CA2188264C - Isolated nucleic acid molecule coding for fibroblast activation protein .alpha. and uses thereof - Google Patents

Abstract

The invention describes the identification and isolation of nucleic acid mol ecules which code for fibroblast activation protein alph a, or "FAP.alpha.". Various applications of the isolated molecules are also des cribed.

Description

~WO 95!29233 -- PCTIUS95104860 21~~264 ISOLATED NUCLEIC ACID MOLECULE CODING FOR FIBROBLAST
ACTIVATION PROTEIN a AND USES THEREOF
FTFT.n OF T_H_E T_NVFTff'T-ON ,. ,.
This invention relates to certain molecules associated with cancer cells and reactive with tumor stromal cells. More ~l0 particularly, it relates to fibroblast activation protein alpha ("FAPa" hereafter). The molecule has previously been identified immunologically, but nucleic acid molecules coding for it had not been isolated or cloned. This, inter ~lis3, is the subject of the invention. The protein has a molecular weight of from about 88 to about 95 kilodaltons as determined by SDS-PAGE. This molecule is characterized by a number of features and properties which are shared by and characteristic of membrane bound enzymes, suggesting very strongly that it, too, is a membrane bound enzyme. The nucleic acid molecules, which are a key part of the invention, are useful both as probes for cell expressing FAPa, and as starting materials for recombinant production of the protein. The recombinant protein can then be used to produce monoclonal antibodies specific for the protein, and are thus useful diagnostic agents themselves. -nA~CROL~D AND PurOR A~Rm The invasive growth of epithelial cancers is associated with characteristic cellular and molecular changes in the supporting stroma. For example, epithelial cancers induce the formation of tumor blood vessels, the recruitment of reactive tumor stromal fibroblasts, lymphoid and phagocytic infiltrates, the release of-peptide mediators and proteolytic enzymes, and the production of an altered extracellular matrix (ECM). See, e.g., Folkman, Adv. Cancer Res. 43: 175-203 (1985); Basset et al., Nature 348: 699-704 (1990); Denekamp et al., Cancer Metastasis Rev. 9: 267-282 (1990): Cullen et al., Cancer Res. 51: 4978-4985 (1991) Dvorak et al., Cancer Cells 3: 77-85 (1991): Liotta et al., Cancer Res. 51: 5054s-5059s (1991); Garin-Chesa et al., J. Histochem. Cytochem. 37: 1767-1776 (1989). A highly consistent molecular trait of the stroma in several common histologic types of epithelial SUBSTITUTE SHEET (RULE 26) cancers is induction of the fibroblast activation protein (FAFa), a cell t~utface glycoprotein with arf observed M" of 95.,000 originally discovered with a monoclonal antibody, m7~b F19, raised against proliferating Cultured fibroblasts, Sae Rattig et al:,w Canoes Res. 46 : 6406-6412 (1986); Rettig et 0 al., ProC: Natl. Aca~. SCi. USA, 85: 3110-3114 (1988): Garin-Chesa et al.., :Proc. Natl. Aced. LISA 87: 7235-7239 (1990):
Rettiq et al . , CatiCer Res... 53: _3327-3335 ( 1993 ) .
