HK1193620B - Novel antibodies inhibiting c-met dimerization, and uses thereof - Google Patents

Abstract

The present inventions relates to novel antibodies inhibiting c-MET dimerization, and uses thereof. Particularly, the present inventions relates to a process for the selection of anti c-Met antibodies capable to inhibit both ligand-dependent and ligand-independent activation of c-Met. More particularly, said process is based on the inhibition of the c-Met dimerization. In another aspect, the present invention concerns such antibodies and compositions comprising such antibodies for the preparation of a medicament to treat cancer. Diagnosis process and kits are also part of the invention.

Description

Novel antibodies that inhibit c-MET dimerization and uses thereof

The present application is a divisional application of the chinese patent application having the title of "novel antibody inhibiting c-MET dimerization and use thereof" with the application number of 200880024368.4, application date of 2008/7/10.

Technical Field

The present invention relates to novel antibodies, in particular monoclonal antibodies of murine, chimeric and humanized origin, capable of binding specifically to the human c-Met receptor and/or of inhibiting specifically the tyrosine kinase activity of said receptor, as well as the amino acid and nucleotide sequences coding for these antibodies. More specifically, the antibodies of the invention are capable of inhibiting c-Met dimerization. The invention also comprises the use of these antibodies as a medicament in the prophylactic and/or therapeutic treatment of malignant tumors or any pathology associated with the overexpression of said receptor, as well as in processes and kits for the diagnosis of diseases associated with the overexpression of c-Met. The invention finally comprises products and/or compositions containing said antibodies 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-malignant agents or agents conjugated to toxins, and their use in the prevention and/or treatment of certain malignancies.

Background

Receptor Tyrosine Kinase (RTK) targeting agents such as trastuzumab, cetuximab, bevacizumab, imatinib, and gefitinib inhibitors have been shown to be advantageous for targeting this protein class for the treatment of selected malignancies.

c-Met is the prototypical member of the RTK subfamily, which also includes RON and SEA. The c-Met RTK family is structurally distinct from other RTK families and is the only known high affinity receptor for hepatocyte growth factor (HGF, also known as scatter factor, SF) [ d.p. bottaro et al, science1991, 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 tissues, and their expression is generally limited to epithelial and mesenchymal derived cells, respectively [ m.f. derezo et al, Oncogene1991, 6: 1997-2003, E.Sonnenberg et al, J.cell.biol.1993, 123: 223-235]. Both are essential for normal mammalian development and have been shown to be particularly important in cell migration, morphological differentiation and organization of three-dimensional tubular structures as well as growth and angiogenesis [ f.baldt et al, nature1995, 376: 768-: 373: 699-: 98-101]. Controlled regulation of c-Met and HGF has 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, Tssugouchi H, J Pharmacol Exp ther.2003,307 (1): 146-51], their dysregulation is related to the progression of malignant tumors.

Aberrant signaling resulting from inappropriate activation of c-Met is one of the most common alterations observed in human malignancies, playing a critical 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 occur through ligand-dependent and independent mechanisms (including c-Met overexpression, and/or paracrine or autocrine activation), or through acquired functional mutations [ j.g. christensen, Burrows j.and Salgia r., Cancer letters.2005, 226: 1-26]. However, oligomerization of the c-Met receptor in the presence or absence of ligand is essential in modulating the binding affinity and binding kinetics of kinases to ATP and tyrosine-containing peptide substrates [ Hays JL, Watowich SJ, Biochemistry,2004Aug17, 43: 10570-8]. Activated c-Met recruits signaling effectors to its multiple docking sites located in the cytoplasmic region, leading to the activation of several key signaling pathways, including Ras-MAPK, PI3K, Src, and Stat3[ Gao CF, Vande Woude GF, Cell res.2005,15 (1): 49-51, Fuge KA, Zhang YW, Vande Woude GF, oncogene.2000,19 (49): 5582-9]. These pathways are essential for proliferation, infiltration and angiogenesis of tumor cells and for escape of apoptosis [ fuse KA, Zhang YW, van de Woude GF, Oncogene,2000,19 (49): 5582-9, Gu H, NeelBG, Trends Cell biol.2003Mar,13 (3): 122-30, Fan S, Ma YX, Wang JA, Yuan RQ, Meng Q, Cao Y, Laterra JJ, Goldberg ID, Rosen EM, oncogene.2000Apr27,19 (18): 2212-23]. Furthermore, c-Met signaling is unique over other RTKs in that it has been reported to bind to focal adhesion complexes and non-kinase binding partners such as α 6 β 4 integrin [ Trusolino L, Bertotti a, ComoglioPM, 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 B1 or semaphorin (semaphorin) [ Giordano S, CorsoS, Conrotto P, Artigiani S, Gilesro G, Barberis D, Tamagnone L, Comoglio PM, Natcell biol.2002,4 (9): 720-4, Conrotto P, Valdmeri D, Corso S, Serini G, Tamagnone L, Comoglio PM, Bussolino F, Giordano S, blood.2005,105 (11): 4321-9, Conrotto P, Corso S, Gamberini S, Comoglio PM, Giordano S, oncogene.2004, 23: 5131-7], which further increases the complexity of this receptor in modulating cellular function. Finally, recent data demonstrate that c-Met may be involved in tumor resistance to gefitinib and erlotinib, suggesting that compounds targeting both EGFR and c-Met in combination may be of importance [ Engelman JA at, Science,2007,316: 1039-43].

Over the past few years, a number of different strategies have been developed to attenuate c-Met signaling in malignant tumor cell lines. These strategies include i) neutralizing antibodies against c-Met or HGF/SF [ Cao B, Su Y, OskarssonM, 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 use of an antagonist of HGF/SF 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 of c-Met to block kinase activity [ Christensen JG, Schreck R, Burrows J, Kurugant 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) an engineered SH2 domain polypeptide that affects access to multiple docking sites, and RNAi or ribozymes that reduce receptor or ligand expression. Most of these methods show selective inhibition of c-Met, leading to tumor inhibition, and suggest that c-Met may be of interest in therapeutic intervention in malignancies.

Some of the molecules produced targeting c-Met are antibodies.

The most widely described are anti-c-Met 5D5 antibodies produced by Genentech [ WO96/38557], which act as potent agonists when added alone in a variety of models, and as antagonists when used as Fab fragments. The monovalent engineered form of this antibody is described as one-armed 5D5 (OA 5D5), produced as a recombinant protein in e.coli, which is also the subject of Genentech patent application [ WO2006/015371 ]. However, this molecule cannot be considered an antibody due to its specific scaffold (scarfold), it also appears to produce mutations that are immunogenic to humans. In terms of activity, this unglycosylated molecule has no effector function, and finally no clear data demonstrates that OA5D5 inhibits dimerization of c-Met. Furthermore, when tested in the G55 in vivo model (a glioblastoma cell line expressing c-Met but not HGF mRNA and protein, which grows independent of ligand), single-arm anti-c-Met had no significant effect on G55 tumor growth, suggesting that OA5D5 acts primarily by blocking HGF binding and is unable to target HGF-independent activated tumors [ Martens t.et al, clin.cancer res.,2006,12 (20): 6144-6152].

Another antibody targeting c-Met described by Pfizer is an antibody that functions by acting "primarily as a c-Met antagonist, and in some cases as a c-Met agonist" [ WO2005/016382 ]. The application does not describe data showing any effect of Pfizer antibodies on c-Met dimerization.

A novel aspect of the invention is the generation of murine monoclonal antibodies that have no intrinsic agonist activity and inhibit c-Met dimerization. In addition to targeting ligand-dependent tumors, this approach also disrupts ligand-independent c-Met activation due to c-Met overexpression or intra-cellular region mutations, which activation still relies on oligomerization for cell signaling. Another aspect of this antibody activity may be the steric hindrance of the interaction between c-Met and its partner, thereby disrupting the function of c-Met. In addition to the functions associated with specifically blocking the c-Met receptor, it is preferred that these antibodies be humanized and engineered as human IgG1 to obtain effector functions such as ADCC and CDC, but are not so limited.

Disclosure of Invention

Surprisingly, the inventors have for the first time sought to generate antibodies capable of binding to c-Met and also capable of inhibiting c-Met dimerization. In the prior art, it is sometimes suggested that antibodies capable of inhibiting dimerization of c-Met with its partner may be interesting antibodies, and if this is true, has never been disclosed, or it is clearly suggested that antibodies are capable of doing so. Furthermore, with respect to the specificity of the antibody, the successful production of such active antibodies is not at all apparent.

In a first aspect, the subject of the invention is a method for producing and selecting the antibodies according to the invention.

More specifically, the present invention relates to a method for selecting an anti-c-Met antibody or one of its functional fragments or derivatives capable of inhibiting ligand-dependent and ligand-independent c-Met activation, said method comprising the steps of:

i) screening the produced antibodies, selecting antibodies capable of specifically binding to c-Met;

ii) evaluating in vitro the antibodies selected in step i) and selecting antibodies capable of inhibiting tumor cell proliferation of at least one tumor type by at least 50%, preferably by at least 60%, 70% or 80%;

iii) testing the antibodies selected in step ii) and selecting antibodies capable of inhibiting c-Met dimerization.

As explained earlier, since this antibody has practical implications for patients of a larger population, inhibition of c-Met dimerization is a primary aspect of the invention. Not only ligand-dependent activated c-Met malignancies (as was the case until the present invention), but also ligand-independent activated c-Met malignancies can be treated by the antibodies produced by the methods described herein.

