U.S. patent application number 13/447626 was filed with the patent office on 2012-08-30 for use of an anti-cd151 antibody in the treatment of cancer.
This patent application is currently assigned to PIERRE FABRE MEDICAMENT. Invention is credited to LILIANE GOETSCH, JEAN-FRAN OIS HAEUW.
Application Number | 20120219569 13/447626 |
Document ID | / |
Family ID | 37866318 |
Filed Date | 2012-08-30 |
United States Patent
Application |
20120219569 |
Kind Code |
A1 |
HAEUW; JEAN-FRAN OIS ; et
al. |
August 30, 2012 |
USE OF AN ANTI-CD151 ANTIBODY IN THE TREATMENT OF CANCER
Abstract
The present invention relates to the use of at least one
antibody, or a functional fragment thereof, which is capable of
binding to the CD151 protein and thereby inhibiting tumour growth,
in the preparation of a medicament intended for the treatment of
cancer. The invention is also directed to a composition for the
treatment of cancer, comprising, as active ingredient, at least one
anti-CD151 antibody, or a functional fragment thereof, which is
capable of binding to the CD151 protein and/or of inhibiting the
development of primary tumours and/or of inhibiting its
metastasis-promoting activity, which antibodies may consist of the
antibodies TS151 and/or TS151r.
Inventors: |
HAEUW; JEAN-FRAN OIS;
(BEAUMONT, FR) ; GOETSCH; LILIANE; (AYZE,
FR) |
Assignee: |
PIERRE FABRE MEDICAMENT
BOULOGNE BILLANCOURT
FR
|
Family ID: |
37866318 |
Appl. No.: |
13/447626 |
Filed: |
April 16, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12311682 |
Apr 7, 2009 |
8178096 |
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PCT/FR2007/001688 |
Oct 15, 2007 |
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13447626 |
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Current U.S.
Class: |
424/174.1 ;
530/387.3; 530/388.85; 530/389.7 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 35/04 20180101; A61P 35/00 20180101; C07K 16/2896 20130101;
C07K 2317/34 20130101 |
Class at
Publication: |
424/174.1 ;
530/389.7; 530/388.85; 530/387.3 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; C07K 16/30 20060101
C07K016/30 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 18, 2006 |
FR |
0609135 |
Claims
1. A composition for the treatment of cancers which overexpress a
human CD151 protein, comprising as an active ingredient, at least
one anti-CD151 antibody which binds to the human CD151 protein,
whereby the at least one anti-CD151 antibody inhibits the
development of primary tumors.
2. The composition of claim 1, wherein the at least one anti-CD151
antibody inhibits primary tumor growth.
3. The composition of claim 1, wherein the at least one anti-CD151
antibody inhibits primary tumor cell proliferation.
4. The composition of claim 1, wherein the at least one anti-CD151
antibody inhibits tumor cell migration.
5. The composition of claim 1, wherein the at least one anti-CD151
antibody inhibits cell invasion by tumor cells.
6. The composition of claim 1, wherein the at least one anti-CD151
antibody inhibits tumor cell adhesion.
7. The composition of claim 1, wherein the at least one anti-CD151
antibody binds to an epitope on the extracellular loop 2 (EC2)
region comprising, at least, the amino acids Glutamine, Arginine
and Aspartic Acid at positions 194, 195 and 196 (QRD.sup.194-196),
respectively, of the CD151 protein.
8. The composition of claim 1, wherein the at least one anti-CD151
antibody is a monoclonal antibody.
9. The composition of claim 1, wherein the at least one anti-CD151
antibody is a binding fragment selected from Fv, scFv, Fab,
F(ab').sub.2, F(ab') and scFv-Fc.
10. The composition of claim 1, wherein the at least one anti-CD151
antibody is a TS151 antibody.
11. The composition of claim 10, wherein the TS151 antibody
comprises: three heavy-chain complementarity determining regions,
CDR-H1 (SEQ ID NO: 7), CDR-H2 (SEQ ID NO: 8) and CDR-H3 (SEQ ID NO.
9); and three light-chain complementarity determining regions,
CDR-L1 (SEQ ID NO: 11), CDR-L2 (SEQ ID NO: 12) and CDR-L3 (SEQ ID
NO: 13).
12. The composition of claim 11, wherein the TS151 antibody
comprises: a heavy chain further comprising the sequence SEQ ID No.
10, and/or a light chain further comprising the sequence SEQ ID No.
14.
13. The composition of claim 1, wherein the at least one anti-CD151
antibody is a TS151r antibody.
14. The composition of claim 13, wherein the TS151r antibody
comprises: three heavy-chain complementarity determining regions,
CDR-H1 (SEQ ID NO: 15), CDR-H2 (SEQ ID NO: 16) and CDR-H3 (SEQ ID
NO. 17); and three light-chain complementarity determining regions,
CDR-L1 (SEQ ID NO: 19), CDR-L2 (SEQ ID NO: 20) and CDR-L3 (SEQ ID
NO: 21).
15. The composition of claim 13, wherein the TS151r antibody
comprises: a heavy chain further comprising the sequence SEQ ID No.
18, and/or a light chain further comprising the sequence SEQ ID No.
22.
16. The composition of claim 1, wherein the at least one anti-CD151
antibody which binds to the human CD151 protein is selected from a
TS151 antibody, a TS151r antibody and combinations thereof.
17. The composition of claim 1, which is administered to a human or
animal subject in an amount effective for the treatment of cancers
which overexpress a human CD151 protein.
18. The composition of claim 1, which is administered to a human or
animal subject in an amount effective for the treatment of colon
cancer.
19. The composition of claim 1, which is administered to a human or
animal subject in an amount effective for the treatment of lung
cancer.
20. The composition of claim 1, which is administered to a human or
animal subject in an amount effective for the treatment of prostate
cancer.
21. The composition of claim 1, which is administered to a human or
animal subject in an amount effective for the treatment of
pancreatic cancer.
22. The composition of claim 1, which is administered to a human or
animal subject in an amount effective for the treatment of primary
tumors.
23. The composition of claim 1 further comprising at least one
pharmaceutically acceptable carrier.
Description
[0001] The present invention relates to a new use of anti-CD151
antibodies capable of inhibiting tumour growth, said antibodies
being especially monoclonal of murine origin, chimaeric and
humanised. According to a particular aspect, the invention relates
to the use of those antibodies, or of functional fragments thereof,
as medicaments for the prophylactic and/or therapeutic treatment of
cancers. Finally, the invention includes products and/or
compositions comprising such antibodies in association, for
example, with anti-cancer agents and/or antibodies or conjugated
with toxins, and use thereof in the prevention and/or treatment of
certain cancers.
BACKGROUND OF THE INVENTION
[0002] CD151, also referred to as PETA-3 or SFA-1, is a membrane
protein belonging to the tetraspanin family (Boucheix and
Rubinstein, 2001, Cell Mol. Life Sci. 58, 1189-1205; Hemler, 2001,
J. Cell Biol. 155, 1103-1107). In humans, CD151 has 253 amino acids
and includes 4 membrane fragments and 2 extracellular domains EC1
(18 amino acids, sequence [40-57]) and EC2 (109 amino acids,
sequence [113-221]) which are also referred to as extracellular
loops. It is to be noted, however, that, in the nucleotide
sequence, two variants of CD151 have been identified hitherto,
namely one having nucleotides A and C at positions 395 and 409,
respectively, (SEQ ID No. 1) [Fitter et al., 1995, Blood 86(4),
1348-1355] and the other having, at the same positions, nucleotides
G and T instead of nucleotides A and C [Hasegawa et al., 1996, J.
Virol. 70(5), 3258-3263]. As a result, a mutation can be observed
in the peptide sequence, namely a mutation of the residues K (Lys)
and P (Pro) at positions 132 and 137, respectively, to the residues
R (Arg) and S (Ser) [Fitter et al., 1995, Blood 86(4),
1348-1355/Hasegawa et al., 1996, J. Virol. 70(5), 3258-3263].
[0003] CD151 is overexpressed in numerous cancers such as, for
example, cancers of the lung [Tokuhara et al., 2001, Clin. Cancer
Res. 7, 4109-4114], colon [Hashida et al., 2003, Br. J. Cancer 89,
158-167], prostate [Ang et al., 2004, Cancer Epidemiol. Biomarkers
Prev. 13, 1717-1721] or pancreas [Gesierich et al., 2005, Clin.
Cancer Res. 11, 2840-2852].
[0004] The use of knock-out mice which do not express CD151 and of
anti-CD151 antibodies and siRNA in order to block, in vitro, the
functionality and expression of CD151 in various types of cell has
allowed it to be shown that CD151 is involved in a number of
phenomena related to cancer, such as cell adhesion (Nishiuchi et
al., 2005, Proc. Natl. Acad. Sci. USA 102, 1939-1944; Winterwood et
al., 2006, Mol. Biol. Cell 17, 2707-2721), cell motility (Kohno et
al., 2002, Int. J. Cancer 97, 336-343), cell migration (Yauch et
al., 1998, Mol. Biol. Cell 9, 2751-2765; Testa et al., 1999, Cancer
Res. 59, 3812-3820; Penas et al., 2000, J. Invest. Dermatol. 114,
1126-1135; Klosek et al., 2005, Biochem. Biophys. Res. Commun 336,
408-416), cell invasion (Kohno et al., 2002, Int. J. Cancer 97,
336-343; Shiomi et al., 2005, Lab. Invest. 85, 1489-1506; Hong et
al., 2006, J. Biol. Chem. 281, 24279-24292) and angiogenesis
(Yanez-Mo et al., 1998, J. Cell Biol. 141, 791-804; Sincock et al.,
1999, J. Cell Sci. 112, 833-844; Takeda et al., 2006, Blood).