;5 . Ttpn~unohistochCmi,.cal.studies such' .a8 those cited supra have shown that FAP_a~ is .tran$iently expressed in ~ certain normal fetal mesenchyma~. tissues but that nortrtal~ adult tissues are generally FA:Pa'. Similarly, ipalignant epithelial. neural and- hamatopoietic cells are FAPa.'. - fiawever, most -of the !o eomunon types of epithelial cancera, including >90~ of breast, lung, skin, pancreas, and colorectal carcirtoaias, contain abundant FAPa' r~aactive stromal fibroblasts. Garin-Chesa et al., Proc. Natl. Aced. Sei. USA 87n 7235-7239 (1990). The FAPa' tumor stxomal tibroblasts almost invariably accompany !5 newly-farmed tumflr blood vessels, forming a distinct cellular compartment interposed between the :tumor capillary endothelium arid the basal aspect of malignant epithelial cell cluster6.
While FARa' stror0.al fibroblasts are found in both primary and metastatic carc;lnomas, banign~and premalignant epithelial t0 lesions, such' as fibroedenomas of the breast and colorectal adenomas only raxely contain FAPa'.stromal cells-. In contrast.
to the stroma-specific localiaatipn of FlvPa in epithelial neoplasms, F~rPa is expressed in the fialignant cells of a large proportion of.ban~a.and soft tissue sarcomas. Rettig et al., t5 prwc. Nati. Aced. Sai, USA 85: 3110-3114 (1988). Finally, FAPa'fibrot~lasts have been detected.in the granulation tissue of healing wounds.(Garin-Chesa et al., sunrn). Eased on the restricted di6tribution pattern of FAPa in normal tissues and its. uniform ex~~ression in the supporting. stroma of many epithelial cancers, clinical trials with "'T-labeled.mAb Fl9 have been initiated in patients with metastatic colon cancer SU~BSTITUTt SHEET (RULE 26) W O 95/29233 ~ ~ ~ PCT/U595104860 (Welt et al., Proc. Am. Assoc. Cancer Res. 33: 319 (1992)) to explore the concept of "tumor stromal targeting" for immunodetection and immunotherapy of epithelial cancers.
The induction of FAPa" fibroblasts at times and sites of tissue remodeling during fetal development, tissue repair, and l10 carcinogenesis is consistent with a fundamental role for this molecule in normal fibroblast physiology. Thus, it is of interest and value to isolate and to clone nucleic acid molecules which code for this molecule. This is one aspect of the invention, which is described in detail together with other features of the invention, in the disclosure which follows.
BRTEF DESORTPTTON OF '~E FTGL~ES -_ Figure 1 shows results obtained from immunoprecipitation studies carried out on detergent extracts of Trans 'SS-labeled cells. The study was designed to immunoprecipitate FAPa and CD26. Cell types were SW872, which is a human sarcoma cell line, COS-FAP, which is a cell line transfected with a vector coding for FAPa, i-.e., pFAP-38, described in the application, and COS-CD26, which is a COS cell line transfected with a CD26 coding plasmid. Extracts were precipitated with anti-FAPa monoclonal antibody F19 , anti-CD26 mAb EF-1 , or a negative control mouse Ig.
Figure 2A presents Northern blot analysis of FAPa expression in a cell line (ovarian cancer SK-OV6), which has FAPa'/CD26' phenotype), as well as two cell lines (fibroblasts WI-38 and GM 05389), which have FAPa'/CD26' phenotype.
Figure 2B shows y-actin expression forthe cell lines of figure 2A.
Figure-3 compares the deduced amino acid sequence for FAPa, and the known sequence of CD26. The alignment has been optimized.
Figure 4 depicts heterodimer formation between FAPa and CD26 in COS-1 transfectants.
Figures 5A-5H, inclusi-ve, display immunohistochemical detection of FAPa and CD26 in various cancers. In figures 5A
and 5B, breast cancer is studied, for-FAPa (figure 5A), and SUBSTITUTE SHEET (RULE 26j w0 951:9233 PC?NS95104860 .