Antibody production can be accomplished by any method known to those skilled in the art, for example, by fusing myeloma cells with spleen cells obtained from immunized mice or any other species compatible with the selected myeloma cells [ Kohler & Milstein,1975, Nature, 256: 495-497]. The immunized animal may comprise a transgenic mouse with human immunoglobulin loci, which can then directly produce human antibodies. Other possible embodiments may consist in using phage display technology to screen libraries.

The screening step i) may be achieved by any method or process known to the skilled person. As non-limiting examples, mention may be made of ELISA, BIAcore, immunohistochemistry, FACS analysis and functional screening. The preferred method consists in screening the c-Met recombinant protein by ELISA and then in analyzing at least one tumor cell line by FACS in order to confirm that the antibodies produced are also able to recognize the receptors native to the tumor cells. This method will be described more precisely in the examples below.

Likewise, step ii) can also be classically effected by known methods or procedures, e.g. using 3H-thymidine or any other DNA stain, MTT, ATP evaluation, etc. A preferred tumor cell model in the present invention may be the BxPC3 model.

By inhibiting c-Met dimerization, it must be understood that it is preferably c-Met homodimerization.

In a preferred embodiment of step iii) of the selection method according to the invention, said step iii) consists in evaluating the antibodies by BRET analysis of cells expressing both c-Met-RLuc/c-Met-YFP and in selecting antibodies capable of inhibiting the BRET signal by at least 30%, preferably 35%, 40%, 45%, 50%, 55% or most preferably 60%.

BRET technology is a known technology, which is a representative technology for protein dimerization [ Angers et al, PNAS,2000, 97: 3684-89].

The BRET technique used in step iii) of the process is well known to those skilled in the art and will be described in detail in the examples below. More specifically, BRET (bioluminescence resonance energy transfer) is a nonradioactive energy transfer occurring between a bioluminescent donor (Renilla Luciferase, Rluc) and a mutant of the fluorescence acceptor, GFP (green fluorescent protein) or YFP (yellow fluorescent protein). In the present case, EYFP (enhanced yellow fluorescent protein) was used. The efficiency of transfer depends on the direction and distance between the donor and the acceptor. Thus, energy transfer is only possible when the two molecules are close (1-10 nm). This property is used to perform protein-protein interaction analysis. Indeed, to study the interaction between the two partners, the first was genetically engineered to fuse it into renilla luciferase and the second into a yellow mutant of GFP. Fusion proteins are typically, but not necessarily, expressed in mammalian cells. In the presence of a substrate whose membrane is permeable (coelenterazine), Rluc emits blue light. If the GFP mutant is close to Rluc above 10nm, energy transfer can occur and an additional yellow signal can be detected. The BRET signal measured is the ratio of light emitted by the acceptor and light emitted by the donor. Thus, when the two fusion proteins are brought into proximity or if the conformational change brings Rluc and GFP mutants closer, the BRET signal will increase.

If BRET analysis is a preferred embodiment, any method known to those skilled in the art can be used to measure c-Met dimerization. Without limitation, the following techniques may be mentioned: FRET (fluorescence resonance energy transfer), HTRF (homogeneous time-resolved fluorescence), FLIM (fluorescence lifetime imaging microscope) or SW-FCCS ((single wavelength fluorescence correlation spectroscopy)).

Other classical techniques such as co-immunoprecipitation, alpha screening, chemical cross-linking, two-hybrid, affinity chromatography, ELISA or remote western blotting (Far western blot) can also be used.

In a second aspect, the subject of the invention is an isolated antibody or one of its functional fragments or derivatives obtained by said method. Said antibody or one of said functional fragments or derivatives thereof is capable of specifically binding to human c-Met and, if necessary, preferably also of inhibiting the natural binding of its ligand HGF and/or of specifically inhibiting the tyrosine kinase activity of said c-Met, said antibody also being capable of inhibiting the dimerization of c-Met. More specifically, the antibodies are capable of inhibiting both ligand-dependent and ligand-independent activation of c-Met.

The expression "functional fragments and derivatives" is defined in detail hereinafter in the present description.

It must be understood here that the invention does not relate to antibodies in their natural form, that is to say that these antibodies are not in their natural environment, but that they can be isolated or obtained from natural sources by purification, or also obtained by genetic recombination, or obtained by chemical synthesis, and therefore they contain unnatural amino acids, as will be further described.

More specifically, according to another aspect of the present invention, it is claimed an antibody, characterized in that it comprises at least one complementarity determining region CDR chosen from the CDRs comprising the amino acid sequences SEQ ID nos. 1 to 17 and 56 to 61, or one of its functional fragments or derivatives.

Any antibody or fragment or derivative comprising at least one CDR whose sequence has at least 80% identity, preferably 85%, 90%, 95% or 98% identity, after optimal alignment with the sequences SEQ ID nos. 1 to 17 and 56 to 61, is to be understood as an equivalent of the invention and is therefore also part of the invention.

By CDR regions or CDRs is meant the hypervariable regions of the heavy and light chains of an immunoglobulin defined by IMGT.

The unique number of IMGT (IMGT unique number) is defined for comparison of variable regions regardless of The type of antigen receptor, chain or species [ Lefranc M. -P., Immunology today18,509(1997)/LefrancM. -P., The Immunologist,7,132-136(1999)/Lefranc, M. -P., Pommie, C, Ruiz, M., Giudicelli, V., Fouluer, E., Truong, L., Thiourein-Contet, V., and Lefranc, Dev.Comp.Immunol,27,55-77 (2003). In the unique numbering of IMGT, CONSERVED amino acids always have the same position, e.g., cysteine 23 (1 st-CYS), tryptophan 41 (CONSERVED-TRP (CONSERVED-TRP)), hydrophobic amino acid 89, cysteine 104 (2 nd-CYS), phenylalanine or tryptophan 118(J-PHE or J-TRP). Unique numbering of IMGT provides for the framework regions (FRl-IMGT: positions 1 to 26, FR 2-IMGT: 39 to 55, FR 3-IMGT: 66 to 104 and FR 4-IMGT: 118 to 128) and their complementary determining regions: CDR 1-IMGT: 27 to 38, CDR 2-IMGT: 56 to 65 and CDR 3-IMGT: standard definition of 105 to 117. Since the gaps represent unoccupied positions, the length of the CDR-IMGT (shown in brackets as being split by a point of merger, e.g., [8.8.13]) is important information. The unique numbering of the IMGT is represented in 2D diagrams, called IMGT colloids de Perles [ Ruiz, m.and leifranc, m. -p., Immunogenetics,53, 857-.

There are 3 heavy chain CDRs and 3 light chain CDRs. The term CDR or CDRs as used herein is used to denote one or several, or even all, of these regions (the regions containing the majority of the amino acid residues responsible for the affinity binding of an antibody to an antigen or an epitope recognized thereby), as the case may be.

"percent identity" between two nucleic acid or amino acid sequences in the sense of the present invention means the percentage of nucleotides or identical amino acid residues between the two sequences to be compared, which percentage is obtained after optimal alignment (optimized alignment), which percentage is purely statistical, and the differences between the two sequences are randomly distributed and distributed over the full length. Traditionally, two nucleic acid or amino acid sequences are compared by aligning them in an optimized manner, which comparison can be made by means of segments or "comparison windows". In addition to manual approaches, optimal alignment of sequences for comparison can be achieved by Smith and Waterman (1981) [ ad. 482], Neddleman and Wunsch (1970) [ j.mol.biol.48: 443], Pearson and Lipman (1988) [ proc.natl.acad.sci.usa 85: 2444) the similarity search method of (3) is performed by means of Computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575Science Dr., Madison, Wis., or otherwise by BLAST N or BLAST P comparison software).

The percent identity between two nucleic acid or amino acid sequences is determined by comparing the two sequences in an optimized manner, and wherein the nucleic acid or amino acid sequences being compared may comprise added or deleted sequences relative to a reference sequence used for optimized alignment between the two sequences. Percent identity is calculated by determining the number of identical positions of identical nucleotides or amino acid residues between two sequences, dividing the number of such identical positions by the total number of positions in the window being compared, and multiplying the result by 100 to obtain the percent identity between the two sequences.

For example, 2sequences may be BLAST using the BLAST program (Tatusova et al, "BLAST 2 sequences-a new tools for composing proteins and nucleotide sequences", FEMSMicyobiol Lett. 174: 247-250), which may be performed at the website http: html, the percentage identity of two sequences to be compared is calculated directly by the program, using those parameters given by default (in particular the parameters "open gap penalty": 5, and "extended gap penalty": 2; chosen as a matrix, for example the "BLOSUM 62" matrix proposed by the program).

Amino acid sequences which have at least 80%, preferably 85%, 90%, 95% or 98% identity with respect to the reference amino acid sequence, those which have certain modifications, in particular deletion, addition or substitution of at least one amino acid, relative to the reference sequence, truncation or elongation are preferred. In the case of substitution of one or more consecutive or non-consecutive amino acids, the substitution is preferably such that the amino acid to be substituted is replaced by an "equivalent" amino acid. The expression "equivalent amino acids" is intended herein to mean any amino acid capable of being substituted by one of more amino acids, the substituted amino acid having the basic structure of the corresponding antibody without substantially modifying its biological activity, and these amino acids are defined hereafter, in particular in the examples. The determination of these equivalent amino acids can be based on their structural homology with the amino acids that they replace, or on comparative tests that can be carried out for the biological activity between the different antibodies.

