincorporated nucleotides in the resulting reaction were ~ 20 removed by multiple ethanol precipitations and the resulting '~ pellet was resuspended in 10 mM TRIS-HCl, pH 7.4: 1 mM DTT.
, The probe was diluted'to a concentration of 2.5 x 105 cpm/~l. , RNA samples to be~hybridiæed (30 ~1) were dried and resuspended in 50 ~1 hybridization mix (20 mM TRIS-HCl, pH 7.4; 500 mM
NaCl; 2 mM EDTA; 78~ formamide; 1 ~lj 2.5 x 105 cpm GRP-R
~- probe). The samples were heated to 80 C for 2 minutes and ' ,,i hybridized 16-18 hours at 43 C.
Unprotected RNA was digested in a reaction consisting ' of 88 units RNase A (United States Biochemical); 20 mM TRIS-;~! 30 HCl, pH 7.4; 300 mM NaCl; and 1 mM EDTA in a final volume of 350 ~1 at 37 C for 30 minutes. The reaction was then made `
l 0.5% in SDS and 0.05 ~g of proteinase K (BRL) was added and ' ,, incubated at 37 C~for 15 minutes. The reaction was then extracted with phenol/chloroform and ethanol precipitated. The ,~ 35 pellet was collected by centri~ugation and resuspended in 5 ~1 i of the following so}ution: 80~ formamide; 50 mM TRIS; 50 mM
~ borate; 11 mM EDTA; O.l~Bromophenol Blue; 0.1% Xylene Cyanol.
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WO92/16623 2 ~ ~ ~ 3 ~ 6 PCT/US92/02091 Samples were denatured for 2 minutes at 95 C prior to electrophoresls on a 6% denaturing polyacrylamide gel. The gel was dried an~ exposed to X-ray film in the presence of an intensifying screen.
- 5 The GRP-R probe used above was derived from a human genomic GRP-R clone which included 299 bp of exon 2 (nucleotides 465-764, Table 2) and extended 301 bp into the second intron. Accordingly, the probe would be protected from ~; ribonuclease digestion by a 299 base region of the GRP-R mRNA.
` 1 0 , , - G~P-R mRNA was detected in cell lines from all ` histological types of lung carcinoma examined, but not all members of any one histological group were found to express GRP-R mRNA. Data from various lung carcinoma cell lines is " 15 summarized in Table lO. A representative autoradiograph of the assay results is shown in Figure 19 and described in more - detail in Example 26. Additionally, the level of GRP-R message varied among expressing cell lines. The highest level of expression was found in the SCLC cell li~e NCI-H345.

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; W092/1~623 2 1 0 ~ 3 ~ ~ PCT~US92/02091 Table 10: GRP- and NMB- receptor mRNA
levels in lung cancer cell lines as determined by RNase protection assay.
Signal strength on resulting autoradiogram was assessed and assigned an arbitrary value relative to other cell lines. See also description in Example 26.
~ .
Cell line and morpholoqical type GRP-receptor NMB-receptor ; , ~ . ............................. .
Small Cell Lung Carcinoma NCI-H60 +
NCI-H69 tr NCI-H146 tr NCI-H209 - ++
NCI-H345 -~+ ++

~ NCI-N510 tr +
: NCI-N592 +
NCI-H889 +
NCI-H1092 +

` Carcinoid ~-~ NCI-H720 + -l NCI-H727 +
Non-Small Cell Lung Carcinoma NCI-H23 - - .
, NCI-H125 tr -i, NCI-H157 ` - --i NCI-H226 NCI-H322 +

NCI-H520 +

', NCI-H1299 +
NCI-H1373 +
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WO 92/166~3 2 ~ 0 5 3 ~ ~ PCr/US~2/02091 EX~E~
Pharmacolo~fical Evidence for Distinct Rece~tors for Bombesin-like Pe~tides Bombesin-like peptides induce an increase in intracellular calcium in the NCI-H345 cell line. Bombesin-stimulated Ca2~ mobilization studies were performed in the human lung carcinoma cell line NCI-H345 using Quin 2-~- fluorescence in order to determine if one or more bombesin receptor subtypes could be active in these cells.
- 10 NCI-H345 SCLC cells were cultured in SIT medium (RP~I
1640, (GIBCO) with 10 mM HEPES (pH 7.4) and 30 nM sodium selenite, 5 ,ug/ml insulin, and 10 ,ug/ml transferrin~. Cells were washed three times in 0.015 M NaPO4, 0.15 M NaCl, 0.01 M
HEPES, pH 7.4, and once in SIT medium. The washed cells were suspended in SIT medium at 1 X 107 cells/ml. These cells were incubated with 5 ,uM of quin-2-acetoxymethyl ester (quin-2;
Molecular Probes, Eugene, OR) at 37 C for 90 minutes. After incubation the cells were washed once and resuspended in SIT
medium without quin-2 at 1 X 107 cells/ml. Approximately, 5 X
106 cells were pelleted and resuspended in 2 ml of HEPES
buffered saline (140 mM NaCl, 5 mM KCl, 1 mM CaC12, 1 mM MgC12, 5 mM glucose, and 20 mM Hepes, pH 7.4) in an Elkay Lab Systems acrylic "ultra-W", four-sidedj 10 mm, 4.5 ml cuvette. Using a Perkin Elmer L5B Luminescence Spectrometer, with an excitation wavelength of 339 nm and an emission wavelength of 492 nm (slits = 5 nm) the change in fluorescence after addition of llgand was measured. The cells in the cuvette were kept at a constant temperature (37 C) and were continuously suæpended with a magnetic stirrer while the fluorescent measurements were taken. Ligand was added to the cells when a stable - fluorescence reading was obtained, usually within 5 minutes.
The inhibitor ~D-Phe6]BN(6-13) ethyl ester was added to the cells five minutes prior to the addition of ligand.~ To ~, determine total~ [Ca2+~]i the~ cells were lysed by addition of 10 JI~35 ~ ~1 1096 Triton-X to obtain~ Fmax. Then, 100 ~1 0.4 M EGTA was added to the cuvettes to determine the fluorescence background (Fmin)~ The [Ca2+]i was calaulated from thè fluorescence measurements using the~;formula:

WO92/166~3 2 i ~ ~ 3 0 ~ PCT/~S9~/02091 (F observed F minimum) X 115 nM
[Ca2'~]i = ~~~~ ~
(Fmaximum F observed) 5 Both bombesin and NMB elicited an immediate calcium response in these cells (Figure 14). In several experiments, ~` the increase in intracellular calcium mediated by NMB was consistently more sustained than that elicited by Tyr4-bombesin. The increase in intracellular calcium was detected at <l nM concentrations of NMB agonist, and maximal at about loo nM for both NMB and Tyr4-bombesin. Either peptide alone could elicit a detectable response at between l and lo nM
levels (Figure 15). These observations indicate that at least . .
part of the calcium mobilization response is mediated by a bombesin receptor subtype that binds NMB at high affinity, ~; pharmacologically similar to the esophageal NMB-R.
Figure 16 shows that approximately 50% of the ~, increase in intracellular calcium elicited by Tyr4-bombesin is - blocked by the GRP-receptor specific antagonist, [D-Phe6]BN(6-13) ethyl ester at 30 nM concentrations, whereas further inhibition of the BN-mediated calcium response is complete only A! after the addition of 1000 to 10,000 nM antagonist. The NMB-i elicited calcium response was insensitive to the antagonist ~ (minimal effects on calcium response at >lO00 nM
;3, 25 concentrations, as shown in Figure 16). These data further . demonstrate that the calcium response to bombesin-like peptides in NCI-H345 is mediated by at least two distinct receptors, and that both the human GRP-preferring and NMB-preferring bombesin receptors are expressed and functional in human lung carcinoma , 30 cells.
Bombesin-like peptides are expressed in human SCLC
:l and are thought to function as autocrine growth factors. These ~3, results show that the SCLC cell line NCI-H345 expresses two pharmacological~ly distinct bombesin-peptide receptors one of which is GRP-preferring and blocked by the antagonist, [D-, Phe6~BN(6-13) ethyl ester and the other which is NMB preferring ~ and was not blocked by the antagonist. A subset of lung ', - ,~

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~' W09~ 623 2 1 ~ ~ 3 ~ 6 PCT/US92/~2~91 carcinoma cell lines examined express either receptor, or both receptors, at levels detectable by a sensitive RNase protection assay, but often below the level of detection by Northern blot analysis of total RNA, see discussion below, Example 26. The low levels of GRP-R and NMB-R mRNA are consistent with bombesin ligand binding studies in lung carcinoma cell lines which showed less than 5000- receptors per cell.

Isolation of Human NMB Receptor The bombesin-stimulated calcium mobilization properties indicated that more than one bombesin receptor subtype exists in NCI-H345. Thus, distinct human GRP-R and NMB-R receptor cDNA clones should be isolatable using murine Swiss 3T3 GRP-R cDNA, see Battey et al. (1991) Proc. Natl.
Acad. Sci. USA 88:395-399, or rat NMB-R cDNA, see Wada et al.
(1991) Neuron 6:421-430, as probes. The isolation of human GRP-R is described in Example 20. Briefly, human genomic NMB-R
clones were isolated from both placental and peripheral blood genomic libraries to compare the sequence of receptor cDNA
clones derived from the NCI-H345 tumor cells with their normal genomic counterparts.

Isolation of human aenomic and cDNA clones Approximately 1 X 106 recombinants from a human-placenta genomic library (Stratagene, La Jolla, CA) and a ; ~ human-peripheral blood genomic library (Promega, Madison, WI) were screened with a 32P-labeled rat neuromedin-B probe containing the coding region. The general procedure described above, see Example 20, for ~isolating the human GRP receptor was followed.
To obtain a human neuromedin B receptor cDNA, ;~ oligonucleotldes~5~' sense primer: 5'GTGGGCGTTCAGTCCTCAGG 3';
3' antisense primer: 5'GTTCTCTCCAGGTAGTGAGTT 3') complementary to sequences from the 5~'- and 3i-untranslated domains that immediately flank the~coding region were synthesized for use as polymerase chain~rea~tion~ PCR)~primers. These primers were - , :

; W092/~6623 2 ~ ~ ~ 3 ~ ~ PCT/US92/~2091 then used in PCR with 20 ng hexamer primed cDNA template reverse transcribed from poly-A~ NCI-H345 mRNA. Buffers and nucleotides were provided in the GeneAmp PCR kit (Perkin-Elmer). The cycling conditions were: 94 C, 1 min; 60 c, 1 min; 72 C, 2.5 min. for 40 cycles. The ends of the resulting products were polished with T4 DNA polymerase, and the 5'-ends phosphorylated with T4 polynucleotide kinase to allow subcloning into the 5' dephosphorylated Sma I site of pGEM-4.
Positive colonies were identified by hybxidization to the rat neuromedin-B receptor probe. Two clones were sequenced.
The entire amino acid coding sequences of the human NMB receptor genomic clones were sequenced on both strands ~` using gene-specific synthetic oligonucleotide primers. See Table 4. Nucleotide sequence analysis was performed using the Sequence Analysis Software Package (Pepplot program for the hydropathy analysis) of the University of Wisconsin Genetics Group and a VAX computer. See Devereux et al. (1984) Nucleic Acids Res. 12:387-395. A hydropathy analysis is shown in Figure 17.
The human G~P-R coding region is contained in three exons, and the predicted amino acid seguence encodes a 3$4-amino acid protein as described above. The human NMB-R is also ~1 contained in three exons, and the predicted amino acid sequence - encodes a 390-amino acid protein. Analysis of two NMB-R cDNA
clones isolated from NCI-H345 revealed that the protein coding -region sequence of these clones was identical to the sequence of the exons found in the human yenomic NMB-R gene. A similar . compaxison of GRP-R sequences from normal and SCLC cell lines '-! is reported above, and shows the same identity. Thus, neither the GRP-R or NMB-R protein coding sequence is structurally altered by somatic mutation in this SCLC cell line.
'! Molecular Genetic AnalYsis of human GRP-R and NMB-R
` Both human GRP-R and NMB-R coding regions show high amino acid identity with their rodent counterparts (GRP-R 90 identity, NMB-R 89~ identity). Hydropathy analysis of the ~ predicted GRP-R and NMB-R proteins reveals seven regions of .~t~ hydrophobic amino acids (Tables 2 and 4; Table 11, boxes) i .