CD26 (fiqure_ 5H). In figures 5C and 5D, malignant fibrous' histiocytoma are studied,. for FAPa (figure 5C), and CD26.
(figuxe 5D). D~trmal sear tissue is examined in figures 58 (FAPa), and SF ~CD26),. Renal cell carcinoma 1s studied in figure 5G (FAPa.), and 5H (CD26).
LO DETAT ~ED, DES~,',$ZPTION OF 'PREFERR.ED Eli80DIliEI~iTS
Exa;m_Rle 1 Fibroblast cell line WI-38 had been observed, previously;
to react w~,th ipAb F19 (Rett7.g et al., Cane. Res. 46:6406.-6412 .
( 19.86 ) ; Rettig et al . , Proc: .Natl . Acad. USA 85 : 3110-3114.
- (1988); Garin-Ctlesa et al.. Froc. Natl. Acad. Sci.. .uSA 87:
. 7235-7239 (1990) R~3ttig et al., Cane. Res. 53: 3327-3335 (1993)). It wa.: used in the experiments which follow.
A cDNA library was prepared from wI-38, using well known techniques and commercially available materials.
2o Specifically, true library was constructed in expression vecfor pCDNAI, using tlxe Fast Track"tIRNA isolation ki't, and Iabrarium*
cDNA phagemid system. once the. library ,was prepared, the Vectors were eleCtroporated into cell line )~.: ~ MC 1061/P3. ' The- pCDNJ~I expression vector contains an antibiotic resistance .
gene, so the ~. co were selectQd. via antibiotic resistance.
The colonies which were resistant were then used in further experiments. z~he plasmid DNA from the. colonies was obtained via alkaline lysis and purification on CsCl" in accordance with Sambrook sa al, Molecular Cloning: a LaboFatory Manual (Cold Spring flarbor Lab, Cold Spring harbor, N:Y. 2d Ed:
1989). The technique. is well. known to, the art, Once the plaxmid DNA was isolated; ,it was used to transfect COs-1' cells, which were then cultured for forty-eight hours, after which these was tested with antibody coated dishes. The mAbs used included F19, as described by Rettig et al . , ( 1986 ), supra.
As COS-1 cells are normally FAPa', any positive r~sults indicated the presence of the coding sequence. The.
4o immunoselection protocol was that of Aruffo et al., Proc.
Natl: Acad. Sri SSA 84: 3365-3389 (1987), SUBSTITUTE SHEET (RULE 26) *Trade-mark wo 9sa9z33. ~ ~rrnrs9sroissfl -s.
Plasmid DNA frog positive clones was: xecod~red, in accordance with Hint, J. Mol. Biol: Z6: 365-369 (196y), reintroduced into .,~. c~li HC.lo61/P3,. and rtcelected ;into COS-1' ce11.8.. . .. ~, :. : ; .. . : -fihe protocol prdsented herein was vfol~lowed_ for tour rounds, After this,- tlle_ plnsmid DNl1'of .50 isolated bacterial colonies was, purified; casing t~he..Qiagen plasmid kit. of ~tha colowies., x7 clones were.-found to~-contaiD identical 2.8 kb inserts, as deter~minad by EcoRI .restriction enayme mapping.
Several of these were. fo~td to contain FAPa-specific cDNA, .via .
transient expressioix in COS-1 cells. ~ and direct i~amunofluorescenee .staining. Oi~e of these clones, i:e., "pFAP.~38" was sei~ected for further study, as elaborated upon , infra .
Exam Once.pFAP..38 had~been idsntigied,.it was tested together with a vector coding: for known well surface marker CD2s ("pCD26."), as well as.with control vector pCDNl~ I..
In these experimapts,.COS-1 cells were transfected with , i one of pFaP..38, pCD26, or pCDNAI. Inter forty-eight hours, .
the. transfectant.~ were tested,, using ~Che well down MHA
roaett~.ng assay fox cell surface antigen expression. In these experiments,'mAb F19., which is FAPa spect.fic, was us~d, together -with mAb EF-1, which .3e CDZ6 sgQCific.. ?rlso used were four other'F.lvPa specific mAbs, i.e., FB23, F852, FH58 and C48.: Also tested were two.oanoer cell lines, which~are known to react with ai1!,b F19 (SWS~2 liposarcoma), or EF,1 (SK-OV6 ovarian cancer).. The.results ere, sit forth in. Table 1, which follows.
SUBSTITUTE SNEcT (RULE 2G) WO 95/19233 . PCT1US95ro486Q
Table 1. Cell surface exprassio~ of multiple FAPQ epitopes and CD26 in human cells and GDS-~. cell transf.ectants.
o Target Cell ~ F19. FBZ3 FB52 FB58 C4$ EF-T
Hyman cgls SW87-2: liposarcoaN ~ . 95i: ~ >95% >95% >95% >959G -SW-OV6 ovarian. cancer - - - - >9.5',G
OS-1 transfa tents . _ COS-pCDNAI corutrol . :. . . _ Co5-pFAP 38 4ox ~ 30x ' 40x . 2ox 2ox -.5. CDS-pC026~. ~ _ , _ v_ 40%