By way of example, possible substitutions are mentioned which can be made which do not lead to a major modification of the biological activity of the corresponding modified antibody.

As a non-limiting example, the following table 1 gives possible alternatives in view of maintaining the biological activity of the modified antibody. The opposite substitution is of course also possible under the same conditions.

TABLE 1

Original residues	Replacement of
		Ala(A)	Val，Gly，Pro
Arg(R)	Lys，His
		Asn(N)	Gln
Asp(D)	Glu
		Cys(C)	Ser
Gln(Q)	Asn
		Glu(G)	Asp
Gly(G)	Ala
		His(H)	Arg
Ile(I)	Leu
		Leu(L)	Ile，Val，Mer
Lys(K)	Arg
		Met(M)	Leu
Phe(F)	Tyr
		Pro(P)	Ala
Ser(S)	Thr，Cys
		Thr(T)	Ser
Trp(W)	Tyr
		Tyr(Y)	Phe，Trp
Val(V)	Leu，Ala

It must be understood here that the invention does not relate to antibodies in their natural form, that is to say that they are not in their natural environment, but that they can be isolated or obtained from natural sources by purification, or obtained by genetic recombination, or obtained by chemical synthesis, and that they may therefore contain unnatural amino acids, as will be further described.

According to the first mode, the antibody will be defined by its heavy chain sequence. More specifically, the antibody according to 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 the CDRs comprising the amino acid sequences SEQ ID nos. 1 to 9 and 56 to 58.

The sequences mentioned are the following:

SEQ ID No.1：GYIFTAYT

SEQ ID No.2：IKPNNGLA

SEQ ID No.3：ARSEITTEFDY

SEQ ID No.4：GYSFTDYT

SEQ ID No.5：INPYNGGT

SEQ ID No.6：AREEITKDFDF

SEQ ID No.7：GYTFTDYN

SEQ ID No.8：INPNNGGT

SEQ ID No.9：ARGRYVGYYYAMDY

SEQ ID No.56：GYTFTSYW

SEQ ID No.57：INPTTGST

SEQ ID No.58：AIGGYGSWFAY

the CDRs of the heavy chain may be randomly selected among the aforementioned sequences, i.e. SEQ ID nos. 1 to 9 and 56 to 58.

According to a preferred aspect, the antibody or one of its functional fragments or derivatives according to the invention comprises a heavy chain comprising at least one CDR selected from CDR-H1, CDR-H2 and CDR-H3, wherein:

-CDR-H1 comprises the amino acid sequence SEQ ID No.1, 4, 7 or 56,

-CDR-H2 comprises the amino acid sequence SEQ ID No.2, 5,8 or 57, and

-CDR-H3 comprises the amino acid sequence SEQ ID No.3, 6, 9 or 58.

According to a first particular embodiment of said aspect, the antibody or one of its functional fragments or derivatives according to the invention comprises a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1 comprises the amino acid sequence SEQ ID No.1, CDR-H2 comprises the amino acid sequence SEQ ID No.2 and CDR-H3 comprises the amino acid sequence SEQ ID No. 3.

More specifically, according to a first embodiment, said antibody or one of its functional fragments or derivative comprises a heavy chain, the sequence of which comprises SEQ ID No. 18.

SEQ ID No.18：EVQLQQSGPELVKPGASVKISCKTSGYIFTAYTMHWVRQSLGESLDWIGGIKPNNGLANYNQKFKGKATLTVDKSSSTAYMDLRSLTSEDSAVYY CARSEITTEFDYWGQGTALTVSS

According to a second particular embodiment of said aspect, the antibody according to the invention, or one of its functional fragments or derivatives, comprises a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1 comprises the amino acid sequence SEQ ID No.4, CDR-H2 comprises the amino acid sequence SEQ ID No.5 and CDR-H3 comprises the amino acid sequence SEQ ID No. 6.

According to said second embodiment, the antibody or one of its functional fragments or derivatives preferably comprises a heavy chain, the sequence of which comprises the amino acid sequence SEQ ID No. 19.

SEQ ID No.19：EVQLQQSGPELVKPGASMKISCKASGYSFTDYTLNWVKQSHGKTLEWIGLINPYNGGTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCAREEITKDFDFWGQGTTLTVSS

According to a third particular embodiment of said aspect, the antibody or one of its functional fragments or derivatives according to the invention comprises a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1 comprises the amino acid sequence SEQ ID No.7, CDR-H2 comprises the amino acid sequence SEQ ID No.8 and CDR-H3 comprises the amino acid sequence SEQ ID No. 9.

According to said third embodiment, the antibody or one of its functional fragments or derivatives preferably comprises a heavy chain, the sequence of which comprises the amino acid sequence SEQ ID No. 20.

SEQ ID No.20：EVLLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGMSLEWIGDINPNNGGTIFNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCARGRYVGYYYAMDYWGQGTSVTVSS

According to a fourth particular embodiment of said aspect, the antibody according to the invention, or one of its functional fragments or derivatives, comprises a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1 comprises the amino acid sequence SEQ ID No.56, CDR-H2 comprises the amino acid sequence SEQ ID No.57 and CDR-H3 comprises the amino acid sequence SEQ ID No. 58.

According to said fourth embodiment, the antibody or one of its functional fragments or derivatives preferably comprises a heavy chain, the sequence of which comprises the amino acid sequence SEQ ID No. 62.

SEQ ID No.62：

QVQLQQSGAELAKPGASVKMSCKASGYTFTSYWMNWVKQRPGQGLEWIGYINPTTGSTDYNQKLKDKATLTADKSSNTAYMQLSSLTSEDSAVYYCAIGGYGSWFAYWGQGTLVTVSA

In the second way, the antibody will now be defined by its light chain sequence. More specifically, 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 the CDRs comprising the amino acid sequences SEQ ID nos. 10 to 17 and 59 to 61.

The sequences mentioned are the following:

SEQ ID No.10：ESVDSYANSF

SEQ ID No.11：RAS

SEQ ID No.12：QQSKEDPLT

SEQ ID No.13：ESIDTYGNSF

SEQ ID No.14：QQSNEDPFT

SEQ ID No.15：ENIYSN

SEQ ID No.16：AAT

SEQ ID No.17：QHFWGPPYT

SEQ ID No.59：SSVSSTY

SEQ ID No.60：TTS

SEQ ID No.61：HQWSSYPFT

the CDRs of the light chain may be randomly selected among the aforementioned sequences, i.e. SEQ ID nos. 10 to 17 and 59 to 61.

According to another preferred aspect, the antibody or one of its functional fragments or derivatives according to the invention comprises a light chain comprising at least one CDR selected from the group consisting of CDR-L1, CDR-L2 and CDR-L3, wherein:

CDR-L1 comprises the amino acid sequence SEQ ID No.10, 13, 15 or 59,

CDR-L2 comprising the amino acid sequence SEQ ID No.11, 16 or 60, and

-CDR-L3 comprises the amino acid sequence SEQ ID No.12, 14, 17 or 61.

According to a first specific embodiment of said further aspect, the antibody according to the invention, or one of its functional fragments or derivatives, comprises a light chain comprising CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 comprises the amino acid sequence SEQ ID No.10, CDR-L2 comprises the amino acid sequence SEQ ID No.11 and CDR-L3 comprises the amino acid sequence SEQ ID No. 12.

More specifically, according to this first embodiment, said antibody or one of its functional fragments or derivative comprises a light chain, the sequence of which comprises the amino acid sequence SEQ ID No. 21.

SEQ ID No. 21 ：DIVLTQSPASLAVSLGQRATISCRASESVDSYANSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSKEDPLTFGSGTKLEMK

According to a second particular embodiment of said further aspect, the antibody according to the invention, or one of its functional fragments or derivatives, comprises a light chain comprising CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 comprises the amino acid sequence SEQ ID No.13, CDR-L2 comprises the amino acid sequence SEQ ID No.11 and CDR-L3 comprises the amino acid sequence SEQ ID No. 14.

According to said second embodiment, said antibody or one of its functional fragments or derivative preferably comprises a light chain, the sequence of said light chain comprising the amino acid sequence SEQ ID No. 22.

SEQ ID No. 22 ：GIVLTQSPASLAVSLGQRATISCRVSESIDTYGNSFIHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDSATYYCQQSNEDPF TFGSGTKLEMK

According to a third specific embodiment of said further aspect, the antibody according to the invention or one of its functional fragments or derivatives comprises a light chain comprising CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 comprises the amino acid sequence SEQ ID No.15, CDR-L2 comprises the amino acid sequence SEQ ID No.16 and CDR-L3 comprises the amino acid sequence SEQ ID No. 17.

According to said third embodiment, the antibody or one of its functional fragments or derivatives preferably comprises a light chain, the sequence of said light chain comprising the amino acid sequence SEQ ID No. 23.

SEQ ID No.23：DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATNLVDGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWGPPYTF GGGTKLEIK

According to a fourth specific embodiment of said further aspect, the antibody according to the invention or one of its functional fragments or derivatives comprises a light chain comprising CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 comprises the amino acid sequence SEQ ID No.59, CDR-L2 comprises the amino acid sequence SEQ ID No.60 and CDR-L3 comprises the amino acid sequence SEQ ID No. 61.

According to said third embodiment, the antibody or one of its functional fragments or derivatives preferably comprises a light chain, the sequence of said light chain comprising the amino acid sequence SEQ ID No. 63.

