NOVEL ANTI-cMET ANTIBODY
The present invention relates to a novel divalent antibody capable of binding
specifically to the human c-Met receptor and/or capable of specifically inhibiting the
tyrosine kinase activity of said receptor, preferably both in a ligand-dependent and in a
ligand-independent manner as well as the amino acid and nucleic acid sequences coding
for said antibody. More preferably said antibody comprises a modified hinge region and
exhibits an improved antagonistic activity. More particularly, the antibody according to
the invention is capable of inhibiting the c-Met dimerization. The invention likewise
comprises the use of said antibody as a medicament for the prophylactic and/or
therapeutic treatment of cancers, preferably for cancer characterized by a ligandindependent
activation of c-Met, or any pathology connected with the overexpression of
said receptor as well as in processes or kits for diagnosis of illnesses connected with the
over-expression of c-Met. The invention finally comprises products and/or
compositions comprising such an antibody in combination with other antibodies and/or
chemical compounds directed against other growth factors involved in tumor
progression or metastasis and/or compounds and/or anti-cancer agents or agents
conjugated with toxins and their use for the prevention and/or the treatment of certain
cancers.
Receptor tyrosine kinase (RTK) targeted agents such as trastuzumab, cetuximab,
bevacizumab, imatinib and gefitinib inhibitors have illustrated the interest of targeting
this protein class for treatment of selected cancers.
c-Met, is the prototypic member of a sub-family of RTKs which also includes
RON and SEA. The c-Met RTK family is structurally different from other RTK families
and is the only known high-affinity receptor for hepatocyte growth factor (HGF), also
called scatter factor (SF) [D.P. Bottaro et al, Science 1991, 251:802-804; L. Naldini et
al, Eur. Mol. Biol. Org. J . 1991, 10:2867-2878]. c-Met and HGF are widely expressed
in a variety of tissue and their expression is normally restricted to cells of epithelial and
mesenchymal origin respectively [M.F. Di Renzo et al., Oncogene 1991, 6:1997-2003;
E. Sonnenberg et al, J . Cell. Biol. 1993, 123:223-235]. They are both required for
normal mammalian development and have been shown to be particularly important in
cell migration, morphogenic differentiation, and organization of the three-dimensional
tubular structures as well as growth and angiogenesis [F. Baldt et al, Nature 1995,
376:768-771; C. Schmidt et al, Nature. 1995:373:699-702; Tsarfaty et al, Science
1994, 263:98-101]. While the controlled regulation of c-Met and HGF have been shown
to be important in mammalian development, tissue maintenance and repair [Nagayama
T., Nagayama M., Kohara S., Kamiguchi H., Shibuya M., Katoh Y., Itoh J., Shinohara
Y., Brain Res. 2004, 5;999(2): 155-66; Tahara Y., Ido A., Yamamoto S., Miyata Y., Uto
H., Hori T., Hayashi K., Tsubouchi H., J Pharmacol Exp Ther. 2003, 307(1): 146-51],
their dysregulation is implicated in the progression of cancers.
Aberrant signalling driven by inappropriate activation of c-Met is one of the
most frequent alteration observed in human cancers and plays a crucial role in
tumorigenesis and metastasis [Birchmeier et al., Nat. Rev. Mol. Cell Biol. 2003, 4:915-
925; L. Trusolino and Comoglio P. M., Nat Rev. Cancer. 2002, 2(4):289-300].
Inappropriate c-Met activation can arise by ligand-dependent and independent
mechanisms, which include overexpression of c-Met, and/or paracrine or autocrine
activation, or through gain in function mutation [J.G. Christensen, Burrows J . and
Salgia R., Cancer Latters. 2005, 226:1-26]. However an oligomerization of c-Met
receptor, in presence or in absence of the ligand, is required to regulate the binding
affinity and binding kinetics of the kinase toward ATP and tyrosine-containing peptide
substrates [Hays JL, Watowich SJ, Biochemistry, 2004 Aug 17, 43:10570-8]. Activated
c-Met recruits signalling effectors to its multidocking site located in the cytoplasm
domain, resulting in the activation of several key signalling pathways, including Ras-
MAPK, PI3K, Src and Stat3 [Gao CF, Vande Woude GF, Cell Res. 2005, 15(1):49-51;
Furge KA, Zhang YW, Vande Woude GF, Oncogene. 2000, 19(49):5582-9]. These
pathways are essential for tumour cell proliferation, invasion and angiogenesis and for
evading apoptosis [Furge KA, Zhang YW, Vande Woude GF, Oncogene, 2000,
19(49):5582-9; Gu H., Neel BG, Trends Cell Biol. 2003 Mar, 13(3): 122-30; Fan S., Ma
YX, Wang JA, Yuan RQ, Meng Q., Cao Y., Laterra JJ, Goldberg ID, Rosen EM,
Oncogene. 2000 Apr 27, 19(1 8):2212-23]. In addition, a unique facet of the c-Met
signalling relative to other RTK is its reported interaction with focal adhesion
complexes and non kinase binding partners such as 6b4 integrins [Trusolino L.,
Bertotti A., Comoglio PM, Cell. 2001, 107:643-54], CD44v6 [Van der Voort R., Taher
TE, Wielenga VJ, Spaargaren M., Prevo R., Smit L., David G., Hartmann G., Gherardi
E., Pals ST, J . Biol. Chem. 1999, 274(10):6499-506], Plexin Bl or semaphorins
[Giordano S., Corso S., Conrotto P., Artigiani S., Gilestro G., Barberis D., Tamagnone
L., Comoglio PM, Nat Cell Biol. 2002, 4(9):720-4; Conrotto P., Valdembri D., Corso
S., Serini G., Tamagnone L., Comoglio PM, Bussolino F., Giordano S., Blood. 2005,
105(1 1):4321-9; Conrotto P., Corso S., Gamberini S., Comoglio PM, Giordano S.,
Oncogene. 2004, 23:5131-7] which may further add to the complexity of regulation of
cell function by this receptor. Finally recent data demonstrate that c-Met could be
involved in tumor resistance to gefitinib or erlotinib suggesting that combination of
compound targeting both EGFR and c-Met might be of significant interest [Engelman
JA et al, Science, 2007, 316:1039-43].
In the past few years, many different strategies have been developed to attenuate
c-Met signalling in cancer cell lines. These strategies include i) neutralizing antibodies
against c-Met or HGF/SF [Cao B., Su Y., Oskarsson M., Zhao P., Kort EJ, Fisher RJ,
Wang LM, Vande Woude GF, Proc Natl Acad Sci U S A. 2001, 98(13):7443-8;
Martens T., Schmidt NO, Eckerich C , Fillbrandt R., Merchant M., Schwall R.,
Westphal M., Lamszus K., Clin Cancer Res. 2006, 12(20):6144-52] or the use of
HGF/SF antagonist NK4 to prevent ligand binding to c-Met [Kuba K., Matsumoto K.,
Date K., Shimura H., Tanaka M., Nakamura T., Cancer Res., 2000, 60:6737-43], ii)
small ATP binding site inhibitors to c-Met that block kinase activity [Christensen JG,
Schreck R., Burrows J., Kuruganti P., Chan E, Le P., Chen J., Wang X., Ruslim L.,
Blake R., Lipson KE, Ramphal J., Do S., Cui JJ, Cherrington JM, Mendel DB, Cancer
Res. 2003, 63:7345-55], iii) engineered SH2 domain polypeptide that interferes with
access to the multidocking site and RNAi or ribozyme that reduce receptor or ligand
expression. Most of these approaches display a selective inhibition of c-Met resulting in
tumor inhibition and showing that c-Met could be of interest for therapeutic intervention
in cancer.
Within the molecules generated for c-Met targeting, some are antibodies. The
most extensively described is the anti-c-Met 5D5 antibody generated by Genentech
[WO 96/38557] which behaves as a potent agonist when added alone in various models
and as an antagonist when used as a Fab fragment. A monovalent engineered form of
this antibody described as one armed 5D5 (OA5D5) and produced as a recombinant
protein in E. Coli is also the subject of a patent application [WO 2006/015371] by
Genentech. However, this molecule that could not be considered as an antibody because
of its particular scaffold, displays also mutations that could be immunogenic in humans.
In terms of activity, this unglycosylated molecule is devoided of effector functions and
finally, no clear data demonstrate that OA5D5 inhibits dimerization of c-Met.
Moreover, when tested in the G55 in vivo model, a glioblastoma cell line that expresses
c-Met but not HGF mR A and protein and that grows independently of the ligand, the
one armed anti-c-Met had no significant effect on G55 tumor growth suggesting that
OA5D5 acts primarily by blocking HGF binding and is not able to target tumors
activated independently of HGF [Martens T. et al, Clin. Cancer Res., 2006,
12(20):6144-6152].
Another antibody targeting c-Met is described by Pfizer as an antibody acting
"predominantly as c-Met antagonist, and in some instance as a c-Met agonist" [WO
2005/016382]. No data showing any effect of Pfizer antibodies on c-Met dimerization is
described in this application.
One of the innovative aspects of the present invention is to generate a chimeric
and/or humanized monoclonal antibody without intrinsic agonist activity and inhibiting
c-Met dimerization. More particularly, an innovative aspect of the present invention is
to generate a chimeric and/or humanized monoclonal antibody with antagonist activity
and inhibiting c-Met dimerization.
In addition of targeting ligand-dependent tumors, this approach will also impair
ligand-independent activations of c-Met due to its overexpression or mutations of the
intra cellular domains which remained dependent to oligomerization for signalling.
Another aspect of the activity of this antibody could be a steric hindrance for c-Met
interaction with its partners that will result in impairment of c-Met functions. This
antibody is humanized and engineered preferentially, but not limited, as human IgGl to
get effector functions such as ADCC and CDC in addition to functions linked to the
specific blockade of the c-Met receptor.
Surprisingly, for the first time, inventors have managed to generate a chimeric
and/or humanized monoclonal antagonist antibody capable of binding to c-Met but also
capable of inhibiting the c-Met dimerization, said monoclonal antibody being divalent
contrary to existing antagonist antibodies directed against c-Met. If it is true that, in the
prior art, it is sometimes suggested that an antibody capable of inhibiting the
dimerization of c-Met with its partners could be an interesting one, it has never been
disclosed, or clearly suggested, an antibody capable of doing so. Moreover, regarding
antibody specificity, it was not evident at all to succeed in the generation of such an
active divalent antibody.
As it was explained before, the inhibition of the c-Met dimerization is a capital
aspect of the invention as such antibodies will present a real interest for a larger
population of patients. Not only ligand-dependent activated c-Met cancer, as it was the
case until the present invention, but also ligand-independent activated c-Met cancer
could be traited with antibodies generated by the process of the present invention.
Antibodies were evaluated by BRET analysis on cells expressing both c-Met-
RLuc/c-Met-YFP and selected on their ability to inhibit at least 40 %, preferably 45 %,
50 %, 55 % and most preferably 60 % of the BRET signal.
The BRET technology is known as being representative of the protein
dimerization [Angers et al, PNAS, 2000, 97:3684-89].
The BRET technology is well known by the man skill in the art and will be
detailed in the following examples. More particularly, BRET (Bioluminescence
Resonance Energy Transfer) is a non-radiative energy transfer occurring between a
bioluminescent donor (Renilla Luciferase (Rluc)) and a fluorescent acceptor, a mutant
of GFP (Green Fluorescent Protein) or YFP (Yellow fluorescent protein). In the present
case EYFP (Enhanced Yellow Fluorescent Protein) was used. The efficacy of transfer
depends on the orientation and the distance between the donor and the acceptor. Then,
the energy transfer can occur only if the two molecules are in close proximity (1-10
nm). This property is used to generate protein-protein interaction assays. Indeed, in
order to study the interaction between two partners, the first one is genetically fused to
the Renilla Luciferase and the second one to the yellow mutant of the GFP. Fusion
proteins are generally, but not obligatory, expressed in mammalian cells. In presence of
its membrane permeable substrate (coelenterazine), Rluc emits blue light. If the GFP
mutant is closer than 10 nm from the Rluc, an energy transfer can occur and an
additional yellow signal can be detected. The BRET signal is measured as the ratio
between the light emitted by the acceptor and the light emitted by the donor. So the
BRET signal will increase as the two fusion proteins are brought into proximity or if a
conformational change brings Rluc and GFP mutant closer.
If the BRET analysis consists in a preferred embodiment, any method known by
the man skilled in the art can be used to measure c-Met dimerization. Without
limitation, the following technologies can be mentioned: FRET (Fluorescence
Resonance Energy Transfer), HTRF (Homogenous Time resolved Fluorescence), FLIM
(Fluorescence Lifetime Imaging Microscopy) or SW-FCCS single wavelength
fluorescence cross-correlation spectroscopy).
Other classical technologies could also be used, such as Coimmunoprecipitation,
Alpha screen, Chemical cross-linking, Double-Hybrid, Affinity
Chromatography, ELISA or Far western blot.
The terms "antibody", "antibodies" or "immunoglobulin" are used
interchangeably in the broadest sense and include monoclonal antibodies (e.g., full
length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies or
multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired
biological activity).
More particularly, such molecule consists in a glycoprotein comprising at least
two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each
heavy chain is comprised of a heavy chain variable region (or domain) (abbreviated
herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant
region is comprised of three domains, CHI, CH2 and CH3. Each light chain is
comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a
light chain constant region. The light chain constant region is comprised of one domain,
CL. The VH and VL regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (CDR), interspersed with regions that are
more conserved, termed framework regions (FR). Each VH and VL is composed of
three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the
following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. The variable regions of
the heavy and light chains contain a binding domain that interacts with an antigen. The
constant regions of the antibodies may mediate the binding of the immunoglobulin to
host tissues or factors, including various cells of the immune system (e.g. effector cells)
and the first component (Clq) of the classical complement system.
The heavy chains of immunoglobulins can be divided into three functional
regions: the Fd region, the hinge region, and the Fc region (fragment crystallizable). The
Fd region comprises the VH and CHI domains and, in combination with the light chain,
forms Fab - the antigen-binding fragment. The Fc fragment is responsible for the
immunoglobulin effector functions, which includes, for example, complement fixation
and binding to cognate Fc receptors of effector cells. The hinge region, found in IgG,
IgA, and IgD immunoglobulin classes, acts as a flexible spacer that allows the Fab
portion to move freely in space relative to the Fc region. The hinge domains are
structurally diverse, varying in both sequence and length among immunoglobulin
classes and subclasses.