[0005] One of the noteworthy properties of the tetraspanins is
their ability to form associations amongst themselves and also with
a large number of other surface molecules so as to form structured
macromolecular complexes. Within those complexes, each tetraspanin
is associated specifically with one or more surface molecules,
thereby forming primary complexes composed of a tetraspanin and a
partner molecule. The tetraspanins are capable of organising
particular microdomains of the plasma membrane from which
microdomains they may recruit their partner molecules, which may be
functionally coupled. The set of interactions involving the
tetraspanins has been referred to as the "network of tetraspanins"
or "Tetraspanin Web".
[0006] CD151 interacts on the surface of cells with various
membrane proteins. In particular, there have been identified highly
stable complexes, resistant to the action of certain detergents,
with laminin receptor integrins, more particularly with the
integrins .alpha.3.beta.1 or .alpha.6.beta.4, whose preferred
ligand is laminin 5 (Yauch et al., 1998, Mol. Biol. Cell 9,
2751-2765; Lammerding et al., 2003, Proc. Natl. Acad. Sci USA 100,
7616-7621). This association involves the extracellular domains of
CD151 and the integrins. The sequence QRD [194-196] of CD151,
located in the EC2 loop, is very important in that association
because mutation of this site causes loss of interaction with
certain integrins (Kazarov et al., 2002, J. Cell Biol. 158,
1299-1309). Functional ternary complexes of CD151/integrin
.alpha.6.beta.4/c-Met (HGF receptor) have moreover been identified
in tumour cells (Klosek et al., 2005, Biochem. Biophys. Res.
Commun. 336, 408-416) Inhibition of the expression of CD151 as a
result of treating cells with an interference RNA results in
inhibition of the cell growth and migration caused by HGF.
[0007] The interactions, within a particular cell, between CD151
and other tetraspanins, necessary for formation of the network of
tetraspanins, are thought to depend on the membrane and cytoplasmic
regions of CD151 because it has been shown that deletion of the EC2
loop does not disrupt the association of CD151 with other
tetraspanins (Berditchevski, 2001, J. Cell Sci. 114,
4143-4151).
[0008] CD151 is capable of regulating the phenomena of cell
adhesion, migration and invasion by modulation of various
signalling pathways such as, for example, the phosphoinositide
pathway via an association with PI4-kinase (Yauch et al., 1998,
Mol. Biol. Cell 9, 2751-2765), the c-Jun signalling pathway via the
phosphorylation de FAK, Src, p38-MAPK and JNK (Hong et al., 2006),
the phosphorylation of integrins by PKC (Zhang et al., 2001, J.
Biol. Chem. 276, 25005-25013) and the activation of GTPases of the
Rho family (Shigeta et al., 2003, J. Cell Biol. 163, 165-176).
[0009] Homophilic-type interactions between cells are also
responsible for an increase in cell motility and in expression of
the metalloproteinase MMP-9 (Hong et al., 2006). Those
intercellular CD 151-CD 151 interactions bring about the activation
of c-Jun via the phosphorylation of FAK, Src, p38-MAPK and JNK.
[0010] Despite the interest in the CD151 protein, only one
therapeutically aimed antibody has been generated to date, namely
the monoclonal antibody 50-6.
[0011] The monoclonal antibody 50-6 (IgG1 isotype) directed to
CD151 was generated in the mouse by subtractive immunisations with
human epidermoid carcinoma HEp-3 cells (Testa et al., 1999, Cancer
Res. 59, 3812-3820).
[0012] The antibody 50-6 is capable of inhibiting, in vitro,
migration of human cervical carcinoma HeLa cells, transfected so as
to overexpress CD151, and of HEp-3 cells and angiogenesis in a
model of chorio-allantoic membrane neovascularisation caused by
bFGF (basic fibroblast growth factor). In vivo it inhibits the
metastases brought about by inoculation of HEp-3 cells in 2 chicken
embryo models (Testa et al., 1999, Cancer Res. 59, 3812-3820). In
these models, the inhibitory activity of the antibody 50-6 is
determined by measurement of the activity of the protein huPA
(human urokinase-type plasminogen activator) in lung extracts.
According to the authors, this assay reflects the presence of human
cells in the lungs. After assaying, the reduction in metastases
(dissemination of HEp-3 cells into the chicken embryo lungs) that
is brought about by the antibody 50-6 is estimated, by comparison
with a control antibody, to be 74% in a so-called "spontaneous
metastasis" model, in which inoculation of the cells is followed by
injection of the antibody, and 57% in a so-called "experimental
metastasis" model, in which the cells and the antibody are
inoculated together. According to the authors, the anti-tumour
properties of the antibody 50-6 that are observed in vivo do not
seem to be related to a cytostatic or cytotoxic effect because the
antibody showed no effect on the in vitro proliferation of HEp-3
cells.
[0013] The hybridoma producing the antibody 50-6 is available at
the ATCC under the reference CRL-2696 (hybridoma initially
deposited under the reference 50-6 [PTA-227]).
BRIEF SUMMARY OF THE INVENTION
[0014] The present invention relates to a new use of anti-CD151
antibodies capable of inhibiting tumour growth, said antibodies
being especially monoclonal of murine origin, chimaeric and
humanised. According to a particular aspect, the invention relates
to the use of those antibodies, or of functional fragments thereof,
as medicaments for the prophylactic and/or therapeutic treatment of
cancers. Finally, the invention includes products and/or
compositions comprising such antibodies in association, for
example, with anti-cancer agents and/or antibodies or conjugated
with toxins, and use thereof in the prevention and/or treatment of
certain cancers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the nucleotide sequence of the CD151 protein
(SEQ ID NO:1) and protein sequence of the CD151 protein (SEQ ID
NO:2), on which sequences there are shown the EC1 and EC2
loops.
[0016] FIG. 2 is a diagram illustrating the structure of the
tetraspanins, to which the CD151 protein belongs, and very
especially the two extracellular loops EC1 and EC2.
[0017] FIG. 3 shows the PBS/control antibody comparison in the A549
orthotopic model.
[0018] FIG. 4 shows evaluation of the in vivo anti-tumour activity
of the TS151 antibody in the orthotopic model. 1.times.10.sup.6
A549 cells are grafted into immunodepressed mice (n=10) by the
intrapleural route. Seven days after grafting, the mice are
treated, by the intraperitoneal route, with a challenge dose of 500
.mu.g of TS151 antibody followed by treatment, twice a week for 5
weeks, with a dose of 250 .mu.g of antibody per mouse. The control
group is injected with PBS according to the same administration
regimen.
[0019] FIG. 5 illustrates expression of the CD151 molecule in
patients suffering from prostate cancer. Each letter corresponds to
study of one patient and for each patient the upper panel
corresponds to the normal tissue adjacent to the tumour and the
lower panel corresponds to the tumour tissue.
[0020] FIG. 6 illustrates expression of the CD151 molecule in
patients suffering from lung cancer. Each letter corresponds to
study of one patient and for each patient the upper panel
corresponds to the normal tissue adjacent to the tumour and the
lower panel corresponds to the tumour tissue.
[0021] FIG. 7 illustrates the in vivo activity of the TS151 and
TS151R antibodies in the PC3 xenograft model. The PC3 cells were
grafted into Swiss Nude mice (n=6) by the subcutaneous route. Five
days after grafting of the cells, the mice receive, by the i.p.
route, a challenge dose of 2 mg/mouse of the antibodies under test
followed by two administrations per week of a dose of 1 mg/mouse of
those antibodies. The tumour volume is evaluated by the formula
.pi./6.times.length.times.width.times.thickness, and a Mann and
Whitney test is carried out for statistical evaluation of the
results.
[0022] FIG. 8 illustrates evaluation of the specificity of the
TS151 and TS151r antibodies for the human form of CD 151 by Western
blot.
[0023] FIG. 9 illustrates inhibition of the adhesion of A549 cells
on laminin 5. A/ Inhibition of cell adhesion by different
anti-integrin antibodies. B/ Inhibition of cell adhesion by a
combination of TS151/anti-integrin .alpha.3 antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0024] According to a general aspect, the present invention is
directed to use of at least one antibody, or a functional fragment
thereof, which is capable of binding to the CD151 protein and
thereby inhibiting tumour growth, in the preparation of a
medicament intended for the treatment of cancer.
[0025] Several experimental studies have shown the major role of
the tetraspanins in the formation of metastases by acting either as
suppressors or as promoters of metastases. Accordingly, the
transfection of tetraspanins such as CD9, CD63 or CD82 reduces the
metastatic potential of cancer lines. In contrast, expression of
the tetraspanins CD151 and Co-029 seems to produce the opposite
effect. These 2 tetraspanins are therefore thought to be promoters
of metastasis. These results are consistent with various clinical
studies which have shown that, in a number of cancers (breast,
lung, oesophagus, stomach, liver, pancreas, colon, prostate,
melanoma . . . ), CD9 and CD82 are less expressed in primary
tumours when there is metastasis and that a reduction in their
expression is predictive of a lower survival rate. In lung cancer,
the combined reduction in the expression of CD9 and CD82 has been
correlated with greater metastatic potential than when expression
of just one of those two antigens is reduced.
[0026] Several retrospective studies have shown that overexpression
of CD151 is associated with aggressiveness of certain cancers, such
as lung, colon and prostate cancers, and that it might be
considered to be a factor for poor prognosis (Tokuhara et al.,
2001, Clin. Cancer Res. 7, 4109-4114; Hashida et al., 2003, Br. J.
Cancer 89, 158-167; Ang et al., 2004, Cancer Epidemiol. Biomarkers
Prev. 13, 1717-1721). In these cases, mean survival is in fact
reduced in those patients having tumours which express CD151,
compared to those having tumours which do not express CD151.
[0027] The overexpression of CD151 in various human tumour lines
(HeLa, RPMI14788, A172, HT1080), brought about by transfection of
the corresponding gene, causes an increase in the motility of, the
migration of and invasion by the transfected cells (Testa et al.,
1999, Cancer Res. 59, 3812-3820; Kohno et al., 2002, Int. J. Cancer
97, 336-343). These phenomena are inhibited in the presence of
anti-CD151 antibodies.