SEQ ID No.63：QIVLTQSPAIMSASPGEKVTLTCSASSSVSSTYLYWYQQKPGSSPKLWIYTTSILASGVPARFSGSGSGTSYSLTISSMETEDAASYFCHQWSSYPFTFGSGTKLDIK

According to a third approach, the antibody will now be defined by its light chain sequence and its heavy chain sequence. The antibody according to the invention or one of its functional fragments or derivatives is characterized in that it comprises a heavy chain and a light chain, said heavy chain comprising the amino acid sequence SEQ ID No.18, 19, 20 or 62 and said light chain comprising the amino acid sequence SEQ ID No.21, 22, 23 or 63.

More specifically, according to the invention, a preferred antibody or one of its functional fragments or derivatives, named 224G11, comprises a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences SEQ ID nos. 1,2 and 3, respectively, and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences SEQ ID nos. 10, 11 and 12, respectively.

In another aspect, antibody 224G11 comprises a heavy chain comprising the amino acid sequence of SEQ ID No.18 and a light chain comprising the amino acid sequence of SEQ ID No. 21.

According to the invention, another preferred antibody or one of its functional fragments or derivatives, designated 227H1, comprises a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences SEQ ID nos. 4,5 and 6, respectively; the light chain comprises CDR-L1, CDR-L2 and CDR-L3, which comprise the amino acid sequences SEQ ID Nos. 13, 11 and 14, respectively.

In another aspect, antibody 227H1 comprises a heavy chain comprising the amino acid sequence of SEQ ID No.19 and a light chain comprising the amino acid sequence of SEQ ID No. 22.

Another preferred antibody or one of its functional fragments or derivatives, designated 223C4, comprises a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences seq id nos. 7, 8 and 9, respectively; the light chain comprises CDR-L1, CDR-L2 and CDR-L3, which comprise the amino acid sequences SEQ ID Nos. 15, 16 and 17, respectively.

In another aspect, antibody 223C4 comprises a heavy chain comprising the amino acid sequence of SEQ ID No.20 and a light chain comprising the amino acid sequence of SEQ ID No. 23.

Another preferred antibody or one of its functional fragments or derivatives, designated 11E1, comprises a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences seq id nos. 56, 57 and 58, respectively; the light chain comprises CDR-L1, CDR-L2 and CDR-L3, which comprise the amino acid sequences SEQ ID Nos. 59, 60 and 61, respectively.

In another aspect, antibody 11E1 comprises a heavy chain comprising the amino acid sequence of SEQ ID No.62 and a light chain comprising the amino acid sequence of SEQ ID No. 63.

According to another aspect, the present invention relates to a murine hybridoma capable of secreting a monoclonal antibody according to the present invention, in particular a murine hybridoma as deposited at Collection National de Cultures de microorganisms (CNCM, National Collection of microorganisms Cultures) (Institut Pasteur, Paris, France).

The monoclonal antibody or one of its functional fragments or derivatives according to the invention is characterized in that said antibody is secreted by the hybridoma deposited under the CNCM accession number CNCM I-3724 (corresponding to 11E1), I-3731 (corresponding to 224G11), I-3732 (corresponding to 227H1) and deposited under 07/06/2007 under the accession number I-3786 (corresponding to 223C4) due to 03/14/2007. These hybridomas are murine hybridomas made by fusing immunized mouse splenocytes with cells of the myeloma cell line (Sp20Ag 14).

Table 2 below recombines the elements (elements) related to the preferred antibodies.

TABLE 2

Table 2 clearly shows that CDR-L2 of antibodies 227H1 and 224G11 are similar. This example clearly supports the claims of the present application, covering antibodies comprising at least one CDR randomly selected from the described CDR sequences.

According to a preferred embodiment, the invention relates to monoclonal antibodies.

The term "monoclonal antibody" or used according to its ordinary meaning refers to an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprised in the population are identical except for a small number of mutations that may naturally occur. In other words, a monoclonal antibody is a homologous antibody that is derived from the proliferation of a single cell clone (e.g., a hybridoma cell, a eukaryotic host cell transfected with DNA encoding the homologous antibody, a prokaryotic host cell transformed with DNA encoding the homologous antibody, etc.), and is generally characterized by a single class or subclass of heavy chains and a single type of light chains. Monoclonal antibodies are highly specific, being directed against a single antigen. In addition, each monoclonal antibody is directed against a single determinant on the antigen, as compared to polyclonal antibody preparations which typically include different antibodies directed against different defined clusters or epitopes.

In the present invention, the terms polypeptide, polypeptide sequence, amino acid sequence, peptide and protein in relation to an antibody compound or its sequence are interchangeable.

According to a likewise particular aspect, the invention relates to a chimeric antibody or one of its functional fragments, characterized in that according to the invention said antibody additionally comprises the light and heavy chain constant regions of an antibody from a species heterologous to murine, in particular human, and in a preferred manner is characterized in that the light and heavy chain constant regions from a human antibody are respectively the kappa and gamma-1, gamma-2 or gamma-4 regions.

In the present application, IgG1 preferably gain effector function, and most preferably ADCC and CDC.

The skilled artisan will recognize that effector functions include, for example, binding to C1 q; complement Dependent Cytotoxicity (CDC); binding to an Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down-regulation of cell surface receptors (e.g., B cell receptors; BCR).

The antibodies of the invention are preferably specific monoclonal antibodies, in particular of murine, chimeric or humanized origin, which can be obtained according to standard procedures well known to those skilled in the art.

In general, for the preparation of monoclonal Antibodies or functional fragments or derivatives thereof, in particular of murine origin, reference may be made, in particular, to the techniques described 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 techniques described by Kohler and Milstein, prepared from hybridomas (Nature, 256: 495-497, 1975).

The monoclonal antibody according to the invention may be obtained, for example, from animal cells immunized against c-Met or one of the fragments of c-Met comprising the epitopes specifically recognized by the monoclonal antibody according to the invention. Said c-Met, or one of said fragments thereof, can be produced, in particular according to the usual working methods, by genetic recombination starting from a nucleotide sequence containing a cDNA sequence coding for c-Met, or by peptide synthesis starting from an amino acid sequence contained in a peptide sequence of c-Met.

The monoclonal antibody of the invention may, for example, be purified on an affinity column to which c-Met or one of the fragments of c-Met containing the epitope specifically recognized by the monoclonal antibody of the invention has been previously immobilized. More specifically, the monoclonal antibody can be purified by chromatography on proteins A and/or G, with or without subsequent ion exchange chromatography, with the aim of removing residual protein contaminants and DNA and LPS, and with or without subsequent Sepharose on its own^TMExclusion chromatography on gel to remove possible aggregates due to the presence of dimers or other multimers. In an even more preferred manner, these techniques may all be used simultaneously or sequentially.

Chimeric or humanized antibodies are also encompassed by the antibodies of the invention.

By chimeric antibody is meant an antibody that contains the natural variable (light and heavy) regions of an antibody from a given species, as well as the light and heavy constant regions of an antibody from a species heterologous to the given species (e.g., mouse, horse, rabbit, dog, cow, chicken, etc.).

The chimeric type antibody or a fragment thereof according to the present invention can be produced by using a gene recombination technique. For example, a chimeric antibody can be prepared by cloning a recombinant DNA containing a promoter and a sequence encoding the variable region of a non-human (particularly murine) monoclonal antibody of the present invention and a sequence encoding the constant region of a human antibody. The chimeric antibody of the present invention encoded by the recombinant gene will be, for example, a mouse-human chimera, the specificity of which is determined by the variable region derived from mouse DNA and the isotype of which is determined by the constant region derived from human DNA. As a method for producing the chimeric antibody, reference may be made, for example, to the literature of Verhoeyn et al (BioEssays, 8: 74,1988), Morrison et al (Proc. Natl. Acad. Sci. USA 82: 6851-6855,1984) ou Ie brevet U.S. Pat. No.4,816,567.

By humanized antibody is meant an antibody that contains CDR regions from a non-human antibody, with the remainder of the antibody molecule being from one (or several) human antibodies. In addition, some residues of the segments of the scaffold (referred to as FR) may be modified to maintain binding affinity (Jones et al, Nature, 321: 522-525,1986; Verhoeyen et al, Science 239: 1534-1536,1988; Riechmann et al, Nature, 332: 323-327, 1988).

Humanized antibodies or fragments thereof of the invention may be prepared by techniques known to those of skill in the art (e.g., as described in Singer et al, J.Immun.150: 2844-2857,1992; Mountain et al, Biotechnology. Gene. Eng. Rev., 10: 1-142,1992; or Bebbington et al, Bio/Technology, 10: 169-175, 1992).

Other humanization methods are known to those skilled in the art, for example the "CDR grafting" method described in the patent applications EP0451261, EP0682040, EP09127, EP0566647 or U.S. Pat. No.5,530,101, U.S. Pat. No.6,180,370, U.S. Pat. No.5,585,089 and U.S. Pat. No.5,693,761 by Protein Design Lab (PDL). The following patent applications may also be mentioned: U.S. Pat. No.5,639,641, U.S. Pat. No.6,054,297, U.S. Pat. No.5,886,152 and U.S. Pat. No.5,877,293.