Immunoprecipitatian studies..-.were ther'i carried. aut to identify the antigen.baiitc~ targeted by the antibodies:. . ~ , v.o Cells were metabolioally labelled with Trans '°S-labe;i, (ICN), extracted with lysis buffer (0.oi H Tris-HC1/0,~15 M
NaCi/0.01 M MgCl=/0,.5% Nonidet P-~6*/aprotinin (20 ug/ml)/2 mH
phenyimethyl- sulfonyl fluoride), and then immunopreaipitated.
The protocols used are all well known, as will be seen. by 35'~ reference to Rettig et al.., Cane. Res. 53. 332?-3335 (1993);
and Felling~r at al:, Canc. Res. 51 : 336-340 (1991~,~
Precipitating mAhs were negatfve control mouse =g, mAb F19, or EF-1. Control tests were carried out 40. with mock transfected COS-l cells.,. Following.
immunoprecipitstion,.the immunoprecipitates were separated on Nt~DOdSO,/PAGE, under reducing conditions. In some experiments,. an additional. test was- carried out to.aatermine whether or not the immunoprecipitated material was 45 glycosylated. 1n these experiments, cell extracts' were fractionated with con A-SEPHAROSE prior to irnmunoprecipitation_ Following imaiunopreeipitation, but prior to fractionation on NaDodSO,/PAGE, these precipitates were SUBSTITUTE SHEET(RULE 26j *~Trade -mark 2:188264 WO 95!29233 PCT/US95/04860 digested with[JN~G~y~ar~ase.
The results 0a('re shown in figure 1. In COS-1 cells, pFAP.38 directs expression of an 88 kd protein species (as determined via SDS-PAGE), which is slightly smaller than the 95 kd FAPa species produced by SW872, or cultured fibroblasts.
. 10 Digestion with N-Glycanase produced peptides of comparable size (i.e., 74 kd versus 75 kd), showing that the glycosylation of the FAPa protein in COS-1 cells is different than in the human cell lines.
FXamTJla 4 Classic Northern blot analysis was then carried out, using the mRNA from FAPa' fibroblast cell lines WI-38 and GM
05389, and FAPa'ovarian cancer cell line SK-OV6. Using the procedures of Sambrook et al., supra, five micrograms of mRNA
from each cell line were tested. The probes used were "P
labelled, and were prepared from a 2.3 kb ECO I fragment of pFAP.38, a 2.4 kb Hind III fragment of CD26, and a 1.8 kb BamHI fragment of Y-actin cDNA. These fragments had been purified from 1% agarose gels.
Figure 2 presents these results. The extracts of FAPa' fibroblast strains show a 2.8 kb FAP mRNA species, but extracts of SK-OV6 do not. A y-actin mRNA species (1.8 kb), is seen in all species.
EXamt~~ P 5 The cDNA identified as coding for FAPa was subjected to 3o more detailed analysis, starting with sequencing. The classic Sanger methodology, as set forth in Proc. Natl. Acad. Sci. USA
74: 5463-5467 (1977), was used to sequence both strands of the cDNA. Once this was secured, an amino acid sequence was deduced therefrom. This information is presented in SEQ ID
NO: 1. The sequence was then compared to the known amino acid sequence of CD26 (Morimoto et al., J. Immunol. 143: 3430-3437 (1989)). Figure 3 presents the comparison, using optimized sequence alignment.- Any gaps in the comparison are indicated by asterisks, while identical amino acids are shown by dashes in the CD26 sequence. A hydrophobic, putative transmembrane sequence is double underlined, while potential N-glycosylation SUBSTITUTE SHEET (RULE 26) WO 95r19t~3 . PC110S95Jb4860 5" sites are single'underlined.-The sequence anitlysis shows. a 2812 base pail irirert,, wherein 2277 base~pairs constitute the ,open reading frame:
This ORF extends from start colon aTG at nucleotide 209, to stop cpdon TAA at ~ 241:6 .
to The deduced peptide is 759 amino acids long, aid has a molecular weight. of 88,233. In cotltrast, N-Glycanase digested, immunopurified F'APa was reported to have an estimated M, of 75,000 on NaDodSO,%PAGE~(Rettig et al., Canc.
xes. 53: 3327-3335 (1993)). A_TlrTA~boX is found 83 base pairs i5 . upstream o~ the start colon. A polyadenylationsignal and a poly-A' tail were found in tyre 5'-untranslated'region of the insert.
A GenBank* dat~r bass search' was then carried out.. The moat closely related genes found were those encoding 20 dipeptidyl peptidase IV homologues (DPPIV: 8C 3.4.14.5), with ' human DPpIv (also known as T-cell activation antigen CD26),~.
showing 61% nucleotide sequence. identity, and 48~ 4:41!!0 acid' sequence identity.
The second set-of related' genes are human, rat, and '.
25 bQVine homologues of DPP7(, a gene, of .unknown function widely expres$ed in brain. and othtr normal tissues.- The pret~icted human DPPX gene prc,duct,shows about 30~ amino acid sequence identity with FAPa and CD26. The FAB~t molecule exhibits structural ~featurer~ typical of type II integral membrane 30 proteins, including a large ~OGH-terminal extracaliular domain, a hydrophobic transme~abrane segment,, , acd a short cytoplas~mic.~tail.., The putative extracellular domain. contains six poteritinil"N~Qlycosylation Sites, 13 cysteina.residues (8 of which are conserved Detwe~a FhPa arid CD26),..and three 35 ~ segments corresponding to highly..conceried catalytic domains characteristic of ,serine proteases,. such as .DPPIV. These conserved sequsnce~s are presented in Table 2, which follows;
Comparisons ta~DBPTV and DPPX were made via Morimoto et al:, Wade e'c al., PrOC. Natl, Aced: Sci. USA 89: 1971201 40 (1992);: YokOtani et al., Human Mol. Genet, 2: iG~37-1439 (1993).
S(JBSTITUTE SHEET (RULE 26) *Trade-mark 2~ 1. 8 8 2 6 4 P~~s95~o~860 ~!t j ~~ 9 Table 2. Putative catalytic domains of FAPa, DPPIV and DPPX.