[0028] According to another aspect, the functional fragments of
antibodies according to the invention consist, for example, of Fv,
scFv (sc standing for single chain), Fab, F(ab').sub.2, Fab' or
scFv-Fc fragments or diabodies, or any fragment whose half-life may
have been extended by chemical modification, e.g. addition of
poly(alkylene)glycol such as poly(ethylene)glycol ("PEGylation")
(the PEGylated fragments being referred to as Fv-PEG, scFv-PEG,
Fab-PEG, F(ab').sub.2--PEG or Fab'-PEG) ("PEG" from the designation
Poly(Ethylene)Glycol), or by incorporation in a liposome,
microspheres or PLGA, said fragments being capable of generally
exerting activity, even partial, of the antibody from which it is
derived.
[0029] Preferably, said functional fragments will be composed of or
will comprise a partial sequence of the variable heavy or light
chain of the antibody from which they are derived, said partial
sequence being sufficient to retain the same binding specificity as
the antibody from which it is derived and an adequate affinity,
preferably equal to at least 1/100th, more preferably at least
1/10th, of that of the antibody from which it is derived.
[0030] Such a functional fragment will comprise at least 5
consecutive amino acids, preferably 10, 15, 25, 50 or 100
consecutive amino acids, from the sequence of the antibody from
which it is derived.
[0031] Preferably, these functional fragments will be fragments of
Fv, scFv, Fab, F(ab').sub.2, F(ab'), scFv-Fc type, or diabodies,
which generally have the same fixing specificity as the antibody
from which they are obtained. According to the present invention,
fragments of antibodies of the invention can be obtained starting
from antibodies as described hereinbefore by methods such as
digestion using enzymes such as pepsin or papain and/or by cleavage
of the disulfide bridges by means of chemical reduction. The
antibody fragments included in the present invention can also be
obtained by genetic recombination techniques that are likewise
well-known to the person skilled in the art or by peptide synthesis
by means of, for example, automatic peptide synthesisers such as
those supplied by the company Applied.
[0032] According to an aspect of the invention, the antibody used
consists of a murine monoclonal antibody.
[0033] Antibodies according to the present invention also include
chimaeric or humanised antibodies.
[0034] A chimaeric antibody is understood as referring to an
antibody which contains a natural variable (light chain and heavy
chain) region derived from an antibody from a given species in
association with the constant light chain and heavy chain regions
of an antibody from a heterologous species to said given
species.
[0035] Chimaeric-type antibodies, or their fragments, used in
accordance with the invention can be prepared using genetic
recombination techniques. For example, the chimaeric antibody may
be produced by cloning a recombinant DNA comprising a promoter and
a sequence coding for the variable region of a non-human,
especially murine, monoclonal antibody according to the invention
and a sequence coding for the constant region of a human antibody.
A chimaeric antibody of the invention encoded by such a recombinant
gene may be, for example, a mouse-human chimaera, the specificity
of that antibody being determined by the variable region derived
from the murine DNA and its isotype determined by the constant
region derived from the human DNA. For methods of preparing
chimaeric antibodies, reference may be made, for example, to the
document Verhoeyn et al. (BioEssays, 8:74, 1988).
[0036] A humanised antibody is understood as referring to an
antibody which contains CDR regions derived from an antibody of
non-human origin, the other parts of the antibody molecule being
derived from one (or more) human antibody/antibodies. In addition,
some of the residues of the segments of the skeleton (referred to
as FR) can be modified in order to preserve the binding affinity
(Jones et al., Nature, 321:522-525, 1986; Verhoeyen et al.,
Science, 239:1534-1536, 1988; Riechmann et al., Nature,
332:323-327, 1988).
[0037] The humanised antibodies or functional fragments thereof can
be prepared by techniques known to the person skilled in the art
(such as, for example, those described in the documents Singer et
al., J. Immun. 150:2844-2857, 1992; Mountain et al., Biotechnol.
Genet. Eng. Rev., 10:1-142, 1992; or Bebbington et al.,
Bio/Technology, 10:169-175, 1992). Such humanised antibodies are
preferred for their use in in vivo prophylactic and/or therapeutic
treatment methods. Other humanisation techniques are also known to
the person skilled in the art, such as, for example, the technique
of "CDR Grafting", described by PDL, which is the subject-matter of
patents EP 0 451 261, EP 0 682 040, EP 0 939 127, EP 0 566 647 or
also U.S. Pat. No. 5,530,101, U.S. Pat. No. 6,180,370, U.S. Pat.
No. 5,585,089 and U.S. Pat. No. 5,693,761. There may also be
mentioned the patents U.S. Pat. No. 5,639,641 or also 6,054,297,
5,886,152 and 5,877,293.
[0038] Surprisingly, and contrary to any expectation on the part of
the person skilled in the art, the present invention describes for
the first time the use of an anti-CD151 antibody as described
hereinbefore, or one of its functional fragments, which is capable
of inhibiting the proliferation of tumour cells and the development
of the primary tumour, independently of its ability to inhibit
angiogenesis and/or the formation of metastases.
[0039] The antibodies described in the Application accordingly have
the ability to inhibit the development of tumours at a very early
stage.
[0040] This anti-tumour activity of the antibodies to which the
present invention relates constitutes a new and unexpected property
for an antibody directed to CD151, because no hitherto described
anti-CD151 antibody has this type of activity. The antibodies to
which the present invention relates accordingly have a different,
and additional, property compared to the previously described
antibodies, especially compared to the 50-6 antibody because the
anti-tumour properties of that antibody are not due to an effect on
the proliferation of tumour cells.
[0041] This result also constitutes the first demonstration of a
link between CD151 and the development of the primary tumour, or
indeed the in vivo proliferation of tumour cells. Only the
pro-metastatic and pro-angiogenic activities of CD151 have in fact
been described hitherto.
[0042] The disordered proliferation of the cells of an organ or
tissue constitutes one of the first stages in cancer. Tumour cells
are cells which are no longer subject to the normal cell growth
constraints within the organ or tissue concerned. Tumour growth is
exponential; and the tumour cells multiply in excessive manner
under the effect of growth and angiogenic factors.
[0043] According to a main aspect, the invention relates to use of
at least one anti-CD151 antibody, or a functional fragment thereof,
which is capable of inhibiting the development of the primary
tumour and the proliferation of the tumour cells.
[0044] More particularly, the present invention relates to the use
of at least one antibody, or a functional fragment thereof, which
is capable of binding to the CD151 protein, in the preparation of a
medicament intended for the treatment of primary tumours.
[0045] In addition, the Applicant is putting forward, without
wishing to be bound by any such theory, that the use of anti-CD151
antibodies in the context of cancer treatment may be of value not
only due to the fact of angiogenesis inhibition but also due to the
fact of inhibition of the metastasis-promoting activity of
CD151.
[0046] The present invention accordingly describes the use of an
antibody as described hereinbefore, or a functional fragment
thereof, which is capable of inhibiting the metastasis-promoting
activity of said CD151 protein within tumour cells.
[0047] More particularly, the Applicant thinks that this inhibition
takes the form of inhibition of the various stages in the
metastatic process, especially cell adhesion, cell migration and/or
cell invasion.
[0048] The classic steps of said promoting activity, and more
particularly of tumour dissemination and the metastatic process,
are as follows:
[0049] 1/ invasion of the underlying tissue by the cells of the
primary tumour, requiring degradation by proteolytic enzymes (such
as metalloproteinases) of the basal membrane and the extracellular
matrix composed of structural proteins such as laminin, collagen or
fibronectin,
[0050] 2/ migration of the tumour cells through the tissues and
into the blood stream,
[0051] 3/ adhesion to the vessel wall and coming to a halt in an
organ,
[0052] 4/ exit from the vessel (new invasion step) and adaptation
to the new environment (proliferation and angiogenesis).
[0053] Cell migration is essential during development of the
embryo. Although cell migration is less substantial in the adult,
certain types of cell such as lymphocytes, macrophages and
fibroblasts, will continue to move around during the immune
response, inflammation and wound healing in the adult in order to
maintain homeostasis. However, at the pathological level, migration
of tumour cells contributes substantially to the progression of
tumours to the metastatic stage. A certain number of chemotactic
factors are responsible for that migration, which factors are
derived either from the tumour cells or from the host. Amongst
those factors there are mentioned growth factors (especially those
which stimulate angiogenesis), collagen degradation peptides,
adhesion proteins such as laminin and fibronectin.
[0054] According to a particular aspect, the invention relates to
use of at least one anti-CD151 antibody, or a functional fragment
thereof, which is capable of inhibiting the cell migration of
tumour cells.
[0055] Invasion is the principal sign of the malignancy of a
tumour, with the latter breaking away from its original site and
moving into neighbouring and distant tissues. The invasive
character is a reflection of loss of the customary properties of a
cell: normally, the cells of most tissues adhere to one another by
structures referred to as desmosomes, by means of adhesive
molecules; in an epithelium, they also adhere to the basal membrane
which limits it in terms of depth. Tumour cells lose those normal
properties and acquire new ones. The links between them are
loosened and the cells free themselves from one another. They
acquire a mobility which allows them to detach themselves from the
primary site and to infiltrate (invade) neighbouring tissues,
sometimes following connective tissue fibres. For epithelia
normally bounded by a basal membrane and for carcinomas derived
therefrom, this membrane is the first obstacle to be crossed. It is
degraded and dissolved by enzymes (proteases, cathepsin) secreted
by the tumour cells. This destruction of the basal membrane is
sometimes accentuated by enzymes normally secreted by white blood
cells and diverted from their customary activities. All those
biological and molecular modifications of cell behaviour are a
condition of invasion.
[0056] According to another aspect, the invention relates to use of
at least one anti-CD151 antibody, or a functional fragment thereof,
which is capable of inhibiting the cell invasion by tumour
cells.
[0057] The cells of the body adhere to one another and to the
extracellular matrix which surrounds them. Cell adhesion is a
ubiquitous mechanism involved in most physiological cell phenomena,
such as the survival, proliferation or differentiation of cells,
but also in various pathological situations such as, for example,
cancer and the phenomenon of metastasis. Various cell surface
proteins are involved in cell adhesion, such as the cadherins or
the integrins.