"functional fragment" of an antibody according to the invention is intended to mean, in particular, an antibody fragment such as Fv, scFv (sc for single chain), Fab, F (ab ')2, Fab', scFv-Fc fragment or diabodies, or any fragment whose half-life can be increased by chemical modification, such as the addition of a poly (alkenyl) diol, such as polyethylene glycol ("PEGylation") (the PEGylated fragment is called Fv-PEG, scFv-PEG, Fab-PEG, F (ab ')2-PEG or Fab' -PEG) ("PEG" stands for polyethylene glycol), or by the addition of liposomes, said fragment having at least one characteristic CDR whose sequence is that of the sequences SEQ ID Nos. 1 to 17 and 56 to 61 according to the invention, and which, in particular, is capable of exhibiting, in a general manner, part of the activity of the antibody from which it is derived, such as, in particular, being capable of recognizing and binding to c-Met, and, if necessary, the ability to inhibit c-Met activity.

Preferably, said functional fragment consists of or comprises a partial sequence of the heavy or light variable chain of the antibody from which they are derived, said partial sequence being sufficient to maintain the same binding specificity and sufficient affinity for c-Met as the antibody from which it is derived, preferably at least equal to 1/100, more preferably at least 1/10 of the antibody from which it is derived. Such functional fragments contain a minimum of 5 amino acids, preferably 6, 7, 8, 9, 10, 12, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which it is derived.

Preferably, these functional fragments are Fv, scFv, Fab, F (ab ')2, F (ab'), scFv-Fc type fragments or diabodies, which generally have the same binding specificity as the antibody from which they are derived. In a more preferred embodiment of the invention, these fragments are selected from bivalent fragments such as F (ab')2 fragments. According to the present invention, the antibody fragment of the present invention can be obtained from the above-described antibody by, for example, enzymatic digestion (e.g., pepsin or papain) and/or chemical reduction cleavage of disulfide bonds. In another embodiment, the antibody fragment encompassed by the present invention can be obtained by techniques of genetic recombination that are also well known to those skilled in the art or by peptide synthesis using, for example, an automated peptide synthesizer (e.g., supplied by Applied Biosystems, etc.).

By "bivalent fragment" it must be understood any antibody fragment comprising two arms, more particularly a F (ab')2 fragment.

More specifically, the invention comprises antibodies or functional fragments thereof, in particular chimeric or humanized antibodies according to the invention obtained by genetic recombination or chemical synthesis.

By "derivative" of an antibody according to the invention is meant a binding protein comprising a protein scaffold and at least one CDR selected from the original antibody to retain binding capacity. Such compounds are well known to those skilled in the art and will be described in more detail in the following specification.

More specifically, according to the invention, the antibody or one of its functional fragments or derivatives is characterized in that said derivative is a binding protein comprising a scaffold to which at least one CDR has been grafted in order to maintain the in situ paracorporeal (paracpic) recognition properties of the original antibody (original antibody).

One or several of the 6 CDR sequences described in the present invention can be placed on a protein scaffold. In this case, the protein scaffold reproduces the protein backbone with the grafted CDR or CDR sequences properly folded, allowing it (or them) to retain their antigenic in situ paracrine recognition properties.

One skilled in the art would know how to select a protein scaffold on which at least one CDR can be grafted from the original antibody. More specifically, it is known that, in order to be selected, such a stent should exhibit several characteristics (Skerra a., j.mol.recogn.,13,2000, 167-:

good conservation in terms of phylogeny,

robust structures with well-known three-dimensional molecular organization (e.g., crystallography or NMR),

-the dimensions are small, and,

no or only a low degree of post-translational modification,

easy to produce, express and purify.

Such a protein scaffold may be, but is not limited to, one whose structure is selected from fibronectin, and is preferably selected from the tenth fibronectin type III domain (FNfn 10), lipocalin (lipocalin), antiporter (anticalin, Skerra a., j.biotechnol,2001,74(4): 257-75), protein Z derived from domain B of staphylococcal protein a, thioredoxin a, or any protein with a repeat domain (such as "ankyrin repeat" (Kohlet al, PNAS,2003, vol.100, No.4,1700-1705), armadillo repeat (armadil repeat), "leucine-rich repeat", or "tetrakainic acid repeat (tetrakainic repeat)").

Also mentioned are scaffolds derived from toxins (e.g. scorpion, insect, plant or mollusc toxins) or protein inhibitors of nicotinamide nitrene oxyntomosynthase (PIN).

As a non-limiting example of such a hybrid construct, it may be mentioned that the CDR-H1 (heavy chain) of the anti-CD 4 antibody (i.e. the 13B8.2 antibody) is inserted into one exposed loop of PIN. The binding properties of the obtained proteins remained similar to the original antibodies (Bes et al, BBRC343,2006, 334-. Mention may also be made of the CDR-H3 (heavy chain) of an anti-lysozyme VHH antibody grafted onto the loop of neocarzinostatin (Nicaise et al, 2004).

In the context of the present invention, without limitation, the CDR of interest to be retained may be CDR-L2, since it is conserved in the two identified antibodies described herein (i.e., 227H1 and 224G 11).

As described above, such a protein scaffold may comprise 1 to 6 CDRs from the original antibody. In a preferred embodiment, without any limitation, the skilled person selects the CDRs of at least one heavy chain, which is known to be particularly involved in the specificity of an antibody. The selection of the CDR(s) of interest by known methods will be apparent to those skilled in the art (BES et al, FEBS letters508,2001, 67-74).

As a proof, these examples are non-limiting and any other known or described stent must also be included in this specification.

According to a new aspect, the present invention relates to an isolated nucleic acid, characterized in that it is a nucleic acid selected from the group consisting of:

a) nucleic acid, DNA or RNA encoding one of the antibodies or one of the functional fragments or "derivatives" thereof according to the invention;

b) a nucleic acid comprising a DNA sequence selected from the group consisting of:

-a core sequence comprising the sequences SEQ ID No.24, SEQ ID No.25, SEQ ID No.26 and the sequences SEQ ID No.33, SEQ ID No.34 and SEQ ID No. 35;

-a core sequence comprising the sequences SEQ ID No.27, SEQ ID No.28, SEQ ID No.29 and SEQ ID No.36, SEQ ID No.34 and SEQ ID No. 37;

-a core sequence comprising the sequences SEQ ID No.30, SEQ ID No.31, SEQ ID No.32 and the sequences SEQ ID No.38, SEQ ID No.39 and SEQ ID No. 40; and

-a core sequence comprising the sequences SEQ ID No.64, SEQ ID No.65, SEQ ID No.66 and the sequences SEQ ID No.67, SEQ ID No.68 and SEQ ID No. 69;

c) a nucleic acid comprising a DNA sequence selected from the group consisting of:

-a core sequence comprising the sequences SEQ ID No.41 and SEQ ID No. 44;

-a core sequence comprising the sequences SEQ ID No.42 and SEQ ID No. 45;

-a core sequence comprising the sequences SEQ ID No.43 and SEQ ID No. 46;

-a core sequence comprising the sequences SEQ ID No.70 and SEQ ID No. 71;

d) RNA nucleic acids corresponding to the nucleic acids defined in b) or c);

e) a nucleic acid complementary to the nucleic acid defined in a), b) and c); and

f) a nucleic acid of at least 18 nucleotides capable of hybridising under high stringency conditions to at least one CDR of sequence SEQ ID nos. 24 to 40 and 64 to 69.

The terms nucleic acid, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, as used without distinction in the present invention, are meant to refer to the precise linkage of nucleotides, modified or unmodified, allowing segments or regions of nucleic acids to be defined, with or without non-natural nucleotides, and also capable of corresponding to double-stranded DNA, single-stranded DNA and transcripts of said DNA.

It must also be understood that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, that is to say in the natural state. The invention relates to nucleic acid molecules which have been isolated and/or purified, that is to say which have been selected directly or indirectly, for example by copying, their environment having been modified at least in part. Thus, isolated nucleic acids obtained by genetic recombination, for example by means of a host cell, or by chemical synthesis are likewise referred to here.

Hybridization under high stringency conditions refers to the selection of temperature conditions and ionic strength conditions in a manner that allows hybridization to occur between two complementary DNA fragments. By way of example, for the purpose of defining the above-mentioned polynucleotide fragments, the highly stringent conditions of the hybridization step are advantageously the following conditions.

DNA-DNA or DNA-RNA hybridization is performed in two steps: (1) prehybridization at 42 ℃ for 3 hours in phosphate buffer (20mM, pH7.5) containing 5 XSSC (1 XSSC corresponds to 0.15M NaCl +0.015M sodium citrate solution), 50% formamide, 7% Sodium Dodecyl Sulfate (SDS), 10XDenhardt's, 5% dextran sulfate, and 1% salmon sperm DNA; (2) the actual hybridization is carried out for 20 hours, the hybridization temperature being determined according to the probe size (i.e.: 42 ℃ C. for probe sizes >100 nucleotides) and then washed twice at 20 ℃ for 20 minutes in 2 XSSC +2% SDS and once at 20 ℃ for 20 minutes in 0.1 XSSC +0.1% SDS. For probe sizes >100 nucleotides, the last wash was performed in 0.1x SSC +0.1% SDS at 60 ℃ for 30 minutes. For larger or smaller oligonucleotides, the skilled artisan can adjust the highly stringent hybridization conditions described above for a defined size of polynucleotide according to the teachings of Sambrook et al (1989, Molecular cloning: a laboratory manual.2nd Ed. Cold spring harbor).

The invention likewise relates to vectors comprising the nucleic acids according to the invention.

The object of the present invention is in particular a cloning and/or expression vector containing the nucleotide sequence according to the invention.