Human FAPa ....WGWSYEI........GTADDNV........DQNHGLS....
Human DPPIV ....WGWSYGG........GTADDNV......,.DEDHCIA....
Mouse DPPIV ....WGWSYGG........GTADDNV.....,..DEDHGIA....
Rat DPPIV ....WGWSYGG........GTADDNV..... " .DEDHGIA....
Yeast DPPIV ....FGWSYGG........GTGDDNV........DSDHSIR....
Human DPPX ....FGKDYGG........pTADEKI........DESHYFT....
Rat DPPX ....FGKDYGG........ATADEKI........DESHYFH....
Bovine DPPX ....FGKDYGG........ATEDEKI......,.DESHYFS....
Fxamn~p 6 An additional set of experiments were carried out to determine whether FAPa related sequences are present in non-human species. To do so, human, mouse, and Chinese hamster genomic DNA was digested using restriction enzymes, and tested, via Southern blotting, using the 2.3 kb fragment, labelled with "P, describes supra. Hybridization was carried out using stringent washing conditions (0.1 x SSC, 0.1%
NaDodSO" 68°C). Cross-hybridization was readily observed with both the mouse and hamster DNA, suggesting the existence of highly conserved FAPa homologues. In control experiments using the CD26 cDNA fragment described supra, no evidence of cross hybridization was observed.
FxamD~e 7 The CD26 molecule shares a number of biochemical and serological properties with FAPB, which is a previously described, FAPa associated molecule having a molecular weight r of 105 kd, and found on cultured fibroblasts and melanocytes (Rettig et al., Canc. Res. 53: 3327-3335 (1993)).
Cotransfection experiments were carried out to determine whether FAPB is a CD26 gene product. To test this, the same protocols were used which were used for transfection with pFAP.38 or pCD26, as described su ra, but using the two SUBSTITUTE SHEET (RULE 26) rcr~s9sroas6o to vectors. The results presented supra showed that cotransfection efficiency was about 40% for each vector, so about 10-20% of cell should be cotransfected.
Following cotransfection, the COS-1 cells were Trans "S-labeled, as described supra, then lysed, also as described l0 supra.
The resulting cell extracts were separated on Con A
SEPHAROSE, and the antigen (FAPa and/or-CD26) were recovered in the Con A-bound fraction. The bound fraction was eluted with 0.25 M a-D-mannopyranoside. Immunoprecipitation was then carried out, as described supra, and the precipitates were separated on NaDodSO,/PAGE, also as discussed supra.
Figure 4 shows these results, together with results from single transfection experiments. Those cells transfected only with pFAP.38 produce FAPa, but not FAPB (determined from mAb F19 immunoprecipitates). They also produce no CD26 antigen (tested with EF-1). Those cells-transfected with pCD26 alone produce CD26 but no FAPa. Cotransfectants produce CD26 ~
FABa/FAP13 heteromers, as determined in the mAb F19 precipitates. This result provides direct evidence that FAPfi is a CD26 gene product.
Examgle 8 - _ It has been observed previously that some cultured human cell types coexpress FAPa and CD26, and- show FAPa/CD26 heteromer -formation. ~ vivo distribution-patterns of-FAPa and CD26, however, as determined in previous immunohistochemical studies, appeared to be non-overlapping.
(See Rettig et al., Proc. Natl. Aced. Sci. USA 85: 3110-3114 ( 1988 ) ; Garin-Chesa et al. , Proc. Natl. Aced. Sci. USA 87:
7235-7329 (1990); Rettig et al., Canc. Aes. 53: 3327-3335 . _ (1993); Stein et al., in Knapp et al., eds. Leukocyte typing IV-white cell differentiation antigens, pp 412-415 (Oxford University Press, N.Y. 1989), pp. 412-415: Mobious et al., J.
Exp. Immunol. 74: 431-437 (1988)). In view of the potential , significance of FAPa/CD26 coassociation, tissue. distribution was reexamined, via side by side immunohistochemical staining of normal tissues and lesional tissues known to contain FAPa' SUBSTITUTE 5HEE7 (RULE 26) fibroblasts or FAPa* malignant cells.
To test the sampl~~,f~t~~y were embedded in OCT compound, frozen in isopentane precool~ed in liquid nitrogen, and stored at -70°C until used. Five micrometer thick sections were cut, mounted on poly-L-lysine coated slides, air dried, and fixed ' 10 in cold acetone (4°C, for 10 minutes). The sections were then tested with mAbs (10-20 ug/ml), using the well known avidin-biotin immmuno-peroxidase method, as described by, e.g., Garin-Chesa et al., J. Histochem. Cytochem. 37: 1767-777F
( 1989 ) ; Gaxin-Chesa et al . , Proc. Natl . Acad. Sci . USA 87:
7235-7239 (1990); Rettig et al., Canc. Res. 53: 3327-3335 (1993); Garin-Chesa et al., Am. J. Pathol. 142: 557-567.
The results are shown in figure 5. Breast, colorectal, pancreas and lung carcinomas showed strong expression of FAPa and not CD26 was found (see figures 5A and 5B). Five FAPa*
sarcomas, including malignant fibrous histocytoma (figures 5C
and 5D) , were tested, and there was no expression of CD26.
Examination of reactive fibroblasts of healing dermal wounds (figures 5E, 5F),- showed abundant expression of both FAPa and CD26. The three renal carcinomas tested (figures 5G, 5H), showed expression of CD26 in malignant epithelium. FAPa was absent from malignant epithelial cells, and showed low expression in the stroma of these carcinomas.