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WO92/]6623 2 ~1 ~ 5 3 ~ 6 PCT/US92/02091 consistent with a seven transmembrane structure typical of G-protein coupled receptors. Comparison of the human GRP-R and NMB-R sequences indicates 55~ identity at the amino acid level (vertical lines, Table 11). There are also two consensus sites of potential protein kinase C phosphorylation in bo~h GRP-R and NMB-R (dotted outline boxes enclose potential phosphorylation - sites in Table 11~. Of interest, the two introns that divide the protein coding region are found in analogous locations in both the GRP-R and NMB-R genes (Table 1 and 4), suggesting that both receptor genes evolved by duplication of a common ancestor.
Several structural features of the human GRP-R and NMB-R
are worthy of note. Comparison of the predicted amino acid sequences of human GRP-R and human NMB-R (Table 11 shows that 15 the third transmembrane domain is extremely well conserved between these two receptor subtypes; 95% identical in this region versus 55% identity for the entire amino acid sequence)O
In contrast, this domain is not particularly well conserved (<25~ identity) when compared to-other known G-protein coupled ~0 receptors. These results suggest that this region may be involved in ligand binding, or other functional properties that would be expected to be similar among closely related receptor subtypes but not common to all members of the G-protein coupled receptor family. The genomic sequences of NMB-R and GRP-R show that the first intron is located at the same position in both genes, immediately carboxy-terminal to the third transmembrane domain (Tables 2 and 4; Asp _Eg Tyr). Several other intron-containing G-protein coupled receptor genes, e.g., substance P
receptor, D2 and D3 dopamine receptors, and opsins, also contain an intron at this location, e.g. Asp Arq Tyr. This conserved structural feature suggests that these members of the . ~-G-protein coupled receptor superfamily evolved from a common ancestor.
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Table 11: Comparison of the derived amino acid sequences from a human GRP-R (upper se~uence) and a human NMB-R
(lower sequence). Overall amino acid identity was 55%
(indicated by vertical lines). Shaded boxes indicate the location of seven predicted hydrophobic transmembrane domains.
Dotted-outline boxes enclose conserved potential sites of protein kinase C ph~sphorylation.

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WO92/1SS23 PCT/US92/02~91 2 ~ 3 ~ 5 EX~MPLE 25 Functional Comparison of Cloned Human `. GRP ReceE~or and NMB Receptor To evaluate the functional properties and pharmacology of the cloned NCI-H345 human GRP-R and NMB-R, XenoPUs oocytes were injected with an ln vitro transcript encompassing the coding region of either the N~I-H345 GRP-R or NMB-R cDNA.
`` 10 RNA was transcribed and capped ln vitro from the GRP-R and NMB-R cDNA clones using T7 or SP6 RNA polymerase as ~ recommended by the manufacturer (Promega). Defolliculated ; oocytes were microinjected with approximately 10 nanograms of mRNA per oocyte, and kept at 20 C in ND solution (96 mM NaCl, 2 mM KCl, 1 mM MgC12, 5 m~ Na+HEPES, 1.8 mM CaC12). After 24 to 48 hours, oocytes were placed in a perfusion chamber and voltage clamped at a holding potential of -60 mV. Ligands were added directly to the chamber, and ligand-dependant Cl currents were measured.
!, 20 In oocytes injected with approximately 10 ng of the ; GRP-R transcript, GRP applied at 10 8 M concentration consistently elicited a depolarizing response which was greater in magnitude than the response to 10 8 M NMB (Figure 18A).
This response was blocked by an antagonist specific for the GRP-R, (~D-Phe6]BN(6-13) ethyl ester) at a 10:1 molar ratio of . antagonist:agonist as shown in Figure 18A. In contrast, 5~ oocytes injected with NMB-R transcr~ipt showed a greater response to 10 8 M NMB than to an equivalent concentration of ~; GRP (Figure 18B). The responses of oocytes injected with NMB-R
- were not blocked by that GRP-receptor specific antagonist, ~D-Phe6]BN(6-13) ethyl ester (Figure 18B). These results are consistent with previous studies o~ rodent bombesin receptor .
¦ subtypes. The oocyte expression studies of cloned GRP-R and NMB-R isolated from NCI-H345 are consistent with the properties 35 of the Ca2+ response el~iclted by bombesin peptide agonists in-intact NCI-H345 cells, where both an antagonist-sensitive I response to bombesin and an antagonist-insensitlve NMB response ~ were observed~(Figure 14).

1 . .

WO92/166~3 2 l a ~3 ~ PcT/US92/020 Repeated application of bombesin peptide agonists results in a rapid desensitization of the responses mediated through either the GRP-R or NMB-R expressed in Xenopus oocytes, or the calcium mobilization response to bombesin observed in NCI-H345. In a previous study of bombesin receptor function in SCLC, the phorbol compound PMA, which activates protein kinase C (PK-C), had no effect on the intracellular Ca~+ concentration in the SCLC cell line NCI-H345, but attenuated the bombesin-stimulated increase in intracellular Ca++. It has been demonstrated that the early cellular responses following stimulation of the Swiss 3T3 GRP receptor by ligand included activation of protein kinase C, as demonstrated by bombesin-stimulated phosphorylation of an 80 kDa protein substrate for PK-C. Taken together, these observations suggest that bombesin receptors are phosphorylated at PK-C recognition sites present in the receptor protein after receptor activation, and that phosphorylation of these sites may desensitize the receptor to subsequent activation. Notably, two consensus PK-C
phosphorylation sites are conserved in both the human GRP-R and ~;
NMB-R sequences (Table 11, dotted outline boxes) in segments of , the protein predicted to be intracellular tthird cytoplasmic ' loop and carboxy terminal domain).
PK-C mediated phosphorylation of one or both of these ~'~ sites may provide a mechanism to transiently desensitize the receptor. Studies using site-directed mutagenesis of the GRP-R
cDNA and NMB-R cDNA to alter these sites are described in Example 28, below.

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WO92/16623 2 1 0 ~ 3 ~ 6 PCT/US92/02091 RNAse Protection Analysis Comparinq the Expression of NMB-R mRNA in Lunq Carcinoma Cells Since NCI-H345 lung carcinoma cells express both functional GRP-R and NMB-R, the patterns of expression for both receptors in a panel of other lung carcinoma cell lines were also examined. GRP-R and NMB-R mRNAs are relatively rare transcripts in NCI-H345 mRNA, detectable by RNA blot analysis only after long autoradiographic exposures. To detect low but significant levels of GRP-R mRNA and NMB-R mRNA, a more sensitive RNase protection assay as described in Example 22 was used to analyze Iung carcinoma mRNA samples for expression of these peptide receptors.
Northern Blot Analvsis Total RNA (10 ~g) was resolved by electrophoresis on agarose/formaldehyde gels, and blotted to nitrocellulose membranes using methodology of Davis et al. (1986). After ~ baking at 80 C, membranes were hybridized to a 32P-labeled `i human beta-actin fragment that contained the entire coding region. Blots were washed at high stringency (65 C in 15 mM
NaCl, 1.5 mM sodium citrate, 0.1% SDS, for two cycIes of 15 ~;1 minutes each).
RNAse Protection Assay The assay was performed according to the procedure described above for the GRP-R transcrip~s.
Lung cancer cell lines were obtained from Dr. J.
Minna and Dr. A. Gazdar. Total ~NA was isolated from cells ~ using guanidine thiocyanate homogenization and CsCl gradient ; purification according to Davis et al. (1986) and as described above. The NMB-R probe was a 400 bp Hind III genomic fragment.
The fragment was cloned into pGEM-4 and transcribed according ~`
to the manufacturers directions (Promega). DNA template was r~ removed by digestion with 5 units RQ1 DNase (Promega). `
Unincorporated nucleotides in the resulting reaction were1 35 removed by multiple ethanol precipitations and the resulting pellet was resuspended in 10 mM TRIS-HCl, pH 7.4; 1 mM DTT.
The pro~e was diluted to a~concentration of 2.5 x 105 cpm/~
RNA samples to be hybridized (30 ~) were dried and resuspended ~ ~ :

.3 W092/1~6~3 ~ 3 ~ 6 PCT/US92/~2091 ~, in 50 ~1 hybridization mix (20 mM TRIS-HCl, pH 7.4; 500 mM
NaC1; 2 mM EDTA; 78% formamide; 1 ~1, 2.5 x 105 cpm NMB-R
probe). The samples were heated to 80 C for 2 minutes and hybridized 16-18 hours at 43 C.
The NMB-R probe used in the RNase protection assay was an approximately 400 bp Hind III fragment of the human genomic NMB-R clone that contained a portion of the second intron and extended 219 bp (nucleotides 771-gso, Table 4) into the third exon. Therefore the probe would be protected by a 219 base region of the NMB-R mRNA.
A representative autoradiograph indicating the results of this assay is shown in Figure 19 (Fig l9A, GRP-R;
Fig l9B, NMB-R) and the data from all lung carcinoma cell lines examined are summarized in Table 10. GRP-R mRNA was detected in 10 of 22 cell lines fram all histological types of lung carcinoma examined. See Table 10. Not all SCLC cell lines express GRP-R (4 of 7). Additionally, the level of GRP-R mRNA
varied among expressing cell lines. The highest level of -3 expression was found in the SCLC cell line NCI-H345. NMB-R
expression was expressed in 5 of 22 lung carcinoma cell lines, with highest levels found in NCI-H209. Expression of one receptor subtype did not exclude expression of the other subtype; both SCLC line NCI-H345 and NCI-HS10 express both GRP-R and NMB-R mRNA.
s 25 Molecular genetic studies of the structure of growth regulatory genes in human lung cancer cells frequently showed i evidence of somatic mutation or gene deletion which alters the s regulation or function of the encoded protein. The nucleotide sequence of several GRP-R and NMB-R cDNA clones isolated from the SCLC cell line NCI-H345 are identical to the sequence of ' the respective genomic clones for these receptors throughout ¦ the protein coding region. Thus, the GRP-dependent growth stimulation observed in lung cancer cells does not require a ~;; structural change in the GRP-R protein or in the NMB-R protein, ~`
~35 i.e., the natural receptor is present and expressed.
¦ Instead, it seems more likely that malignant cells may be stimulated to grow by the normal intracellular signals , ~ ',~

W092/16623 21~ ~ 3 0 6 PCT/US92tO2091 evoked by ligand-dependant activation of bombesin-like peptide receptors. It has been reported that many different putative G-protein coupled neuropeptide receptors, e.g., vasupressin, bradykinin, cholecystokinin, galanin, and neurotensin, can transiently increase intracellular calcium in SCLC. A previous study shows that individual SCLC cell lines have great heterogeneity in response to a particular neuropeptide, but great similarity in possessing the capacity to increase intracellular calcium in response to at least one neuropeptide.
Receptors for these neuropeptides are all G-protein coupled, and potentially activate a similar signal transduction pathway which may be important to the growth or cellular economy of SCLC.
The antagonist [D-Phe6]BN(6-13) ethyl ester at 500 nM
concentrations only partially inhibits the calcium response elicited by 50 nM [Tyr4]BN in NCI-H345 SCLC cells, consistent with the conclusion from molecular genetic studies that the bombesin response is mediated by both the antagonist sensitive GRP-R and the relatively insensitive NMB-R. It is noted that very high concentrations of antagonist (10 nM) can completely -i block the NCI-H345 calcium response to 50 nM ~Tyr~]BN, while similar high levels of antagonist do not block responses ~,; elicited from the cloned NMB-R expressed alone in Xeno~us oocytes under similar circumstances. The explanations for this ~ 25 difference in sensitivity is not clear at present. The co-; expression of GRP-R and NMB-R in some way probably increases ~ the antagonist sensitivity of the NMB-R mediated calcium -l response to [Tyr4]BN in the NCI-H345 cells. Additional studies of the nature of responses elicited by bombesin peptides in cells expressing both GRP-R and NMB-R will determine whether or~
not the two receptors appear to generate responses independently, or interact in some more complex fashion.
Although GRP ligand expression is confined to SCLC
~` cell lines, GRP-R and NMB-R mRNA expression is not restricted to SCLC lung carcinoma cell lines. Since these non-SCLC cell ¦ lines do not express preproGRP mRNA, autocrine growth stimulation of the GRP-R seems unlikely in these non-SCLC cell i : ~
1( ~ ~ .. ' .

WO92/16623 2 i O ~ 3 0 ~ P~T/US92/02091 lines. Elevated levels of bombesin-like peptides have been - noted in the bronchial secretions of heavy smokers. Bombesin-; like peptides synthesized by other cells in the lung known to ~ -express GRP, e.g., pulmonary endocrine cells, are likely to act in a paracrine fashion to stimulate the growth of some non-SCLC
tumors expressing bombesin receptors. GRP-R expression is probably important at some stage in the pathogenesis of these particular non-SCLC tumors. Reversal or blockage of these tumors may result upon therapeutic administration of various reagents made available herein.
`~ At least one SCLC line (NCI-N417) reported to show `~; bombesin-dependent growth expressed no detectable mRNA for either GRP-R or NMB-R. This result might be due to the fact that GRP-R and/or NMB-R mRNA is present, but below the level of detection by RNase protection assay. An alternate explanation is that these cells express a bombesin receptor subtype that has not yet been identified. Probes to isolate such receptors are provided herein, and methods for their use are described, e.g., in Example 29.
EXAMPLE_28 l Mutaqenesis of GRP-R or NMB-R
J`j In vitro or site directed mutagenesis methods are described in standard references, see e.g., Sambrook et al.
(1989) or Ausubel et al. (1987 and Supplements), each of which is incorporated herein by reference. Mutagenesis may be directed towards analysis of varioùs different activities and functions of the receptors. In particular, mutagenesis of ~, post-translational modifications sites is of interest to determine, e.g., the effect of glycosylation on various activitiés. Fusion proteins will be made by standard ~:
techniques, typically by recombinant methods. Mutagenesis or replacemenk of segments homologous to identified phospAorylation sites of other ~-protein linked receptors wilI
be performed. Activities of interest include ligand binding, G-protein linkage, phosphorylation activities, and Ca++
sequestration. Standard assays for each activity are known and ~ 9~/16623 2 ~ O ~ 3 ~ ~ PCT/US92/02091 will be used to specifically identify the structural features which correlat~ with them.