According to crystallographic studies, the immunoglobulin hinge region can be
further subdivided structurally and functionally into three regions: the upper hinge, the
core, and the lower hinge (Shin et al, Immunological Reviews 130:87, 1992). The
upper hinge includes amino acids from the carboxyl end of CHI to the first residue in
the hinge that restricts motion, generally the first cysteine residue that forms an
interchain disulfide bond between the two heavy chains. The length of the upper hinge
region correlates with the segmental flexibility of the antibody. The core hinge region
contains the inter-heavy chain disulfide bridges. The lower hinge region joins the amino
terminal end of, and includes residues in the CH2 domain. The core hinge region of
human IgGl contains the sequence Cys-Pro-Pro-Cys that, when dimerized by disulfide
bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring
flexibility. Conformational changes permitted by the structure and flexibility of the
immunoglobulin hinge region polypeptide sequence may affect the effector functions of
the Fc portion of the antibody.
The term « Monoclonal Antibody » is used in accordance with its ordinary
meaning to denote an antibody obtained from a population of substantially
homogeneous antibodies, i.e. the individual antibodies comprising the population are
identical except for possible naturally occurring mutations that may be present in minor
amounts. In other words, a monoclonal antibody consists in a homogenous antibody
resulting from the proliferation of a single clone of cells (e.g., hybridoma cells,
eukaryotic host cells transfected with DNA encoding the homogenous antibody,
prokaryotic host cells transformed with DNA encoding the homogenous antibody, etc.),
and which is generally characterized by heavy chains of a single class and subclass, and
light chains of a single type. Monoclonal antibodies are highly specific, being directed
against a single antigen. Furthermore, in contrast to polyclonal antibodies preparations
that typically include different antibodies directed against different determinants, or
epitope, each monoclonal antibody is directed against a single determinant on the
antigen.
In the present description, the terms polypeptides, polypeptide sequences, amino
acid sequences, peptides and proteins attached to antibody compounds or to their
sequence are interchangeable.
The invention relates to a monoclonal antibody, or a divalent functional
fragment or derivative thereof, capable to inhibit the c-Met dimerization and comprising
a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 with respectively the amino
acid sequences SEQ ID Nos. 1, 2 and 3 or a sequence having at least 80 % identity after
optimum alignment with sequences SEQ ID Nos. 1, 2 and 3; and a light chain
comprising CDR-L1, CDR-L2 and CDR-L3 with respectively the amino acid sequences
SEQ ID Nos. 5, 6 and 7 or a sequence having at least 80% identity after optimum
alignment with sequences SEQ ID Nos. 5, 6 or 7, said antibody being further
characterized in that it also comprises a hinge region comprising the amino acid
sequence SEQ ID No. 56.
In a preferred embodiment, the present invention is directed to a monoclonal
antibody, or a divalent functional fragment or derivative thereof, capable to inhibit the
c-Met dimerization,
said antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3
with respectively the amino acid sequences SEQ ID Nos. 1, 2 and 3; and a light chain
comprising CDR-L1, CDR-L2 and CDR-L3 with respectively the amino acid sequences
SEQ ID Nos. 5, 6 and 7, said antibody further comprising a hinge region comprising the
amino acid sequence SEQ ID No. 56;
for use for the prevention or the treatment of a patient in need thereof having a cancer
characterized by ligand-independent activation of c-Met, preferably a cancer further
characterized by overexpression of c-Met, said c-Met overexpression resulting more
preferably from genie amplification of c-Met, and, also more preferably, resulting in
ligand-independent activation of c-Met.
In this aspect, the present invention comprises a method for the prevention or the
treatment of a patient in need thereof having a cancer characterized by ligandindependent
activation of c-Met, preferably a cancer further characterized by
overexpression of c-Met, said c-Met overexpression resulting more preferably from
genie amplification of c-Met, and, also more preferably, resulting in ligand-independent
activation of c-Met, said method comprising the step of administering a composition
comprising a monoclonal antibody, or a divalent functional fragment or derivative
thereof, capable to inhibit the c-Met dimerization, said antibody comprising a heavy
chain comprising CDR-H1, CDR-H2 and CDR-H3 with respectively the amino acid
sequences SEQ ID Nos. 1, 2 and 3; and a light chain comprising CDR-L1, CDR-L2 and
CDR-L3 with respectively the amino acid sequences SEQ ID Nos. 5, 6 and 7, said
antibody further comprising a hinge region comprising the amino acid sequence SEQ
ID No. 56.
In this particular aspect and in a more preferred embodiment, said cancer
characterized by ligand-independent activation of c-Met, and preferably further
characterized by overexpression of c-Met, resulting more preferably from genie
amplification of c-Met, and, also more preferably, resulting in ligand-independent
activation of c-Met, is selected from the group consisting of renal cell carcinoma and
gastric cancer.
More particularly, the invention relates to a monoclonal antibody, or a divalent
functional fragment or derivative thereof, as above described characterized in that said
hinge region comprises the amino acid sequence SEQ ID No. 57.
In other words, the invention relates to a monoclonal antibody, or a divalent
functional fragment or derivative thereof, capable to inhibit the c-Met dimerization and
comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 with
respectively the amino acid sequences SEQ ID Nos. 1, 2 and 3 or a sequence having at
least 80% identity after optimum alignment with sequences SEQ ID Nos. 1, 2 and 3;
and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 with respectively the
amino acid sequences SEQ ID Nos. 5, 6 and 7 or a sequence having at least 80%
identity after optimum alignment with sequences SEQ ID Nos. 5, 6 or 7, said antibody
being further characterized in that it also comprises a hinge region comprising the
amino acid sequence SEQ ID No. 57.
More particularly, the invention relates to a monoclonal antibody, or a divalent
functional fragment or derivative thereof, as above described characterized in that it also
comprises a hinge region comprising the amino acid sequence SEQ ID No. 21.
In other words, the invention also relates to a monoclonal antibody, or a divalent
functional fragment or derivative thereof, capable to inhibit the c-Met dimerization and
comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 with
respectively the amino acid sequences SEQ ID Nos. 1, 2 and 3 or a sequence having at
least 80% identity after optimum alignment with sequences SEQ ID Nos. 1, 2 and 3;
and a light chain comprising CDR-Ll, CDR-L2 and CDR-L3 with respectively the
amino acid sequences SEQ ID Nos. 5, 6 and 7 or a sequence having at least 80%
identity after optimum alignment with sequences SEQ ID Nos. 5, 6 or 7, said antibody
being further characterized in that it also comprises a hinge region comprising the
amino acid sequence SEQ ID No. 21.
As it will be apparent for the man skilled in the art, the consensus sequences
SEQ ID Nos. 57 and 2 1 are comprised in the consensus sequence SEQ ID No. 56.
Table 1
For SEQ ID No. 56:
X I P , R , C , X5 D , C , G , X8: H , V , K , - X12: P , -
X2 K , C , R , X 6 K , C , - X9: T , C , E , P , X14: P , T
X3 S , C , D , X 7 T , C , - Xll: P , I
The expression "functional fragments and derivatives" will be defined in details
later in the present specification.
By CDR regions or CDR(s), it is intended to indicate the hypervariable regions
of the heavy and light chains of the immunoglobulins as defined by IMGT.
The IMGT unique numbering has been defined to compare the variable domains
whatever the antigen receptor, the chain type, or the species [Lefranc M.-P.,
Immunology Today 18, 509 (1997); Lefranc M.-P., The Immunologist, 7, 132-136
(1999); Lefranc, M.-P., Pommie, C , Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L.,
Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol, 27, 55-77 (2003)]. In the
IMGT unique numbering, the conserved amino acids always have the same position, for
instance cysteine 23 (lst-CYS), tryptophan 4 1 (CONSERVED-TRP), hydrophobic
amino acid 89, cysteine 104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or JTRP).
The IMGT unique numbering provides a standardized delimitation of the
framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT:
66 to 104 and FR4-IMGT: 118 to 128) and of the complementarity determining regions:
CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps
represent unoccupied positions, the CDR-IMGT lengths (shown between brackets and
separated by dots, e.g. [8.8.13]) become crucial information. The IMGT unique
numbering is used in 2D graphical representations, designated as IMGT Colliers de
Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002); Kaas, Q.
and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)], and in 3D structures in
IMGT/3Dstructure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and
MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)].
Three heavy chain CDRs and 3 light chain CDRs exist. The term CDR or CDRs
is used here in order to indicate, according to the case, one of these regions or several,
or even the whole, of these regions which contain the majority of the amino acid
residues responsible for the binding by affinity of the antibody for the antigen or the
epitope which it recognizes.
By "percentage of identity" between two nucleic acid or amino acid sequences
in the sense of the present invention, it is intended to indicate a percentage of
nucleotides or of identical amino acid residues between the two sequences to be
compared, obtained after the best alignment (optimum alignment), this percentage being
purely statistical and the differences between the two sequences being distributed
randomly and over their entire length. The comparisons of sequences between two
nucleic acid or amino acid sequences are traditionally carried out by comparing these
sequences after having aligned them in an optimum manner, said comparison being able
to be carried out by segment or by "comparison window". The optimum alignment of
the sequences for the comparison can be carried out, in addition to manually, by means
of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math.
2:482], by means of the local homology algorithm of Neddleman and Wunsch (1970) [J.
Mol. Biol. 48: 443], by means of the similarity search method of Pearson and Lipman
(1988) [Proc. Natl. Acad. Sci. USA 85:2444), by means of computer software using
these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or else by
BLAST N or BLAST P comparison software).
The percentage of identity between two nucleic acid or amino acid sequences is
determined by comparing these two sequences aligned in an optimum manner and in
which the nucleic acid or amino acid sequence to be compared can comprise additions
or deletions with respect to the reference sequence for an optimum alignment between
these two sequences. The percentage of identity is calculated by determining the
number of identical positions for which the nucleotide or the amino acid residue is
identical between the two sequences, by dividing this number of identical positions by
the total number of positions in the comparison window and by multiplying the result
obtained by 100 in order to obtain the percentage of identity between these two
sequences.
For example, it is possible to use the BLAST program, "BLAST 2 sequences"
(Tatusova et al, "Blast 2 sequences - a new tool for comparing protein and nucleotide
sequences", FEMS Microbiol Lett. 174:247-250) available on the site
http://www.ncbi.nlm.nih.gov/ gorf/bl2.html, the parameters used being those given by
default (in particular for the parameters "open gap penalty": 5, and "extension gap
penalty": 2; the matrix chosen being, for example, the matrix "BLOSUM 62" proposed
by the program), the percentage of identity between the two sequences to be compared
being calculated directly by the program.
By amino acid sequence having at least 80 %, preferably 85 %, 90 %, 95 % and
98 % identity with a reference amino acid sequence, those having, with respect to the
reference sequence, certain modifications, in particular a deletion, addition or
substitution of at least one amino acid, a truncation or an elongation are preferred. In the
case of a substitution of one or more consecutive or nonconsecutive amino acid(s), the
substitutions are preferred in which the substituted amino acids are replaced by
"equivalent" amino acids. The expression "equivalent amino acids" is aimed here at
indicating any amino acid capable of being substituted with one of the amino acids of
the base structure without, however, essentially modifying the biological activities of
the corresponding antibodies and such as will be defined later, especially in the
examples. These equivalent amino acids can be determined either by relying on their
structural homology with the amino acids which they replace, or on results of
comparative trials of biological activity between the different antibodies capable of
being carried out.
By way of example, mention is made of the possibilities of substitution capable
of being carried out without resulting in a profound modification of the biological
activity of the corresponding modified antibody.
As non limitative example, the following table 2 is giving substitution
possibilities conceivable with a conservation of the biological activity of the modified
antibody. The reverse substitutions are also, of course, possible in the same conditions.
Table 2
It must be understood here that the invention does not relate to the antibodies in
natural form, that is to say they are not in their natural environment but that they have
been able to be isolated or obtained by purification from natural sources, or else
obtained by genetic recombination, or by chemical synthesis, and that they can then
contain unnatural amino acids as will be described further on.
It must also be understood, as previously mentioned, that the invention concerns
more particularly a chimeric and/or a humanized divalent antibody, or any divalent
functional fragment or derivative, with an antagonistic activity. Divalent antibodies of
the prior art are agonists or partial agonists. The monoclonal antibody of the invention,
including a modified hinge as previously described, i.e. including a hinge region
comprising the amino acid sequence SEQ ID No. 56, 57 or 21, is novel and presents the
particularity to have a improved antagonistic activity compared to the chimeric or
humanized antibody 224G11 without such a modified hinge as it will appear from the
following examples.
Contrary to the prior art, inventors have obtained an improved antagonistic
activity without modifying the format of the antibody. Actually, in the closest prior art
represented by the antibody 5D5, it has been necessary to develop a monovalent
fragment of the antibody to generate an antagonistic activity. In the present application,
by the use of the hinge of the invention, it is possible for the first time to obtain a full
divalent antibody with increased antagonistic activity, and this contrary to the general
knowledge.
In a preferred embodiment, the antibody of the invention comprises a hinge
region comprising an amino acid sequence selected from the group consisting of SEQ
ID Nos. 22 to 28 and 58 to 72, or a sequence having at least 80% identity after optimum
alignment with sequences SEQ ID Nos. 22 to 28 and 58 to 72.
For more clarity, the following tables 3 and 4 regroup the amino acids and
nucleotides sequences of the different preferred hinges of the invention.
Table 3
Table 4
According a first approach, the antibody will be defined by its heavy chain
sequence. More particularly, the antibody of the invention, or one of its functional
fragments or derivatives, is characterized in that it comprises a heavy chain comprising
at least one CDR chosen from CDRs comprising the amino acid sequences SEQ ID
Nos. 1 to 3.
The mentioned sequences are the following ones:
SEQ ID No. 1: GYIFTAYT
SEQ ID No. 2 : IKPNNGLA
SEQ ID No. 3: ARSEITTEFDY
According to a preferred aspect, the antibody of the invention, or one of its
functional fragments or derivatives, comprises a heavy chain comprising at least one,
preferably two, and most preferably three, CDR(s) chosen from CDR-Hl, CDR-H2 and
CDR-H3, wherein:
- CDR-Hl comprises the amino acid sequence SEQ ID No. 1,
- CDR-H2 comprises the amino acid sequence SEQ ID No. 2,
- CDR-H3 comprises the amino acid sequence SEQ ID No. 3.
In a second approach, the antibody will be now defined by its light chain
sequence. More particularly, according to a second particular aspect of the invention,
the antibody, or one of its functional fragments or derivatives, is characterized in that it
comprises a light chain comprising at least one CDR chosen from CDRs comprising the
amino acid sequence SEQ ID Nos. 5 to 7.
The mentioned sequences are the following ones:
SEQ ID No. 5: ESVDSYANSF
SEQ ID No. 6 : RAS
SEQ ID No. 7 : QQSKEDPLT
According to another preferred aspect, the antibody of the invention, or one of
its functional fragments or derivatives, comprises a light chain comprising at least one,
preferably two, and most preferably three, CDR(s) chosen from CDR-Ll, CDR-L2 and
CDR-L3, wherein:
- CDR-Ll comprises the amino acid sequence SEQ ID No. 5,
- CDR-L2 comprises the amino acid sequence SEQ ID No. 6,
- CDR-L3 comprises the amino acid sequence SEQ ID No. 7.