The vectors of the invention preferably contain elements which allow the expression and/or secretion of the nucleotide sequence in a defined host cell. Thus, the vector must contain a promoter, translation initiation and termination signals, and appropriate transcriptional regulatory regions. It must be able to be maintained in a stable manner in the host cell and may optionally have specific signals directing secretion of the translated protein. The skilled person can select and optimize different elements as a function of the host cell used. To this effect, the nucleotide sequences of the present invention may be inserted into an autonomously replicating vector in the selected host, or be an integrating vector for the selected host.

Such vectors are prepared by methods currently used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods (e.g., lipofection, electroporation, heat shock, or chemical methods).

The vector of the present invention is, for example, a plasmid or a virus-derived vector. They can be used to transform host cells to clone or express the nucleotide sequences of the present invention.

The invention likewise comprises a host cell transformed by a vector according to the invention or comprising a vector according to the invention.

The host cell may be selected from prokaryotic or eukaryotic systems, such as bacterial cells, likewise yeast cells or animal cells, in particular mammalian cells. It is likewise possible to use insect cells or plant cells.

The invention likewise relates to animals, other than humans, which comprise at least one transformed cell according to the invention.

According to another aspect, the subject of the invention is a method for producing an antibody according to the invention or one of its functional fragments, characterized in that it comprises the following phases:

a) culturing the cells of the invention in a culture medium and under suitable culture conditions; and

b) harvesting said antibody or one of its functional fragments thus produced from said culture medium or said cultured cells.

The transformed cells of the invention may be used in a method of producing a recombinant polypeptide of the invention. The methods for the preparation of the polypeptides in recombinant form according to the invention are characterized in that they are cells transformed with the vectors according to the invention and/or the vectors according to the invention, these methods being themselves encompassed by the invention. Preferably, cells transformed with the vector of the invention are cultured under conditions that allow expression of the polypeptide, and the recombinant polypeptide is harvested.

As already indicated, the host cell may be selected from prokaryotic or eukaryotic systems. In particular, in such prokaryotic or eukaryotic systems, it is possible to identify the nucleotide sequences according to the invention and to make them easy to secrete. The vectors of the invention with such sequences can therefore be advantageously used for the production of recombinant proteins whose secretion is desired. Indeed, the fact that these recombinant proteins of interest are present in the supernatant of the cell culture, rather than inside the host cell, will facilitate their purification.

It is likewise possible to prepare the polypeptides according to the invention by chemical synthesis. This production method is likewise subject matter of the invention. The person skilled in the art is aware of methods for chemical synthesis, for example classical synthesis by concentrating fragments or by solution using Solid phase techniques [ Steward et al,1984, Solid phase peptide synthesis, Pierce chem. company, Rockford,111,2nd ed., (1984) ] or using partial Solid phase techniques. Polypeptides obtained by chemical synthesis and capable of comprising the corresponding unnatural amino acid are also encompassed by the invention.

Antibodies or one of their functional fragments or derivatives obtainable by the process according to the invention are likewise encompassed by the invention.

The invention also relates to an antibody according to the invention as a medicament.

The invention also relates to a pharmaceutical composition comprising a compound consisting of one of the antibodies according to the invention or of one of its functional fragments, in the form of an active principle, preferably in admixture with excipients and/or pharmaceutically acceptable carriers.

Another complementary embodiment of the invention consists in a composition, such as the one described above, which additionally comprises, as a combination product, an anti-tumor antibody for simultaneous, separate or sequential use.

Most preferably, the second anti-tumor antibody may be selected from the group consisting of anti-IGF-IR, anti-EGFR, anti-HER 2/neu, anti-VEGFR, anti-VEGF, etc., an antibody or any other anti-tumor antibody known to one skilled in the art. Obviously, the use of a functional fragment or derivative of the above mentioned antibody as a second antibody is part of the present invention.

An anti-EGFR antibody such as antibody C225(Erbitux) is selected as the most preferred antibody.

By "simultaneous use" is understood both the compounds of the composition according to the invention to be administered in a single and in the same dosage form.

By "separately used" is understood two compounds of the composition of the invention that are administered in different dosage forms at the same time.

By "sequential use" is understood two compounds of the composition according to the invention, each in a different dosage form, to be administered sequentially.

In a general manner, the compositions of the present invention substantially increase the effectiveness of the treatment of malignant tumors. In other words, the therapeutic effect of the anti-c-Met antibody of the invention is enhanced in an unexpected manner by administration of a cytotoxic agent. Another major subsequent advantage produced by the composition according to the invention relates to the possibility of using lower effective doses of the active principle, which avoids or reduces the risk of side effects, in particular the effects of cytotoxic agents.

In addition, the compositions of the present invention may achieve the desired therapeutic effect more quickly.

The composition according to the invention is also characterized in that it additionally comprises, as a combined product, a cytotoxic/cytostatic agent for simultaneous, separate or sequential use.

By "anti-malignant-tumor therapeutic agent" or "cytotoxic/cytostatic agent" is meant a substance that, when administered to a subject, treats or prevents the development of a malignant tumor in the subject. As non-limiting examples of such agents, mention may be made of alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, antiangiogenic agents, antiestrogens, antiandrogens or immunomodulators.

Such formulations are cited, for example, in the page of VIDAL (2001 edition) directed to compounds related to the cancerology and hematology column "cytotoxins", which are cited in this document as preferred cytotoxic agents.

More specifically, according to the present invention, the following formulations are preferred.

By "alkylating agent" is meant any substance capable of cross-linking or alkylating any molecule, preferably a nucleic acid, such as DNA, in a cell. Examples of alkylating agents include nitrogen mustards such as mechlorethamine, chlorambucil (chlorambucol), melphalan, phenylaminoxidic acid (chlorohydrate), bromopropylpiperazine, prednimustine, disodium phosphate or estramustine phosphate; oxazophorines such as cyclophosphamide, hexamethylmelamine, chloroacetohydrofamide, sulfonfamide or ifosfamide; aziridine or ethylenimine such as thiotriamide, triethylene or altetramine; nitrosoureas such as carmustine, streptozocin, fotemustine or lomustine; alkylene sulfonates such as busulfan, busulfan or improsulfan; triazenes such as dacarbazine; or platinum complexes such as cisplatin, oxaliplatin and carboplatin.

By "antimetabolite" is meant an agent that blocks cell growth and/or metabolism by affecting certain activities, typically DNA synthesis. Examples of antimetabolites include methotrexate, 5-fluorouracil, flucoside, 5-fluorodeoxyuracil, capecitabine, cytarabine (cytarabine), fludarabine, cytarabine (cytosine arabine), 6-mercaptopurine (6-MP), 6-mercaptoguanine (6-TG), 2-chlorodeoxyadenosine (chlorodesoxyyadenosine), 5-azacytidine, 2-difluorodeoxycytidine, cladribine, deoxycoformycin, and pentostatin.

"antitumor antibiotic" refers to a compound that can prevent or inhibit DNA, RNA and/or protein synthesis. Examples of antitumor antibiotics include doxorubicin, daunorubicin, noroxytetracycline, valrubicin, mitoxantrone hydrochloride, dactinomycin, mithramycin, mitomycin C, bleomycin, and procarbazine.

"mitotic inhibitors" prevent the normal progression of the cell cycle and mitosis. In general, microtubule inhibitors or taxanes (such as paclitaxel and docetaxel) are capable of inhibiting mitosis. Vinca alkaloids (such as vinblastine, vincristine, vindesine and vinorelbine) also inhibit mitosis.

"chromatin function inhibitor" or "topoisomerase inhibitor" refers to a substance that inhibits the normal function of a chromatin modeling protein (e.g., topoisomerase I or topoisomerase II). Examples of chromatin function inhibitors include camptothecin and its derivatives such as topotecan or irinotecan for topoisomerase I and etoposide, etoposide phosphate and etoposide for topoisomerase II.

By "anti-angiogenic agent" is meant any drug, compound, substance or formulation that inhibits the growth of blood vessels. Exemplary anti-angiogenic agents include, but are not limited in any way to, propylenimine, marimastat, batimastat, prinomastat, tanostat, ilomastat, CGS-27023A, bromocloperaquine, COL-3, neovastat, BMS-275291, thalidomide, CDC501, DMXAA, L-651582, squalamine, endostatin, SU5416, SU6668, alpha-interferon, EMD121974, interleukin-12, IM862, angiostatin, and vitaxin.

"anti-estrogen" or "anti-estrogen agent" refers to any substance that reduces/antagonizes or inhibits the action of estrogen. Examples of antiestrogens are tamoxifen, toremifene, raloxifene, droloxifene, oxifene (iodoxyfene), anastrozole, letrozole and exemestane.

"antiandrogen" or "antiandrogen" refers to any substance that reduces, antagonizes, or inhibits the action of androgens. Examples of antiandrogens are flutamide, nilutamide, bicalutamide, spironolactone, cyproterone acetate, finasteride and cimetidine.

An "immunomodulator" is a substance that stimulates the immune system.

Examples of immunomodulators include interferons, interleukins such as aldesleukin (aldesleukin), OCT-43, denine interleukin (denileukin diflox) and interleukin-2, tumor necrosis factors such as tasonermine (tasonermine) or other immunomodulators such as lentinan, cizose, roquinmerac, pidotene, methoxypolyethyleneglycol succinamide adenosine deaminase, thymosin preparations, poly I: c or levamisole in combination with 5-fluorouracil.