Isolation of Homoloqous Receptors The present invention provides at least four full length probes for additional recPptors for bombesin-like peptides. In particular, genes for a mouse GRP receptor, a rat neuromedin B receptor, and human GRP and NMB receptors are provided. These nucleic acids, or fragments thereof, can be used alone or in combination to screen other DNA sources for sequences having various levels of homology. In particular, the third transmembrane segment has shown high homology among the various receptors for bombesin-like peptides, but other fragments may also be used. Low stringency hybri~ization of GRP-R and NMB-R probes to Eco RI digested human genomic DNA -shows at least six novel fragments which hybridize to either or both probes, but are not the earlier identified human GRP-R or NMB-R gene. See Figure 20. These fragments likely encode exons of additional receptor subtypes for bombesin-like peptides. Genomic cloning, sequencing, and analysis of expression, as applied above, will establish the nature of these hybridi~zing fragments.
Fifteen micrograms of human genomic DNA were cut with Eco RI, and the fragments resolved by electrophoresis and capillary transferred to nitrocellulose. The nitrocellulose filter was hybridized to a mouse GRP-R cDNA probe (comprising the entire open reading frame of the cDNA) labeled by nick translation to a specific activity of about 300 cpm/pg.
Hy`oridization buffer was 40% formamide, 5X SSC, 20 mM TRIS, l X
Denhart's solution, 20 micrograms per ml denatured salmon sperm DNA, lO6 cpm/ml denatured labeled probe. The hybridization was incubated overnight :at 37 C. The filter was washed twice in 2 X~SSC, 0.1% SDS at room temperature, and twice for fifteen ~ ;
minutes in O.l X SSC, 0.1% SDS at 37 C. The blot was exposed `-to XAR-5 film for several days. Six novel bands are detected, see Figure 20.

~ : -:
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W092/~66~3 2 l ~ ~3 a~ PCT/US92/0209 Based upon the positive hybridization results on the filter, conditions ~or a library screen were determined and clones isolated. The sequence of one isolated clone is presented in Table 12. The nucleotide sequence is entered as 5 SEQ ID NO: 9 and the corresponding amino acid sequence is SEQ
ID NO: 10. This receptor gene sequence has about 60%
nucleotide homology with human RlBP, and its corresponding amino acid sequence has about 50% amino acid identity. Table 13 presents an amino acid sequence comparison between the two.

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WO~/16623 ~ $ PCT/US92/02091 Table 12: A nucleotide sequence of a human R3BP. The putative coding region has an initiation codon which begins at nucleotide 172 and a termination codon UGA which ends at nucleotide 1371.

~ 10 151 AAGACACAGT CTTCAGAAGA AATGGCTCAA AGGCAGCCTC ACTCACCTAA
: 201 TCAGACTTTA ATTTCAATCA CAAATGACAC AGAATCATCA AGCTCTATGG

!
. 451 CTTTTACTTC TGCTAACTTG TGTGCCAGTG GATGCAACTC ACTACCTTGC

.. . .

,'! 601 GACAGATACA AGGCAGTTGT GAAGCCACTT GAGCGACAGC CCTCCAATGC :~
.

701 TATTTGCTCT ACCTGAGGCT ATATTTTCAA ATGTATACAC TTTTCGAGAT : -~7` 751 CCCAATAAAA ATATGACATT TGAATCATGT ACCTCTTATC CTGTCTCTAA : .
. 801 GAAGCTCTTG CAAGAAATAC ATTCTCTGCT GTGCTTCTTA GTGTTCTACA
? :

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lOOl TGGCTCTGTT TGCCCTCTGC TGGTTGCCAA ATCACCTCCT GTACCTCTAC

~ 1251 CTCTCTTACC ACCCTGGCTG TGATGGGAAC GGTCCCGGGC ACTGGGAGCA
j 1301 TACAGATGTC TGAAATTAGT GTGACCTCGT TCACTGGGTG TAGTGTGAAG

;. 10 1451 TTTTTGTTGT TTGAAAAGTG TGTTGAAATC TTAGGAGTGA AGGATCCCTA
15Ql TAAGTAAGTA AAATACAAAC CATTACTTTC TTCAAAGTAC AAATAGTAAT
`1551 GTCATCGGCT TCTAATAAAT GAGCCCACTA GTGCAGAAAG ACAGTTTATA
`' 1601 TATGCC .
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WO 92tl66'3 21~ 5 ~ ~ ~ pcT/us92/o2n9l .,; , Table 13: A comparlson of amino acid sequences of human R3BP and human RlBP (GRP-R). rrhe R3BP is a~ove, RlBP is below.

1 .M~QRQPHSPNQTLISITNDTESSSSMVSNDNTNKGWSGDNSPGIEALCAI 50 . -~ MALNDCFLLNL~.VDH~MXCNISSHS.. ADLPVNDDWS... HPGI.. LYVI 43 .. . . . ...
51 YITYAV IaVGILGNAILIKVFFKTKSMQTVPNIFITSLAFGDLLLLLTC 100 44 PAVYGVIILIGLIGNITLIKIFCTVKSMRNVPNLFISSLALGDLLL~ITC 93 .. . . .
101 VP~DATHYLAEGWIFGRIGCKVLSFIRLTSVGVSVFTLTILSADRYKA W 150 .~
' .IIII.:II1: 111111111::.II.IIIII1.111111 11111111:1 ,.

151 KPLERQPSNAILKTCVKAG~VWIVSMIFALPEAIFSNVYTFRDPNKNMTF 200 144 RPMDIQASHALMKICLKAAFIWIISMLL~IPEA~ SDLHPF~EESTNQTF 193 201 ESCTSYPVS ~ LQEIHSLLCFLVFYIIPLSIISVYYSLIARTLYKSTLN 250 ::

, .
251 IPTEEQSHU~KQIESRXRIARTVLVLVALFALCWIPNHLLYLYHSFTSQT 300 244 LPVEGNIHV~CKQIESRKRLAKTVL~FVGLFAFCWLPNHVIYLYRSYHYSE 293 301 YVDPSAMHFIFTIFSRvLAFsNsCvNPFALywLsKsFQKHFKAQLFccKA 350 --11 1 11: .1 .I:lll.lllllllllt:lllll.l:l-.ll:ll-: -299 .VDTSMLHFVTSICARLLAFTNSCVNPFALYLLSKSFRRQFNTQLLCCQP 342 .. . . ..
3jl ERPEPPVADTSLTTLAVMGTVPGTGSIQMSEISVTSFTGCSVKQ~EDRF* 400 343 GLIIRSHS5GRSTT....... CMTSLRSTNPSVATFS~INGNICHERY. 383 1 ; , 1, :

WO9~ 623 ~ P~T/US92/02091 Amplification methods, e.g., polymerase chain reaction techniques, may also be used with these probes to isolate and purify additional receptors.
Alternatively, other screening methods using antibodies or activity assays will be used to verify or assist in the isolation of new receptors. Expression of receptor may ; be screened by antibodies or endocrine stimulation of cells expressing the appropriate receptor sequences.
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and -modifications can be made thereto without departing from the spirit or scope of the appended claims.

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W~92/16623 PCT/~S9~/02091 21~3~

Table 14: In the claims, the ~ollowing SEQ ID NO:
correspondences are intended: .
: ' .
SEQ ID NO: corresponds to which is :
' 1 Table 1 mouse RlBP tGRP-R) nucleic acid 2 Table 1 mouse RlBP (GRP-R) amino acid :
. 3 Table 2 human RlBP (GRP-R) nucleic acid 10 4 Table 2 human RlBP (GRP-R) amino acid ; 5 Table 3 rat R2BP (NMB-R) nucleic acid 6 Table 3 rat R2BP (NMB-R) amino acid 7 Table 4 human R2BP (NMB-R) nucleic acid

8 Table 4 human R2BP (NMB-R) amino acid 15 9 Table 12 human R3BP nucleic acid ~` 10 human R3BP amino acid :~ ..` ' ,. '' .~. .

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W O 92/16623 ,.j PCT/U~92/02091 SEQUEtNCE LISTING .

(1) GENERAL INFORMATION:
(i) APPLICANT: Battey Jr., James F.
Corjay, Nartha H.
Fathi, Zahra : Feldman, Richard I.
Harkin~, Richard No Sla~tery, Timothy K.
Wada, Etsuko WU, JamQ~ M. .
: 15 (ii) TITLE OF INVEtNTION: RECEPTORS FOR BOMBESIN-LI~E PEPTIDES
(iii) NUMBER OF SEQUENCES: 10 :
:~ (iv) CORRESPONDENCE ADDRESS: .:.
IA) ADDRESSEE: Edwin P. Chinq (B) STREET: 1501 Harbor Bay Parkway :
~C) CITY: Alam~da :~ (D) STATE: CA . .
~E) COUNTRY: USA
(F) ZIP: 94501 (v) CO~PUTER READABLE FORM:
. (A) MEDIUM TYPE: Floppy di~k -~ (B) COMPUTER: IBM PC compatible .i 30 (c) OPERATING SYSTEM: PC-DOS/MS-DOS
~ (D) SOFTWARE: P~tentIn Rel~a~e ~1.0, Ver~ion ~1.25 .!1 ' .
(vi) CURRENT APPLICATION DATA:
,~ (A) APPLICATION NUMBER:
~, 35 ~B) FILING DATE:
:,tj (C) CLASSIFICATION: -t . : (vii) PRIOR APPLICATION DATA:
,t (A) APPLICATION MUMBER: U5 07/426,150 t 40 . (B) FILING DATEo 24-OCT-1939 .~
. (vii) PRIOR APPLICATION DATA:
,.J (A) APPLICATION NUMBER: US 07/533,659 (B) FILING DATE: 05-JUN-1990 viii) ATTORNEY/AGENT INFO~MATION:
~A) NAME: Ching, Edwin P.
' (B) REGISTRATION NUNBER: 34090 ..
tC) REFERENCElDOCRET NUMBER: A-0092C

ix) TELECOMMUNICATION INFORWAiTION~
' ~A) TELEP~ONE: 415-266-7476 .~ (B) TELEFAX: 415-266-7400 -~
:3 ~ ::

~ 55 : ": ~ .:
, , :

W O 92/16623 2 1 ~ 5 3 ~ ~ PC~/U~92/020gl (2) INFORMATION FOR SEQ ID NO:l:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 1700 ba~e p~ir~
( B ) TYPE: nucleic acid ~C) STRANDEDNESS: double ~D) TOPOLOGY: linear (ii) ~OLECULE TYPE: cDNA to mRNA
(iii) T~YPOTHETIC~L: NO
(vi) ORIaINAL SOURCE:
(A) ORGANISM: Mu~ mu~culu~ .
(H) CELL LINE: Swis~ 3T3 (vii) IMMEDIATE SOURCE:
(A) LIBRARY: Lambda GT10 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3~8..1532 , ~
(xi) SEQUENCE DESC~IPTION: SEQ ID NO:1:
AAAAcTGCAG CCAGAGAGAC TCAGTCTAGG ATGGAGGTAG GAAGAGCTGA GACAAAGTGG 60 -GCTTAATTCT A~GCTTTTCT TCAGGCTGAG TTTCTGTTGC TTGTTAACTT AGTGAATGTA 120 30 ~ - :
CAGATGTATT GCTTGCTGGT GGTGTGAAGG CTGGGACAGA ACCAACATCA ACA~ACTGAG 180 ~ CCTTCAGCGC CTAACTGAAA AACCCAGAAG TTACAAAGCA GCATCTTGAA GGCGCATTTG 360 i~ AAGAGAGAAG CTTTGAG ATG GCT CCA AAT AAT TGT TCC CAC CTG AAC TTG 410 40. Met Ala Pro A~n Asn Cy~ Ser Hi~ Leu A~n Leu 1 5 10 .:
, 1 Asp Val Asp Pro Phe Leu Ser Cy~ A~n A~p Thr Phe A~n Gln Ser Leu :,4515 20 25 ::
.
AGT CCC,CCC AAG ATG GAC A~C TGG TTT CAC CCG GGC TTC ATC TAT GTC 506 Ser Pro Pro Ly~ Met Anp A~n Trp Phe Hi~ Pro Gly Phe Ile Tyr Val ~.
30 : 35 40 50 ~ --: ATC CCT GCA GTT TAT GGG CTT ATC ATC GTG ATA GGT CT$ ATT GGC AAC 554 : Ile Pro Ala Val Tyr Gly Leu Ile Ile Val Ile Gly Leu Ile Gly A~n - ::

: -, ~ _ ''.