The murine hybridoma capable of secreting monoclonal antibodies according to
the present invention, especially hybridoma of murine origin, was deposited at the
CNCM (Institut Pasteur, Paris, France) on 03/14/2007 under the number CNCM 1-3731.
In the present application, IgGl are preferred to get effector functions, and most
preferably ADCC and CDC.
The skilled artisan will recognize that effector functions include, for example,
Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding;
antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down
regulation of cell surface receptors (e.g. B cell receptor; BCR).
The antibodies according to the present invention are preferably specific
monoclonal antibodies, especially of murine, chimeric or humanized origin, which can
be obtained according to the standard methods well known to the person skilled in the
art.
In general, for the preparation of monoclonal antibodies or their functional
fragments or derivatives, especially of murine origin, it is possible to refer to techniques
which are described in particular in the manual "Antibodies" (Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor
NY, pp. 726, 1988) or to the technique of preparation from hybridomas described by
Kohler and Milstein (Nature, 256:495-497, 1975).
The monoclonal antibodies according to the invention can be obtained, for
example, from an animal cell immunized against the c-Met, or one of its fragments
containing the epitope specifically recognized by said monoclonal antibodies according
to the invention. Said c-Met, or one of its said fragments, can especially be produced
according to the usual working methods, by genetic recombination starting with a
nucleic acid sequence contained in the cDNA sequence coding for the c-Met or by
peptide synthesis starting from a sequence of amino acids comprised in the peptide
sequence of the c-Met.
The monoclonal antibodies according to the invention can, for example, be
purified on an affinity column on which the c-Met or one of its fragments containing the
epitope specifically recognized by said monoclonal antibodies according to the
invention has previously been immobilized. More particularly, said monoclonal
antibodies can be purified by chromatography on protein A and/or G, followed or not
followed by ion-exchange chromatography aimed at eliminating the residual protein
contaminants as well as the DNA and the LPS, in itself followed or not followed by
exclusion chromatography on Sepharose™ gel in order to eliminate the potential
aggregates due to the presence of dimers or of other multimers. In an even more
preferred manner, the whole of these techniques can be used simultaneously or
successively.
The antibody of the invention, or a divalent functional fragment or derivative
thereof, consists preferably of a chimeric antibody.
By chimeric antibody, it is intended to indicate an antibody which contains a
natural variable (light chain and heavy chain) region derived from an antibody of a
given species in combination with the light chain and heavy chain constant regions of
an antibody of a species heterologous to said given species (e.g. mouse, horse, rabbit,
dog, cow, chicken, etc.).
The antibodies or their fragments of chimeric type according to the invention
can be prepared by using the techniques of genetic recombination. For example, the
chimeric antibody can be produced by cloning a recombinant DNA containing 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 human antibody. A chimeric antibody of the invention encoded by
such a recombinant gene will be, for example, a mouse-man chimera, the specificity of
this 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 the
methods of preparation of chimeric antibodies, it is possible, for example, to refer to the
documents Verhoeyn et al. (BioEssays, 8:74, 1988), Morrison et al. (Proc. Natl. Acad.
Sci. USA 82:6851-6855, 1984) or US 4,816,567.
More particularly, said antibody, or a functional fragment or derivative thereof,
comprises a chimeric heavy chain variable domain of sequence comprising the amino
acid sequence SEQ ID No. 46 or a sequence having at least 80 % identity after optimum
alignment with the sequence SEQ ID No. 46.
SEQ ID No. 46: EVQLQQSGPELVKPGASVKISCKTSGYIFTAYTMHWVRQSLGE
SLDWIGGIKPNNGLANYNQKFKGKATLTVDKSSSTAYMDLRSLTSEDSAVYYC
ARSEITTEFD YWGQGTALTVS S
More particularly, said antibody, or a functional fragment or derivative thereof,
comprises a chimeric light chain variable domain of sequence comprising the amino
acid sequence SEQ ID No. 47 or a sequence having at least 80 % identity after optimum
alignment with the sequence SEQ ID No. 47.
SEQ ID No. 47: DIVLTQSPASLAVSLGQRATISCRASESVDSYANSFMHWYQQKP
GQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSKEDP
LTFGSGTKLEMKR
More particularly, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [IgG2chim],
comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID
No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID
No. 47, and a hinge region comprising the amino acid sequence SEQ ID No. 22.
In the present application, the use of square brackets is not necessary and, as en
example, the reference [224G1 1] [IgG2chim] must be considered as identical to
224G1 HgG2chim. In a same way, to indicate that the antibody is a murine one, the
expression murine or the letter m can be added; to indicate that the antibody is a
chimeric one, the expression chim or the letter c can be added and ; to indicate that the
antibody is a humanized one, the expression hum, hz, Hz or the letter h can be added.
As an example, the chimeric antibody 224GlIgG2 can be referred as c224GHIgG2,
c224Gl l[IgG2], c[224Gl l]IgG2, c[224Gl l][IgG2], 224G1 HgG2chim,
224G1 l[IgG2chim], [224G1 l]IgG2chim or [224G1 l][IgG2chim].
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [TH7chim],
comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID
No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID
No. 47, and a hinge region comprising the amino acid sequence SEQ ID No. 28.
In the present application, the reference TH7 must be considered as identical to
C7A6-9 or TH7C7A6-9. The symbol D means deletion.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [MHchim],
comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID
No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID
No. 47, and a hinge region comprising the amino acid sequence SEQ ID No. 23.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [MUP9Hchim],
comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID
No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID
No. 47, and a hinge region comprising the amino acid sequence SEQ ID No. 26.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [MMCHchim],
comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID
No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID
No. 47, and a hinge region comprising the amino acid sequence SEQ ID No. 24.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [CI], comprises a
heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a
light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a
hinge region comprising the amino acid sequence SEQ ID No. 58.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [C2], comprises a
heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a
light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a
hinge region comprising the amino acid sequence SEQ ID No. 59.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [C3], comprises a
heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a
light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a
hinge region comprising the amino acid sequence SEQ ID No. 60.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [C5], comprises a
heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a
light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a
hinge region comprising the amino acid sequence SEQ ID No. 61.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [C6], comprises a
heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a
light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a
hinge region comprising the amino acid sequence SEQ ID No. 62.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [C7], comprises a
heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a
light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a
hinge region comprising the amino acid sequence SEQ ID No. 63.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [C9], comprises a
heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a
light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a
hinge region comprising the amino acid sequence SEQ ID No. 64.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [D 1-3], comprises a
heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a
light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a
hinge region comprising the amino acid sequence SEQ ID No. 65.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [C7A6], comprises a
heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a
light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a
hinge region comprising the amino acid sequence SEQ ID No. 66.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [C6A9], comprises a
heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a
light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a
hinge region comprising the amino acid sequence SEQ ID No. 67.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [C2A5-7], comprises
a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a
light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a
hinge region comprising the amino acid sequence SEQ ID No. 68.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [C5A2-6], comprises
a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a
light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a
hinge region comprising the amino acid sequence SEQ ID No. 69.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [C9A2-7], comprises
a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a
light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a
hinge region comprising the amino acid sequence SEQ ID No. 70.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G11] [D5-6-7-8],
comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID
No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID
No. 47, and a hinge region comprising the amino acid sequence SEQ ID No. 7 1.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [IgGl/IgG2],
comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID
No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID
No. 47, and a hinge region comprising the amino acid sequence SEQ ID No. 72.
The antibody of the invention, or a divalent functional fragment or derivative
thereof, consists preferably of a human antibody.
The term "human antibody" includes all antibodies that have one or more
variable and constant region derived from human immunoglobulin sequences. In a
preferred embodiment, all of the variable and constant domains (or regions) are derived
from human immunoglobulin sequence (fully human antibody). In other words, it
includes any antibody which have variable and constant regions (if present) derived
from human germline immunoglobulin sequences, i.e. which possesses an amino acid
sequence which corresponds to that of an antibody produced by a human and/or has
been made using any techniques for making human antibodies known by the man skill
in the art.
In one embodiment, the human monoclonal antibodies are produced by a
hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g.,
a transgenic mouse, having a genome comprising a human heavy chain transgene and a
light chain transgene fused to an immortalized cell.
As example for such transgenic mouse, it can be mentioned the
XENOMOUSE™ which is an engineered mouse strain that comprises large fragments
of the human immunoglobulin loci and is deficient in mouse antibody production
(Green at al, 1994, Nature Genetics, 7:13-21). The XENOMOUSE™ produces an
adult-like human repertoire of fully human antibodies, and generate antigen-specific
human monoclonal antibodies. A second generation XENOMOUSE™ contains
approximately 80% of the human antibody repertoire (Green & Jakobovits, 1998, J .
Exp. Med., 188:483-495).
Any other technique known by the man skill in the art, such as phage display
technique, can also be used for the generation of human antibody according to the
invention.
The antibody of the invention, or a divalent functional fragment or derivative
thereof, consists preferably of a humanized antibody.
By the expression "humanized antibody", it is intended to indicate an antibody
which contains CDR regions derived from an antibody of nonhuman origin, the other
parts of the antibody molecule being derived from one (or from several) human
antibodies. Moreover, some of the residues of the segments of the skeleton (called FR)
can be modified in order to conserve the affinity of the binding (Jones et al., Nature,
321:522-525, 1986; Verhoeyen et al, Science, 239:1534-1536, 1988; Riechmann et al,
Nature, 332:323-327, 1988).
The humanized antibodies according to the invention or their fragments 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).
Other humanization method are known by the man skill in the art as, for
example, the "CDR Grafting" method described by Protein Design Lab (PDL) in the
patent applications EP 0 451261, EP 0 682 040, EP 0 9127, EP 0 566 647 or US
5,530,101, US 6,180,370, US 5,585,089 and US 5,693,761. The following patent
applications can also be mentioned: US 5,639,641; US 6,054,297; US 5,886,152 and US
5,877,293.
More particularly, said antibody, or a functional fragment or derivative thereof,
comprises a humanized heavy chain variable domain of sequence comprising the amino
acid sequence SEQ ID No. 4 or a sequence having at least 80 % identity after optimum
alignment with the sequence SEQ ID No. 4.
SEQ ID No. 4 : QVQLVQSGAEVKKPGASVKVSCKASGYIFTAYTMHWVRQAPG
QGLEWMGWIKPNNGLANYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVY
YCARSEITTEFDYWGQGTLVTVSS
More particularly, said antibody, or a functional fragment or derivative thereof,
comprises a humanized light chain variable domain selected from the group of
sequences comprising the amino acid sequence SEQ ID No. 8, 9 or 10 or a sequence
having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 8,
9 or 10.
SEQ ID No. 8 : DIVLTQSPDSLAVSLGERATINCKSSESVDSYANSFMHWYQQKP
GQPPKLLIYRASTRESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQSKEDP
LTFGGGTKVEIKR
SEQ ID No. 9 : DIVMTQSPDSLAVSLGERATINCKSSESVDSYANSFMHWYQQKP
GQPPKLLIYRASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDP
LTFGGGTKVEIKR
SEQ ID No. 10: DIVMTQSPDSLAVSLGERATINCKSSESVDSYANSFLHWYQQKP
GQPPKLLIYRASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDP
LTFGGGTKVEIKR
More particularly, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [IgG2Hzl],
comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID
No. 4, a light chain variable domain comprising the amino acid sequence SEQ ID No. 8,
and a hinge region comprising the amino acid sequence SEQ ID No. 22.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [IgG2Hz2],
comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID
No. 4, a light chain variable domain comprising the amino acid sequence SEQ ID No. 9,
and a hinge region comprising the amino acid sequence SEQ ID No. 22.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [IgG2Hz3],
comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID
No. 4, a light chain variable domain comprising the amino acid sequence SEQ ID
No. 10, and a hinge region comprising the amino acid sequence SEQ ID No. 22.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [TH7Hzl],
comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID
No. 4, a light chain variable domain comprising the amino acid sequence SEQ ID No. 8,
and a hinge region comprising the amino acid sequence SEQ ID No. 28.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [TH7z2], comprises
a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 4, a
light chain variable domain comprising the amino acid sequence SEQ ID No. 9, and a
hinge region comprising the amino acid sequence SEQ ID No. 28.
In another aspect, a preferred antibody, or a divalent functional fragment or
derivative thereof, according to the invention and named [224G1 1] [TH7Hz3],
comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID
No. 4, a light chain variable domain comprising the amino acid sequence SEQ ID
No. 10, and a hinge region comprising the amino acid sequence SEQ ID No. 28.
In another aspect, antibodies of the invention can be described by their total
heavy and light chains, respectively.
As example, the antibody [224G1 1] [IgG2chim] of the invention comprises a
complete heavy chain comprising the amino acid sequence SEQ ID No. 50, or a
sequence having at least 80 % identity after optimum alignment with the sequence SEQ
ID No. 50, and a complete light chain comprising the amino acid sequence SEQ ID
No. 52, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [TH7chim] of the invention
comprises a complete heavy chain comprising the amino acid sequence SEQ ID No. 51,
or a sequence having at least 80 % identity after optimum alignment with the sequence
SEQ ID No. 51, and a complete light chain comprising the amino acid sequence SEQ
ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [CI] of the invention comprises a
complete heavy chain comprising the amino acid sequence SEQ ID No. 88, or a
sequence having at least 80 % identity after optimum alignment with the sequence SEQ
ID No. 88, and a complete light chain comprising the amino acid sequence SEQ ID No.
52, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [C2] of the invention comprises a
complete heavy chain comprising the amino acid sequence SEQ ID No. 89, or a
sequence having at least 80 % identity after optimum alignment with the sequence SEQ
ID No. 89, and a complete light chain comprising the amino acid sequence SEQ ID No.
52, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [C3] of the invention comprises a
complete heavy chain comprising the amino acid sequence SEQ ID No. 90, or a
sequence having at least 80 % identity after optimum alignment with the sequence SEQ
ID No. 90, and a complete light chain comprising the amino acid sequence SEQ ID No.
52, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [C5] of the invention comprises a
complete heavy chain comprising the amino acid sequence SEQ ID No. 91, or a
sequence having at least 80% identity after optimum alignment with the sequence SEQ
ID No. 91, and a complete light chain comprising the amino acid sequence SEQ ID No.
52, or a sequence having at least 80% identity after optimum alignment with the
sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [C6] of the invention comprises a
complete heavy chain comprising the amino acid sequence SEQ ID No. 92, or a
sequence having at least 80 % identity after optimum alignment with the sequence SEQ
ID No. 92, and a complete light chain comprising the amino acid sequence SEQ ID No.
52, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [C7] of the invention comprises a
complete heavy chain comprising the amino acid sequence SEQ ID No. 93, or a
sequence having at least 80 % identity after optimum alignment with the sequence SEQ
ID No. 93, and a complete light chain comprising the amino acid sequence SEQ ID No.
52, or a sequence having at least 80% identity after optimum alignment with the
sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [C9] of the invention comprises a
complete heavy chain comprising the amino acid sequence SEQ ID No. 94, or a
sequence having at least 80 % identity after optimum alignment with the sequence SEQ
ID No. 94, and a complete light chain comprising the amino acid sequence SEQ ID No.
52, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [D 1-3] of the invention comprises a
complete heavy chain comprising the amino acid sequence SEQ ID No. 95, or a
sequence having at least 80 % identity after optimum alignment with the sequence SEQ
ID No. 95, and a complete light chain comprising the amino acid sequence SEQ ID No.
52, or a sequence having at least 80% identity after optimum alignment with the
sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [C7A6] of the invention comprises a
complete heavy chain comprising the amino acid sequence SEQ ID No. 96, or a
sequence having at least 80 % identity after optimum alignment with the sequence SEQ
ID No. 96, and a complete light chain comprising the amino acid sequence SEQ ID No.
52, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [C6A9] of the invention comprises a
complete heavy chain comprising the amino acid sequence SEQ ID No. 97, or a
sequence having at least 80 % identity after optimum alignment with the sequence SEQ
ID No. 97, and a complete light chain comprising the amino acid sequence SEQ ID No.
52, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [C2A5-7] of the invention
comprises a complete heavy chain comprising the amino acid sequence SEQ ID No. 98,
or a sequence having at least 80 % identity after optimum alignment with the sequence
SEQ ID No. 98, and a complete light chain comprising the amino acid sequence SEQ
ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [C5A2-6] of the invention
comprises a complete heavy chain comprising the amino acid sequence SEQ ID No. 99,
or a sequence having at least 80 % identity after optimum alignment with the sequence
SEQ ID No. 99, and a complete light chain comprising the amino acid sequence SEQ
ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [C9A2-7] of the invention
comprises a complete heavy chain comprising the amino acid sequence SEQ ID No.
100, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 100, and a complete light chain comprising the amino acid
sequence SEQ ID No. 52, or a sequence having at least 80 % identity after optimum
alignment with the sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [D5-6-7-8] of the invention
comprises a complete heavy chain comprising the amino acid sequence SEQ ID No.
101, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 101, and a complete light chain comprising the amino acid
sequence SEQ ID No. 52, or a sequence having at least 80% identity after optimum
alignment with the sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [IgGl/IgG2] of the invention
comprises a complete heavy chain comprising the amino acid sequence SEQ ID
No. 102, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 102, and a complete light chain comprising the amino acid
sequence SEQ ID No. 52, or a sequence having at least 80 % identity after optimum
alignment with the sequence SEQ ID No. 52.
As another example, the antibody [224G1 1] [IgG2Hzl] of the invention
comprises a complete heavy chain comprising the amino acid sequence SEQ ID No. 36,
or a sequence having at least 80 % identity after optimum alignment with the sequence
SEQ ID No. 36 and a complete light chain comprising the amino acid sequence SEQ ID
No. 38, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 38.
As another example, the antibody [224G1 1] [IgG2Hz2] of the invention
comprises a complete heavy chain comprising the amino acid sequence SEQ ID No. 36,
or a sequence having at least 80 % identity after optimum alignment with the sequence
SEQ ID No. 36 and a complete light chain comprising the amino acid sequence SEQ ID
No. 39, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 39.
As another example, the antibody [224G1 1] [IgG2Hz3] of the invention
comprises a complete heavy chain comprising the amino acid sequence SEQ ID No. 36,
or a sequence having at least 80 % identity after optimum alignment with the sequence
SEQ ID No. 36 and a complete light chain comprising the amino acid sequence SEQ ID
No. 40, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 40.
As another example, the antibody [224G1 1] [TH7Hzl] of the invention
comprises a complete heavy chain comprising the amino acid sequence SEQ ID No. 37,
or a sequence having at least 80 % identity after optimum alignment with the sequence
SEQ ID No. 37 and a complete light chain comprising the amino acid sequence SEQ ID
No. 38, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 38.
As another example, the antibody [224G1 1] [TH7Hz2] of the invention
comprises a complete heavy chain comprising the amino acid sequence SEQ ID No. 37,
or a sequence having at least 80 % identity after optimum alignment with the sequence
SEQ ID No. 37 and a complete light chain comprising the amino acid sequence SEQ ID
No. 39, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 39.
As another example, the antibody [224G1 1] [TH7Hz3] of the invention
comprises a complete heavy chain comprising the amino acid sequence SEQ ID No. 37,
or a sequence having at least 80 % identity after optimum alignment with the sequence
SEQ ID No. 37 and a complete light chain comprising the amino acid sequence SEQ ID
No. 40, or a sequence having at least 80 % identity after optimum alignment with the
sequence SEQ ID No. 40.
Other examples of antibodies, or derivatives thereof, according to the invention
comprises complete heavy chains comprising an amino acid sequence selected in the
group consisting of SEQ ID Nos. 88 to 102 (corresponding nucleotide sequences are
SEQ ID Nos. 103 to 117).
By "functional fragment" of an antibody according to the invention, it is
intended to indicate in particular an antibody fragment, such as Fv, scFv (sc for single
chain), Fab, F(ab') 2, Fab', scFv-Fc fragments or diabodies, or any fragment of which
the half-life time would have been increased by chemical modification, such as the
addition of poly(alkylene) glycol such as poly(ethylene) glycol ("PEGylation")
(pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab') 2-PEG or Fab'-PEG)
("PEG" for Poly(Ethylene) Glycol), or by incorporation in a liposome, said fragments
having at least one of the characteristic CDRs of sequence SEQ ID Nos. 1 to 3 and 5 to
7 according to the invention, and, especially, in that it is capable of exerting in a general
manner an even partial activity of the antibody from which it is descended, such as in
particular the capacity to recognize and to bind to the c-Met, and, if necessary, to inhibit
the activity of the c-Met.
Preferably, said functional fragments will be constituted or will comprise a
partial sequence of the heavy or light variable chain of the antibody from which they are
derived, said partial sequence being sufficient to retain the same specificity of binding
as the antibody from which it is descended and a sufficient affinity, preferably at least
equal to 1/100, in a more preferred manner to at least 1/10, of that of the antibody from
which it is descended, with respect to the c-Met. Such a functional fragment will
contain at the minimum 5 amino acids, preferably 6, 7, 8, 9, 10, 12, 15, 25, 50 and 100
consecutive amino acids of the sequence of the antibody from which it is descended.
Preferably, these functional fragments will be fragments of Fv, scFv, Fab,
F(ab') 2, F(ab'), scFv-Fc type or diabodies, which generally have the same specificity of
binding as the antibody from which they are descended. In a more preferred
embodiment of the invention, these fragments are selected among divalent fragments
such as F(ab') 2 fragments. According to the present invention, antibody fragments of the
invention can be obtained starting from antibodies such as described above by methods
such as digestion by enzymes, such as pepsin or papain and/or by cleavage of the
disulfide bridges by chemical reduction. In another manner, the antibody fragments
comprised in the present invention can be obtained by techniques of genetic
recombination likewise well known to the person skilled in the art or else by peptide
synthesis by means of, for example, automatic peptide synthesizers such as those
supplied by the company Applied Biosystems, etc.
By "divalent fragment", it must be understood any antibody fragments
comprising two arms and, more particularly, F(ab') 2 fragments.
By "derivatives" of an antibody according to the invention, it is meant a binding
protein comprising a protein scaffold and at least on of the CDRs selected from the
original antibody in order to maintain the binding capacity. Such compounds are well
known by the man skilled in the art and will be described in more details in the
following specification.
More particularly, the antibody, or one of its functional fragments or derivatives,
according to the invention is characterized in that said derivative consists in a binding
protein comprising a scaffold on which at least one CDR has been grafted for the
conservation of the original antibody paratopic recognizing properties.
One or several sequences through the 6 CDR sequences described in the
invention can be presented on a protein scaffold. In this case, the protein scaffold
reproduces the protein backbone with appropriate folding of the grafted CDR(s), thus
allowing it (or them) to maintain their antigen paratopic recognizing properties.
The man skilled in the art knows how to select the protein scaffold on which at
least one CDR selected from the original antibody could be grafted. More particularly, it
is known that, to be selected, such scaffold should display several features as follow
(Skerra A., J . Mol. Recogn., 13, 2000, 167-187):
phylogenetically good conservation,
robust architecture with a well known three-dimensional molecular
organization (such as, for example, crystallography or NMR),
small size,
no or only low degree of post-translational modifications,
easy to produce, express and purify.
Such protein scaffold can be, but without limitation, structure selected from the
group consisting in fibronectin and preferentially the tenth fibronectin type III domain
(FNfnlO), lipocalin, anticalin (Skerra A., J . Biotechnol, 2001, 74(4):257-75), the
protein Z derivative from the domain B of staphylococcal protein A, thioredoxin A or
any protein with repeated domain such as "ankyrin repeat" (Kohl et al., PNAS, 2003,
vol. 100, No.4, 1700-1705), "armadillo repeat", "leucin-rich repeat" or
"tetratricopeptide repeat".
It could also be mentioned scaffold derivative from toxins (such as, for example,
scorpion, insect, plant or mollusc toxins) or protein inhibitors of neuronal nitric oxyde
synthase (PIN).
As non limitative example of such hybrid constructions, it can be mentioned the
insertion of the CDR-H1 (heavy chain) of an anti-CD4 antibody, i.e. the 13B8.2
antibody, into one of the exposed loop of the PIN. The binding properties of the
obtained binding protein remain similar to the original antibody (Bes et al., BBRC 343,
2006, 334-344). It can also be mentioned the grafting of the CDR-H3 (heavy chain) of
an anti-lyzozyme VHH antibody on a loop of neocarzinostatine (Nicaise et al., 2004).
As above mentioned, such protein scaffold can comprise from 1 to 6 CDR(s)
from the original antibody. In a preferred embodiment, but without any limitation, the
man skilled in the art would select at least a CDR from the heavy chain, said heavy
chain being known to be particularly implicated in the antibody specificity. The
selection of the CDR(s) of interest will be evident for the man of the art with known
method (BES et al, FEBS letters 508, 2001, 67-74).
As an evidence, these examples are not limitative and any other scaffold known
or described must be included in the present specification.
According to a novel aspect, the present invention relates to an isolated nucleic
acid, characterized in that it is chosen from the following nucleic acids:
a) a nucleic acid, DNA or RNA, coding for an antibody, or one of its functional
fragments or derivatives, according to the invention;
b) a nucleic acid sequence comprising the sequences SEQ ID No. 11, SEQ ID
No. 12, SEQ ID No. 13 and the sequences SEQ ID No. 15, SEQ ID No. 16 and SEQ ID
No. 17;
c) a nucleic acid sequence comprising the sequences SEQ ID No. 14 and SEQ
ID No. 18, 19 or 20;
d) the corresponding RNA nucleic acids of the nucleic acids as defined in b) or
c);
e) the complementary nucleic acids of the nucleic acids as defined in a), b) and
c); and
f a nucleic acid of at least 18 nucleotides capable of hybridizing under
conditions of high stringency with at least one of the CDRs of sequence SEQ ID Nos.
11 to 13 and 15 to 17.
According to still another aspect, the present invention relates to an isolated
nucleic acid, characterized in that it is chosen from the following nucleic acids:
- a nucleic acid, DNA or RNA, coding for an antibody, or one of its functional
fragments or derivatives, according to the present invention and wherein the nucleic
sequence coding for the hinge region of said antibody comprises or has a sequence
selected from the group consisting of the sequences SEQ ID Nos. 29 to 35 and SEQ ID
Nos. 73 to 87.
By nucleic acid, nucleic or nucleic acid sequence, polynucleotide,
oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be
employed indifferently in the present invention, it is intended to indicate a precise
linkage of nucleotides, which are modified or unmodified, allowing a fragment or a
region of a nucleic acid to be defined, containing or not containing unnatural
nucleotides, and being able to correspond just as well to a double-stranded DNA, a
single-stranded DNA as to the transcription products of said DNAs.
It must also be understood here that the present invention does not concern the
nucleotide sequences in their natural chromosomal environment, that is to say in the
natural state. It concerns sequences which have been isolated and/or purified, that is to
say that they have been selected directly or indirectly, for example by copy, their
environment having been at least partially modified. It is thus likewise intended to
indicate here the isolated nucleic acids obtained by genetic recombination by means, for
example, of host cells or obtained by chemical synthesis.
A hybridization under conditions of high stringency signifies that the
temperature conditions and ionic strength conditions are chosen in such a way that they
allow the maintenance of the hybridization between two fragments of complementary
DNA. By way of illustration, conditions of high stringency of the hybridization step for
the purposes of defining the polynucleotide fragments described above are
advantageously the following.
The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1)
prehybridization at 42°C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5
x SSC ( 1 x SSC corresponds to a 0.15 M NaCl + 0.015 M sodium citrate solution),
50 % of formamide, 7 % of sodium dodecyl sulfate (SDS), 10 x Denhardt's, 5 % of
dextran sulfate and 1 % of salmon sperm DNA; (2) actual hybridization for 20 hours at
a temperature dependent on the size of the probe (i.e.: 42°C, for a probe size > 100
nucleotides) followed by 2 washes of 20 minutes at 20°C in 2 x SSC + 2% of SDS, 1
wash of 20 minutes at 20°C in 0.1 x SSC + 0.1 % of SDS. The last wash is carried out
in 0.1 x SSC + 0.1 % of SDS for 30 minutes at 60°C for a probe size > 100 nucleotides.
The hybridization conditions of high stringency described above for a polynucleotide of
defined size can be adapted by the person skilled in the art for oligonucleotides of
greater or smaller size, according to the teaching of Sambrook et al. (1989, Molecular
cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor).
The invention likewise relates to a vector comprising a nucleic acid according to
the present invention.
The invention aims especially at cloning and/or expression vectors which
contain a nucleotide sequence according to the invention.
The vectors according to the invention preferably contain elements which allow
the expression and/or the secretion of the translated nucleotide sequences in a
determined host cell. The vector must therefore contain a promoter, signals of initiation
and termination of translation, as well as appropriate regions of regulation of
transcription. It must be able to be maintained in a stable manner in the host cell and can
optionally have particular signals which specify the secretion of the translated protein.
These different elements are chosen and optimized by the person skilled in the art as a
function of the host cell used. To this effect, the nucleotide sequences according to the
invention can be inserted into autonomous replication vectors in the chosen host, or be
integrative vectors of the chosen host.
Such vectors are prepared by methods currently used by the person skilled in the
art, and the resulting clones can be introduced into an appropriate host by standard
methods, such as lipofection, electroporation, thermal shock, or chemical methods.