[0058] Preferably, the use according to the invention is based
mainly on the inhibition of cell adhesion.
[0059] According to yet another aspect, the invention relates to
use of at least one anti-CD151 antibody, or a functional fragment
thereof, which is capable of inhibiting the cell adhesion of tumour
cells.
[0060] As has been mentioned hereinbefore, the CD151 protein
belongs to the tetraspanin family and, by virtue thereof, has 2
extracellular domains EC1 (18 amino acids, sequence [40-57]) and
EC2 (109 amino acids, sequence [113-221]), also referred to as
extracellular loops.
[0061] According to the present invention, the antibodies used are
capable of binding to at least one epitope located in the
extracellular domain Preferably, said antibody will become fixed to
the EC1 and/or EC2 loops.
[0062] More particularly, according to a preferred embodiment of
the invention, there is described the use of at least one
anti-CD151 antibody, or a functional fragment thereof, which is
capable of binding to an epitope included in the extracellular loop
1 (EC 1) and/or 2 (EC2), preferably EC2, corresponding to the amino
acids 40-57 (SEQ ID No. 6) and 113-221 (SEQ ID No. 4),
respectively, of the CD151 protein.
[0063] The EC1 loop [40-57] contains 18 amino acids and has a
theoretical weight of 2002.2 Da.
[0064] The EC2 loop [113-221] has an N-glycosylation site (residue
Asn159) and 6 cysteine residues forming 3 disulfide bridges. A
structural model of the EC2 loop of the tetraspanins, and
especially of CD151, has been proposed on the basis of the
three-dimensional structure of the EC2 loop of the tetraspanin CD81
(Seigneuret et al., 2001, J. Biol. Chem. 276, 40055-40064).
According to that model, the tetraspanins have a common, relatively
conserved scaffold composed of 3 .alpha. helices and a specific
variable domain. For CD151, that scaffold is thought to be composed
of the regions [113-157] and [209-221], and the variable domain is
thought to be composed of the region [158-208].
[0065] The variable domain of the EC2 loop is thought to be more
especially involved in the specific interactions of CD151 with
proteins of the integrin family. Directed mutation experiments have
especially shown the importance of the region [193-208], and more
precisely of the tripeptide QRD [194-196] and the cysteine residue
at position 192, in the association of CD151 with certain laminin
receptor integrins such as integrins .alpha.3.beta.1 or
.alpha.6.beta.4 (Kazarov et al., 2002, J. Cell Biol. 158,
1299-1309).
[0066] Still more preferably, the present invention is directed to
use of at least one anti-CD151 antibody, or a functional fragment
thereof, which is capable of binding to an epitope of the EC2
region comprising, at least, the amino acids Glutamine, Arginine
and Aspartic Acid at positions 194, 195 and 196 (QRD.sup.194-196),
respectively, of the CD151 protein.
[0067] According to another aspect, it must be understood that the
invention consists mainly of use of at least one anti-CD151
antibody, or a functional fragment thereof, which consists of a
monoclonal antibody.
[0068] A "monoclonal antibody" is to be understood as an antibody
derived from a population of substantially homogeneous antibodies.
More especially, the individual antibodies of a population are
identical with the exception of a few possible mutations that may
be produced naturally and that may be present in minimal amounts.
In other words, a monoclonal antibody consists of a homogeneous
antibody resulting from the proliferation of just one cell clone
(for example, a hybridoma, a eukaryotic host cell transfected with
a DNA molecule coding for the homogeneous antibody, a prokaryotic
host cell transfected with a DNA molecule coding for the
homogeneous antibody etc.) and which is usually characterised by
heavy chains of one and the same class and sub-class and light
chains of just one type. Monoclonal antibodies are highly specific
and are directed to a single antigen. In addition, in contrast to
preparations of polyclonal antibodies which customarily comprise
different antibodies directed to different determinants, or
epitopes, each monoclonal antibody is directed to a single epitope
of the antigen.
[0069] According to a particular embodiment of the invention, the
monoclonal antibody used is selected from the antibodies TS151 and
TS151r. In the remainder of the description, the expressions TS151r
and TS151R are interchangeable.
[0070] The present invention accordingly describes use of at least
one anti-CD151 antibody, or a functional fragment thereof, said
antibody consisting of the TS151 antibody and/or the TS151r
antibody.
[0071] More particularly, the TS151 antibody is defined in that it
comprises at least: [0072] the 3 heavy-chain CDRs CDR-H1, CDR-H2
and CDR-H3 of sequence SEQ ID No. 7, 8 and 9, respectively; and
[0073] the 3 light-chain CDRs CDR-L1, CDR-L2 and CDR-L3 of sequence
SEQ ID No. 11, 12 and 13, respectively.
[0074] According to another embodiment, the antibody TS151 is
characterised in that it comprises a heavy chain comprising the
sequence SEQ ID No. 10 and a light chain comprising the sequence
SEQ ID No. 14. Table 1 below provides a summary of those
elements.
TABLE-US-00001 TABLE 1 Antibody Light chain Heavy chain SEQ ID No.
TS151 CDR-H1 7 CDR-H2 8 CDR-H3 9 CDR-L1 11 CDR-L2 12 CDR-L3 13
Complete (variable 10 domain) Complete (variable 14 domain)
[0075] As for the antibody TS151r, the latter is defined in that it
comprises, at least: [0076] the 3 heavy-chain CDRs CDR-H1, CDR-H2
and CDR-H3 of sequence SEQ ID No. 15, 16 and 17, respectively; and
[0077] the 3 light-chain CDRs CDR-L1, CDR-L2 and CDR-L3 of sequence
SEQ ID No. 19, 20 and 21, respectively.
[0078] According to another embodiment, the antibody TS151r is
characterised in that it comprises a heavy chain comprising the
sequence SEQ ID No. 18 and a light chain comprising the sequence
SEQ ID No. 22. Table 2 below provides a summary of those
elements.
TABLE-US-00002 TABLE 2 Antibody Light chain Heavy chain SEQ ID No.
TS151r CDR-H1 15 CDR-H2 16 CDR-H3 17 CDR-L1 19 CDR-L2 20 CDR-L3 21
Complete (variable 18 domain) Complete (variable 22 domain)
[0079] According to another embodiment, Table 3 hereinbelow
summarises the nucleotide sequences of the antibodies TS151 and
TS151r.
TABLE-US-00003 TABLE 3 Antibody Light chain Heavy chain SEQ ID No.
TS151 CDR-H1 23 CDR-H2 24 CDR-H3 25 CDR-L1 27 CDR-L2 28 CDR-L3 29
Complete (variable 26 domain) Complete (variable 30 domain) TS151r
CDR-H1 31 CDR-H2 32 CDR-H3 33 CDR-L1 35 CDR-L2 36 CDR-L3 37
Complete (variable 34 domain) Complete (variable 38 domain)
[0080] Details of the generation of said antibodies are given in
Example 1. These 2 antibodies are directed to different epitopes
because TS151 recognises CD151 whether it is associated with
integrins or is free on the surface of the cell (Chometon et al.,
2006, Exp. Cell Res. 312, 983-995), whereas TS151r does not
recognise CD151-integrin complexes (Serru et al., 1999, Biochem. J.
340, 103-111; Geary et al., 2001, Tissue Antigens 58, 141-153;
Kazarov et al., 2002, J. Cell Biol. 158, 1299-1309; Sterk et al.,
2002, J. Cell Sci. 115, 1161-1173). The epitope recognised by
TS151r is located in the EC2 loop and comprises the residues Q194,
R195 and D196 (Kazarov et al., 2002, J. Cell Biol. 158, 1299-1309).
This antibody is therefore directed, at least in part, at that site
on CD151 which is involved in interactions with the integrins. The
C192 residue may also be involved in the recognition of CD151 by
TS151r (Kazarov et al., 2002, J. Cell Biol. 158, 1299-1309). The
epitope of the TS151 antibody, although being different to that of
TS151r, has not been precisely determined
[0081] Treating human keratinocytes (the epithelial line HaCaT)
with the TS151r antibody brings about a loss of cell-cell contact,
rearrangement of the cytoskeleton, intracellular redistribution of
the integrin .alpha.6.beta.4 and an increase in the migration of
cells on laminin 1 (Chometon et al., 2006, Exp. Cell Res. 312,
983-995).
[0082] More particularly, the antibody preferably used consists of
the antibody TS151.
[0083] Preferably, use of the anti-CD151 antibodies in the context
of cancer treatment is of value very especially in cancers
overexpressing that same CD151 receptor.
[0084] Such cancers consist of colon cancer [Hashida et al., Br. J.
Cancer 89 (2003):158-167], lung cancer, preferably non-small-cell
lung cancer [Tokuhara et al., Clin. Cancer Res. 7
(2001):4109-4114], prostate cancer Ping et al., Cancer Epidemiol.
Biomarkers 13 (2004):17] and pancreatic cancer [Gesierich et al.,
Clin. Cancer Res. 11 [2005):2840-2852].
[0085] The present invention accordingly claims use of an antibody
as described hereinbefore in the treatment of cancer, said cancer
preferably consisting of colon, lung, prostate or pancreatic
cancers.
[0086] The invention relates also to a pharmaceutical composition
comprising, as active ingredient, a compound consisting of an
antibody, or one of its derivative compounds or functional
fragments, to which there is preferably added an excipient and/or a
pharmaceutically acceptable carrier.
[0087] More especially, the invention is directed to use of an
antibody according to the invention in the preparation of a
pharmaceutical composition additionally comprising at least one
pharmaceutically acceptable carrier.