':::

WO 92/16S23 2 ~ ~ ~ 3 ~ ~ PCI`/US9~/02091 ATC ACG ~TC ATC AAG ATC TTC TGC ACG GTC AAG TCC ATC CGA AAC GTG 602 ; Ile Thr L~u Ile Ly~ Ile Phe cyn Thr Val Ly~ Ser MHt Arg A~n Val Pro A~n Leu Phe Ile Ser Ser Leu Ala Leu Gly A~p Leu Leu Leu Leu Val Thr Cy~ Ala Pro Val A~p Ala Ser Ly~ Tyr Leu Al~ Asp Arg Trp CTA TTT GGC AGA ATT GGC TGC AAA CTG ATC CCC TTT ~A CAA CTT ACT 746 Leu Phe Gly Arg Ile G}y Cys Ly~ Leu Ile Pro Phe Ile Gln Leu Thr ~ Ser Val Gly Val Ser Val Phe Thr Leu Thr Ala Leu Ser Ala Aap Arg `` 125 130 135 ~: 20 TAC AAA GCC ATT GTA CGG CCA ATG GAT.ATC CAG GCA TCC CAT GCC CTG 842 Tyr Ly~ Ala Ile Val Arg Pro 2~et Aap Ile Gln ~la Ser ~ Ala Leu ,.~
~5 ATG AAG ATC TGT CTC AAA GCT GCT TTG ATC TGG ATT GTC TCT ATG TTG 890 ~ Met Lys Ile Cy~ Leu Ly~ Ala Ala Leu Ile Trp Ile Val Ser Met Leu .~ 160 165 170 . .
-~ TTG GCC ATC CCA GAG GCT GTG TTT TCT GAC CTC CAC CCC TTC CAT GTG 938 :1 30 Leu Ala Ile Pro Glu Ala Val Phe Ser Asp Leu Hi~ Pro Phe His Val :" 175 180 185 AAA GAT ACC AAC CA~ ACC TTC ATT AGT TGT GCC CCC TAC CCA CAC TCC 986 Ly~ ABP Thr Asn Gln Thr Phe Ile Ser CYB Ala Pro Tyr Pro Hi~ Ser ,'l35 190 195 200 1 AAT GAG CTA CAC CCT AAA ATC CAT TCC ATG GCT TCC TTT CTG GTT TTC 1034.l A~n Glu Leu Hi~ Pro Lyn Ile Hia Ser Met Ala Ser Phe Leu Val Phe .,! : 205 ` 210 : 215 `~

¦ Tys Val Ile Pro Leu Ala Ile Ile Ser Val Tyr ~yr Tyr Phe Ile Ala :l 220 225 230 235 .
j 45 CGA AAT CTG ATT CAG AGT GCC TAC AAT CTT CCC GTG GA~ GGC AAT ATA 1130 5 l Arg Asn Leu Ile Gln Ser Ala Tyr A~n ~eu Pro Val Glu Gly.Aan Ile f 240 245 250 .,, . " . ;

Hi~ Val Lys Lyu Gln Ile Glu Ser Arg Lya Arg Leu Ala Lys Thr Val :
~: 255 . : : :260 265 .~ .
j CTG GTG TTT GTG GGC CTC TTT GCC TTC TGC TGG CTC CCC AAC QT GTC 1226 .I Leu Val Phe Val Gly Leu Phe Ala Phe Cy~ Trp Leu Pro A~n Hia Val ~ 55 270 : ~ 275 280 ." ~ .
.. ..
,~ ~

WO 92/16623 2 ~ ~ 5 ~ ~ 6 PCr/US92/02091 Ile Tyr Leu Tyr Arg Ser Tyr Hi~ Tyr Ser Glu Val A~p Thr Ser Met . 5 CTC CAC TTT GTC ACC AGC ATC TGT GCC CAC CTC CTG GCC TTC ACC AAC 1322 Leu Hi0 Phe Val Thr Ser Ile Cy~ Ala Hi~ LQU Leu A1A Phe Thr A~n 300 305 . 310 315 Ser Cy~ Val ~n Pro Phe Ala Leu Tyr Leu Leu Ser Ly~ Ser Phe Ar~

~ ~ .
AAG CAG TTC A~C ACT CAA C~T CTC TGC TGC Q G CCT ZGC CTG ATG AAC 1418 Lys Gln Phe ARn Thr.Gln Leu L~u Cy0 Cy~ ~ln Pro Gly Lau M~t A~n 335 3~0 3~5 : AGG TCC CAC AGC ACA GGC AGA AGT ACC ACC TGC ATG ACC TCC TTC AAG 1466 , Arg Ser Hi~ Ser Thr Gly Arg Ser Thr Thr Cy~ Met Thr Ser Phe Ly~

Ser Thr Ann Pro Ser Ala Thr Phe Ser Leu Ile A~n Arg A~n Ile Cy~

~, 25 CAT GAG GGG TAT GTC TAGACTAAAC TTCAACCTTG CCTCTAAAGG AACTCCTGGT 1569 Hi~ Glu Gly Tyr Val ~ 380 385 :
.' ATTGTTCTAC AGATGTCCAG GGGCCCTGAG ATTGATTGTT GTCTCTATAT CTTCTGAAGA 1629 CTCTTCAGGG GGATGAGTGA TACAGACGGA TGGGAAAGAT GTCCAAATGC ACCAA~CACC 1689 ,:
i, ATTGTATCTC A 1700 ..
~ 35 . ~
,1 ~2) INFORHATION FOR SEQ ID NO:2: : .
i) SEQUENCE CHARACTERISTICS:
~i ~ (A) LENGTH: 384 amino acid~ .
' 40 ~B) TYPE: amino acid :::.
`~ ~D) TOPOLOGY: linear . (ii) ~O~ECULE TYPE: protein ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ;~
Met Ala Pro A~n Asn Cyc Ser Hi~ Leu A~n Leu Acp Val A~p Pro Phe 1 5 ~ ~ 10 15 , 50 Leu Ser Cy~ A~n Aap Thr Phe A~n Gln Ser Leu Ser Pro Pro Lya ~et ` 30 -:
1 Acp A~n Trp Phe Hi~ Pro Gly Phe Ile Tyr Val Ile Pro Ala Val Tyr ~-.
35 40 45 :.:~
~: 55 W O 92/16623 2 ~ ~ 5 3 ~ ~ PC~r/U~92/02091 134 ~
Gly Leu Il~ Ile V~l Ile Gly L~u Ile Gly A~n Ile Thr L~u Ile Ly~

Ile Phe cy~ Thr Val Ly Ser Met Arg A~n Val Pr;: Asn Leu Phe Ile ser Ser Leu Ala Leu Gly AEip Leu Leu Leu Leu Val Thr Cya Ala Pro 85 gO 95 , Val A~p Ala Ser Lya Tyr Leu Ala Aap Arg Trp Leu Phe Gly Arg Ile : 100 105 110 .
Gly Cya Ly~ Leu Ile Pro Phe Ile Gln Leu Thr Ser Val Gly Val Ser : 115 120 125 ' Val Phe Thr Leu Thr Ala I,eu Ser Ala A~p Arg Tyr Lys Ala Ile Val .. 130 135 140 . ~rg Pro Met Aap Ile Gln Ala Ser Hi~ Ala Leu ~et Ly~ Ile Cya Leu '~20 145 150 155 160 Ly~ Ala Ala Leu Ile Trp Ile Val Ser Met Leu L~u Ala Ila Pro Glu , 165 170 175 ,~ 25 Ala Val Phe Ser Asp Leu His Pro Phe Hi~ Val Ly~ A~p Thr Aan Gln . .
. 180 185 190 i Thr Phe Ile Ser Cya Ala Pro Tyr Pro Hia Ser. Aan Glu Leu Hia Pro .! 30 Ly~ Ile Hi~ Ser Met Ala Ser Phe Leu Val Phe Tyr Val Ile Pro Leu Ala Ile Ile Ser Val Tyr Tyr Tyr Phe Ile Ala Arg Asn Leu Ile Gln i : .
225 230 235 240 : -1 ':
3 ~ Ser Ala Tyr Asn Leu Pro Val Glu Gly AE~n Ile Hi~ Val Lya Lya Gln ~:
245 250 255 :
!~ 40 ~ Ile Glu Ser Arg Lya Arg Leu Ala Lyn Thr Val Leu Val Phe Val Gly `I 260 265 270 ~; Leu Phe Ala Phe Cya Trp Leu Pro A~n HL~ Val Ile Tyr Leu Tyr Arg Ser Tyr HLa Tyr Ser Glu Val Aap Thr Ser Met Leu Hin Phe Val Thr j 290 295 300 ~ .
Ser Ile CYB Ala Hia~Leu Leu Ala Phe ~hr Aun Ser Cya Val A~ Pro Phe Ala Leu Tyr Leu Leu Ser Lya Ser Phe Ar^g Lya Gln Phe A~n Thr 325~ ~ 330 : ` 335 . .
;55~ Gln Leu Leu Cy8 Cya Gln Pro Gly:Leu Met: Aan Arg Ser Hi~ Ser Thr 3~0 ~ ~ 34~5 . 350 :: ~
1: - ~ : ~ : ., W O 92/16623 2 1 ~ 5 3 ~ ~ PCT/~S92/02091 ~ .

Gly Arg Ser Thr Thr Cy~ Met Thr Ser Phe Ly~ S0r Thr A~n Pro S~r Ala Thr Phe Ser Leu Ile Asn Arg Asn Ile Cy~ Hi~ Glu Gly Tyr Val (2) INFORMATION FOR SEQ ID NO:3:
~i) SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 1726 ba~e pair~
(B) TYPE: nucleic acid ~ ~C) STRANDEDNESS: do~ble - (D) TOPOLOGY: linear .~ 15 (ii) MOLECULE TYPE: cDNA to mRNA
~iii) HYPOTHETICAL: NO
:. 20 (vi) ORIGINAL SOURCE:
(A) ORCANISM: Homo ~apien~
- (G) CELL TYPE: Small cell lung carcinoma . (H) CELL LINE: NCI-~345 ~vii) IM~EDIATE SOURCE:
(A) LI~RARY: Lambda GT10 (ix) FEATURE:
.~ (A) NAME/KEY: CDS , .
.30 (B) LOCATION: 399.. 1553 ,;
.- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
~;i 35 CCAGATTCTA AATATCAGGA AAGACGCTGT GGGAAAATAG CAGGCCAAAA GTTCTTAGTA 60 ~`~' ' , .'l TTTGAATACC ATAGTTACTA TATATGTACT CAGAGTATTT TTATTAAAGA AGGCAAAGAG 300 CCCGGCATAG ATCTTATCTT CATCTTCACT CGGTTGCAAA ATCAATAGTT AAGAAATAGC 360 :~
ATCTAAGGGA ACTTTTACGT GGGAAAAAAA ATCTAGAG ATG GCT CTA AAT GAC ~13 ~i 1 5 -. :
:

Cy8 Phe Leu Leu A~n Leu Glu Val ADP Hi~ Phe Met ~i~ Cy~ A~n Ile , 10 ~ . 15 20 :1; 55 , ~):

.~ .
$ : ...
.', :