The vectors according to the invention are, for example, vectors of plasmidic or
viral origin. They are useful for transforming host cells in order to clone or to express
the nucleotide sequences according to the invention.
The invention likewise comprises the host cells transformed by or comprising a
vector according to the invention.
The host cell can be chosen from prokaryotic or eukaryotic systems, for example
bacterial cells but likewise yeast cells or animal cells, in particular mammalian cells. It
is likewise possible to use insect cells or plant cells.
The invention likewise concerns animals, except man, which comprise at least
one cell transformed according to the invention.
According to another aspect, a subject of the invention is a process for
production of an antibody, or one of its functional fragments according to the invention,
characterized in that it comprises the following stages:
a) culture in a medium and appropriate culture conditions of a host cell
according to the invention; and
b) the recovery of said antibodies, or one of their functional fragments, thus
produced starting from the culture medium or said cultured cells.
The cells transformed according to the invention can be used in processes for
preparation of recombinant polypeptides according to the invention. The processes for
preparation of a polypeptide according to the invention in recombinant form,
characterized in that they employ a vector and/or a cell transformed by a vector
according to the invention, are themselves comprised in the present invention.
Preferably, a cell transformed by a vector according to the invention is cultured under
conditions which allow the expression of said polypeptide and said recombinant peptide
is recovered.
As has been said, the host cell can be chosen from prokaryotic or eukaryotic
systems. In particular, it is possible to identify nucleotide sequences according to the
invention, facilitating secretion in such a prokaryotic or eukaryotic system. A vector
according to the invention carrying such a sequence can therefore advantageously be
used for the production of recombinant proteins, intended to be secreted. In effect, the
purification of these recombinant proteins of interest will be facilitated by the fact that
they are present in the supernatant of the cell culture rather than in the interior of the
host cells.
It is likewise possible to prepare the polypeptides according to the invention by
chemical synthesis. Such a preparation process is likewise a subject of the invention.
The person skilled in the art knows the processes of chemical synthesis, for example the
techniques employing solid phases [Steward et al, 1984, Solid phase peptide synthesis,
Pierce Chem. Company, Rockford, 111, 2nd ed., (1984)] or techniques using partial
solid phases, by condensation of fragments or by a classical synthesis in solution. The
polypeptides obtained by chemical synthesis and being able to contain corresponding
unnatural amino acids are likewise comprised in the invention.
The antibodies, or one of their functional fragments or derivatives, capable of
being obtained by a process according to the invention are likewise comprised in the
present invention.
The invention also concerns the antibody of the invention as a medicament.
The invention likewise concerns a pharmaceutical composition comprising by
way of active principle a compound consisting of an antibody, or one of its functional
fragments according to the invention, preferably mixed with an excipient and/or a
pharmaceutically acceptable vehicle.
Another complementary embodiment of the invention consists in a composition
such as described above which comprises, moreover, as a combination product for
simultaneous, separate or sequential use, an anti-tumoral antibody.
Most preferably, said second anti-tumoral antibody could be chosen through
anti-IGF-IR, anti-EGFR, anti-HER2/neu, anti-VEGFR, anti-VEGF, etc., antibodies or
any other anti-tumoral antibodies known by the man skilled in the art. It is evident that
the use, as second antibody, of functional fragments or derivatives of above mentioned
antibodies is part of the invention.
As a most preferred antibody, anti-EGFR antibodies are selected such as for
example the antibody C225 (Erbitux).
"Simultaneous use" is understood as meaning the administration of the two
compounds of the composition according to the invention in a single and identical
pharmaceutical form.
"Separate use" is understood as meaning the administration, at the same time, of
the two compounds of the composition according to the invention in distinct
pharmaceutical forms.
"Sequential use" is understood as meaning the successive administration of the
two compounds of the composition according to the invention, each in a distinct
pharmaceutical form.
In a general fashion, the composition according to the invention considerably
increases the efficacy of the treatment of cancer. In other words, the therapeutic effect
of the anti-c-Met antibodies according to the invention is potentiated in an unexpected
manner by the administration of a cytotoxic agent. Another major subsequent advantage
produced by a composition according to the invention concerns the possibility of using
lower efficacious doses of active principle, which allows the risks of appearance of
secondary effects to be avoided or to be reduced, in particular the effects of the
cytotoxic agent.
In addition, this composition according to the invention would allow the
expected therapeutic effect to be attained more rapidly.
The composition of the invention can also be characterized in that it comprises,
moreover, as a combination product for simultaneous, separate or sequential use, a
cytotoxic/cytostatic agent.
By "anti-cancer therapeutic agents" or "cytotoxic/cytostatic agents", it is
intended a substance which, when administered to a subject, treats or prevents the
development of cancer in the subject's body. As non limitative example of such agents,
it can be mentioned alkylating agents, anti-metabolites, anti-tumor antibiotics, mitotic
inhibitors, chromatin function inhibitors, anti-angiogenesis agents, anti-estrogens, antiandrogens
or immunomodulators.
Such agents are, for example, cited in the 2001 edition of VIDAL, on the page
devoted to the compounds attached to the cancerology and hematology column
"Cytotoxics", these cytotoxic compounds cited with reference to this document are cited
here as preferred cytotoxic agents.
More particularly, the following agents are preferred according to the invention.
"Alkylating agent" refers to any substance which can cross-link or alkylate any
molecule, preferably nucleic acid (e.g., DNA), within a cell. Examples of alkylating
agents include nitrogen mustard such as mechlorethamine, chlorambucol, melphalen,
chlorydrate, pipobromen, prednimustin, disodic-phosphate or estramustine;
oxazophorins such as cyclophosphamide, altretamine, trofosfamide, sulfofosfamide or
ifosfamide; aziridines or imine-ethylenes such as thiotepa, triethylenamine or
altetramine; nitrosourea such as carmustine, streptozocin, fotemustin or lomustine;
alkyle-sulfonates such as busulfan, treosulfan or improsulfan; triazenes such as
dacarbazine; or platinum complexes such as cis-platinum, oxaliplatin and carboplatin.
"Anti-metabolites" refer to substances that block cell growth and/or metabolism
by interfering with certain activities, usually DNA synthesis. Examples of ant i
metabolites include methotrexate, 5-fluoruracil, floxuridine, 5-fluorodeoxyuridine,
capecitabine, cytarabine, fludarabine, cytosine arabinoside, 6-mercaptopurine (6-MP),
6-thioguanine (6-TG), chlorodesoxyadenosine, 5-azacytidine, gemcitabine, cladribine,
deoxycoformycin and pentostatin.
"Anti-tumor antibiotics" refer to compounds which may prevent or inhibit DNA,
RNA and/or protein synthesis. Examples of anti-tumor antibiotics include doxorubicin,
daunorubicin, idarubicin, valrubicin, mitoxantrone, dactinomycin, mithramycin,
plicamycin, mitomycin C, bleomycin, and procarbazine.
"Mitotic inhibitors" prevent normal progression of the cell cycle and mitosis. In
general, microtubule inhibitors or taxoides such as paclitaxel and docetaxel are capable
of inhibiting mitosis. Vinca alkaloid such as vinblastine, vincristine, vindesine and
vinorelbine are also capable of inhibiting mitosis.
"Chromatin function inhibitors" or "topoisomerase inhibitors" refer to
substances which inhibit the normal function of chromatin modeling proteins such as
topoisomerase I or topoisomerase II. Examples of chromatin function inhibitors include,
for topoisomerase I, camptothecine and its derivatives such as topotecan or irinotecan,
and, for topoisomerase II, etoposide, etoposide phosphate and teniposide.
"Anti-angiogenesis agent" refers to any drug, compound, substance or agent
which inhibits growth of blood vessels. Exemplary anti-angiogenesis agents include, but
are by no means limited to, razoxin, marimastat, batimastat, prinomastat, tanomastat,
ilomastat, CGS-27023A, halofuginon, COL-3, neovastat, BMS-275291, thalidomide,
CDC 501, DMXAA, L-651582, squalamine, endostatin, SU5416, SU6668, interferonalpha,
EMD121974, interleukin-12, IM862, angiostatin and vitaxin.
"Anti-estrogen" or "anti-estrogenic agent" refer to any substance which reduces,
antagonizes or inhibits the action of estrogen. Examples of anti-estrogen agents are
tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, anastrozole, letrozole, and
exemestane.
"Anti-androgens" or "anti-androgen agents" refer to any substance which
reduces, antagonizes or inhibits the action of an androgen. Examples of anti-androgens
are flutamide, nilutamide, bicalutamide, sprironolactone, cyproterone acetate,
finasteride and cimitidine.
"Immunomodulators" are substances which stimulate the immune system.
Examples ofimmunomodulators include interferon, interleukin such as
aldesleukine, OCT-43, denileukin diflitox and interleukin-2, tumoral necrose fators such
as tasonermine or others immunomodulators such as lentinan, sizofiran, roquinimex,
pidotimod, pegademase, thymopentine, poly I:C or levamisole in conjunction with 5-
fluorouracil.
For more detail, the man skill in the art could refer to the manual edited by the
"Association Francaise des Enseignants de Chimie Therapeutique" and entitled "Traite
de chimie therapeutique", vol. 6, Medicaments antitumoraux et perspectives dans le
traitement des cancers, edition TEC & DOC, 2003.
Can also be mentioned as chemical agents or cytotoxic agents, all kinase
inhibitors such as, for example, gefitinib or erlotinib.
In a particularly preferred embodiment, said composition as a combination
product according to the invention is characterized in that said cytotoxic agent is
coupled chemically to said antibody for simultaneous use.
In order to facilitate the coupling between said cytotoxic agent and said antibody
according to the invention, it is especially possible to introduce spacer molecules
between the two compounds to be coupled, such as poly(alkylene) glycols like
polyethylene glycol, or else amino acids, or, in another embodiment, to use active
derivatives of said cytotoxic agents into which would have been introduced functions
capable of reacting with said antibody according to the invention. These coupling
techniques are well known to the person skilled in the art and will not be expanded upon
in the present description.
The invention relates, in another aspect, to a composition characterized in that
one, at least, of said antibodies, or one of their functional fragments or derivatives, is
conjugated with a cell toxin and/or a radioelement.
Preferably, said toxin or said radioelement is capable of inhibiting at least one
cell activity of cells expressing the c-Met, in a more preferred manner capable of
preventing the growth or the proliferation of said cell, especially of totally inactivating
said cell.
Preferably also, said toxin is an enterobacterial toxin, especially Pseudomonas
exotoxin A.
The radioelements (or radioisotopes) preferably conjugated to the antibodies
employed for the therapy are radioisotopes which emit gamma rays and preferably
iodine131 , yttrium90, gold1 9, palladium 100 , copper67, bismuth217 and antimony211 . The
radioisotopes which emit beta and alpha rays can likewise be used for the therapy.
By toxin or radioelement conjugated to at least one antibody, or one of its
functional fragments, according to the invention, it is intended to indicate any means
allowing said toxin or said radioelement to bind to said at least one antibody, especially
by covalent coupling between the two compounds, with or without introduction of a
linking molecule.
Among the agents allowing binding in a chemical (covalent), electrostatic or
noncovalent manner of all or part of the components of the conjugate, mention may
particularly be made of benzoquinone, carbodiimide and more particularly EDC (1-
ethyl-3-[3-dimethyl-aminopropyl]-carbodiimide hydrochloride), dimaleimide, dithiobisnitrobenzoic
acid (DTNB), N-succinimidyl S-acetyl thio-acetate (SATA), the bridging
agents having one or more phenylazide groups reacting with the ultraviolets (U.V.) and
preferably N-[-4-(azidosalicylamino)butyl]-3 '-(2'-pyridyldithio)-propionamide (APDP),
N-succinimid-yl 3-(2-pyridyldithiopropionate (SPDP), 6-hydrazino-nicotinamide
(HYNIC).
Another form of coupling, especially for the radioelements, can consist in the
use of a bifunctional ion chelator.
Among these chelates, it is possible to mention the chelates derived from EDTA
(ethylenediaminetetraacetic acid) or from DTPA (diethylenetriaminepentaacetic acid)
which have been developed for binding metals, especially radioactive metals, and
immunoglobulins. Thus, DTPA and its derivatives can be substituted by different
groups on the carbon chain in order to increase the stability and the rigidity of the
ligand-metal complex (Krejcarek et al. (1977); Brechbiel et al. (1991); Gansow (1991);
US patent 4,831,175).
For example diethylenetriaminepentaacetic acid (DTPA) and its derivatives,
which have been widely used in medicine and in biology for a long time either in their
free form, or in the form of a complex with a metallic ion, have the remarkable
characteristic of forming stable chelates with metallic ions and of being coupled with
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).
Likewise preferably, said at least one antibody forming said conjugate according
to the invention is chosen from its functional fragments, especially the fragments
amputated of their Fc component such as the scFv fragments.
As already mentioned, in a preferred embodiment of the invention, said
cytotoxic/cytostatic agent or said toxin and/or a radioelement is coupled chemically to at
least one of the elements of said composition for simultaneous use.
The present invention comprises the described composition as a medicament.
The present invention moreover comprises the use of the composition according
to the invention for the preparation of a medicament.
In another aspect, the invention deals with the use of an antibody, or one of its
functional fragments or derivatives, and/or of a composition as above described for the
preparation of a medicament intended to inhibit the growth and/or the proliferation of
tumor cells.
Another aspect of the invention consists in the use of an antibody, or one of its
functional fragments or derivatives and/or of a composition, as described above or the
use above mentioned, for the preparation of a medicament intended for the prevention
or for the treatment of cancer.
Is also comprised in the present invention a method intended to inhibit the
growth and/or the proliferation of tumor cells in a patient comprising the administration
to a patient in need thereof of an antibody, or one of its functional fragments or
derivatives according to the invention, an antibody produced by an hybridoma
according to the invention or a composition according to the invention.
The present invention further comprises a method for the prevention or the
treatment of cancer in a patient in need thereof, comprising the administration to the
patient of an antibody, or one of its functional fragments or derivatives according to the
invention, an antibody produced by an hybridoma according to the invention or a
composition according to the invention.
In a particular preferred aspect, said cancer is a cancer chosen from prostate
cancer, osteosarcomas, lung cancer, breast cancer, endometrial cancer, glioblastoma or
colon cancer.
As explained before, an advantage of the invention is to allow the treatment of
HGF dependent and independent Met-activation related cancers.
In a particular aspect, the invention comprises the use of an antibody, or one of
its functional fragments or derivatives and/or of a composition, as described above or
the use above mentioned, for the preparation of a medicament intended for the
prevention or for the treatment of a patient in need thereof having a cancer characterized
by overexpression of c-Met (for example, due to a genie amplification of c-Met)
resulting in a ligand-independent constitutive activation of said c-Met receptor .