[0088] In the present description, a pharmaceutically acceptable
carrier is understood as referring to a compound or combination of
compounds included in a pharmaceutical composition which does not
give rise to secondary reactions and which, for example, makes it
possible to facilitate the administration of the active
compound(s), to increase the life and/or efficacy thereof in the
body, to increase the solubility thereof in solution or to improve
its storage. Such pharmaceutically acceptable carriers are
well-known and will be adapted by the person skilled in the art as
a function of the nature and mode of administration of the selected
active compound(s).
[0089] Preferably, those compounds will be administered by a
systemic route, especially the intravenous route, by the
intramuscular, intradermal, intraperitoneal or subcutaneous route,
or by the oral route. More preferably, the composition comprising
the antibodies according to the invention will be administered on a
plurality of occasions staggered over time.
[0090] Their optimal modes of administration, dosage regimens and
galenic forms can be determined according to criteria generally
taken into consideration in establishing a suitable treatment for a
patient such as, for example, the age or bodyweight of the patient,
the severity of his or her general condition, the tolerability of
the treatment and the secondary effects established.
[0091] According to the invention there is described a composition
for the treatment of cancer, characterised in that it comprises, as
active ingredient, at least one anti-CD151 antibody, or a
functional fragment thereof, which is capable of binding to the
CD151 protein.
[0092] According to the invention there is described a composition
for the treatment of cancer, characterised in that it comprises, as
active ingredient, at least one anti-CD151 antibody, or a
functional fragment thereof, which is capable of binding to the
CD151 protein and/or of inhibiting its metastasis-promoting
activity.
[0093] According to the invention there is also described a
composition for the treatment of cancer, characterised in that it
comprises, as active ingredient, at least one anti-CD151 antibody,
or a functional fragment thereof, which is capable of inhibiting
the development of primary tumours.
[0094] According to another aspect of the invention, there is
described a composition comprising at least one anti-CD151
antibody, or a functional fragment thereof, said at least one
antibody being a monoclonal antibody selected from the antibodies
TS151 or TS151r.
[0095] According to yet another aspect of the invention, there is
claimed a composition which comprises a combination of the
antibodies TS151 and TS151r, or of functional fragments
thereof.
[0096] The literature shows that the CD151 protein is overexpressed
in cancers and, very especially, in colon carcinomas [Hashida et
al., Br. J. Cancer 89 (2003): 158-167], non-small-cell lung cancers
[Tokuhara et al., Clin. Cancer Res. 7 (2001): 4109-4114], prostate
cancers [Ping et al., Cancer Epidemiol. Biomarkers 13 (2004):
1717-1721] and pancreatic cancers [Gesierich et al., Clin. Cancer
Res. 11 (2005): 2840-2852].
[0097] Of course, the above list is given solely by way of
illustration and any cancer must be understood as overexpressing
the CD151 protein and therefore as being capable of being treated
in accordance with the present invention.
[0098] Another, complementary embodiment of the invention consists
of a composition as described hereinbefore which additionally
comprises, as a combination product for simultaneous, separate or
time-staggered use, a cytotoxic/cytostatic agent and/or a
monoclonal antibody.
[0099] The present invention accordingly relates also to a
composition as described hereinbefore, characterised in that it
additionally comprises, as a combination product for simultaneous,
separate or time-staggered use, at least one cytotoxic/cytostatic
agent and/or a cell toxin and/or a radioelement and/or a monoclonal
antibody.
[0100] "Simultaneous use" is understood as the administration of
the two compounds of the composition according to the invention
contained in one and the same pharmaceutical form.
[0101] "Separate use" is understood as the administration, at the
same time, of the two compounds of the composition according to the
invention contained in separate pharmaceutical forms.
[0102] "Time-staggered use" is understood as the successive
administration of the two compounds of the composition according to
the invention, each contained in a separate pharmaceutical
form.
[0103] In general manner, the composition according to the
invention considerably increases the efficacy of the cancer
treatment. In other words, the therapeutic effect of the antibody
according to the invention is potentiated in unexpected manner by
the administration of a cytotoxic agent. Another major subsequent
advantage produced by a composition according to the invention
relates to the possibility of using lower effective doses of active
ingredient, which makes it possible to avoid or reduce the risks of
secondary effects appearing, especially the effect of the cytotoxic
agent. Moreover, this composition according to the invention should
make it possible to achieve the expected therapeutic effect more
rapidly.
[0104] "Anti-cancer therapeutic agents" or "cytotoxic agents"
should be understood as substances which, when administered to a
patient, treat or prevent the development of the cancer in the
patient. By way of non-limiting example of such agents there may be
mentioned "alkylating" agents, antimetabolites, anti-tumour
antibiotics, mitotic inhibitors, chromatin function inhibitors,
anti-angiogenesis agents, anti-oestrogens, anti-androgens or
immunomodulators.
[0105] Such agents are, for example, mentioned in the VIDAL, on the
page devoted to compounds used in oncology and haematology in the
column "Cytotoxiques" (English: cytotoxic agents); such cytotoxic
compounds mentioned by way of reference to that document are
mentioned here as preferred cytotoxic agents.
[0106] "Alkylating agents" refer to any substance which is capable
of covalently binding to or alkylating any molecule, preferably a
nucleic acid (e.g.: DNA), within a cell. As examples of such
alkylating agents there may be mentioned nitrogen mustards such as
mechlorethamine, chlorambucil, melphalan hydrochloride, pipobroman,
prednimustine disodium phosphate or estramustine; oxazophorines
such as cyclophosphamide, altretamine, trofosfamide, sulfofosfamide
or ifosfamide; aziridines or ethylene-imines such as thiotepa,
triethyleneamine or altetramine; nitrosoureas such as carmustine,
streptozocin, fotemustine or lomustine; alkyl sulfonates such as
busulfan, treosulfan or improsulfan; triazenes such as dacarbazine;
and also platinum complexes such as cisplatin, oxaliplatin or
carboplatin.
[0107] "Antimetabolites" refer to substances which block cell
growth and/or cell metabolism by interfering with certain
activities, generally DNA synthesis. By way of example of
antimetabolites there may be mentioned methotrexate,
5-fluorouracil, floxuridine, 5-fluorodeoxyuridine, capecitabine,
cytarabine, fludarabine, cytosine arabinoside, 6-mercaptopurine
(6-MP), 6-thioguanine (6-TG), chlorodeoxyadenosine, 5-azacytidine,
gemcitabine, cladribine, deoxycoformycin and pentostatin.
[0108] "Anti-tumour antibiotics" refer to compounds which can
prevent or inhibit the synthesis of DNA, of RNA and/or of proteins.
Examples of such anti-tumour antibiotics include doxorubicin,
daunorubicin, idarubicin, valrubicin, mitoxantrone, dactinomycin,
mithramycin, plicamycin, mitomycin C, bleomycin and
procarbazine.
[0109] "Mitotic inhibitors" prevent the normal progression of the
cell cycle and mitosis. In general, the microtubule inhibitors or
"taxoids" such as paclitaxel and docetaxel are capable of
inhibiting mitosis. The vinca alkaloids such as vinblastine,
vincristine, vindesine and vinorelbine are also capable of
inhibiting mitosis.
[0110] "Chromatin function inhibitors" or "topoisomerase
inhibitors" refer to substances which inhibit the normal function
of chromatin remodelling proteins such as topoisomerases I and II.
Examples of such inhibitors include, for topoisomerase I,
camptothecin and also its derivatives such as irinotecan or
topotecan and, for topoisomerase II, etoposide, etiposide phosphate
and teniposide.
[0111] "Anti-angiogenesis agents" refer to any drug, compound,
substance or agent which inhibits the growth of blood vessels.
Examples of anti-angiogenesis agents include, without any
limitation, razoxin, marimastat, batimastat, prinomastat,
tanomastat, ilomastat, CGS-27023A, halofuginone, COL-3, neovastat,
BMS-275291, thalidomide, CDC 501, DMXAA, L-651582, squalamine,
endostatin, SU5416, SU6668, interferon-alpha, EMD121974,
interleukin-12, IM862, angiostatin and vitaxin.
[0112] "Anti-oestrogens" or "anti-oestrogen agents" refer to any
substance which reduces, antagonises or inhibits the action of
oestrogens. Examples of such agents are tamoxifen, toremifene,
raloxifene, droloxifene, iodoxyfene, anastrozole, letrozole and
exemestane.
[0113] "Anti-androgens" or "anti-androgen agents" refer to any
substance which reduces, antagonises or inhibits the action of an
androgen. Examples of anti-androgens are flutamide, nilutamide,
bicalutamide, spironolactone, cyproterone acetate, finasteride and
cimitidine.
[0114] Immunomodulators are substances which stimulate the immune
system. Examples of such immunomodulators include interferons,
interleukins such as aldesleukin, OCT-43, denileukin diflitox or
interleukin-2, tumour necrosis factors such as tasonermin, or other
types of immunomodulators such as lentinan, sizofuran, roquinimex,
pidotimod, pegademase, thymopentin, poly I:C, or levamisole in
combination with 5-fluorouracil.
[0115] For further details, the person skilled in the art will be
able to refer to the manual published by the French Association of
Teachers of Therapeutic Chemistry entitled "Traite de chimie
therapeutique, Vol. 6, Medicaments antitumoraux et perspectives
dans le traitement des cancers, ed. TEC & DOC, 2003".
[0116] Preferred monoclonal antibodies are selected from isolated
antibodies which are capable of specifically inhibiting the
tyrosine kinase activities of the receptors IGF-IR, EGFR, HER2/neu,
cMET, VEGFR, VEGF, etc. (or any other anti-tumour antibody known to
the person skilled in the art), or functional fragments or
derivative compounds thereof, and capable of inhibiting the
proliferative and/or anti-apoptotic and/or angiogenic and/or
metastatic dissemination-causing activities promoted by said
receptors.
[0117] In an especially preferred embodiment, said composition in
the form of a combination product according to the invention is
characterised in that said cytotoxic agent is chemically bound to
said antibody for simultaneous use.
[0118] In an especially preferred embodiment, said composition
according to the invention is characterised in that said
cytotoxic/cytostatic agent is selected from spindle inhibitor or
stabiliser agents, preferably vinorelbine and/or vinflunine and/or
vincristine.