W O ~2~16623 2 ~ PCT/USg2/0209 ~36 Ser Ser Hig Ser Ala A~p Leu Pro Val A~n Aap A~p Trp S~r His Pro 5G&G ATC CTC TAT GTC ATC CCT GCA GTT TAT GGG GTT ATC ATT CTG ATA 557 :: Gly Ile Leu Tyr Val Ile Pro A}a Val Tyr Gly Val Ile Ile Leu Ile 0Gly Leu Ile Gly A~n Ile Thr Leu Ile Ly~ Ile Phe Cy~ Thr Val Lys ~ TCC ATG CGA AAC GTT CCA AAC CTG TTC ATT TCC AGT CTG GCT TTG GGA 653 ï Ser Met Arg A~n Val Pro A~n L~u Phe Ile Ser Ser Leu Ala Leu Gly "~ 1570 75 80 85 . .
:~ GAC CTG CTC CTC CTA ATA ACG TGT GCT CCA GTG GAT GCC AGC AGG TAC 701 Aap Leu Leu Leu Leu Ile Thr Cys Ala Pro Val Asp Ala Ser Arg Tyr :. 20 Leu Ala Asp Arg Trp Leu Phe Gly Arg Ile Gly Cys Ly~ Leu Ile Pro ~1 `.
~05 110 115 ~; Phe Ila Gln Leu Thr Ser Val Gly Val Ser Val Phe~Thr Leu Thr Ala `` 120 125 130 30Leu Ser Ala Asp Arg Tyr Lys Ala Ile Val Arg Pro Met A~p Ile Gln 135 140 145 ..
~ `GCC TCC CAT GCC CTG ATG AAG ATC TGC CTC AAA GCC GCC TTT ATC TGG 893 'I Ala Ser His Ala Leu ~et Lys Ile Cy~ Leu Ly~ Ala Al~ Phe Ile Trp i~ ATC ATC TCC ATG CTG CTG GCC ATT CCA GAG GCC GTG TTT TCT GAC CTC 941 ..
Ile Ile Ser ~et Leu Leu Ala Ile Pro Glu Ala Val Phe Ser Asp Leu . .
. 170 175 180 , 40 -His Pro Phe His Glu Glu Ser Thr Asn Gln Thr Phe Ile Ser Cys Ala ` Pro Tyr Pro His Ser Aan Glu Leu His Pro Ly~ Ile His Ser Met Ala ~. .
~ `TCC TTT CTG GTC TTC TAC GTC ATC CCA CTC TCG ATC ATC TCT GTT TAC 1085 ,j 50 Ser Phe Leu Val Phe Tyr Val~Ile Pro Leu Ser Ile I}e Ser Yal Tyr ' 215 : ~ 220 225 :, 'TAC TAC TTC ATT GCT AAA AAT CTG ATC CAG AGT GCT TAC AAT CTT CCC 1133 : :
3Tyr Tyr Phe Ile Ala Lys A~n Leu Ile Gln Ser Ala Tyr Aan Leu Pro :-:.:
1 55 230 235 ~ : 240 245 , : :
,: :

~: :
.
,' ~ : :

~0 92~16623 2 ~ O ~ PCT/US9~/0~091 GTG GAA GG4 AAT ATA GAT GTC AAG ~AG CAG ArT GAA TCC CGG AAG CGA 1181 Val Glu Gly Asn Ila Hl~ Val Ey~ Lys Gln Il~ Glu Ser Arg Ly~ Arg 250 255 2~0 Leu Ala Ly~ Thr Val Leu Val Phe Val Gly Leu Phe Ala Phe Cy~ Trp Leu Pro A~n Hiu V~l Ile Tyr Leu Tyr Arg S~r Tyr ~i0 Tyr S~r Glu 280 285 290~' GTG GAC ACC TCC ATG CTC CAC TTT GTC ACC A~C ATC TGT GCC CGC CTC 1325 Val A~p Thr s~r Met L~u HiB Phe Vnl Thr S~r Il~ Cy~ Al~ Arg Leu Leu Ala Phe Thr Asn Ser Cy~ Val A~n Pro Phe Ala L~u Tyr Leu Leu 31~ 315 320 325 AGC AAG AGT TTC AGG A~ CAG TTC AAC ACT CAG CTG CTC TGT TGC CAG 1421 Ser Ly~ Ser Phe Arg Lys Gln Phe A~n Thr Gln Leu Leu Cy~ CYB Gln :: `

Pro Gly Leu Ile Ile Arg Ser ~i~ Ser Thr Gly Arg.Ser Thr Thr Cy~ .

ATG ACC TCC CTC AAG A&T ACC AAC CCC TCC GTG GCC ACC TTT AGC CTC 1517 Met Thr Ser Leu Ly~ Ser Thr A~n Pro Ser Val Al~ Thr Phe Ser Leu ATC AAT GGA AAC ATC TGT CAC GAG CG& TAT GTC TAGATTGACC CTTGATTTTG 1570 ::.
Ile Asn Gly A~n Ile Cy~ Hin Glu Arg Tyr Val CTGTGCCCTC CAAAGAGCCT TCAGAATGCT CCTGAGTGGT GTAGGTGG4& GTGGGGAGGC 1690 (2) INFORMATION FOR:SEQ ID NO:4: .
~i) SEQUENCE CHARACTERISTICS: .
~A) LENGTH: 384 ~mino acid~ ~.
(B) TYPE: amino acid ..
~D) TOPOLOGY: linear .~
. : .
~ii) MOL~CULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
5 Met Ala Leu A~n A~p Cy~ Phe Leu Leu A~n Leu Glu Val Aap ~i~ Phe . ., ::, W~ 92JI6623 . P~ 92~'~2091 2~0~3~6 Met ~i~ Cyn A~n Ile S~r Ser ~}i0 Ser Al~ A~p L~u Pro V~l A~n A~p A~p ~rp Ser ~i~ Pro Gly Ile L~u Tyr V~l Ils Pro Ala Val Tyr Gly Val Ile Ile Leu Ile Gly Leu Ile Gly A~n lle Thr Leu Ile LyE~ Ile Phe Cys Thr Val Ly~ Ser Met Arg A~n Val Pro A~n Leu Phe Ile Ser ~`
Ser Leu Ala L0u Gly A~p Leu L~u L~3u Leu Ile Thr Cy~ Ala Pro Val : 85 gO 95 . 15 ABP Ala Ser Arg Tyr L~u Ala Aap Arg Trp Leu Phe Gly Arg Ile Gly 100 10~ 110 Cy~ Ly~ Leu Ile Pro Phe Ile Gln Leu Thr Ssr YA1 Gly V~l Ser Val Phe Thr Leu Thr Ala Leu Ser Ala A~p Arg Tyr Lys Ala Il0 Val ~rg , 130 135 140 Pro ~let A~p Ile &ln Ala Ser Hi~ Ala Leu Met Ly~ Ile Cy~ Leu Lys `` 145 150 155 160 : ~ .
.. Ala Ala Phe Ile Trp Ile Ile Ser Met Leu L~u Ala Ile Pro Glu Ala ~' 30 '! Yal Phe Ser A~p Leu Hi~ Pro Phe Hi~ Glu Glu Ser Thr A~n Gln Thr Phe Ile Ser Cyç~ Ala Pro Tyr Pro Hia Ser A~n Glu Lau Hi~ Pro Ly~

, ; Ile Hi~ Ser M~at Ala Ser Phe Leu Val Phe Tyr Val Ile Pro Leu Ser :'J 40 Ile Ile Ser Yal Tyr Tyr Tyr Phe Ile Ala Ly~ A3n Leu Ile Gln Ser Ala Tyr Al3n Leu Pro Val Glu Gly Asn Ile Hi~ Val Ly0 Ly~ Gln Ile ~, Glu Ser Arg Ly~ Arg Leu Ala Ly~ ~hr Val Leu Val Phe Val Gly Leu ~, Phe Ala Phe Cy~ Trp Leu Pro Asn Hi~ Val Ile Tyr Leu Tyr Arg Ser ~1 50 ~ 27~5 : 280 ~ 285 . .
,`1 Tyr Hi~ Tyr Ser Glu Val Asp Thr Ser ~et Leu Hi~: Phe Val Thr Ser :~~ 290 . 295 300 :
Ile Cy~ Ala Arg Leu Leu Ala Phe Thr A~n Ser Cy~ Va} A~n Pro Phe :, 305 310: 315 320 ,J

~: . ~ ':
'~ ' : ' 'o W O 92~l6623 PCT/US92/020g1 ~ 2i~53û~

Ala Lau Tyr Leu L~u Ser Lya Ser Phe Arg Ly~ Gln Ph~ A~n Thr aln ~ .

Leu Leu Cy~ Cy~ Gln Pro Gly Leu Ile Ile Arg Ser Hi~ Ser Thr Gly ~: .

Arg Ser Thr Thr Cy~ Met Thr Ser Leu LYB Ser Thr A~n Pro Ser Val Ala Thr Phe Ser Leu Ile Asn Gly A~n Ile Cy~ Glu Arg Tyr Val t2) INFORMATION FOR SEQ ID NO:5:
EQUENCE CHARACT~RIS~ICS: - :
(A) LENGTH: 1584 base pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: double -.:~
tD) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: N0 .
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Rattu~ rattu~ .
(F) TISSUE TYPE: E~oph~gu~
3 0 ( ix ) FEATURE:
(A) NAME/KEY: CDS ..
(B) LOCATION: 132.. 1304 .
.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GGTGGCTCAG TTCCAGGAGC CACAAACTTG CCAGGATCAG AGACAATCAA CTA~ACCCAG 60 . ~.
. .
GTCGTACTCA CCGCACTTTC GAGACGCGCG:AGTGCAGGAA AACTCCCGCG AATCCCCTGG 120 GAAAGGACAC C ATG CCC CCC AG~ TCT CTC CCC AAC CTC TCC TTG CCC ACC 170 ,.. ...
Met Pro Pro Arg Ser Leu Pro A~n Leu Ser Leu Pro Thr GAG GCG AGC GAG AGC GAG TTG GAA CCC GAG GTG TGG GAA AAT GAT TTC 218 -:
Glu Ala Ser.Glu Ser Glu Leu Glu Pro Glu Val Trp Glu Ann A~p Phe CTG~CCT GAC TCA GAC GGG ACC ACC GCG GAG TTG GTA ATC CGC TGT GTG 266 Leu Pro A~p Ser A~p Gly Thr Thr AIa Glu Leu Val Ile Arg CYB Val 30 35 ~ ~0 45 Ile Pro Ser Leu Tyr Leu Ile Ile Ile Ser Val Gly L~u Leu Gly A~n 55 ~ 50 ~ -. S5 60 .

, ::

,. . . .

W O 92/16623 210 ~ 3 ~ ~ PCT/US92/n2091 ATC ATG C~G GTG A~G ATA TTC CTC ACC AAC AGC ACC ATG CGG AGT GTC 362 Ile M~t L~u Val Ly&i Ile Phe L~u Thr Aiin s~r Thr M~t Arg Ser Val ~5 70 75 CCC AAC ATC TTC ATC ~CT AAC CTG GCT GCG GaA GAC CTG CTG CTG CTG 410 Pro Asn I1Q Phe I1e Ser A0n LeU A1a Ala Gly A~p L~u Leu L~u Leu CT& ACC TGC GTC CCA GTG GAT GCC TCC CGA TAC TTC TTT GAT GAA TGG 458 LeU Thr Cy0 Val Pro Val Asp A1a Ser Arg Tyr Phe Phe Asp Glu Trp g5 100 105 GTG TTC GGC AaG CTG GGC TGC AA~ CTC ATC CCA GCC ATC CAG CTC ACC 506 Val Phe Gly LyEi L~u Cly CYB Lys L~u Ile Pro Ala Il~ Gln Leu Thr TCG GTG GGG GTT TCC GTG TTC ACT CTC ACG GCC CTC AGC GCT GAC AGG 554 :.Ser Val Gly Val Ser Val Phe Thr Leu Thr Ala Leu Ser Ala A0p Arg ' 130 135 140 Tyr Arg Ala Ile Val A0n Pro Met A~ip Met Gln Thr Ser Gly Val Val Leu Trp Thr Ser Leu Ly~ Ala Val Gly Ile Trp Val.Val Ser Val Leu Leu Ala Val Pro Glu Ala Val Phe Ser Glu Val Ala Arg Ile Gly Ser : 175 180 185 Ser Asp Asn Ser Ser Phe Thr Ala Cys Ile~ Pro Tyr Pro Gln Thr Asp GAG TTA CAT CCA J~G ATC CAC TCA GTG CTC ATT TTT CTT GTC TAT TTC . 794 Glu Leu Hi0 Pro Lys Ile Hi~i S ir Yal Leu Ile Phe Leu Val Tyr Phe ~0 Leu Ile Pro Leu Val Ile Ile Ser Ile Tyr Tyr Tyr His Ile Ala Ly~
~ 225 230 235 ACT TTA ATT AGA AGT GCA CAC AAT CTT CCT GGA GAA TAC AAT GAA CAT 890 .
Thr Leu lle Arg Ser Ala His A0n Leu Pro Gly Glu Tyr Asn Glu Hi Thr Lys Ly~ Gln Met Glu Thr Arg Ly6i Arg Leu Ala Lys Ile Val Leu ~: 255 ~ 260 ~ ~ 265 GTG TTT GTG GGC TGC TTT GTC TTC TGC Tt~G TTT CCC AAC CAC ATC CTC 986 Val Phe Val Gly Cys Phe Val Ph~ Cys Trp Phe Pro Asn Elin Ile Leu 270 275 ~ 280 285 , :

.: .
:. .' ', , - : ~.', WOg2/16623 21~3a~, PC~/US92/02091 Tyr Leu Tyr Arg Ser Phe A0n Tyr Ly~ Glu Il~ Aap Pro S2r L0u Gly ~90 295 300 HiB Met Ile Val Thr Leu Val Ala Arg Val L~u Ser Phe S~r A0n Ser TGT GTC AAC CCG TTT GCT CTT TAC CTG CTC AGT GA~ AGC TTC AGG AAG 1130 CYB Val A~n Pro Phe Ala Leu Tyr Leu Leu Ser Glu Ser Phe Arg Ly~ :