Preferably, said cancer characterized by a genie amplification of c-Met resulting
in a ligand-independent constitutive activation of said c-Met receptor is selected from
the group consisting of renal cell carcinoma and gastric cancer.
The invention, in yet another aspect, encompasses a method of in vitro diagnosis
of illnesses induced by an overexpression or an underexpression of the c-Met receptor
starting from a biological sample in which the abnormal presence of c-Met receptor is
suspected, said method being characterized in that it comprises a step wherein said
biological sample is contacted with an antibody of the invention, it being possible for
said antibody to be, if necessary, labeled.
Preferably, said illnesses connected with an abnormal presence of c-Met
receptor in said diagnosis method will be cancers.
Said antibody, or one of its functional fragments, can be present in the form of
an immunoconjugate or of a labelled antibody so as to obtain a detectable and/or
quantifiable signal.
The antibodies labelled according to the invention or their functional fragments
include, for example, antibodies called immunoconjugates which can be conjugated, for
example, with enzymes such as peroxidase, alkaline phosphatase, beta-D-galactosidase,
glucose oxydase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme,
malate dehydrogenase or glucose 6-phosphate dehydrogenase or by a molecule such as
biotin, digoxygenin or 5-bromodeoxyuridine. Fluorescent labels can be likewise
conjugated to the antibodies or to their functional fragments according to the invention
and especially include fluorescein and its derivatives, fluorochrome, rhodamine and its
derivatives, GFP (GFP for "Green Fluorescent Protein"), dansyl, umbelliferone etc. In
such conjugates, the antibodies of the invention or their functional fragments can be
prepared by methods known to the person skilled in the art. They can be coupled to the
enzymes or to the fluorescent labels directly or by the intermediary of a spacer group or
of a linking group such as a polyaldehyde, like glutaraldehyde,
ethylenediaminetetraacetic acid (EDTA), diethylene-triaminepentaacetic acid (DPTA),
or in the presence of coupling agents such as those mentioned above for the therapeutic
conjugates. The conjugates containing labels of fluorescein type can be prepared by
reaction with an isothiocyanate.
Other conjugates can likewise include chemoluminescent labels such as luminol
and the dioxetanes, bio-luminescent labels such as luciferase and luciferin, or else
radioactive labels such as iodine123 , iodine , iodine1 , iodine133 , bromine ,
technetium 9"1, indium111 , indium113"1, gallium67, gallium68, ruthenium95, ruthenium97,
ruthenium 103 , ruthenium 105 , mercury107 , mercury203 , rhenium99"1, rhenium 101 , rhenium 105 ,
scandium47, tellurium 12lm, tellurium 1 m, tellurium 1 5m, thulium 165 , thulium 167 ,
thulium 168 , fluorine18 , yttrium1 9, iodine131 . The methods known to the person skilled in
the art existing for coupling the therapeutic radioisotopes to the antibodies either
directly or via a chelating agent such as EDTA, DTPA mentioned above can be used for
the radioelements which can be used in diagnosis. It is likewise possible to mention
labelling with Na[I 125] by the chloramine T method [Hunter W.M. and Greenwood F.C.
(1962) Nature 194:495] or else with technetium99111 by the technique of Crockford et al.
(US patent 4,424,200) or attached via DTPA as described by Hnatowich (US patent
4,479,930).
Thus, the antibody, or a functional fragment or derivative thereof, according to
the invention can be employed in a process for the detection and/or the quantification of
an overexpression or of an underexpression, preferably an overexpression, of the c-Met
receptor in a biological sample, characterized in that it comprises the following steps:
a) the contacting of the biological sample with an antibody, or a functional
fragment or derivative thereof, according to the invention; and
b) the demonstration of the c-Met/antibody complex possibly formed.
In a particular embodiment, the antibody, or a functional fragment or derivative
thereof, according to the invention, can be employed in a process for the detection
and/or the quantification of the c-Met receptor in a biological sample, for the monitoring
of the efficacy of a prophylactic and/or therapeutic treatment of c-Met-dependent
cancer.
More generally, the antibody or a functional fragment or derivative thereof,
according to the invention can be advantageously employed in any situation where the
expression of the c-Met- receptor must be observed in a qualitative and/or quantitative
manner.
Preferably, the biological sample is formed by a biological fluid, such as serum,
whole blood, cells, a tissue sample or biopsies of human origin.
Any procedure or conventional test can be employed in order to carry out such a
detection and/or dosage. Said test can be a competition or sandwich test, or any test
known to the person skilled in the art dependent on the formation of an immune
complex of antibody-antigen type. Following the applications according to the
invention, the antibody or a functional fragment or derivative thereof can be
immobilized or labelled. This immobilization can be carried out on numerous supports
known to the person skilled in the art. These supports can especially include glass,
polystyrene, poly-propylene, polyethylene, dextran, nylon, or natural or modified cells.
These supports can be either soluble or insoluble.
By way of example, a preferred method brings into play immunoenzymatic
processes according to the ELISA technique, by immunofluorescence, or radio
immunoassay (RIA) technique or equivalent.
Thus, the present invention likewise comprises the kits or sets necessary for
carrying out a method of diagnosis of illnesses induced by an overexpression or an
underexpression of the c-Met receptor or for carrying out a process for the detection
and/or the quantification of an overexpression or of an underexpression of the c-Met
receptor in a biological sample, preferably an overexpression of said receptor,
characterized in that said kit or set comprises the following elements:
a) an antibody, or a functional fragment or derivative thereof, according to the
invention;
b) optionally, the reagents for the formation of the medium favorable to the
immunological reaction;
c) optionally, the reagents allowing the demonstration of c-Met/antibody
complexes produced by the immunological reaction.
A subject of the invention is likewise the use of an antibody or a composition
according to the invention for the preparation of a medicament intended for the specific
targeting of a biologically active compound to cells expressing or overexpressing the c-
Met receptor.
It is intended here by biologically active compound to indicate any compound
capable of modulating, especially of inhibiting, cell activity, in particular their growth,
their proliferation, transcription or gene translation.
A subject of the invention is also an in vivo diagnostic reagent comprising an
antibody according to the invention, or a functional fragment or derivative thereof,
preferably labelled, especially radiolabelled, and its use in medical imaging, in
particular for the detection of cancer connected with the expression or the
overexpression by a cell of the c-Met receptor.
The invention likewise relates to a composition as a combination product or to
an anti-c-Met/toxin conjugate or radioelement, according to the invention, as a
medicament.
Preferably, said composition as a combination product or said conjugate
according to the invention will be mixed with an excipient and/or a pharmaceutically
acceptable vehicle.
In the present description, pharmaceutically acceptable vehicle is intended to
indicate a compound or a combination of compounds entering into a pharmaceutical
composition not provoking secondary reactions and which allows, for example,
facilitation of the administration of the active compound(s), an increase in its lifespan
and/or in its efficacy in the body, an increase in its solubility in solution or else an
improvement in its conservation. These pharmaceutically acceptable vehicles are well
known and will be adapted by the person skilled in the art as a function of the nature
and of the mode of administration of the active compound(s) chosen.
Preferably, these compounds will be administered by the systemic route, in
particular by the intravenous route, by the intramuscular, intradermal, intraperitoneal or
subcutaneous route, or by the oral route. In a more preferred manner, the composition
comprising the antibodies according to the invention will be administered several times,
in a sequential manner.
Their modes of administration, dosages and optimum pharmaceutical forms can
be determined according to the criteria generally taken into account in the establishment
of a treatment adapted to a patient such as, for example, the age or the body weight of
the patient, the seriousness of his/her general condition, the tolerance to the treatment
and the secondary effects noted.
Other characteristics and advantages of the invention appear in the continuation
of the description with the examples and the figures wherein:
Figure 1: Effect of irrelevant IgGl Mabs from mouse and human origin and PBS
on c-Met receptor phosphorylation on A549 cells.
Figures A and 2B: Effect of murine and humanized 224G1 1 Mabs produced as
a human IgGl/kappa isotype on c-Met receptor phosphorylation on A549 cells.
Figure 2A: agonist effect calculated as percentage versus maximal stimulation of c-Met
phosphorylation by HGF [100 ng/ml].
Figure 2B: antagonist effect calculated as percentage of inhibition of the maximal
stimulation of c-Met phosphorylation by HGF [100 ng/ml].
Figures 3A and 3B: Comparison between murine 224G1 1 Mab and chimeric
224G1 1 Mabs containing various engineered hinge regions, on c-Met receptor
phosphorylation on A549 cells.
Figure 3A: agonist effect calculated as percentage versus maximal stimulation of c-Met
phosphorylation by HGF [100 ng/ml].
Figure 3B: antagonist effect calculated as percentage of inhibition of the maximal
stimulation of c-Met phosphorylation by HGF [100 ng/ml].
Figures 4A and 4B: Comparison between murine 224G1 1 Mab and chimeric and
humanized 224G1 1 Mabs produced as a human IgG2/kappa isotype, on c-Met receptor
phosphorylation on A549 cells.
Figure 4A: agonist effect calculated as percentage versus maximal stimulation of c-Met
phosphorylation by HGF [100 ng/ml].
Figure 4B: antagonist effect calculated as percentage of inhibition of the maximal
stimulation of c-Met phosphorylation by HGF [100 ng/ml].
Figures 5A and 5B: Comparison between murine 224G1 1 Mab and chimeric and
humanized 224G1 1 Mabs produced as an engineered hinge mutant TH7IgGl /kappa, on
c-Met receptor phosphorylation on A549 cells.
Figure 5A: agonist effect calculated as percentage versus maximal stimulation of c-Met
phosphorylation by HGF [100 ng/ml].
Figure 5B: antagonist effect calculated as percentage of inhibition of the maximal
stimulation of c-Met phosphorylation by HGF [100 ng/ml] .
Figures 6A and 6B, Figures 7A and 7B, Figures 8A and 8B, Figures 9A and 9B,
Figures 10A and 10B: BRET models with Figures A: c-Met dimerization model; and
Figures B: c-Met activation model.
Figure 11: c-Met recognition by chimeric and humanized 224G1 1 forms.
Figure 12: Effect of murine and chimeric antibodies on HGF-induced
proliferation of NCI-H441 cells in vitro. NCI-H441 cells were plated in serum-free
medium. 24 hours after plating m224Gll and [224Gl l]chim were added either in
absence or in presence of HGF. Black arrows indicate the wells plated with cells alone
either in absence or in presence of HGF. A murine IgGl (mlgGl) was introduced
as an isotype control.
Figure 13: In vivo comparison of murine and IgGl chimeric 224G1 1 Mabs on
the NCI-H441 xenograft model.
Figures 14A and 14B: Effect of the murine 224G1 1 Mab and of various
chimeric and humanized versions of this antibody on HGF-induced proliferation of
NCI-H441 cells in vitro. NCI-H441 cells were plated in serum-free medium. Twenty
four hours after plating antibody to be tested were added either in absence or in
presence of HGF. In panel (Figure 14A), the murine m224Gl l , chimeric IgGl
[224Gl l]chim, humanized IgGl [224G1 1] [Hzl], [224G1 1] [Hz2], [224G1 1] [Hz3]
versions were shown. In panel (Figure 14B), the murine m224Gl 1 and various chimeric
IgGl forms ([224G1 1] chim, [224G1 1] [MH chim], [224G1 1] [MUP9H chim],
[224G1 1] [MMCH chim], [224G1 1] [TH7 chim]) were presented. Black arrows
indicate the wells plated with cells alone either in absence or in presence of HGF.
A murine IgGl was introduced as a negative control for agonist activity. The m5D5 was
used as a dose-dependent full agonist control.
Figure 15: Effect of the murine 224G1 1 Mab and of various chimeric and
humanized versions of this antibody on HGF-induced proliferation of NCI-H441 cells
in vitro. NCI-H441 cells were plated in serum-free medium. Twenty four hours after
plating antibody to be tested were added either in absence or in presence of HGF. The
murine m224Gl l , [224G1 1] chim, [224G1 1] [TH7 chim]) IgGl chimeric forms and
[224G1 1] [TH7 Hzl], [224G1 1] [TH7 Hz3],) were presented. Black arrows indicate the
wells plated with cells alone either in absence or in presence of HGF. A murine
IgGl was introduced as a negative control for agonist activity. The m5D5 was used as a
dose-dependent full agonist control.
Figures 16A-16C: In vivo comparison of murine, chimeric and humanized
224G1 1 Mabs on the NCI-H441 xenograft model.
Figure 17A: agonist effect calculated as percentage versus maximal stimulation
of c-Met phosphorylation by HGF [100 ng/ml] .
Figure 17B: antagonist effect calculated as percentage of inhibition of the
maximal stimulation of c-Met phosphorylation by HGF [100 ng/ml].
Figure 18: BRET models with c-Met activation model.
Figures 19A-19B: Effect of m224Gl l and h224Gl l on c-Met degradation on
A549 cells. A) Mean of 4 independent experiments +/- s.e.m. B) Western blot image
representative of the 4 independent experiments performed.
Figures 20A-20B: Effect of m224Gl l and h224Gl l on c-Met degradation on
NCI-H441 cells. A) Mean of 4 independent experiments +/- s.e.m. B) Western blot
image representative of the 4 independent experiments performed.
Figure 2 1: Set up of an ELISA to evaluate c-Met shedding.
Figure 22: In vitro evaluation of c-Met shedding on NCI-H441 cells treated for 5
days with m224Gl 1. mlgGl is an irrelevant antibody used as an isotype control.
Figure 23: In vitro evaluation of c-Met shedding on amplified Hs746T, MKN45
and EBC-1 cell lines treated for 5 days with m224Gl 1. mlgGl is an irrelevant antibody
used as an isotype control. PMA is a shedding inducer used as a positive control.
Figure 24: In vitro evaluation of c-Met shedding on NCI-H441 and amplified
Hs746T, MK 45 and EBC-1 cell lines treated for 5 days with m224Gl 1. mlgGl is an
irrelevant antibody used as an isotype control. PMA is a shedding inducer used as a
positive control.
Figure 25: Study of intrinsic phosphorylation of h224Gl 1 on Hs746T cell line.
Figures 26A-26B: Study of intrinsic phosphorylation of h224Gl 1 on NCI-H441
cell line. A) phospho-ELISA and B) Western analysis.
Figures 27A-27B: Study of intrinsic phosphorylation of h224Gl l on Hs578T
cell line. A) phospho-ELISA and B) Western analysis.
Figures 28A-28B: Study of intrinsic phosphorylation of h224Gl l on NCI-H125
cell line. A) phospho-ELISA and B) Western analysis.
Figures 29A-29B: Study of intrinsic phosphorylation of h224Gll on T98G cell
line. A) phospho-ELISA and B) Western analysis.
Figures 30A-30B: Study of intrinsic phosphorylation of h224Gl 1 on MDA-MB-
231 cell line. A) phospho-ELISA and B) Western analysis.