The foregoing examples describe an isolated nucleic acid molecule which codes for fibroblast activating protein alpha ("FAPa"). The expression product of the sequence is a protein which, on SDS-PAGE, shows a molecular weight of about 75 kd.
Deduced amino acid sequence, as provided in SEQ ID NO: 1, for one form of the molecule, yields a molecular weight of about 88 kd. It is to be understood that, as described, FAPa may be glycosylated, with the type and amount of glycosylation varying, depending upon the type of cell expressing the molecule. The experiment described herein shows this.
The invention also comprehends the production of expression vectors useful in producing the FAPa molecule. In their broadest aspect, these vectors comprise a FAPa coding sequence, operably linked to a promoter. Additional elements SUBSTITUTE SHEET (RULE 2B) 2-18p264 may be a part of the expression vector, such as genes which confer antibiotic resistance, amplifiable genes, and so forth.
The coding sequences and vectors may also be used to prepare cell lines, wherein the coding sequence or expression vector is used to transfect or to transform a recipient host.
The type of cell used may be prokaryotic, such as ~. coli, or ' eukaryotes, such as yeast, CHO, COS, or other cell types.
The identification of nucleic acid molecules such as that set forth in SEQ ID NO: 1 also enables the artisan to identify and to isolate those nucleic acid molecules which hybridize to it under stringent conditions. "Stringent condition" as used herein, refers -to those parameters set forth supra, whereby both murine and hamster sequences were also identified.- It will be recognized by the skilled artisan that these conditions afford a degree of stringency which canbe achieved using parameters which vary from those recited. Such variance is apprehended by the expression "stringent conditions".
The ability of nucleic acid molecules to hybridize to complementary molecules also enables-the artisan to identify cells which express FAPa, via the use of a nucleic acid hybridization assay. One may use the sequences described in the invention to hybridize to complementary sequences, and thus identify them. In this way, one can target mRNA, e.g., which is present in any cell expressing the FAPa molecule.
It is of course understood that the nucleic acid molecules of the invention are also useful in the production of recombinant FAPa. The recombinant protein may be used, e.g., as a source of an immunogen for generation of antibodies akin to known mAb F19, and with the same uses. Similarly, the recombinant protein, and/or cells which express the molecule on its surface, may be used in assays to determine antagonists, agonists, or other molecules which interact with , the FAPa molecule. Such molecules may be, but are- not necessarily limited to, substrates, inhibiting molecules, antibodies, and so forth. This last feature of the invention should be considered in light of the observed structural resemblances to membrane bound enzymes. This type of molecule SUBSTITUTE SHEET (RULE 26) W O 95129233 2 ~ g 8 2 6 4 PCT/US95/04860 is associated with certain properties which need not be described in detail here. It will suffice to say that inhibition or potentiation of these properties as associated with FAPa is a feature of this invention. For example, one may identify substrates or the substrate for the FAPa ' 10 molecule, via the use of recombinant cells or recombinant FAPa per se. The substrates can be modified to improve their effect, to lessen their effect, or simply to label them with detectable signals so that they can be used, e.g., to identify cells which express FAPa. Study of the interaction of substrate and FAPa, as well as that between FAPa and any molecule whatsoever, can be used to develop and/or to identify agonists and antagonists of the FAPa molecule.
Other aspects of the invention will be clear to the skilled artisan, and need not be set forth here.
The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.
SU6ST!TUTE SHEET (RULE 26) WO 95!29233 PCTlUS95104860 (1) GENERAL INFORMATION:
(i) APPLICANTS: Rettig, Wolfgang J.; Scanlan, Matthew J.;
Garin-Chesa, Pilar; Dld, Lloyd J.
(ii) TITLE OF INVENTION: I50LATED NUCLEIC ACID MOLECULECODING ' FOR FIBROBLAST ACTIVATION PROTEIN a AND USES THEREOF
(iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Felfe & Lynch (B) STREET: 805 Third Avenue (C) CITY: New York City (D) STATE: New York 2p (E) COUNTRY: USA
(F) ZIP: 10022 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette, 5.25 inch, 360 kb storage (B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: Wardperfect (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/230,491 (B) FILING DATE: 20-APRIL-1994 (C) CLASSIFICATION: 435 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Hanson, Norman D.
(B) REGISTRATION NUMBER: 30,946 (C) REFERENCE/DOCKET NUMBER: LUD 330 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 688-9200 (B) TELEFAX: (212) 838-3884 SUBSTITU T E SHEET (RULE 26j WO 95129233 2 ~ .8 8 2 6 4 PCT~595104860 _ 15 (2) INFORMATION SEQ ID 1:
FO(2 N0:

(i) SEQUENCE
CHARACTERISTICS:

(A) LENGTH:
2812 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
double (D) TOPOLOGY:
linear (xi) SEQUENCE SEQ ID 1:
DESCRIPTION: N0:

SUBSTITUTE SHEET (RULE 26) WO 95/29233 ~ 18 8 ~ 6 4 t PCT/US95f04860 GGTGGATGGT CGAGGAACAG CTTTCCAAGG TGACAAACTC CTCTATGCAG TGTATCGAAA 1980 ' to TTTCATTGAT GAAAAAAGAA TAGCCATATG GGGCTGGTCC TATGAGATAC GTTTCATCAC 2100 SUBSTITUTE SHEET (RULE 26j

Claims (7)

WE CLAIM:

1. Isolated nucleic acid molecule which codes for mammalian FAP.alpha. having a molecular weight from about 88 to about 95 kilodaltons as determined by SDS-PAGE, the complement of which hybridizes to the isolated nucleic acid molecule set forth in SEQ ID NO: 1, under stringent conditions, (0.1 × .
SSC, 0.1% NaDodSO4, 68°C).

2. The isolated nucleic acid molecule of claim 1, wherein said FAP.alpha.
consists of a sequence of the amino acids encoded by the nucleotide sequence set forth in SEQ ID NO: 1.

3. The isolated nucleic acid molecule of claim 1, consisting of the nucleotide sequence of SEQ ID NO: 1.

4. Expression vector comprising the isolated nucleic acid molecule of claim 1, operably linked to a promoter.

5. Cell line transformed or transfected by the isolated nucleic acid molecule of claim 1.

6. Cell line transformed or transfected by the expression vector of claim 4.

7. Method for determining presence of mRNA corresponding to a gene which encodes FAP.alpha. in a cell comprising contacting said cell with the isolated nucleic acid molecule of claim 1 and determining hybridization of said isolated nucleic acid molecule to a complementary sequence in said cell as a determination of presence of mRNA for said FAP.alpha..