His Phe A0n Ser Gln Le~ Cy~ Cy~ Gly Gln Ly~ S~r Tyr Pro Glu Arg TCT ACC AGC TAC CTC CTC AGC TCT TC~ GCA GTA AGA ATG ACT TCT CTG 1226 Ser Thr Ser Tyr Leu Leu Ser Ser Ser Ala Val Arg Met Thr 5er L~u AAA AGC AAC GCG AAG A~T GTG GTG ACC AAT TCT GTC CTG CTC AAC GGA 1~74 Ly~ Ser A~n Ala LYR ~sn Val Val Thr A~n Ser Val Leu Leu Aen Gly 370 3~5 380 Hi~ ser Thr Ly~ Gln Glu Ile Ala Leu 385 390 .
CATCCTCGGG ~AATACCATT TTCACAACTT TTCCATTATT ATTGAGCGAA GCAGAGCTAA 1381 ,, AATTCACATA TATCTCCTGC TAACATCGGT TTACACATTC CCTTGGGATT TAAGACATTC 1561 .:

(2) INFORMATION POR SEQ ID No:6:
(i3 SEQUENCE CHARACTERISTICS:
(A) LENGTH: 390 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: linoar (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESC~IPTION: SEQ ID NO:6:
Met Pro Pro Ar~ Ser Leu Pro A~n Leu Ser Leu Pro Thr Glu Ala Ser Glu Ser Glu Leu Glu Pro Glu Val Trp Glu Aen Aep Phe Leu Pro A~p 20 25 30 .
'; , . :
-W 0 92/l6623 2 1 ~ ~ 3 ~ 6 Per~U~92/020 1~2 ser Aap Gly Thr Thr ~la G1U LQU Val Ile Axg Cy~ Vnl Ile Pro Ser Leu Tyr Leu Ile Ile Ile Ser Val Gly Leu Leu Gly A~n Ile Met Leu Val LYD Ile Phe Leu Thr A~n Ser Thr Met Arg Ser Val Pro Asn Il~
0 Phe Ile Ser A~n Leu Ala Ala Gly A~p Leu Leu Leu Leu L~u Thr Cy~

Val Pro Val A_p Ala Ser Arg Tyr Phe Phe A~p Glu Trp Val Phe Gly Ly~ Leu Gly Cya Lye Leu Ile Pro Ala Ile Gln Leu Thr Ser Val Gly Val Ser Val Phe Thr Leu Thr Ala ~eu Ser Al~ ARP Arg Tyr Arg Ala Ile Val ARn Pro ~et A~p Met Gln Thr Ser Gly Val Val Leu Trp Thr Ser Leu Lys Ala Val Cly Ile Trp Val Val Ser Val Leu L~u Ala Val 165 170 . 175 2ro Glu Ala Val Phe Ser Glu V~l Ala Arg Ile Gly Ser Ser A~p A~n 180 185 190 .
Ser Ser Phe Thr Ala Cy~ Ile Pro Tyr Pro Gln Thr A~p Glu Leu Hi~

Pro Lys Ile Hi~ Ser Val Leu Ile Phe Leu Val Tyr Phe Leu Ile Pro Leu Val Ile Ile Ser Ile Tyr Tyr Tyr Hi~ Ile Ala Lys Thr Leu Ile Arg Ser Ala Hi~ A~n Leu Pro Gly GIu Tyr Aun Glu Hi~ Thr Ly~ Ly~

Gln Met Glu Thr Arg Ly~ Arg Leu Ala Lya Ile Val Leu Val Phe Val Gly Cy~ Phe Val Phe Cy~ Trp Phe Pro A~n Hi~ Ile Leu Tyr Leu Tyr , .:
275 280 285 .
Arg Sor Phe ARn Tyr Ly~ Glu Ile A~p:Pro Ser Leu Gly HiR Met Ile 230 ~ ~ 295 300 Val Thr Leu Val Ala Arg Val Leu Ser Phe Ser A~n Ser Cy~ Val A~n ~.
305 310 315 320 .
Pro Phe Ala Leu Tyr Leu Lzu Ser Glu Ser Phe Arg Ly~ Hi~ Phe A~n :
325 ~ : 330 3~5 . ;
::
- : ' ` , ' :

WO 92/1~623 2 1 ~ ~ 3 ~ ~ PC~ ;92/~

Ser Gln L~u CYB CY~ Gly Gln I.y~ Ser Tyr Pro Glu Arg Ser Thr Ser 340 345 350 :.
Tyr Leu Leu Ser Ser Ser Ala Val Arg ~et Thr Ser Leu Ly~ Sar Asn Ala Lya Asn V~l Val Thr A~n Ser Val Leu Leu A~n Gly Hi~ Ser Thr 370 375 3~0 Ly~ Gln Glu Ile Ala Leu (2) INFORMATION FOR SEQ ID NO:7: .
. . ; . : .
~i) SEQU~NCE CHAR~CTERISTICS:
~A) LENGTH: 1352 ba~e pair~
~8) ~YPE: nucl~ic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECUL~ TYPE: cDNA to mRNA
. (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapien~
(G) CELL TYP~: Small cell lung carcinoma (H) CELL LINE: NCI-~345 ..
3~ ,.
(ix) FEATURE:
~A) NAME/XEY: CDS
(B) LOCATION: 140..1312 .... .

(xi) SEQUENCE DESCRIPTION: SEQ;ID NO:7:
GTGCTGTGAG GCTTGCCCGC GGAC~GTAAA CTTGCAGGGG CGAGAGGGAG GGACATCGAT 60 :
TAAACCTAAA TCGTGGGCGT TCAGTCCTCA GGGCACCGAG CGCGTGAAaA CTCCAGCGGA 120 -.
~ CTCTGCTGGA AAGGAGATC ATG CCC TCT AAG TCT CTT TCC AAC CTC TCG GTG 172 : Met Pro Ser Ly~ Ser Leu Ser A~n Leu Ser Val . .
I 5 10 ~ :
4S .. :
ACC ACC GGC GCG AAT GAG AGC GGT TCC GTT CCC GAG GGG TGG GAA AGG 220 .:
: Thr Thr Gly Ala A~n Glu Ser Gly Ser Val Pro Glu Gly Trp Glu Arg :~` 50 GAT TTC CTG CCG GCC TCG GAC GGG ACC ACC ACG GAG TTG GTG ATC CGC 268 A~p Phe Leu Pro Ala~Ser A~p Gly Thr Thr Thr GLu Leu Val Ile Asg : :
30 ~ 35 ~ 40 .
,, Cy~ Val Ile Pro Ser Leu Tyr Leu Leu Ile Ile Thr Val Gly ~u Leu : 45 ~ 50 55 : ~ _ : .;
. ~.:

WO 92t~6623 2 1 ~ 5 3 ~ ~ P~/US92/02091 GGC A~C ATC ATG CTG GTt; ~G ATC TTC ATC ACC AAC AGC GCC ATG AGG 364 Gly A~n I1Q ~Set Leu V~l Lys Ile Ph~3 Ile Thr A~n ser ~la M~t Arg AGC GTC CCC AAC ATC TTC ATC TCT ~AC CTG GCG GCC GGG GAC TTG CTG 412 Ser Val Pro A~n Ile Phe Ile Ser A~n Leu Ala Ala Gly A~p Leu Leu Leu Leu Luu Thr CYEI Val Pro Val Asp Ala Ser Arg Tyr Phe Phe A~p Glu Trp M~t Phe Gly Ly~ V~ll Gly Cy~ Ly~ L~u Ile Pro V~l Il~ Gln CTC ACT TCC GTG GGG GTT TCC GTG TTC ACT CTC ACT GCC CTC AGC GCC 556 . .
LQU Thr Ser Val Gly Val Ser Val Phe Thr Leu Thr Aia Le~u Sç~r Ala GAC AGG TAC AGA GCC ATC GTT AAC ccc ATG GAC ATG CAG ACG TCA GGG 604 A~p Arg Tyr Arg Ala Ile Val A~n Pro Met Asp Met Gln Thr Ser Gly 140 145 150 155~
GCA TTG CTG CGG ACC TGT GTG AAG GCC AI'G GGT ATC TGG GTG GTC TCC 652 Ala LeU LeU Arg Thr Cy~ Val LYB Ala Met Gly Ile Trp Val Val Ser GTG TTG CTG GCA GTT CCC GAP~ GCG GTG TTT TCA GAA GTG GCT CGC ATC 700 Val Leu Leu Ala Val Pro Glu Ala Val Phe S~sr Glu Val Ala Arg Ile 175 180 185 .
AGT AGC TTC; GAT AAT AGC AGC TTC ACA GCA TGT ATC CCA TAC CCT CAA 748 Ser Ser Leu A~p Asn Ser Ser Phe Thr Ala Cy~ Ile Pro Tyr Pro Gln -....... :

Thr ABP Glu Leu Hia Pro Lys Ile HiB Ser Val Leu Ile Phe LeU Val : :
2q5 210 215 Tyr Phe Leu Ile Pro Leu A1A Ile Ile Ser Ile Tyr Tyr Tyr Hi~ Ile 220 225 23~ 235 Ala Lys Thr ~eu Ile Lys Ser Ala Hl ~ A~n T,eu Pro Gly Glu Tyr Aun 2~0 245 250 ..
GAa CAT ACC AAA AAA CAG ATG GAA ACA CGG AAA CGC CTG GCT AAA ATT 940 Glu HiB Thr I,y~ LYB Gl~ Me~t Glu Thr Arg Lya Arg Leu Ala Lys }le ..
255 260~ 265 Val Leu Val Phe Val Gly Cy~ Phe Ile Phe CYB Trp Phe Pre A~n Hi~
270 275 280 .
.. :.

. '.
.

WO92/16623 2~a~3~ Pcr/US92/020~

Ila Lsu Tyr ~t Tyr Arg S~r Phs Aan Tyr A~n Glu Il~ A~p Pro Ser CTA GGC CAC ATG ATT GTC ACC TTA GTT GCC CGG GTT CTC AGT TTT &GC 1084 Leu Gly Hi~ Met Ile Val Thr Leu Val Ala Arg Val Leu Ser Phe Gly ..

A~n ser Cy~ Val A~n Pro Phe Ala Leu Tyr Leu L~u Ser Glu Ser Phe AGG AGG ~AT TTC AAC AGC CAA CTG TGC TGT GGG.AGG AAG TCC TAT CAA 1180 Arg Arg Ri~ Phe A~n Ser Gln L~u cy~ Cy~ Gly Arg ~y~ Ser Tyr Gln GAG AGA GGA ACC AGC TAC CTA CTC AGC TCT TCA GCG GTG CGT ATG AC~ 1228 Glu Arg Gly Thr Ser Tyr L0u L~u Ser 5er Ser Ala Val Arg Met Thr TCT CTG A~A AGC AAT GCT AAG AAC ATG GTG ACC AAT TCT GTT TTA CTA 1276 Ser Leu Ly~ Ser A~n Ala Ly~ Asn ~et Val Thr A~n Ser Val Leu Leu AAT GGG CAC AGC ATG AAG CAG GA~ ATG GCA ATG TGATTTTGGC CATTCAACTC 1329 A~n Gly Hi~ Ser Met Lya Gln Glu Met Ala Met 380 385 3~0 .

:.
. . .
(2) INFORMATION FOR SEQ ID NO:8: .
: .' ~i) SEQUENCE CHARACTERISTICS: .
(A) LENGTH: 390 amino acid~
(8) TYPE: amino acid .
- (D) TOPOLOGY: linear :~
(ii) MOLECULE TYPE: protein .
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Pro Ser Ly~ Ser Leu Ser Aun Leu Ser Val Thr Thr Gly Ala A~n ~.`

..
Glu Ser Gly Ser Val Pro Glu Gly Trp Glu Arg A~p Phe Leu Pro Ala 20 . 25 30 .
Ser A~p Gly Thr Thr Thr Glu Leu Val Ile arg Cy~ Val Ile Pro Ser : 50 35 40 ~ 45 -,:
: Leu Tyr Leu Leu Ile~Ile Thr Val Gly Leu Leu Gly A~n Ile Met Leu 50 55 60 .: .
~.::.' Val Ly~ Ile Phe Ile Thr Ann Ser ~la Net Arg Ser Val Pro A~n Ile .
65 70~ ~ 75 80 : ~ -WO 92/1~23 2 1 ~ 6 PCI'/VS92/0~09 Ph~ Ile Ser A~n Leu Ala Ala Gly A~p Leu Leu Leu Leu Leu Thr CYB

Val Pro Val Asp Ala Ser Arg Tyr Phe Phe Asp Glu Trp Met Phe Gly Ly~ Val Gly Cya Ly~ Leu Ile Pro Val Ile Gln Leu Thr Ser Val Gly Val Ser Val Phe Thr Leu Thr Ala Leu Ser Ala A~p Arg Tyr Arg Ala Ile Val A~n Pro ~et Asp Met Gln Thr Ser Gly Ala L~u ~u Arg Thr 145 150 1~5 160 Cys Val Ly~ Ala ~et Gly Ile Trp Val Val Ser Val Leu Leu Ala V21 Pro Glu Ala Val Phe Ser Glu Val Ala Arg Ile Ser Ser Leu A~p A~n 180 185 190 ..
Ser Ser Phe Thr Ala Cys Ile Pro Tyr Pro Gln Thr Asp Glu Leu His ..