Figures 31A-31B: Study of intrinsic phosphorylation of h224Gl l on PC3 cell
line. A) phospho-ELISA and B) Western analysis.
Figure 32: Study of intrinsic phosphorylation of h224Gl 1 on HUVEC cells.
Figure 33: In vivo comparison of the wild type murine 224G1 1 antibody with a
chimeric hinge-engineered 224G1 l[C2D5-7] Mabs on the NCI-H441 xenograft model.
Figures 34A-34B: ADCC induction by h224Gl l on both Hs746T and NCIH441
cells. 5 1Cr-labeled Hs746T (A) or NCI-H441 (B) cells loaded (bold squares) or
not (empty squares) with h224Gl l were mixed with different ratio of human NK cells
and incubated for 4 hr. Cells were harvested and cpm of 5 1Cr released by lysis was
counted. The results are plotted as percentage of lysis against the effector/target cell
ratio. NL for non loaded cells.
Figures 35A-35C: h224Gl l staining in tumor xenograft which expressed
various level of c-Met (A: Hs746T amplified cell line for c-Met, B: NCI-H441 high
level of c-Met expression and C: MCF-7 low level of c-Met).
Figures 36A-36D: In vivo activity of h224Gl l on the Hs746T xenograft model.
A) FACS analysis showing c-Met expression on Hs746T cells compared to other cell
lines expressing c-Met. B) QRTPCR showing c-Met amplification. C) c-Met
phosphorylation in absence or in presence of HGF. D) In vivo model.
Figures 37A-37D: In vivo activity of h224Gl 1 on the MK -45 xenograft model.
A) FACS analysis showing c-Met expression on MK 45 cells compared to other cell
lines expressing c-Met. B) QRTPCR showing c-Met amplification. C) c-Met
phosphorylation in absence or in presence of HGF. D) In vivo model.
Figures 38A-38D: In vivo activity of h224Gl l on the EBC-1 xenograft model.
A) FACS analysis showing c-Met expression on EBC-1 cells compared to other cell
lines expressing c-Met. B) QRTPCR showing c-Met amplification. C) c-Met
phosphorylation in absence or in presence of HGF. D) In vivo model.
Figures 39A-39H: IHC examination of Hs746T tumors in mice treated with the
h224Gl l Mab compared to control-treated tumors. (A) and (B) panels correspond to
isotype control antibodies. (C) and (D) show the expression of c-Met on tumor sections
from mice treated with PBS or h224Gl l respectively demonstrating that a chronic
treatment with h224Gl 1 induced a dramatic down regulation of c-Met. Ki-67 staining is
significantly decreased on treated tumors (F) compared to PBS control samples (E). A
significant apoptosis was observed in tumor treated with h224Gl l (H) while no
apoptosis was noticed in control tumors (G).
Example 1: Generation of antibodies against c-Met
To generate anti-c-Met antibodies 8 weeks old BALB/c mice were immunized
either 3 to 5 times subcutaneously with a CHO transfected cell line that express c-Met
on its plasma membrane (20x1 06 cells/dose/mouse) or 2 to 3 times with a c-Met
extracellular domain fusion protein (10-15 mg/dose/mouse) (R&D Systems, Catalog #
358MT) or fragments of this recombinant protein mixed with complete Freund adjuvant
for the first immunization and incomplete Freund adjuvant for the following ones.
Mixed protocols in which mice received both CHO-cMet cells and recombinant proteins
were also performed. Three days before cell fusion, mice were boosted i.p. or i.v. with
the recombinant protein or fragments. Then spleens of mice were collected and fused to
SP2/0-Agl4 myeloma cells (ATCC) and subjected to HAT selection. Four fusions were
performed. In general, for the preparation of monoclonal antibodies or their functional
fragments, especially of murine origin, it is possible to refer to techniques which are
described in particular in the manual "Antibodies" (Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726,
1988) or to the technique of preparation of hybridomas described by Kofiler and
Milstein (Nature, 256:495-497, 1975).
Obtained hybridomas were initially screened by ELISA on the c-Met
recombinant protein and then by FACS analysis on A549 NSCLC, BxPC3 pancreatic,
and U87-MG glioblastoma cell lines to be sure that the produced antibodies will be able
to also recognize the native receptor on tumor cells. Positive reactors on these 2 tests
were amplified, cloned and a set of hybridomas was recovered, purified and screened
for its ability to inhibit in vitro cell proliferation in the BxPC3 model.
For that purpose 50 000 BxPC3 cells were plated in 96 well plates in RPMI
medium, 2 mM L. Glutamine, without SVF. 24 hours after plating, antibodies to be
tested were added at a final concentration ranging from 0.0097 to 40 mg/ml 60 min
before addition of 100 ng/ml of hHGF. After 3 days, cells were pulsed with 0.5 m of
[ H]thymidine for 16 hours. The magnitude of [ H]thymidine incorporated into
trichloroacetic acid-insoluble DNA was quantified by liquid scintillation counting.
Results were expressed as raw data to really evaluate the intrinsic agonistic effect of
each Mab.
Then antibodies inhibiting at least 50% cell proliferation were evaluated for their
activity on c-Met dimerization and activation BRET analysis on transfected cells. c-Met
receptor activity was quantified by measuring the Gabl signalling molecule recruitment
on activated c-Met. For that purpose, CHO stable cell lines expressing C-Met-Rluc or
C-Met-Rluc and C-Met-Kl 100A-YFP for c-Met dimerization or C-Met-Rluc and a
mutated form of Gabl [Maroun et al, Mol. Cell. Biol, 1999, 19:1784-1799] fused to
YFP for c-Met activation were generated. Cells were distributed in white 96 well
microplates in DMEM-F12/FBS 5 % culture medium one or two days before BRET
experiments. Cells were first cultured at 37°C with C0 2 5 % in order to allow cell
attachment to the plate. Cells were then starved with 200 mΐ DMEM/well overnight.
Immediately prior to the experiment, DMEM was removed and cells quickly washed
with PBS. Cells were incubated in PBS in the presence or absence of antibodies to be
tested or reference compounds, 10 min at 37°C prior to the addition of coelenterazine
with or without HGF in a final volume of 50 mΐ . After incubation for further 10 minutes
at 37°C, light-emission acquisition at 485 nm and 530 nm was initiated using the
Mithras luminometer (Berthold) (ls/wave length/well repeated 15 times).
BRET ratio has been defined previously [Angers et al., Proc. Natl. Acad. Sci.
USA, 2000, 97:3684-3689] as: [(emission at 530 nm) - (emission at 485 nm) X CfJ /
(emission at 485 nm), where Cf corresponds to (emission at 530 nm) / (emission at
485 nm) for cells expressing Rluc fusion protein alone in the same experimental
conditions. Simplifying this equation shows that BRET ratio corresponds to the ratio
530/485 nm obtained when the two partners were present, corrected by the ratio
530/485 nm obtained under the same experimental conditions, when only the partner
fused to R. reniformis luciferase was present in the assay. For the sake of readability,
results are expressed in milliBRET units (mBU); mBU corresponds to the BRET ratio
multiplied by 1000.
After this second in vitro test, the antibody 224G1 1 i) without intrinsic activity
as a whole molecule in the functional test of proliferation, ii) inhibiting significantly
BxPC3 proliferation and iii) inhibiting c-Met dimerization was selected. In the
experiments, the 5D5 Mab, generated by Genentech, and available at the ATCC, was
added as a control for the intrinsic agonistic activity.
Example 2: Humanization process of mouse 224G11 Mab by CDR-grafting
1°) Humanization of the light chain variable domain (VL)
As a preliminary step, the nucleotide sequence of 224G1 1 VL was compared to
the murine germline gene sequences included in the IMGT database
(http://imgt.cines.fr). Murine IGKV3-5*01 and IGKJ4*01 germline genes showing a
sequence identity of 99.31 % for the V region and 94.28 % for the J region,
respectively, have been identified. Regarding these high homologies, the 224G1 1VL
nucleotide sequence has been used directly to search for human homologies, instead of
corresponding mouse germlines.
In a second step, the human germline gene displaying the best identity with the
224G1 1VL has been searched to identify the best human candidate for the CDR
grafting. For optimization of the selection, alignments between the amino acid
sequences have been performed. The human IGKV4-1*01 germline gene yielded a
sequence identity of 67.30 %, but showed a different length for CDR1 (10 amino acids
in 224G1 1 VL and 12 amino acids in IGKV4-1*01). For the J region, the human
IGKJ4*02 germline gene (sequence identity of 77.14 %) was selected.
In a next step, mouse 224G1 1 VL CDR regions were engrafted into the above
selected human framework sequences. Each amino acid position was analyzed for
several criteria such as participation in VH/VL interface, in antigen binding or in CDR
structure, localization of the residue in the 3D structure of the variable domain, CDR
anchors, residues belonging to the Vernier zone. Three humanized versions,
corresponding to SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10 were constructed,
and containing respectively four (4, 39, 40, 84), two (39, 40) or one (40) murine
residues in their FR regions and the CDRs corresponding to mouse 224G1 1 VL.
2°) Humanization of the heavy chain variable domain (VH)
As a preliminary step, the nucleotidic sequence of the 224G11 VH was
compared to the murine germline genes sequences included in the IMGT database
(http://imgt.cines.fr).
Murine IGHV1-18*01, IGHD2-4*01 and IGHJ2*01 germline genes with a
sequence identity of 92.70 % for the V region, 75.00 % for the D region and 89.36 %
for the J region, respectively, have been identified. Regarding these high homologies, it
has been decided to use directly the 224G1 1VH nucleotide sequences to search for
human homologies, instead of corresponding mouse germlines.
In a second step, the human germline gene displaying the best identity with the
224G1 1 VH has been searched to identify the best human candidate for the CDR
grafting. To this end, the nucleotidic sequence of 224G1 1VH has been aligned with the
human germline genes sequences belonging to the IMGT database. The human IGHV1-
2*02 V sequence exhibited a sequence identity of 75.00 % at the nucleotide level and
64.30 % at the amino acid level. Looking for homologies for the J region led to the
identification of the human IGHJ4*04 germline gene with a sequence identity of
78.72 %.
In a next step, mouse 224G1 1 VH CDR regions were engrafted into the above
selected human framework sequences. Each amino acid position was analyzed for
several criteria such as participation in VH/VL interface, in antigen binding or in CDR
structure, localization of the residue in the 3D structure of the variable domain, CDR
anchors, residues belonging to the Vernier zone. One fully humanized form,
corresponding to SEQ ID 4 was constructed; it contains exclusively human residues in
its FR regions and the CDRs corresponding to mouse 224G1 1 VH.
Example 3 : Engineering of improved hinge mutants
It is well known by the skilled artisan that the hinge region strongly participates
in the flexibility of the variable domain of immunoglobulins (see Brekke et al., 1995;
Roux et al., 1997). During the chimerization process of 224G11 Mab, the mouse
constant domain IGHG1 was replaced by the equivalent IGHG1 portion of human
origin. Since the amino acid sequence of the hinge region were highly divergent,
"murinization" of the hinge region was performed in order to keep its length and
rigidity. Since the human IGHG2 hinge region corresponds to the closest homologue of
the mouse IGHG1 hinge, this sequence was as well considered. A series of 7 different
hinge sequences were constructed (SEQ ID Nos. 22 to 28) by incorporating portions of
the mouse IGHG1 and the human IGHG2 hinges into the human IGHG1 hinge portion.
Another series of hinge mutants was designed and constructed (SEQ ID Nos. 58
to 72) to evaluate the influence of either an additional cysteine and its position along the
hinge domain, deletion of 1, 2, 3 or 4 amino acids along the hinge domain and a
combination of these two parameters (cysteine addition and amino acid deletion).
Example 4 : Production of humanized 224G11 Mab and engineered hinge
Mab formats
All above described Mab forms containing either chimeric, humanized and/or
engineered hinge regions were produced upon transient transfection and by using the
HEK293/EBNA system with a pCEP4 expression vector (InVitrogen, US).
The entire nucleotide sequences corresponding to the humanized versions of the
variable domain of 224G1 1 Mab light (SEQ ID No. 18, SEQ ID No. 19 and SEQ ID
No. 20) and heavy (SEQ ID No. 14) chains were synthesized by global gene synthesis
(Genecust, Luxembourg). They were subcloned into a pCEP4 vector (InVitrogen, US)
carrying the entire coding sequence of the constant domain [CH1-Hinge-CH2-CH3] of a
human IgGl or IgG2 immunoglobulin. Modification of the hinge region was performed
by exchanging a {Nhell-Bcll} restriction fragment by the equivalent portion carrying
the desired modifications, each respective {Nhel-Bcll} fragment being synthesized by
global gene synthesis (Genecust, LU). All cloning steps were performed according to
conventional molecular biology techniques as described in the Laboratory manual
(Sambrook and Russel, 2001) or according to the supplier's instructions. Each genetic
construct was fully validated by nucleotide sequencing using Big Dye terminator cycle
sequencing kit (Applied Biosystems, US) and analyzed using a 3100 Genetic Analyzer
(Applied Biosystems, US).
Suspension-adapted HEK293 EBNA cells (InVitrogen, US) were routinely
grown in 250 ml flasks in 50 ml of serum-free medium Excell 293 (SAFC Biosciences)
supplemented with 6 mM glutamine on an orbital shaker ( 110 rpm rotation speed).
Transient transfection was performed with 2.106 cells/ml using linear 25 kDa
polyethyleneimine (PEI) (Polysciences) prepared in water at a final concentration of
1 mg/ml mixed and plasmid DNA (final concentration of 1.25 mg/ml for heavy to light
chain plasmid ratio of 1:1). At 4 hours post-transfection, the culture was diluted with
one volume of fresh culture medium to achieve a final cell density of 106 cells/ml.
Cultivation process was monitored on the basis of cell viability and Mab production.
Typically, cultures were maintained for 4 to 5 days. Mabs were purified using a
conventional chromatography approach on a Protein A resin (GE Healthcare, US).
All different forms of Mabs were produced at levels suitable with functional
evaluations. Productivity levels are typically ranging between 15 and 30 mg/1 of
purified Mabs.
Example 5: Evaluation of c-Met phospshorylation status by a Phospho-c-
Met-specific ELISA assay
This functional assay allows to monitor modulation c-Met phosphorylation
status either by Mabs alone or in the co-presence of HGF.