[0119] In order to facilitate binding between said cytotoxic agent
and said antibody according to the invention, it will be possible,
especially, to introduce spacer molecules between the two compounds
to be bound, e.g. poly(alkylene)glycols such as polyethyleneglycol,
or also amino acids, or, in another embodiment, to use active
derivatives of said cytotoxic agents into which there will have
been introduced functions capable of reacting with said antibody
according to the invention. These binding techniques are well known
to the person skilled in the art and will not be elaborated upon in
the present description.
[0120] According to another aspect, the invention relates to a
composition characterised in that one, at least, of said
antibodies, or one of their derivative compounds or functional
fragments, is conjugated with a cell toxin and/or a
radioelement.
[0121] Preferably, said toxin or said radioelement is capable of
preventing the growth or proliferation of the tumour cell,
especially of totally inactivating said tumour cell.
[0122] Preference is also given to said toxin being an
enterobacterial toxin, especially Pseudomonas exotoxin A.
[0123] The radioelements (or radioisotopes) employed in therapy,
preferably conjugated with the antibody, are radioisotopes which
emit gamma rays, preferably iodine.sup.131, yttrium.sup.90,
gold.sup.199, palladium.sup.100, copper.sup.67, bismuth.sup.217 and
antimony.sup.211. Radioisotopes which emit beta and alpha rays may
also be used in therapy.
[0124] A toxin or radioelement conjugated with at least one
antibody, or a functional fragment thereof, according to the
invention is understood to refer to any means making it possible to
bind said toxin or said radioelement to said at least one antibody,
especially by covalent binding between the two compounds, with or
without introduction of a linking molecule.
[0125] Among the agents allowing chemical (covalent), electrostatic
or non-covalent linkage of all or some of the conjugate's elements
there may be mentioned, very especially, benzoquinone, carbodiimide
and, more especially, EDC
(1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride),
dimaleimide, dithiobis-nitrobenzoic acid (DTNB), N-succinimidyl
S-acetyl thioacetate (SATA), agents referred to as "bridging"
agents having one or more groups, with one or more phenylazide
groups, reacting with ultraviolet (UV) and very preferably
N-[-4-(azidosalicylamino)butyl]-3'-(2'-pyridyldithio)propionamide
(APDP), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and
6-hydrazino-nicotinamide (HYNIC).
[0126] Another form of binding, very especially for radioelements,
can consist of using a bifunctional ion chelator.
[0127] Among those chelators there may be mentioned the chelators
derived from EDTA (ethylenediaminetetraacetic acid) or DTPA
(diethylenetriaminepentaacetic acid) that have been developed for
binding metals, especially radioactive metals, and immunoglobulins.
Accordingly, DTPA and its derivatives can be substituted with
different groups on the carbon chain so as to increase the
stability and rigidity of the ligand-metal complex (Krejcarek et
al. (1977); Brechbiel et al. (1991); Gansow (1991); U.S. Pat. No.
4,831,175).
[0128] For example, DTPA (diethylenetriaminepentaacetic acid) and
its derivatives, which has long been used very widely in medicine
and biology either in its free form or in the form of a complex
with a metal ion, has the noteworthy characteristic of forming
stable chelates with metal ions and of being bound to proteins of
therapeutic or diagnostic interest such as antibodies for the
development of radioimmunoconjugates in cancer therapy (Meases et
al., (1984); Gansow et al. (1990)).
[0129] The present invention additionally comprises use of the
composition according to the invention in the preparation of a
medicament.
[0130] The present invention is accordingly directed more
especially to use of a composition as described hereinbefore in the
preparation of a medicament intended for the treatment of cancer.
Among the cancers which may be prevented and/or treated preference
is given to colon, lung, prostate or pancreatic cancer.
[0131] In addition, according to an especially innovative and
advantageous aspect, the present invention is directed to use of a
composition as described hereinbefore in the preparation of a
medicament intended for the treatment of primary tumours.
[0132] The invention relates also to the use of an antibody
according to the invention in the preparation of a medicament
intended for the specific targeting of a biologically active
compound at cells expressing or overexpressing the CD151
receptor.
[0133] A biologically active compound is understood herein as
referring to any compound capable of modifying, especially
inhibiting, the activity of cells, especially their growth, their
proliferation, or the transcription or translation of genes.
[0134] Other characteristics and advantages of the invention will
emerge in the remainder of the description with the Examples and
Figures, for which the legends are given hereinbelow.
Example 1
Generation of the TS151r and TS151 Antibodies
[0135] Generation of the TS151r antibody
[0136] In order to generate the TS151r antibody, BALB/c mice were
immunised by the intraperitoneal route using 10.sup.7 HeLa cells.
After 3 immunisations and a final booster injection, the spleen
cells of a mouse were fused to P3X63AG8 myeloma cells by customary
techniques described by Kohler and Milstein (5.times.10.sup.7
spleen cells/3.times.10.sup.7 myeloma cells). The supernatants of
the hybridomas resulting from the fusion were then screened for
their ability to recognise HeLa cells by means of flow cytometry
and then for their ability to immunoprecipitate CD151 starting from
a lysate of HeLa cells prepared in the presence of the detergent
Brij 97 and to bring about the co-immunoprecipitation of CD9. The
TS151r antibody was found to have those various properties.
[0137] Generation of the TS151 Antibody
[0138] In order to generate the TS151 antibody, BALB/c mice were
immunised by the intraperitoneal route using 10.sup.7 Jurkat cells
and then 10.sup.7 HEL cells (2 immunisations). After a final
booster injection using protein complexes containing the protein
ADAM10 which were obtained from lysates of Jurkat and HEL cells,
the spleen cells were fused to P3X63AG8 myeloma cells by customary
techniques described by Kohler and Milstein (5.times.10.sup.7
spleen cells/3.times.10.sup.7 myeloma cells). The supernatants of
the hybridomas resulting from the fusion were firstly screened for
their ability to recognise Jurkat and HEL cells by means of flow
cytometry. The TS151 antibody was then selected with respect to its
ability to immunoprecipitate CD151 starting from a cell lysate
prepared in the presence of the detergent Brij 97 and to bring
about the co-immunoprecipitation of other tetraspanins.
Example 2
In Vivo Evaluation of the Anti-Tumour Activity of the TS151 and
TS151r Antibodies in an A549 Orthotopic Model
[0139] Material and Method
[0140] After confirmation of the expression of the CD151 protein
(data not shown), the A549 cells obtained from the ATCC are
routinely cultured in F12K medium, 10 mM glutamine, 10% FCS. These
cells are divided 2 days before grafting so that they will be in
the exponential phase of growth. For grafting, 7-week-old
immunodepressed mice are anaesthetised before being administered
1.times.10.sup.6 A549 cells by the intrapleural route. The primary
tumour develops rapidly and in 4 days invades the structures
adjacent to the injection site including the mediastinum, the lungs
and the diaphragm. In order to mimic the disease better, starting
treatment is not commenced until 7 days after implanting the cells,
by the intraperitoneal route. After injection of a challenge dose
of 500 .mu.g/mouse, the purified TS151 antibody is administered
twice a week for 5 weeks, at a dose of 250 .mu.g/mouse. A group of
mice to which PBS is administered is introduced as a control, given
that previously carried out experiments showed that administration
of an IgG1 isotype control had no impact on the survival of the
animals.
[0141] FIG. 3, which was obtained from preliminary data, shows the
specificity of the activity observed with the anti-CD151 antibody:
treatment of the animals with a murine IgG1 (mIgG1) used as isotype
control shows that this has no impact on the survival of animals
injected with the PBS used as carrier for those antibodies.
[0142] The evaluation parameter for this model is the survival of
the animals, and the anti-tumour activity is expressed by
calculation of the T/C %=median survival of the treated
animals/median survival of the animals from the control group X
100. It has been established that a T/C % greater than or equal to
125% is indicative of activity of the product.
[0143] Results
[0144] FIG. 4 shows anti-tumour activity of the TS151 antibody,
with a calculated T/C % of 140%.
Example 3
Comparison of the In Vivo Anti-Tumour Activity of the TS151 and
50-6 Antibodies in an A549 Orthotopic Model
[0145] Material and Method
[0146] The protocol employed is the same as that of Example 2
hereinbefore.
[0147] Results
[0148] The data obtained clearly demonstrate that the TS151
antibody has anti-tumour activity which is clearly greater than
that shown by the 50-6 antibody, the latter having a T/C % of only
118, that is to say less than the threshold value of 125% (data not
shown).
[0149] The results obtained, namely the T/C % calculated in the
manner defined hereinbefore, are shown in Table 4 below.
TABLE-US-00004 TABLE 4 TS151 antibody 50-6 antibody T/C % 140
118
Example 4
Study of Expression of the CD151 Molecule
[0150] The expression of the CD151 protein was researched by
immunohistochemistry in samples of human tissues obtained from
patients suffering from prostate cancers or lung cancer. For these
patients, slides of normal tissues adjacent to the tumour were
available and were therefore included in order to calibrate the
level of expression in the tumour tissues versus normal
tissues.
[0151] For these experiments, commercially available slides of the
"Tissue array" type are used. After deparaffinisation, antigen
unmasking is performed at 30.degree. C. with the aid of an
enzymatic solution containing pepsin (Labvision ref. AP-9007-005).
This step is followed by a step of removal of endogenous
peroxidases by incubation of the sections in a solution of hydrogen
peroxide (Sigma) 0.3% in water. Saturation of the non-specific
sites is then carried out with a solution of Ultra-V-Block
(Labvision, ref. TA-125-UB) and labelling is carried out using a
commercially available murine anti-CD151 antibody (Serotech, Ref.
MCA 1856) used at a final concentration of 5 .mu.g/ml. A murine
IgG1 isotype control antibody (DakoCytomation, Ref. X0931) is used
as a negative experimental control. Labelling visualisation is
performed using the Envision Dual Link visualisation system
(DakoCytomation, Ref. K4061) and the reference of the DAB
peroxidase substrate is S3309 from DakoCytomation.
