Note: Descriptions are shown in the official language in which they were submitted.
~WO 95!29233 -- PCTIUS95104860
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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
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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
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(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
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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),
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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.
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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%
3
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
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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
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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).
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Table 2. Putative catalytic domains of FAPa, DPPIV and DPPX.
625 701 733
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
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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'
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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
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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
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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.
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(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:
AAGAACGCCC CCAAAATCTGTTTCTAATTTTACAGAAATCTTTTGAAACTTGGCACGGTA60
TTCAAAAGTC CGTGGAAAGAAAAAAACCTTGTCCTGGCTTCAGCTTCCAACTACAAAGAC120
AGACTTGGTC CTTTTCAACGGTTTTCACAGATCCAGTGACCCACGCTCTGAAGACAGAAT180
TAGCTAACTT TCAAAAACATCTGGAAAAATGAAGACTTGGGTAAAAATCGTATTTGGAGT240
TGCCACCTCT GCTGTGCTTGCCTTATTGGTGATGTGCATTGTCTTACGCCCTTCAAGAGT300
TCATAACTCT GAAGAAAATACAATGAGAGCACTCACACTGAAGGATATTTTAAATGGAAC360
ATTTTCTTAT AAAACATTTTTTCCAAACTGGATTTCAGGACAAGAATATCTTCATCAATC42D
TGCAGATAAC AATATAGTACTTTATAATATTGAAACAGGACAATCATATACCATTTTGAG480
TAATAGAACC ATGAAAAGTGTGAATGCTTCAAATTACGGCTTATCACCTGATCGGCAATT540
TGTATATCTA GAAAGTGATTATTCAAAGCTTTGGAGATACTCTTACACAGCAACATATTA600
CATCTATGAC CTTAGCAATGGAGAATTTGTAAGAGGAAATGAGCTTCCTCGTCCAATTCA660
GTATTTATGC TGGTCGCCTGTTGGGAGTAAATTAGCATATGTCTATCAAAACAATATCTA720
TTTGAAACAA AGACCAGGAGATCCACCTTTTCAAATAACATTTAATGGAAGAGAAAATAA780
AATATTTAAT GGAATCCCAGACTGGGTTTATGAAGAGGAAATGCTTCCTACAAAATATGC840
TCTCTGGTGG TCTCCTAATGGAAAATTTTTGGCATATGCGGAATTTAATGATAAGGATAT900
ACCAGTTATT GCCTATTCCTATTATGGCGATGAACAATATCCTAGAACAATAAATATTCC960
ATACCCAAAG GCTGGAGCTAAGAATCCCGTTGTTCGGATATTTATTATCGATACCACTTA1020
CCCTGCGTAT GTAGGTCCCCAGGAAGTGCCTGTTCCAGCAATGATAGCCTCAAGTGATTA1080
TTATTTCAGT TGGCTCACGTGGGTTACTGATGAACGAGTATGTTTGCAGTGGCTAAAAAG1140
AGTCCAGAAT GTTTCGGTCCTGTCTATATGTGACTTCAGGGAAGACTGGCAGACATGGGA1200
TTGTCCAAAG ACCCAGGAGCATATAGAAGAAAGCAGAACTGGATGGGCTGGTGGATTCTT1260
TGTTTCAAGA CCAGTTTTCAGCTATGATGCCATTTCGTACTACAAAATATTTAGTGACAA1320
GGATGGCTAC AAACATATTCACTATATCAAAGACACTGTGGAAAATGCTATTCAAATTAC1380
AAGTGGCAAG TGGGAGGCCATAAATATATTCAGAGTAACACAGGATTCACTGTTTTATTC1440
TAGCAATGAA TTTGAAGAATACCCTGGAAGAAGAAACATCTACAGAATTAGCATTGGAAG1500
CTATCCTCCA AGCAAGAAGTGTGTTACTTGCCATCTAAGGAAAGAAAGGTGCCAATATTA1560
CACAGCAAGT TTCAGCGACTACGCCAAGTACTATGCACTTGTCTGCTACGGCCCAGGCAT1620
CCCCATTTCC ACCCTTCATGATGGACGCACTGATCAAGAAATTAAAATCCTGGAAGAAAA1680
CAAGGAATTG GAAAATGCTTTGAAAAATATCCAGCTGCCTAAAGAGGAAATTAAGAAACT1740
SUBSTITUTE SHEET (RULE 26)
WO 95/29233 ~ 18 8 ~ 6 4 t PCT/US95f04860
16
TGAAGTAGAT GAAATTACTT TATGGTACAA GATGATTCTT CCTCCTCAAT TTGACAGATC 1800
AAAGAAGTAT CCCTTGCTAA TTCAAGTGTA TGGTGGTCCC TGCAGTCAGA GTGTAAGGTC 1860
TGTATTTGCT GTTAATTGGA TATCTTATCT TGCAAGTAAG GAAGGGATGG TCATTGCCTT 1920
GGTGGATGGT CGAGGAACAG CTTTCCAAGG TGACAAACTC CTCTATGCAG TGTATCGAAA 1980 '
GCTGGGTGTT TATGAAGTTG AAGACCAGAT TACAGCTGTC AGAAAATTCA TAGAAATGGG 2040
to TTTCATTGAT GAAAAAAGAA TAGCCATATG GGGCTGGTCC TATGAGATAC GTTTCATCAC 2100
TGGCCCTTGC ATCTGGAACT GGTCTTTTCA AATGTGGTAT AGCAGTGGCT CCAGTCTCCA 2160
GCTGGGAATA TTACGCGTCT GTCTACACAG AGAGATTCAT GGGTCTCCCA ACAAAGATGA 2220
TAATCTTGAG CACTATAAGA ATTCAACTGT GATGGCAAGA GCAGAATATT TCAGAAATGT 2280
AGACTATCTT CTCATCCACG GAACAGCAGA TGATAATGTG CACTTTCAAA ACTCAGCACA 2340
GATTGCTAAA GCTCTGGTTA ATGCACAAGT GGATTTCCAG GCAATGTGGT ACTCTGACCA 2400
GAACCACGGC TTATCCGGCC TGTCCACGAA CCACTTATAC ACCCACATGA CCCACTTCCT 2460
AAAGCAGTGT TTCTCTTTGT CAGACTAAAA ACGATGCAGA TGCAAGCCTG TATCAGAATC 2520
TGAAAACCTT ATATAAACCC CTCAGACAGT TTGCTTATTT TATTTTTTAT GTTGTAAAAT 2580
GCTAGTATAA ACAAACAAAT TAATGTTGTT CTAAAGGCTG TTAAAAAAAA GATGAGGACT 2640
CAGAAGTTCA AGCTAAATAT TGTTTACATT TTCTGGTACT CTGTGAAAGA AGAGAAAAGG 2700
GAGTCATGCA TTTTGCTTTG GACACAGTGT TTTATCACCT GTTCATTTGA AGAAAAATAA 2760
TAAAGTCAGA AGTTCAAAAA AAAAAAAAAA AAAAAAAAAA GCGGCCGCTC GA 2812
SUBSTITUTE SHEET (RULE 26j