A549 cells were seeded in a 12MW plate in complete growth medium [F12K +
10 % FCS]. Cells were starved for 16 hours before stimulation with HGF [100 ng/ml],
and each Mab to be tested was added at its final concentration of 30 mg/ml 15 minutes
prior to ligand stimulation. Ice-cold lysis buffer was added 15 minutes after the addition
of HGF to stop the phosphorylation reaction. Cells were scaped mechanically and cell
lysates were collected by centrifugation at 13000 rpm for 10 min. at 4°C and correspond
to the supernatant phase. Protein content was quantified using a BCA kit (Pierce) and
stored at -20°C until use. The phosphorylation status of c-Met was quantified by
ELISA. A goat anti-c-Met Mab (R&D, ref AF276) was used as a capture antibody
(overnight coating at 4°C) and after a saturation step with a TBS-BSA 5% buffer ( 1
hour at room temperature (RT)), 25 mg of protein lysates were added to each well of the
coated 96MW plate. After a 90 minutes incubation at RT, plates were washed four time
and the detection antibody was added (anti-phospho-c-Met Mab, directed against the
phopshorylated Tyr residues at position 1230, 1234 and 1235). After an additional 1
hour incubation and 4 washes, an anti-rabbit antibody coupled to HRP (Biosource) was
added for 1 hour at RT, and the luminescence detection was performed by adding
Luminol. Luminescence readings were on a Mithras LB920 multimode plate reader
(Berthold).
Both basal and HGF [100 ng/ml]-induced c-Met receptor phosphorylation level
were unaffected neither by PBS treatment, nor by the addition of mouse or human Mabs
which do not target human c-Met receptor (Figure 1). On the other hand, mouse (m)
224G1 1 Mab strongly inhibited HGF [100 ng/ml]-induced c-Met phosphorylation
(Figure 2B) without altering by itself receptor phosphorylation (Figure 2A).
Surprisingly, the chimeric form of 224G1 1 Mab (224G1 lchim/IgGl), meaning variable
domain (VH+VL) from m224Gll combined with human constant domain IgGl /kappa
yielded strong (17 % of maximal HGF effect, Figure 2A) agonist activity associated
with a reduced antagonist efficacy (54 % inhibition of HGF maximal effect compared to
the m224Gl l that yields 75% inhibition of HGF maximum effect, Figure 2B). Three
humanized forms of 224G1 1 Mab, [224G1 l]Hzl/IgGl, [224G1 l]Hz2/IgGl and
[224Gl l]Hz3/IgGl, also constructed on a human IgGl/kappa backbone, yielded also
decreased antagonist efficacy and significant agonist activity ( 11 to 24 % of maximal
HGF level) as compared to mouse 224G1 1 (Figures 2A and 2B). A series of engineered
versions of the heavy chain hinge domain were constructed and assayed in the c-Met
receptor phosphorylation assay. As shown in Figure 3A, an important reduction of the
agonist effect associated with the hlgGl /kappa isotype was observed for both the IgG2-
based construct and for engineered IgGl/kappa constructs [MH, MUP9H and TH7]. A
concomitant increase in antagonist efficacy was as well obtained. The hlgGl/kappabased
TH7 hinge mutant, with the most human sequence, was selected to complete the
humanization process. In a next step, three humanized versions of 224G1 1 Mab variable
domain were generated by combination to either a human IgG2/kappa or an
IgGl /kappa-based TH7 engineered hinge constant domain. For the hIgG2/kappa
humanized constructs, the humanized version Hz3 yielded strong agonism (Figure 4A),
and for all three humanized versions, the antagonist efficacy was below that observed
with murine 224G1 1 Mab and comparable to the chimeric hlgGl -based Mab (56-57 %
inhibition of HGF effect, Figure 4B). On the other hand, combination of the three
humanized versions Hzl, Hz2 or Hz3 to the engineered IgGl/TH7 mutant almost fully
restored the properties of mouse 224G1 1 Mab in terms of weak agonist activity (5-6 %
of HGF effect) and strong antagonist efficacy (68 to 72 % inhibition of HGF effect) of
c-Met receptor phosphorylation (Figures 5A and 5B). These variants were highly
improved as compared to chimeric IgGl -based 224G1 1 Mab but also to IgG2-based
humanized forms.
A second series of engineered versions of the heavy chain hinge domain was
constructed and assayed in the c-Met receptor phosphorylation assay. As shown in
figure 17A, all those new versions (c224Gl l[C2], c224Gl l[C3], c224Gl l[C5],
c224Gl l[C6], c224Gl l[C7], c224Gl 1[D 1-3], c224Gll[C7A6], c224Gl 1[C6A9],
c224Gl l[C2A5-7], c224Gl l[C5A2-6], c224Gl l[C9A2-7] and c224Gl 1[D5-6-7-8])
exhibited weaker agonist effect than c224Gl l since their agonism activities are
comprised between 6 and 14% of the HGF effect compared to 23% for c224Gl l . As
c224Gl 1[TH7], all those new versions exhibited a concomitant increase in antagonist
efficacy [figure 17B]. Those results showed that engineering of the heavy chain domain
by point mutation and/or deletion could modify agonistic/antagonistic properties of an
antibody.
Example 6: BRET analysis
In a first set of experiments, it had been control that irrelevant mouse IgGl,
human IgGl and human IgG2 had no effect of HGF induced BRET signal in both
BRET models (representative experiment out of 12 independent experiments; Figure 6).
These Mabs are forthwith cited as controls.
The effect of a IgGl chimeric form of mouse 224G1 1 Mab ([224Gll]chim) on
both c-Met dimerization and c-met activation BRET model was evaluated. While mouse
224G1 1 Mab inhibited 59.4% of the HGF induced BRET signal on c-Met dimerization
model, [224Gl l]chim Mab inhibited only 28.9% (Figure 7A). [224Gl l]chim antibody
was also less effective in inhibiting HGF induced c-Met activation since [224Gl l]chim
and m224Gl l antibodies inhibited respectively 34.5% and 56.4% of HGF induced
BRET signal (Figure 7B). Moreover, m224Gl 1 alone had no effect on c-Met activation
while [224Gl l]chim had a partial agonist effect on c-Met activation corresponding to
32.9% of the HGF induced signal. This partial agonist effect of the [224Gl l]chim was
also seen on c-Met dimerization BRET model since [224Gl l]chim alone induced a
BRET increase corresponding to 46.6% of HGF-induced signal versus 21.3% for
m224Gl 1 (Figure 7A).
In Figures 8A and 8B, hinge mutated chimeric forms of 224G1 1 antibody
showed a greater inhibitory effect on HGF induced BRET signal than [224Gl l]chim
since they showed a 59.7%, 64.4%, 53.2% and 73.8% inhibition of the HGF induced
activation BRET signal (Figure 8B) and 61.8%, 64.4% 52.5% and 64.4% inhibition of
the HGF induced c-Met dimerization BRET signal (Figure 8A) for [224G1 1][MH
chim], [224G1 1][MUP9H chim], [224G1 1][MMCH chim] and [224G1 1][TH7 chim]
respectively. Contrary to [224G1 l]chim, which had a partial agonist effect on c-Met
activation, hinge mutated chimerical forms of 224G1 1 antibody showed no significant
effect on c-Met activation alone (5.1%>, 7.6%>, -2.0%> and -6.9%> respectively) as
observed for m224G 11.
In Figure 9B, like the [224G1 1] [TH7 chim], the 3 humanized versions of
224G1 1 IgGl antibody with the TH7 hinge induced no significant increased of BRET
signal in activation model when tested alone and showed a strong inhibition of HGF
induced BRET signal: 59.9%, 41.8% and 57.9% for the Hzl, Hz2 and Hz3 forms
respectively. Moreover, [224G1 1] [TH7 Hzl], [224G1 1] [TH7 Hz2] and [224G1 1]
[TH7 Hz3] inhibited HGF induced BRET signal on dimerization model of 52.2%,
35.8% and 49.4% respectively (figure 9A).
Contrary to [224G1 l]chim, the chimeric form of 224G1 1 IgG2 antibody
([224G1 1] [IgG2 chim]) showed no partial agonist effect alone and inhibited 66.3% of
the HGF effect on c-Met activation model (Figure 10B). On c-Met dimerization model,
[224G1 1] [IgG2 chim] inhibited 62.4% of the HGF induced BRET signal (Figure 10A).
The agonist efficacy of the second series of engineered versions of the heavy
chain hinge domain was evaluated in c-Met activation BRET model (Figure 18). In
contrast to c224Gl l , which had a partial agonist effect on c-Met activation,
c224Gl l[C2], c224Gl l[C3], c224Gl l[C5], c224Gl l[C6], c224Gl l[C7],
c224Gl l[Al-3], c224Gl 1[C7A6], c224Gll[C6A9], c224Gl l[C2A5-7],
c224Gl 1[C5A2-6], c224Gl 1[C9A2-7] and c224Gl 1[D5-6-7-8] hinge mutated chimeric
forms of 224G1 1 antibody showed no significant effect on c-Met activation alone.
Example 7: c-Met recognition by chimeric and humanized 224G11 forms
A direct ELISA has been set up to determine the binding ability of the various
chimeric and humanized forms on the recombinant c-Met. Briefly recombinant dimeric
c-Met from R&D Systems was coated at 1.25 mg/ml on 96-well Immunlon II plates.
After an overnight incubation at 4°C, wells were saturated with a 0.5% gelatine/PBS
solution. Plates were then incubated for 1 hour at 37°C before addition of 2 fold
dilutions of antibodies to be tested. Plates were incubated an additional hour before
addition of a goat anti-mouse IgG HRP for detecting the murine antibody and a goat
anti-human Kappa light chain HRP for chimeric and humanized antibody recognition.
Plates were incubated for one hour and the peroxydase substrate TMB Uptima was
added for 5 mn before neutralization with H2SO4 1M. Results presented in Figure 11
showed that all tested forms were comparable for c-Met recognition.

CLAIMS
1. A monoclonal antibody, or a divalent functional fragment or derivative
thereof, capable to inhibit the c-Met dimerization,
said antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3
with respectively the amino acid sequences SEQ ID Nos. 1, 2 and 3; and a light chain
comprising CDR-L1, CDR-L2 and CDR-L3 with respectively the amino acid sequences
SEQ ID Nos. 5, 6 and 7, said antibody further comprising a hinge region comprising the
amino acid sequence SEQ ID No. 56;
for use for the prevention or the treatment of a patient in need thereof having a cancer
characterized by ligand-independent activation of c-Met.
2. The antibody of claim 1, or a divalent functional fragment or derivative
thereof, characterized in that said hinge region comprises the amino acid sequence
SEQ ID No. 57.
3. The antibody of claim 1, or a divalent functional fragment or derivative
thereof, characterized in that said hinge region comprises the amino acid sequence
SEQ ID No. 21.
4. The antibody of claim 1, or a divalent functional fragment or derivative
thereof, characterized in that said hinge region comprises an amino acid sequence
selected from the group consisting of SEQ ID Nos. 22 to 28 and SEQ ID Nos. 58 to 72.
5. The antibody of claim 1, or a divalent functional fragment or derivative
thereof, for use for the prevention or the treatment of a patient in need thereof having a
cancer characterized by overexpression of c-Met.
6. The antibody of claim 1, or a divalent functional fragment or derivative
thereof, for use for the prevention or the treatment of a patient in need thereof having a
cancer characterized by overexpression of c-Met resulting from genic amplification of
c-Met.
7. The antibody of claim 6, or a divalent functional fragment or derivative
thereof, for use for the prevention or the treatment of a patient in need thereof having a
cancer characterized by overexpression of c-Met resulting from genic amplification of
c-Met and resulting in ligand-independent activation of c-Met.
8. The antibody of claim 6, or a divalent functional fragment or derivative
thereof, for use for the prevention or the treatment of a patient in need thereof having a
cancer characterized by overexpression of c-Met resulting from genic amplification of
c-Met, wherein said cancer is selected from the group consisting of renal cell carcinoma
and gastric cancer.
9. The antibody of claim 1, or a divalent functional fragment or derivative
thereof, characterized in that it consists of a chimeric antibody.
10. The antibody of claim 1, or a divalent functional fragment or derivative
thereof, characterized in that it consists of a human antibody.
11. The antibody of claim 1, or a divalent functional fragment or derivative
thereof, characterized in that it consists of a humanized antibody.
12. The antibody of claim 11, or a divalent functional fragment or derivative
thereof, characterized in that it comprises a heavy chain variable domain of sequence
comprising the amino acid sequence SEQ ID No. 4; and a light chain variable domain
of sequence comprising the amino acid sequence SEQ ID No. 8, 9 or 10.
13. The antibody of claim 12, or a divalent functional fragment or derivative
thereof, characterized in that it comprises a heavy chain variable domain of sequence
comprising the amino acid sequence SEQ ID No. 4; a light chain variable domain of
sequence comprising the amino acid sequence SEQ ID No. 8; and a hinge region
comprising the amino acid sequence SEQ ID No. 22.
14. The antibody of claim 12, or a divalent functional fragment or derivative
thereof, characterized in that it comprises a heavy chain variable domain of sequence
comprising the amino acid sequence SEQ ID No. 4; a light chain variable domain of
sequence comprising the amino acid sequence SEQ ID No. 9; and a hinge region
comprising the amino acid sequence SEQ ID No. 22.
15. The antibody of claim 12, or a divalent functional fragment or derivative
thereof, characterized in that it comprises a heavy chain variable domain of sequence
comprising the amino acid sequence SEQ ID No. 4; a light chain variable domain of
sequence comprising the amino acid sequence SEQ ID No. 10; and a hinge region
comprising the amino acid sequence SEQ ID No. 22.
16. The antibody of claim 12, or a divalent functional fragment or derivative
thereof, characterized in that it comprises a heavy chain variable domain of sequence
comprising the amino acid sequence SEQ ID No. 4; a light chain variable domain of
sequence comprising the amino acid sequence SEQ ID No. 8; and a hinge region
comprising the amino acid sequence SEQ ID No. 28.
17. The antibody of claim 12, or a divalent functional fragment or derivative
thereof, characterized in that it comprises a heavy chain variable domain of sequence
comprising the amino acid sequence SEQ ID No. 4; a light chain variable domain of
sequence comprising the amino acid sequence SEQ ID No. 9; and a hinge region
comprising the amino acid sequence SEQ ID No. 28.
18. The antibody of claim 12, or a divalent functional fragment or derivative
thereof, characterized in that it comprises a heavy chain variable domain of sequence
comprising the amino acid sequence SEQ ID No. 4; a light chain variable domain of
sequence comprising the amino acid sequence SEQ ID No. 10; and a hinge region
comprising the amino acid sequence SEQ ID No. 28.
19. The antibody of claims 1 to 18 as a medicament.
20. A composition for the prevention or the treatment of a patient in need
thereof having a cancer characterized by ligand-independent activation of c-Met, said
composition comprising by way of active principle a compound consisting of an
antibody, or a divalent functional fragment or derivative thereof, as claimed in one of
claims 1 to 19.
21. The composition of claim 20 as a medicament.
22. The use of an antibody, or a divalent functional fragment or derivative
thereof, of claims 1 to 19 or of a composition of claims 20 or 2 1 for the preparation of a
medicament for the prevention or the treatment of a patient in need thereof having a
cancer characterized by ligand-independent activation of c-Met.