Pro Lyu Il~ Hi~ Ser Val Leu Ile Phe Leu Val Tyr Phe Leu Ile Pro Leu Ala Ile Ile Ser Ile Tyr Tyr Tyr Hi~ Ile Ala Ly~ Thr Leu Ile 225 230 235 240 .. :
LYB Ser Ala ~is A~n Leu Pro Gly Glu Tyr A~n Glu His Thr LYB LYB

Gln Met Glu Thr Arg Ly~ Arg Leu Ala Lys Il~ V~l L~u Val Phe Val 260 265 270 . .
Gly Cys Phe Ile Phe Cya Trp Phe Pro A~n Hi~ }le Leu Tyr Het Tyr :~

Arg Ser Phe Asn Tyr Asn Glu Ile A~p Pro Ser Leu Gly H1B Met Ile Val Thr Leu Val Ala Arg Val Leu Ser Phe Gly asn Ser Cys Val Asn 305 310 ~ 315 320 Pro Phc Ala Leu Tyr Leu Leu Ser Glu Ser Phe Arg Arg His Phe A~n 325 330 . 335 . ..
Ser Gln Leu CYB Cys Gly Arg LYB Ser Tyr Gln Glu Arg Gly Thr Ser . Tyr Leu Leu Ser Ser Ser Ala Val Arg Met Thr Ser Leu Lys Ser A~n 355 360 365 ..
. ~ .
Ala Lya A~n ~et Val Thr A~n Ser Yal Lau Leu A~n Gly ~i8 Ser Met 370 ~375 : 380 :

W O 9~/16623 2 ~ O ~ 3 0 6PCT/~2~ gl Ly~ Gln Glu ~et Ala M~t ~2) INFORMATION FOR SEQ ID NO:9:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 1606 ba~e pair~
~B) TYPE: nucleic acid ~C) STRANDEDNESS: double ~D) TOPOLOaY: linear ~ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
~vi) ORIGINAL SOURCE:
~A) ORGANISM: Homo ~apien~
~ix) FEATURE:
~A) NAME/KEY: CDS , tB) LOCATION: 172..1371 25(xi) SE~UENCE DESCRIPTION: S~Q ID NO:9:
GAAACACAGA ACTaAAGCAA AGGAGTATCT GGATGTCTTG GATTTTCTTC CCATTCTGTT 60 30 . :
- CTGCATTTGA ACTGAGAAGA AGAaATATTA AAGACACAGT CTTCAGAAGA A ATG GCT 177 ..
-- Met Ala . .
':
35CAA AGG CAG CCT CAC TCA CCT AAT CAG ACT TTA ATT TCA ATC ACA AAT 225 :
Gln Arg Gln Pro Hi~ Ser Pro A~n Gln Thr Leu Ile Ser Ile Thr A~n :
5 10 15 -.-:~
GAC ACA GAA TCA TCA AGC TCT ATG-GTT TCT AAC GAT AAC ACA AAT A~A . 27340Asp Thr Glu Ser Ser Ser Ser Met Val Sex A~n A~p A~n Thr A~n LYB ~.
20 ~5 30 Gly Trp Ser Gly Asp A~n Ser Pro Gly Ile Glu Ala Leu Cy~ Ala Ile 4535 40 45 50 .:

Tyr Ile Thr Tyr Ala Val Ile Ile Ser Val Gly Ile Leu Gly A~n Ala 55 ~ 60 65 5~ ~

Ile Leu Ile Ly~ Vai Phe Phe LYB Thr LYB Ser ~et Gln Thr Val Pro ~ , :

WO g2/16623 2 1 Q ~ 3 ~ 6 PCT/US92/02091 ~n Ile Phe Ile Thr Ser Leu Ala Phe Gly A~p L~u Leu Leu Leu Lau 85 gO 95 Thr Cyn Val Pro Val Anp Ala Thr Hin Tyr Leu Ala Glu Gly Trp Leu Phe Gly Arg Ile Gly CYB Lys Val Leu Ser Phe Ile Arg Leu Thr Ser .. :
115 120 125 130 :.

Val Gly Val Ser Val Phe Thr Leu Thr Ile Leu Sar Ala A~p Ar~ Tyr ~ :

Ly~ Ala Val Val Ly~ Pro Leu Glu Arg Gln Pro Ser A~n Ala Ile Leu Ly~ Thr Cy~ Val Ly~ Ala Gly Cy~ Val Trp Ile V~l ser Met Ile Phe 165 170 175 ..

Ala Leu Pro Glu Ala Ile Phe Ser Asn Val Tyr Thr Phe Arg A~p Pro .. 180 185 190 Asn.Lys ~Bn Met Thr Phe Glu Ser Cy~ Thr S~r Tyr Pro Val Ser Ly~ ~
195 200 205 21Q ~;

Ly~ Leu Leu Gln Glu Ile ~i~ Ser Leu Leu CYB Phe Leu Val Phe Tyr -.
35: 215 220 225 ~ .
: ATT:ATT CCA CTC TCT ATT ATC TCT GTC TAC TAT TCC TTG ATT GCT AGG 897 Ile Ile Pro Leu Ser Ile Ilè Ser Val Tyr Tyr Ser Leu Ile Ala Arg .

: . . .
: ~ ACC CTT TAC A~A AGC ACC:CTG AAC ATA CCT ACT GAG GAA CAA AGC CAT 945 ..
: Thr Leu Tyr Ly~ Ser Thr Leu A~n I le Pro Thr Glu Glu Gln Ser ~i~

`45 GCC CGT AAG CAG ATT CAA TCC CGA AAG AGA ATT GCC AGA ACG GTA TTG 993 : .
Ala Arg Lyo Gln Ile Glu Ser Arg LYB arg Il~ Ala Arg Thr Val Leu GTG TT5 GTG GC~ CTG TTT GCC CTC:TGC TGG TTG CCA AAT CAC CTC CTG 1041 Val Leu Val Ala Leu Phe Ala Leu Cy~Trp Leu Pro A~n Hi~ Leu Leu : 275 280 : ~ 285 2gO

. Tyr Leu Tyr ~iB Ser Phe Thr Ser Gln Thr Tyr Val Asp Pro Ser Ala ~ ~ : 295 ~: ~ 300 305 ~ -:~ .

WO 92/16623 210 5 3 0 ~ P~/US92/02091 ATG cAT TTC ATT TTC ACC A~T TTC TCT CG~ G'ST ~TG GCT TTC At:C aAT 1137 Mat Hi~ Phe Il~ Phe Thr Ile Ph~ Ser Arg Val Leu Al~ Phe Ser A~n TCT TGC GTA AF~C CCC ~TT GCT CTC TAC TGG CTG AGC I~AA AGC TTC CAG 1185 ser cya Val A~n Pro ~he Ala Leu Tyr Trp L~u ser Lyn s~r Phe Gln 325 330 335 : .
AAG CAT TTT A~A GCT CAG TTG TTC TGT TGC AAG GCG GAG CGG CCT GAG 1233 Ly~ Hi~ Phe Ly~ Ala Gln Leu Phe Cyn Cy Ly~ Ala Glu Arg Pro Glu CGT CCT GTT GcT GAC ACC TCT CT~ ACC ACC CTG GCT GTG ATG GGA ACG 1281 Pro Pro Val Ala A~p Thr Ser L~u Thr Thr Leu Al~ V~l H~t Gly Thr GTC CCG GGC ACT GGG AGC ~TA CAG ATG TCT GAA ATT AGT GTG ACC TCG 1329 Val Pro Gly Thr Gly Ser Ile Gln ~et Ser Glu Ile Ser Val Thr Ser TTC ACT GGG TGT AGT GTG A~G CAG GCA GAG GAC AGA TTC TAGCTTTTCA 1378 Phe Thr Gly CYB Ser Val Ly~ Gln Ala Glu A~p Arg Phe 390 395 400 .. ~
AGGAAAAATG CTGCTTCTCC TCCCAGCGTG TG~ATCCGAC TCTA~GCTGT GTGCAG&TGT 1438 ...
ATGGTGTCCA GATTTTTGTT GTTTGAAAAG TGTGTTGAAA TCTTAGGAGT GAAGGATCCC 1498 :
TATAAGTAAG TAAAATACA~ ACCATTACTT TCTTCAAAGT ACAAATAGTA ATGTCATCGG 1558 .
:.:

.: .
~2) INFORMATION FOR SEQ ID NO:10: ..
(i) SEQUENCE CHARACTERISTICS:
(A) LE~5TH: 399 amino acid~
(B) TYPE: amino acid ~D) TOPOLO&Y: linear (ii) MOLECULE TYPE. protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:lO:
4S Met Ala Gln Arg Gln Pro Hi~ Ser Pr~ A~n Gln Thr Leu Ile Ser Ile 1 5 lO 15 Thr Asn A~p Thr Glu Ser Ser Ser Ser Met Val Ser A~n A~p A~n Thr A~n Ly~ Gly Trp Ser Gly A~p A~n Ser Pro Gly I}e Glu Ala Leu Cy~

Ala Ils Tyr Ile Thr Tyr Ala Val Ile Ile Ser Val Gly Ile Leu Gly ;~ 55 ~ 50 ~ 55 60 .:
~' ' , WO 92/1~623 2 ~1 0 ~ 3 ~ ~ PCT/US92~2~9~

Asn Alzl Ila Leu Ils Ly~ Val Phe Phe Ly0 Thr Ly0 Ser Met G1n Thr Val Pro Asn Ile Phe Ile Thr Ser Leu Ala Phe Gly A~p Leu Leu Leu Leu Leu Thr Cy~ Val Pro Val Asp Al~ Thr Hi~ Tyr Leu Ala Glu Gly 100 10~ 110 Trp Leu Phe Gly Arg Ile Gly Cy~ Ly~ Val Leu Ser Phe Ile Arg Lsu Thr Ser Val Gly Val Ser Val Phe Thr Leu Thr Ile I.eu S~r Ala A~p Arg Tyr Ly~ Ala Val Val Ly~ Pr~ Leu Glu Arg Gln Pro Ser A~n Ala Ile Leu Lya Thr Cy~ Val Lys Ala Gly Cy~ Val Trp Ile Val Ser 2~et Ile Phe Ala Leu Pro Glu Ala Il~? Phe Ser A~n Val Tyr Thr Phe Arg ~ .
180 185 190 ~ ..
2 5 Asp Pro Aan Lya A~n Met Thr Phe Glu Ser Cyn Thr Ssr Tyr Pro Val 195 200 205 .
Ser Lya Lys Leu Leu Gln Glu Ile Hia Ser Leu Leu Cy~ Phe Leu Val . . :
Phe Tyr Ile Ile Pro Leu Ser Ile Ile Ser Val Tyr Tyr Ser Leù Ile . .
225 230 ~35 240 Ala Arg Thr Leu l~yr LYB Ser Thr Leu Aan Ile Pro Thr Glu Glu Gln : Ser Hi~ Ala Arg LyEI Gln Ile Glu Ser Arg Lya Arg Ile Ala Arg Thr 260 : 265 270 Val Leu Val Leu Val Ala Leu Phe Ala Leu Cy~ Trp Leu Pro A~n Hia 275 . 2~0 285 ~ .
Leu Leu Tyr Leu Tyr ElLfl Ser Phe Thr Ser Gln Thr ~yr Val Aap Pro Ser Ala Met His Phe Ile Phe Thr Ile Phe Ser Arg Val Leu Ala Phe 305 310 315 320 . :
Ser A~n Ser Cy~ Val Aan Pro Phe Ala Leu Tyr Trp LQU Ser Ly~ Ser 325 ~ 330 335 Phe Gln Ly~ Hi~ Phe Ly~ Ala Gln Leu Phe Cya Cy~ Ly~ Ala Glu .arg 340 345 350 . ..
5S Pro Glu Pro Pro Val Ala Aap Thr ~ Ser Leu Thr Thr Leu Ala Val Met~
~; 355 360 ~ ~ 365 :

- ,: .

WO 92~66~3 2 1 0 5 3 ~ ~ ~J~S~2~ 9~ : .
~ ' , . '.

Gly Thr Val Pro Gly Thr Gly Ser Ile Gln Met Ser Glu Il~ S~r Val Thr Ser Phe Thr Gly Cy~ Ser Val Ly~ 51n Ala Glu A~p Arg Phe .

-: ` :

:
'~ .
'..'~-. .

Claims (86)

WHAT IS CLAIMED IS:

1. A DNA segment coding for a polypeptide having an amino acid sequence corresponding to a human gastrin releasing peptide-receptor, or a unique portion thereof.