For more details, the person skilled in the art can refer to the manual entitled "mineral de chip therapeutics, vol.6, medical instruments et perspectives days additive sites producers" edited by "Association framework des Ensegmentandsde chip therapeutics", TEC & DOC edition, 2003.

Also mentioned as chemical or cytotoxic agents are all kinase inhibitors, for example gefitinib (gefitinib) or erlotinib (erlotinib).

In a particularly preferred embodiment, said composition as a combination product according to the invention is characterized in that said cytotoxic agent is chemically coupled to said antibody for simultaneous use.

In order to facilitate the coupling of the cytotoxic agent and the antibody according to the invention, in particular, a spacer molecule, such as a poly (alkenyl) glycol like polyethylene glycol or other amino acid, may be introduced between the two compounds to be coupled, or in another embodiment, an active derivative of the cytotoxic agent into which a function capable of reacting with the antibody according to the invention has been introduced. These coupling techniques are well known to those skilled in the art and will not be expanded upon in this specification.

In another aspect, the invention relates to a composition characterized in that at least one of said antibodies or one of its functional fragments or derivatives is bound to a cytotoxin and/or a radioactive element.

Preferably, said toxin or said radioactive element is capable of inhibiting the activity of at least one cell expressing c-Met cells, and in a more preferred manner of preventing the growth or proliferation of said cells, in particular of completely inactivating said cells.

Also preferably, the toxin is a bacterial enterotoxin (enterotoxin), in particular pseudomonas exotoxin a.

Preferred radioactive elements (or radioisotopes) for binding to antibodies for use in therapy are gamma-emitting radioisotopes, preferably iodine¹³¹Yttrium, yttrium⁹⁰Gold, gold¹⁹⁹Palladium, palladium¹⁰⁰Copper, copper⁶⁷Bismuth, bismuth²¹⁷And antimony²¹¹. Radioisotopes that emit both beta and alpha radiation are also useful in therapy.

By toxin or radioactive element binding to at least one antibody or one of its functional fragments, it is meant according to the invention any means allowing said toxin or said radioactive element to bind to said at least one antibody, in particular by covalent cross-linking between the two compounds, with or without the introduction of a linking molecule.

Among the formulations which allow chemically (covalently), electrostatically, non-covalently binding all or part of the components of the conjugate, mention may be made in particular of benzoquinone, carbodiimide and more particularly of EDC (1-ethyl-3- [3-dimethyl-aminopropy ] -carbodiimide hydrochloride, 1-ethyl-3- [3-dimethyl-aminopropyl ] -carbodiimide hydrochloride, bismaleimide (dimaleimide), dithio-dinitrobenzoic acid (dithiobis-nitrobenzoic acid (DTNB)), N-succinimidyl S-acetyl thioester (N-succinimidyl S-acetoxy-acetate (SATA), linkers having one or more azidobenzene groups reactive with UV light (U.V.), preferably N- [ -4- (azidosalicylamino) butyl ] -3'- (2' -pyridyldithio) -propionamide (APDP) (N-) [ -4- (azidosalicylamino) butyl ] -3'- (2' -pyridyldithio) -propioamide), N-succinimidyl3- (2-pyridyldithio) propionate (SPDP) (N-succinimidyl 3- (2-pyridyldithio) propiolate), 6-Hydrazinonicotinamide (HYNIC) (6-hydrazino-nicotinoamide).

Another form of coupling, particularly for radioactive elements, consists in using bifunctional ion chelators.

Among these chelating agents, mention may be made of those derived from EDTA (ethylenediaminetetraacetic acid) or from DTPA (diethylenetriaminepentaacetic acid), both of which have been developed for binding metals, in particular radioactive metals, and immunoglobulins. Thus, DTPA and its derivatives can be substituted with different groups on the carbon chain to increase the stability and rigidity of the ligand-metal complex (Krejcarek et al (1977); Brechbiel et al (1991); Gansow (1991); U.S. Pat. No.4,831,175).

For example, diethylenetriaminepentaacetic acid (DTPA) and its derivatives have long been widely used in medicine and biology (either in free form or as complexes with metal ions), which have the remarkable characteristics of forming stable chelates with metal ions and with proteins of therapeutic or diagnostic interest (e.g., antibodies), for the development of radioimmunoconjugates (Meases et. al., (1984); Gansow et. al., (1990)).

Also preferably, said at least one antibody forming said conjugate of the invention is selected from functional fragments thereof, in particular fragments from which their Fc component has been removed, such as scFv fragments.

As already mentioned, in a particular embodiment of the invention, said cytotoxic/cytostatic agent or said toxin and/or radioactive element is chemically coupled to at least one component of said composition for simultaneous use.

The invention includes the described compositions as medicaments.

Furthermore, the invention includes the use of a composition according to the invention for the preparation of a medicament.

In another aspect, the invention relates to the use of an antibody or one of its functional fragments or derivatives, and/or a composition as described above, for the preparation of a medicament for inhibiting the growth and/or proliferation of tumor cells.

Another aspect of the invention is the use of the antibody or one of its functional fragments or derivatives and/or the composition as described above, or the use as described above, for the preparation of a medicament for the prevention or treatment of malignant tumors.

The invention also includes a method of inhibiting the growth and/or proliferation of tumor cells in a patient, comprising administering to a patient in need thereof an antibody or one of its functional fragments or derivatives according to the invention, an antibody produced by a hybridoma according to the invention or a composition according to the invention.

The invention further comprises a method for the prevention or treatment of a malignant tumor in a patient in need thereof, which method comprises administering to the patient an antibody according to the invention or one of its functional fragments or derivatives, an antibody produced by a hybridoma according to the invention or a composition according to the invention.

In a particularly preferred aspect, the malignancy is a malignancy selected from prostate cancer, osteosarcoma, lung cancer, breast cancer, endometrial cancer, glioblastoma, or colon cancer.

As explained previously, one advantage of the present invention is to allow the treatment of malignancies associated with HGF-dependent and independent Met activation.

Another aspect of the invention comprises a method for the in vitro diagnosis of a disease induced by the overexpression or underexpression of the c-Met receptor, starting from a biological sample suspected of having an abnormal presence of the c-Met receptor, said method being characterized in that it comprises a step of contacting said biological sample with an antibody according to the invention, possibly labelled, if necessary.

Preferably, the disease associated with the abnormal presence of the c-Met receptor in said diagnostic method is a malignancy.

The antibody or one of its functional fragments may be present in the form of an immunoconjugate or a labeled antibody to obtain a detectable and/or quantifiable signal.

The labeled antibody or functional fragment thereof according to the present invention includes, for example, an antibody called an immunoconjugate, which antibody can be conjugated, for example, to an enzyme such as peroxidase, alkaline phosphatase, β -D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, or glucose-6-phosphate dehydrogenase, or via a molecule such as biotin, digoxgenin, or 5-bromodeoxyuridine. Fluorescent labels may also be conjugated to the antibodies or functional fragments thereof according to the present invention and include, inter alia, fluorescein and its derivatives, fluorochromes (fluorochromes), rhodamine and its derivatives, GFP (GFP stands for green fluorescent protein), dansyl, umbelliferone, etc. In such conjugates, the antibodies or functional fragments thereof of the present invention may be prepared by methods known to those skilled in the art. They may be coupled directly to the enzyme or fluorescent label, or via an intervening spacer group or linking group such as a polyaldehyde (polyaldehyde) such as glutaraldehyde, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), or to the enzyme or fluorescent label in the presence of a coupling agent such as those mentioned above for the therapeutic conjugates. A label containing a fluorescein type can be prepared by reacting with an isothiocyanate.

Other conjugates may also include chemiluminescent labels such as luminol and diepoxy propane (dioxetanes), bioluminescent labels such as luciferase and luciferin, or other radioactive labels such as iodine¹²³Iodine, iodine¹²⁵Iodine, iodine¹²⁶Iodine, iodine¹³³Bromine, bromine⁷⁷Technetium, technetium^99mIndium, indium¹¹Indium, indium^113mGallium, gallium⁶⁷Gallium, gallium⁶⁸Ruthenium (II) and (III)⁹⁵Ruthenium (II) and (III)⁹⁷Ruthenium (II) and (III)¹⁰³Ruthenium (II) and (III)¹⁰⁵Mercury, mercury¹⁰⁷Mercury, mercury²⁰³Rhenium^99mRhenium¹⁰¹Rhenium¹⁰⁵Scandium (III)⁴⁷Tellurium^121mTellurium^122mTellurium^125mThulium, thulium¹⁶⁵Thulium, thulium¹⁶⁷Thulium, thulium¹⁶⁸Fluorine¹⁸Yttrium, yttrium¹⁹⁹Iodine, iodine¹³¹. Existing methods known to those skilled in the art for coupling therapeutic radioisotopes to antibodies, either directly or via chelating agents such as the above-mentioned EDTA, DTPA, can be used for the radioactive elements that can be used in diagnostics. It may also be mentioned that by the chloramine-T method [ Hunter w.m. and greenwood f.c. (1962) Nature 194: 495]With Na [ I125 ]]Labeling, or another technique using Crockford et al (U.S. Pat. No.4,424,200), with technetium 99m, or labeling via DTPA as described by Hnatowich (U.S. Pat. No.4,479,930).

The antibody or functional fragment thereof according to the invention can therefore be used in a method for detecting and/or quantifying the overexpression or underexpression (preferably overexpression) of the c-Met receptor in a biological sample, characterized in that it comprises the following steps:

a) contacting said biological sample with one of the antibodies or functional fragments thereof according to the invention;

b) the possible formation of c-Met/antibody complexes was demonstrated.