[0152] The results presented in FIG. 5 show that a number of
patients developing prostate tumours exhibit overexpression of the
CD151 molecule. This overexpression may be very significant for 20%
of the patients studied (patients A and C) or moderate (patients A
and D). It is to be noted that, except at the level of the
endothelial cells, the corresponding normal prostatic tissues do
not express CD151 or express it only a little and that, where it is
expressed, it seems to be limited to glandular type structures.
Patient E exhibits an example of a tumour not expressing CD151.
[0153] In the case of the lung cancer (FIG. 6), moderate (patient
A) to marked (patient B) expression is observed in certain cells of
normal pulmonary tissue. However, the tumour tissue exhibits a very
high density of heavily labelled cells (patients A and B). Patient
C exhibits an example of a tumour not expressing CD151.
Example 5
Effect of the TS151 and TS151R Antibodies on In Vivo Growth of the
PC3 Tumour Implanted Subcutaneously in the Nude Mouse
[0154] Given the results obtained by immunohistochemistry on
prostate tissue arrays, the evaluation of anti-CD151 antibodies on
a PC3 tumour xenograft was planned. The PC3 line is an
androgen-independent prostate line obtained from the ATCC and
cultured in F12K medium+10% FCS+L-Glutamine. For evaluation,
5.times.10.sup.6 PC3 cells are implanted in the right flank of
Swiss Nude mice. Five days after implantation, the animals are
randomised on the basis of tumour volume and assigned to 3
comparable groups. The tumour volume of the selected grafted animal
group is between 41 and 47 mm.sup.3 (volume calculated by the
formula .pi./6.times.length.times.width.times.thickness) on day 0
of treatment. The animals are then given the purified antibodies
under test or PBS. The antibody doses and the frequency of
injections is as follows: challenge dose 2 mg/dose of antibody;
maintenance dose 1 mg/dose twice a week.
[0155] The results presented in FIG. 7 show that the two antibodies
tested (TS151 and TS151R) behave similarly and they very
significantly inhibit growth of the PC3 tumour implanted in a
sub-cutaneous position in the Swiss Nude mouse. Table 5 below
summarises the statistical analyses of these results.
TABLE-US-00005 TABLE 5 D 0 D 3 D 7 D 10 D 14 D 17 D 20 D 24 D 28 D
31 D 35 Control/TS151 Mann-Whitney p = p = p = p = p = p = p = p =
p = p = p = (Wilcoxon) 0.132 0.937 0.065 0.589 0.002 0.002 0.002
0.002 0.002 0.002 0.002 Control/TS151R Mann-Whitney p = p = p = p =
p = p = p = p = p = p = p = (Wilcoxon) 0.485 0.180 0.180 0.589
0.240 0.026 0.004 0.002 0.002 0.002 0.002
[0156] Studies carried out in parallel and presented in FIG. 8 show
that the TS151 and TS151R antibodies recognise the human CD151
molecule specifically, without any cross-reaction with the murine
receptor. This observation accordingly suggests that the activity
observed in the xenograft model in the Nude mouse can be attributed
solely to a direct effect on the grafted human tissue and
consequently excludes any possibility of the TS151 and TS151R
antibodies interfering with stromal cells of the tumour or murine
endothelial cells. Moreover, TS151 and TS151R are both murine IgG1
antibodies and, as a consequence and as is known to the person
skilled in the art, there is little likelihood that the observed
activity is due to effector functions of the ADCC and CDC type,
which are more especially mediated by murine IgG2a-type antibodies
in the mouse.
[0157] This set of results is therefore in agreement with a
mechanism of action which is directly due to inhibition of tumour
cell proliferation in vivo by the TS151 and TS151R antibodies.
Example 6
Specificity of the TS151 and TS151r Antibodies
[0158] The specificity of the TS151 and TS151r antibodies was
evaluated by Western blot. Lysates of human and murine lung,
pancreas and colon tissues (Biochain, 10 .mu.g of total proteins)
and also increasing amounts of HT-29 cell lysate (10, 20 and 50
.mu.g of total proteins) were placed on a 4-12% acrylamide gel
(BioRad). After electrophoresis (non-reductive conditions), the
proteins were transferred onto nitrocellulose membranes. The
transfer membranes were then incubated with the purified TS151 and
TS151r antibodies and then with a rabbit anti-mouse Ig polyclonal
antibody coupled to peroxidase (GE Healthcare) before ECL-type
visualisation.
[0159] The TS151 and TS151r antibodies exhibit specificity for the
human form of CD151 as borne out by the recognition, by Western
blot, of CD151 in lysates of HT-29 cells and of different tissues
of human origin (FIG. 8). The absence of reactivity with murine
CD151 in the lysates of various tissues sampled from the mouse
confirms the specificity of the TS151 and TS151r antibodies for the
human form of CD151.
Example 7
Inhibition of Cell Adhesion
[0160] The experiments of tumour cell adhesion on laminin 5, the
ligand of the integrins .alpha.3.beta.1 and .alpha.6.beta.4 with
which CD151 is capable of associating, are carried out in 96-well
plates. After immobilisation of laminin 5 (Chemicon, 200 .mu.l at 1
.mu.g/ml) for 1 hour at 37.degree. C., the wells are saturated with
BSA at 2 mg/ml (200 .mu.l, 1 hour at 37.degree. C.). A549 cells in
suspension are labelled using 5-chloromethylfluorescein diacetate
(CMFDA, Invitrogen) and are then added at a rate of 100000 cells
(100 .mu.l) per well in the presence or absence of antibody (100
.mu.l). After incubation at 37.degree. C. for 15, 30 or 60 minutes,
the cells that have not adhered are removed. After reading the
chemoluminescence with a luminometer (Mithras, Berthold), the
percentage of adhering cells is determined with the aid of a range
of CMFDA-labelled cells. The anti-CD151 antibody TS151 and the
anti-integrin .alpha.3 antibody P1B5, the anti-.alpha.6 antibody
NKI-Go3 and the anti-.beta.4 antibody ASC-3 (Chemicon) are
evaluated at a final concentration of 20 .mu.g/ml. The antibody
9G4, directed to a membrane protein of Escherichia coli, is used as
isotype control.
[0161] The anti-integrin .alpha.3 antibody P1B5 inhibits adhesion
of A549 cells on laminin 5 (FIG. 9A), whereas the anti-integrin
.alpha.6 antibody NKI-Go3 and the anti-integrin .beta.4 antibody
ASC-3 do not inhibit adhesion of the A549 cells on this same
ligand. However, a loss of inhibition as a function of time is
found, the inhibition brought about by P1B5 being greater than 90%
at 15 minutes but falling to about 20% after 1 hour. Association of
the P1B5 antibody with the anti-integrin .alpha.6 antibody NKI-Go3
or the anti-integrin .beta.34 antibody ASC-3 allows marked
inhibition of adhesion to be maintained after 1 hour, the
inhibition being greater than 90% for the association with the
anti-.alpha.6 antibody and being about 70% for the combination with
the anti-.beta.4 antibody. These results show that the A549 cells
adhere on laminin 5 for an initial period by means of the integrin
.alpha.3.beta.1 and then for a second period by means of the
integrin .alpha.6.beta.4.
[0162] The TS151 anti-CD 151 antibody does not inhibit the adhesion
of the A549 cells on laminin 5 when it is used on its own (FIG.
9B). The effect of a combination of TS151 with P1B5 on adhesion of
the A549 cells was then evaluated and compared with the previously
mentioned combinations. This TS151/P1B5 combination gives a result
which is comparable to the association of
anti-.alpha.6/anti-.alpha.3 and anti-.beta.4/anti-.alpha.3
antibodies, there being observed maintenance of inhibition of
adhesion of the order of 80% after 1 hour. The TS151 antibody
should therefore be capable of inhibiting the adhesion of A549
cells by way of an antagonist effect on the integrin
.alpha.6.beta.4.