2. The DNA segment according to Claim 1, wherein said DNA
segment has the sequence shown in SEQ ID NO: 7, allelic or species variation thereof, or a unique portion thereof.

3. The DNA segment according to Claim 1, wherein said DNA
segment encodes the amino acid sequence set forth in SEQ ID NO:
8, allelic or species variation thereof, or a unique portion thereof.

4. A polypeptide free of proteins with which it is naturally associated and having an amino acid sequence corresponding to a human gastrin releasing peptide-receptor, or a unique portion thereof.

5. A polypeptide bound to a solid support and having an amino acid sequence corresponding to a human gastrin releasing peptide-receptor, or a unique portion thereof.

6. The polypeptide according to Claim 4 or 5, wherein said polypeptide has the amino acid sequence set forth in SEQ ID NO:
8, allelic or species variation thereof, or a unique portion thereof.

7. A recombinant DNA molecule comprising a vector and the DNA
segment according to Claim 1.

8. A cell that contains the recombinant DNA molecule according to Claim 7.

9. A method of producing a polypeptide having an amino acid sequence corresponding to human gastrin releasing peptide-receptor comprising culturing the cell according to Claim 8 under conditions such that said DNA segment is expressed and said polypeptide thereby produced, and isolating said polypeptide.

10. An antibody having binding affinity to a recombinant human gastrin releasing peptide-receptor, or unique portions thereof.

11. The antibody according to Claim 10, wherein said receptor has the amino acid sequence set forth in SEQ ID NO: 8, allelic or species variation thereof, or a unique portion thereof.

12. A DNA segment coding for a polypeptide having an amino acid sequence corresponding to a neuromedin-B-preferring bombesin receptor, or a unique portion thereof.

13. The DNA segment according to Claim 12, wherein said DNA
segment has the sequence shown in SEQ ID No:5, allelic or species variation thereof, or a unique portion thereof.

14. The DNA segment according to Claim 12, wherein said DNA
segment encodes the amino acid sequence set forth in SEQ ID NO:
6, allelic or species variation thereof, or a unique portion thereof.

15. A polypeptide free of proteins with which it is naturally associated and having an amino acid sequence corresponding to a neuromedin-B-preferring bombesin receptor, or a unique portion thereof.

16. A polypeptide bound to a solid support and having an amino acid sequence corresponding to a neuromedin-B-preferring bombesin receptor, or a unique portion thereof.

17. The polypeptide according to Claim 15 or 16, wherein said polypeptide has the amino acid sequence set forth in SEQ ID NO:
6, allelic or species variation thereof, or a unique portion thereof.

18. A recombinant DNA molecule comprising a vector and the DNA
segment according to Claim 12.

19. A cell that contains the recombinant DNA molecule according to Claim 18.

20. A method of producing a polypeptide having an amino acid sequence corresponding to neuromedin-B-preferring bombesin receptor comprising culturing the cell according to Claim 19 under conditions such that said DNA segment is expressed and said polypeptide thereby produced, and isolating said polypeptide.

21. An antibody having binding affinity to a recombinant neuromedin-B-preferring bombesin receptor, or unique portions thereof.

22. The antibody according to Claim 21, wherein said receptor has the amino acid sequence set forth in SEQ ID NO: 6, allelic or species variation thereof, or a unique portion thereof.

23. A recombinant or substantially pure nucleic acid comprising. a sequence exhibiting substantial homology to a nucleotide sequence encoding a receptor, or a fragment thereof, for a bombesin-like peptide.

24. A nucleic acid of Claim 23 further comprising sequence encoding a second polypeptide, or fragment thereof.

25. A vector, cell, or organism comprising a nucleic acid of Claim 23.

26. A recombinant or substantially pure polypeptide comprising a region exhibiting substantial identity to an amino acid fragment of a receptor for a bombesin-like peptide.

27. A polypeptide of Claim 26 comprising a fragment of a second polypeptide.

28. A subcellular structure, cell, or organism comprising a protein of Claim 26.

29. A method of producing a receptor, or fragment thereof, for a bombesin-like peptide comprising expressing a nucleic acid of Claim 23.

30. A method of screening for a compound having binding affinity to a receptor for a bombesin-like peptide comprising the steps of:
a) producing said receptor by a method of Claim 29, and b) assaying for the binding of said compound to said receptor.

31. An antibody having binding affinity for a receptor for a bombesin-like peptide or fragment thereof.

32. A method of simultaneously modulating a biological activity of a plurality of subtypes of receptors for bombesin-like peptides, comprising contacting said receptors with a compound which modulates said activity upon contacting said receptors.

33. An antibody exhibiting specificity of binding to at least one receptor for a bombesin-like peptide selected from the group consisting of:
a) a mouse R1BP, or fragment thereof;
b) a human R1BP, or fragment thereof;
c) a rat R2BP, or fragment thereof;
d) a human R2BP, or fragment thereof; and e) a human R3BP, or fragment thereof.

34. A method of modulating biological activity of a receptor for a bombesin-like peptide comprising contacting said receptor with a composition selected from the group consisting of:
a) an antibody which binds to said receptor;
b) a known agonist or antagonist to a receptor for a non-GRP bombesin-like peptide; and c) a ligand binding fragment from a receptor for a bombesin-like peptide.

35. A method of treating a host having cancer or exhibiting abnormal expression of a receptor for a bombesin-like peptide, comprising administering to said host a therapeutically effective amount of a composition comprising:
a) an antibody which binds to a receptor for a bombesin-like peptide;
b) an agonist or antagonist to a receptor for a non-GRP
bombesin-like peptide; or c) a ligand binding receptor, or fragment thereof, for a bombesin-like peptide.

36. A method of diagnosing for cancer in a host organism, comprising the steps of:
a) contacting a sample from said host with a specific binding reagent to:
i) a gene encoding a receptor for a bombesin-like peptide; or ii) a receptor for a bombesin-like peptide; and b) measuring the level of binding of said reagent to said sample.

37. A method of evaluating binding affinity of a test compound to a receptor for a bombesin-like peptide, said method comprising the steps of:
a) contacting a sample containing said receptor with i) a labeled compound having a known affinity for said receptor; and;
ii) said test compound; and b) measuring the level of bound labeled compound, said amount being inversely proportional to the amount of test compound which bound to said receptor.

38. A kit for determining the amount of a receptor for a bombesin-like peptide in a sample, comprising a compartment with a labeled compound having a known binding affinity for said receptor.

39. A kit for assaying antibody against a receptor for a bombesin-like peptide in a sample, comprising compartments having a said receptor and an antibody detection means.

40. A compound known to modulate activity of a receptor for a bombesin-like peptide, selected by a method of:
a) contacting said compound with isolated or recombinant receptor, or fragment thereof, for a bombesin-like peptide; and b) evaluating the effect on biological activity by said contacting.

41. Isolated DNA encoding the gastrin releasing peptide receptor or fragment thereof encoding a biologically active gastrin releasing peptide receptor polypeptide.

42. Isolated DNA which encodes a biologically active protein having gastrin releasing peptide receptor activity and which is capable of hybridizing with the DNA of SEQ ID NO: 1.

43. The DNA of Claim 42 wherein said protein has the amino acid sequence of SEQ ID NO: 2.

44. Isolated DNA encoding proteins which are homologous to the gastrin releasing peptide receptor, and said DNA being isolated using gastrin releasing peptide receptor cDNA as a probe.

45. The DNA sequence according to Claims 41, 42, or 44 characterized in that it further comprises the respective regulatory sequences in the 5' and 3' flanks.

46. A DNA sequence hybridizing to a DNA sequence according to Claims 41, 42, or 44 and containing mutations selected from the group consisting of nucleotide substitutions, nucleotide deletions, nucleotide insertions and inversions of nucleotide stretches and coding for a protein having gastrin releasing peptide receptor activity.

47. A recombinant DNA molecule characterized in that it comprises a DNA sequence according to Claims 41, 42, or 44.

48. A recombinant DNA molecule characterized in that it comprises a DNA sequence according to Claims 41, 42, or 44 that is operably linked to a genetic control element.

49. The recombinant DNA molecule of Claim 48, characterized in that said control element is selected from the group consisting of procaryotic promoter systems and eucaryotic expression control systems.

50. The recombinant molecule of Claim 47 wherein said molecule is an expression vector for expressing eucaryotic cDNA coding for the gastrin releasing peptide receptor in a procaryotic or eucaryotic host, said vector being compatible with said host and wherein the eucaryotic cDNA coding for the gastrin releasing peptide receptor is inserted into said vector such that growth of the host containing said vector expresses said cDNA.

51. A host characterized in that the recombinant DNA molecule according to Claim 47 has been introduced into said host, and which expresses the protein encoded by said DNA.

52. The host of Claim 51 which is selected from the group consisting of: procaryotes including gram negative and gram positive organisms including E. coli; lower eucaryotes including yeasts; and higher eucaryotes including animal cells and mammalian cells including human.

53. A recombinant protein which is encoded by a DNA sequence according to Claim 47 and which is substantially free of protein or cellular contaminants, other than those derived from the recombinant host.

54. A pharmaceutical composition comprising the recombinant protein of Claim 53 and a conventional pharmaceutically acceptable carrier and/or diluent.

55. A vector comprising DNA encoding the gastrin releasing peptide receptor or a fragment thereof encoding a biologically active gastrin releasing peptide receptor polypeptide.

56. The vector of Claim 55 wherein said DNA is under the control of a viral promoter.

57. The vector of Claim 55 which further comprises DNA
encoding a selection marker.

58. The vector of Claim 55 wherein said DNA encodes a predetermined, site-specific mutant gastrin releasing peptide receptor which has greater than about 50% amino acid homology with the gastrin releasing peptide receptor of SEQ ID NO: 2 and which exhibits biological activity in common with the gastrin releasing peptide receptor of SEQ ID NO: 2.

59. A cell from a multicellular organism transformed with the vector of Claim 55.

60. The cell of Claim 59 which is a mammalian cell.

61. A method comprising culturing the cell of Claim 59 in a nutrient medium, permitting the receptor to accumulate in the culture and recovering the receptor from the culture.

62. The method of Claim 61 wherein the receptor is recovered from the culture medium.

63. Antibodies having binding affinity to the recombinant gastrin releasing peptide receptor, or fragments thereof.

64. The antibodies of Claim 63 which are raised against the gastrin releasing peptide receptor, or fragments thereof.

65. The antibodies of Claims 63 or 64 wherein said receptor has the amino acid sequence of SEQ ID NO: 2.

66. The antibodies of Claim 65 wherein said fragments are selected from the group consisting of the following partial amino acid sequences: residues 1-39, inclusive; residues 64-77, inclusive; residues 98-115, inclusive; residues 138-157, inclusive; residues 176-209, inclusive; residues 236-266, inclusive: residues 288-300, inclusive; and residues 330-385, inclusive.

67. The antibodies of Claim 63 which are non-neutralizing antibodies.

68. The antibodies of Claim 63 which are neutralizing antibodies.

69. The antibodies of Claim 63 which are conjugated to toxins.

70. The antibodies of Claim 63 which are conjugated to radionuclides.

71. A kit for determining the concentration of gastrin releasing peptide receptor in a sample comprising a labeled compound having known binding affinity for the gastrin releasing peptide receptor, recombinant gastrin releasing peptide receptor, and a means for separating bound from free labeled compound.

72. The kit of Claim 71 wherein said means for separating is a solid phase for immobilizing the gastrin releasing peptide receptor.

73. The kit of Claim 71 wherein said labeled compound is a ligand.

74. The kit of Claim 73 wherein said ligand is gastrin releasing peptide.

75. The kit of Claim 71 wherein said labeled ligand is an antibody.

76. The kit of Claim 72 wherein said solid phase contains a capture molecule.

77. The kit of Claim 76 wherein said capture molecule is an antibody to the gastrin releasing peptide receptor.

78. A kit for determining the binding affinity of a test compound to the gastrin releasing peptide receptor comprising a test compound, a labeled compound having known binding affinity for the gastrin releasing peptide receptor, recombinant gastrin releasing peptide receptor and a means for separating bound from free labeled compound.

79. The kit of Claim 78 wherein said means for separating is a solid phase for immobilizing the solubilized gastrin releasing peptide receptor.

80. The kit of Claim 78 wherein said labeled compound is a ligand.

81. The kit of Claim 80 wherein said ligand is gastrin releasing peptide.

82. The kit of Claim 78 wherein said labeled ligand is an antibody.

83. The kit of Claim 79 wherein said solid phase contains a capture molecule.

84. The kit of Claim 83 wherein said capture molecule is an antibody to the gastrin releasing peptide receptor.

85. A method of treating patients having a disease or disorder associated with abnormal expression or abnormal triggering of the gastrin releasing peptide receptor comprising administering antibodies having binding affinity to the recombinant gastrin releasing peptide receptor.

86. A method of treating patients having a disease or disorder associated with abnormal expression or abnormal triggering of the gastrin releasing peptide receptor comprising administering recombinant gastrin releasing peptide receptor, or fragments thereof.