Sequence CWU 1
1
381759DNAHomo sapiens 1atgggtgagt tcaacgagaa gaagacaaca tgtggcaccg
tttgcctcaa gtacctgctg 60tttacctaca attgctgctt ctggctggct ggcctggctg
tcatggcagt gggcatctgg 120acgctggccc tcaagagtga ctacatcagc
ctgctggcct caggcaccta cctggccaca 180gcctacatcc tggtggtggc
gggcactgtc gtcatggtga ctggggtctt gggctgctgc 240gccaccttca
aggagcgtcg gaacctgctg cgcctgtact tcatcctgct cctcatcatc
300tttctgctgg agatcatcgc tggtatcctc gcctacgcct actaccagca
gctgaacacg 360gagctcaagg agaacctgaa ggacaccatg accaagcgct
accaccagcc gggccatgag 420gctgtgacca gcgctgtgga ccagctgcag
caggagttcc actgctgtgg cagcaacaac 480tcacaggact ggcgagacag
tgagtggatc cgctcacagg aggccggtgg ccgtgtggtc 540ccagacagct
gctgcaagac ggtggtggct ctttgtgggc agcgagacca tgcctccaac
600atctacaagg tggagggcgg ctgcatcacc aagttggaga ccttcatcca
ggagcacctg 660agggtcattg gggctgtggg gatcggcatt gcctgtgtgc
aggtctttgg catgatcttc 720acgtgctgcc tgtacaggag tctcaagctg gagcactac
7592253PRThomo sapiens 2Met Gly Glu Phe Asn Glu Lys Lys Thr Thr Cys
Gly Thr Val Cys Leu1 5 10 15Lys Tyr Leu Leu Phe Thr Tyr Asn Cys Cys
Phe Trp Leu Ala Gly Leu 20 25 30Ala Val Met Ala Val Gly Ile Trp Thr
Leu Ala Leu Lys Ser Asp Tyr 35 40 45Ile Ser Leu Leu Ala Ser Gly Thr
Tyr Leu Ala Thr Ala Tyr Ile Leu 50 55 60Val Val Ala Gly Thr Val Val
Met Val Thr Gly Val Leu Gly Cys Cys65 70 75 80Ala Thr Phe Lys Glu
Arg Arg Asn Leu Leu Arg Leu Tyr Phe Ile Leu 85 90 95Leu Leu Ile Ile
Phe Leu Leu Glu Ile Ile Ala Gly Ile Leu Ala Tyr 100 105 110Ala Tyr
Tyr Gln Gln Leu Asn Thr Glu Leu Lys Glu Asn Leu Lys Asp 115 120
125Thr Met Thr Lys Arg Tyr His Gln Pro Gly His Glu Ala Val Thr Ser
130 135 140Ala Val Asp Gln Leu Gln Gln Glu Phe His Cys Cys Gly Ser
Asn Asn145 150 155 160Ser Gln Asp Trp Arg Asp Ser Glu Trp Ile Arg
Ser Gln Glu Ala Gly 165 170 175Gly Arg Val Val Pro Asp Ser Cys Cys
Lys Thr Val Val Ala Leu Cys 180 185 190Gly Gln Arg Asp His Ala Ser
Asn Ile Tyr Lys Val Glu Gly Gly Cys 195 200 205Ile Thr Lys Leu Glu
Thr Phe Ile Gln Glu His Leu Arg Val Ile Gly 210 215 220Ala Val Gly
Ile Gly Ile Ala Cys Val Gln Val Phe Gly Met Ile Phe225 230 235
240Thr Cys Cys Leu Tyr Arg Ser Leu Lys Leu Glu His Tyr 245
2503327DNAhomo sapiens 3gcctactacc agcagctgaa cacggagctc aaggagaacc
tgaaggacac catgaccaag 60cgctaccacc agccgggcca tgaggctgtg accagcgctg
tggaccagct gcagcaggag 120ttccactgct gtggcagcaa caactcacag
gactggcgag acagtgagtg gatccgctca 180caggaggccg gtggccgtgt
ggtcccagac agctgctgca agacggtggt ggctctttgt 240gggcagcgag
accatgcctc caacatctac aaggtggagg gcggctgcat caccaagttg
300gagaccttca tccaggagca cctgagg 3274109PRThomo sapiens 4Ala Tyr
Tyr Gln Gln Leu Asn Thr Glu Leu Lys Glu Asn Leu Lys Asp1 5 10 15Thr
Met Thr Lys Arg Tyr His Gln Pro Gly His Glu Ala Val Thr Ser 20 25
30Ala Val Asp Gln Leu Gln Gln Glu Phe His Cys Cys Gly Ser Asn Asn
35 40 45Ser Gln Asp Trp Arg Asp Ser Glu Trp Ile Arg Ser Gln Glu Ala
Gly 50 55 60Gly Arg Val Val Pro Asp Ser Cys Cys Lys Thr Val Val Ala
Leu Cys65 70 75 80Gly Gln Arg Asp His Ala Ser Asn Ile Tyr Lys Val
Glu Gly Gly Cys 85 90 95Ile Thr Lys Leu Glu Thr Phe Ile Gln Glu His
Leu Arg 100 105554DNAhomo sapiens 5tggacgctgg ccctcaagag tgactacatc
agcctgctgg cctcaggcac ctac 54618PRThomo sapiens 6Trp Thr Leu Ala
Leu Lys Ser Asp Tyr Ile Ser Leu Leu Ala Ser Gly1 5 10 15Thr
Tyr78PRTmus musculus 7Gly Tyr Thr Phe Thr Asp Tyr Ser1 588PRTmus
musculus 8Ile Asn Thr Glu Thr Gly Glu Pro1 5913PRTmus musculus 9Ala
Arg Arg Glu Tyr Gly Asn Tyr Tyr Gly Met Glu Tyr1 5 1010120PRTmus
musculus 10Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro
Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Asp Tyr 20 25 30Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly
Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr
Tyr Thr Asp Asp Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr
Ser Ala Asn Thr Ala Tyr65 70 75 80Leu Arg Ile Asn Asn Leu Lys Asn
Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg Arg Glu Tyr Gly Asn
Tyr Tyr Gly Met Glu Tyr Trp Gly Gln 100 105 110Gly Thr Ser Val Thr
Val Ser Ser 115 1201110PRTmus musculus 11Gln Ser Val Ser Thr Ser
Thr Phe Ser Tyr1 5 10123PRTmus musculus 12Ser Ala Ser1139PRTmus
musculus 13Gln His Ser Trp Glu Ile Pro Leu Thr1 514111PRTmus
musculus 14Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser
Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val
Ser Thr Ser 20 25 30Thr Phe Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro
Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Ser Ala Ser Asn Leu Glu
Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile His65 70 75 80Pro Val Glu Glu Glu Asp Thr Ala
Thr Tyr Phe Cys Gln His Ser Trp 85 90 95Glu Ile Pro Leu Thr Phe Gly
Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110158PRTmus musculus 15Gly
Tyr Thr Phe Thr Ser Ser Trp1 5168PRTmus musculus 16Ile His Pro Asn
Ser Gly Asn Thr1 51716PRTmus musculus 17Ala Arg Gly Asp Asp Ala Tyr
Tyr Ser Gly Leu Tyr Ala Met Asp Tyr1 5 10 1518123PRTmus musculus
18Gln Val Gln Leu Gln Gln Pro Gly Ser Val Val Val Arg Pro Gly Ala1
5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Ser 20 25 30Trp Met His Trp Ala Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Gln Ile His Pro Asn Ser Gly Asn Thr Asn Tyr Asn
Glu Lys Phe 50 55 60Lys Val Lys Ala Thr Leu Thr Ile Asp Thr Ser Ser
Ser Thr Ala Tyr65 70 75 80Val Asp Leu Asn Ser Leu Thr Ser Gly Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Asp Asp Ala Tyr Tyr Ser
Gly Leu Tyr Ala Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Val
Thr Val Ser Ser 115 1201910PRTmus musculus 19Gln Ser Val Ser Thr
Ser Arg Tyr Ser Tyr1 5 10203PRTmus musculus 20Tyr Ala Ser1219PRTmus
musculus 21Gln His Ser Trp Glu Ile Pro Tyr Thr1 522111PRTmus
musculus 22Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser
Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val
Ser Thr Ser 20 25 30Arg Tyr Ser Tyr Met His Trp Tyr Gln Gln Ile Gln
Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu
Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Asn Ile His65 70 75 80Pro Val Glu Glu Glu Asp Thr Ala
Thr Tyr His Cys Gln His Ser Trp 85 90 95Glu Ile Pro Tyr Thr Phe Gly
Gly Gly Thr Thr Leu Glu Ile Lys 100 105 1102324DNAmus musculus
23ggttatacct tcacagacta ttca 242424DNAmus musculus 24ataaacactg
agactggtga gcca 242539DNAmus musculus 25gctagaaggg agtatggtaa
ctactatggt atggagtac 3926360DNAmus musculus 26cagatccagt tggtgcagtc
tggacctgag ctgaagaagc ctggagagac agtcaagatc 60tcctgcaagg cttctggtta
taccttcaca gactattcaa tgcactgggt gaagcaggct 120ccaggaaagg
gtttaaagtg gatgggctgg ataaacactg agactggtga gccaacatat
180acagatgact tcaagggacg gtttgccttc tctttggaaa cctctgccaa
cactgcctat 240ttgcggatca acaacctcaa aaatgaggac acggctacat
atttctgtgc tagaagggag 300tatggtaact actatggtat ggagtactgg
ggtcaaggaa cctcagtcac cgtctcgtca 3602730DNAmus musculus
27caaagtgtca gtacatctac ctttagttat 30289DNAmus musculus 28tctgcatcc
92927DNAmus musculus 29cagcacagtt gggagattcc gctcacg 2730333DNAmus
musculus 30gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca
gagggccacc 60atctcatgca gggccagcca aagtgtcagt acatctacct ttagttatat
acactggtac 120caacagaaac caggacagcc acccaaactc ctcatcaagt
ctgcatccaa cctagaatct 180ggggtccctg ccaggttcag tggcagtggg
tctgggacag acttcaccct caccatccat 240cctgtggagg aggaggatac
tgcaacatat ttctgtcagc acagttggga gattccgctc 300acgttcggtg
ctgggaccaa gctggagctg aaa 3333124DNAmus musculus 31ggctacacct
tcaccagctc ctgg 243224DNAmus musculus 32attcatccta atagtggtaa tact
243348DNAmus musculus 33gcaagagggg atgatgctta ctacagcggg ctctatgcta
tggactac 4834369DNAmus musculus 34caggtccaac tgcagcagcc tgggtctgtg
gtggtgaggc ctggagcttc agtgaaactg 60tcctgcaagg cttctggcta caccttcacc
agctcctgga tgcactgggc gaagcagagg 120cctggacaag gccttgagtg
gattggacag attcatccta atagtggtaa tactaattac 180aatgagaagt
tcaaggtcaa ggccacactg actatagaca catcctccag cacagcctac
240gtggatctca acagcctgac atctggggac tctgcggtct attactgtgc
aagaggggat 300gatgcttact acagcgggct ctatgctatg gactactggg
gtcagggaac ctcagtcacc 360gtctcctca 3693530DNAmus musculus
35caaagtgtca gtacatctag gtatagttat 30369DNAmus musculus 36tatgcatcc
93727DNAmus musculus 37caacacagtt gggagattcc gtacacg 2738333DNAmus
musculus 38gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca
gagggccacc 60atctcatgca gggccagcca aagtgtcagt acatctaggt atagttatat
gcactggtac 120caacagatac aaggacagcc acccaaactc ctcatcaagt
atgcatccaa cctagaatct 180ggggtccctg ccaggttcag tggcagtggg
tctgggacag acttcaccct caacatccat 240cctgtggagg aggaggatac
tgcaacatat cactgtcaac acagttggga gattccgtac 300acgttcggag
gggggaccac gctggaaata aaa 333
* * * * *