In a particular embodiment, the antibodies or functional fragments thereof according to the invention can be used in a method for detecting and/or quantifying the c-Met receptor in a biological sample, for monitoring the effect of a prophylactic and/or therapeutic treatment of a c-Met-dependent malignancy.

More generally, the antibodies or functional fragments thereof according to the invention can be advantageously used in any situation where the expression of the c-Met receptor has to be observed in a qualitative and/or quantitative manner.

Preferably, the biological sample is formed from a biological fluid, such as serum, whole blood, cells, a tissue sample or living tissue of human origin.

Any procedure or routine test may be used to perform such detection and/or administration (dosage). The assay may be a competitive or sandwich assay, or any assay known to those skilled in the art that relies on the formation of antigen-antibody immune complexes. After the use according to the invention, the antibody or one of its functional fragments can be immobilized or labeled. Such immobilization can be carried out on a number of supports known to those skilled in the art. These supports may include, in particular, glass, polystyrene, polypropylene, polyethylene, dextran, nylon or natural or modified cells. These supports may be soluble or insoluble.

By way of example, a preferred method of performing the immunoenzymatic method is according to the ELISA technique, by immunofluorescence or Radioimmunoassay (RIA) technique or equivalent.

The invention therefore likewise comprises a kit or set (set) for carrying out a diagnostic method for diseases induced by the overexpression or underexpression of the c-Met receptor, or for carrying out a method for the detection and/or quantification of the overexpression or underexpression of the c-Met receptor (preferably of the overexpression of said receptor) in a biological sample, characterized in that said kit or set comprises the following components:

a) an antibody according to the invention or one of its functional fragments;

b) optionally, reagents for forming a mediator to facilitate the immune response;

c) alternatively, the reagent may demonstrate the c-Met/antibody complex produced by the immune response.

The subject of the invention is also the use of the antibodies or compositions according to the invention for the preparation of a medicament for the specific targeting of biologically active compounds to cells expressing or overexpressing the c-Met receptor.

By biologically active compound is meant herein any compound capable of modulating, in particular inhibiting, the activity of a cell, in particular its growth, its proliferation, transcription or gene translation.

Subject of the present invention is also an in vivo diagnostic agent comprising an antibody or one of its functional fragments (preferably labeled, in particular radiolabeled, or one of its functional fragments) according to the invention, and its use in medical imaging, in particular for the detection of malignancies associated with the expression or overexpression of the c-Met receptor by cells.

The invention also relates to a composition as a combination product according to the invention as a medicament or to an anti-c-Met/toxin conjugate or a radioactive element.

Preferably, said composition is mixed as a product or a conjugate according to the invention with excipients and/or pharmaceutically acceptable carriers.

In the present specification, a pharmaceutically acceptable carrier refers to a compound or combination of compounds that enters a pharmaceutical composition without eliciting side effects and that allows, for example, facilitating administration of the active compound, increasing its lifetime and/or its effects in vivo, increasing its solubility in solution or improving its preservation. Such pharmaceutically acceptable carriers are well known to those skilled in the art and may be adjusted depending on the nature and mode of administration of the active compound selected.

Preferably, these compounds are administered by the systemic route, in particular by the intravenous route, intramuscular, intradermal, intraperitoneal or subcutaneous route or by the oral route. In a more preferred mode, the composition comprising the antibody of the invention is administered several times in a continuous manner.

Their mode of administration, dosage and optimized dosage form may be determined according to criteria that generally need to be considered in determining a treatment appropriate for the patient (e.g., the age or weight of the patient, the severity of his/her general condition, tolerance to the treatment and observed side effects).

Drawings

Further features and advantages of the invention are explained in the description of embodiments and in the drawings, in which:

FIG. 1: examples of FACS profiles of selected anti-c-Met antibodies;

fig. 2A and 2B: the c-Met-targeting antibody inhibits the proliferation of BXPC3 in vitro;

FIG. 3: inhibition of c-Met dimerization;

FIG. 4: a protein recognized by an anti-c-Met antibody;

fig. 5A and 5B: "epitope mapping" of 11E1 and 5D5 by BIAcore analysis;

fig. 6A and 6B: the effect of Mab on c-Met phosphorylation;

fig. 7A and 7B: replacement of radiolabeled HGF by an anti-c-Met antibody;

FIG. 8: anti-c-Met antibody inhibited infiltration [ in this figure, SVF denotes Fetal Calf Serum (FCS) ];

FIG. 9: the effect of anti-c-Met antibodies on wound healing;

fig. 10A and 10B: scattering measurement;

FIG. 11: three-dimensional luminal formation (tubulogenesis) assay;

fig. 12A and 12B: the effect of antibodies on spheroid formation;

FIG. 13: in vivo activity of anti-c-Met Mab in U87MG xenograft model;

FIG. 14: HGF expression of a panel of tumor cell lines;

fig. 15A and 15B: characteristics of the NCI-H441 cell line; FIG. 15A corresponds to quantitative RT-PCR analysis and FIG. 15B corresponds to FACS analysis;

FIG. 16: in vivo activity of anti-c-Met antibodies on the NCI-H441 xenograft model;

FIG. 17A: alignment of 224G11VL with murine IGKV3-5 x 01 germline genes;

FIG. 17B: alignment of 224G11VL with murine IGKJ4 x 01 germline genes;

FIG. 18A: 224G11VL alignment with human IGKV3-l l x 01 and IGKV4-1 x 01 germline genes;

FIG. 18B: alignment of 224G11VL with human IGKJ4 × 02 germline genes;

FIG. 19A: a humanized version of 224G11VL based on IGKV3-l l x 01 with the mentioned mutations;

FIG. 19B: a humanized version of 224G11VL based on IGKV4-l × 01 with the mentioned mutations;

FIG. 20A: alignment of 224G11VH with murine IGHV1-18 x 01 germline genes;

FIG. 20B: alignment of 224G11VH with murine IGHD2-4 x 01 germline gene;

FIG. 20C: alignment of 224G11VH with murine IGHJ 2x 01 germline genes;

FIG. 21A: alignment of 224G11VH with human IGHV1-2 x 02 germline genes;

FIG. 21B: 224G11VH to human IGHJ4 x 01 germline gene alignment;

FIG. 22: humanized 224G11VH with the mentioned mutations;

FIG. 23A: 227H1VL alignment with murine IGKV3-5 x 01 germline genes;

FIG. 23B: 227H1VL alignment with murine IGKJ4 x 01 germline genes;

FIG. 24A: 227H1VL alignment with human IGKV3-11 x 01 and IGKV4-l x 01 germline genes;

FIG. 24B: 227H1VL alignment with human IGKJ4 × 02 germline genes;

FIG. 25A: a humanized version based on IGKV3-11 x 01 of 227H1VL with the mentioned mutations;

FIG. 25B: a humanized version based on IGKV4-l × 01 of 227H1VL with the mentioned mutations;

FIG. 26A: 227H1VH alignment with murine IGHV1-18 x 01 germline genes;

FIG. 26B: 227H1VH alignment with murine IGHD1-1 × 02 germline genes;

FIG. 26C: 227H1VH alignment with murine IGHJ 2x 01 germline genes;

fig. 27A: 227H1VH alignment with human IGHV1-2 x 02 germline genes;

FIG. 27B: 227H1VH alignment with human IGHJ4 x 01 germline gene;

FIG. 28: humanized 227H1VH with the mentioned mutations;

fig. 29A: alignment of 223C4VL with murine IGKV12-46 x 01 germline genes;

FIG. 29B: alignment of 223C4VL with murine IGKJ 2x 01 germline genes;

FIG. 30A: alignment of 223C4VL with human IGKVl-NLl x 01 germline gene;

FIG. 30B: alignment of 223C4VL with human IGKJ 2x 01 germline gene;

FIG. 31: humanized 223C4VL with the mentioned mutations;

fig. 32A: alignment of 223C4VH with murine IGHVl-18 x 01 germline genes;

FIG. 32B: alignment of 223C4VH with murine IGHD6-3 x 01 germline gene;

FIG. 32C: alignment of 223C4VH with murine IGHJ4 x 01 germline gene;

fig. 33A: alignment of 223C4VH with human IGHV1-2 x 02 germline genes;

FIG. 33B: alignment of 223C4VH with human IGHD1-26 x 01 germline gene;

FIG. 33C: alignment of 223C4VH with human IGHJ6 x 01 germline gene; and

FIG. 34: humanized 223C4VH with the mentioned mutations;

FIG. 35: in an established xenograft NCI-H441 tumor model, murine 224G11Mab alone or with NovisanCombined anti-tumor activity;

FIG. 36: evaluation of HUVEC proliferation by anti-c-Met Mab;

FIG. 37: evaluation of anti-c-Met Mab formation of HUVEC luminal-like structures;

fig. 38A: 11E1VL alignment with murine IGKV4-79 x 01 germline genes;

FIG. 38B: 11E1VL alignment with murine IGKJ4 x 01 germline genes;

FIG. 39A: 11E1VL alignment with human IGKV3D-7 x 01 germline genes;

FIG. 39B: 11E1VL alignment with human IGKJ4 × 02 germline genes;

FIG. 40: a humanized version of 11E1VL with the mentioned mutations;

fig. 41A: 11E1VH alignment with murine IGHV1-7 x 01 germline genes;

FIG. 41B: 11E1VH alignment with murine IGHD4-l x 01 germline genes;

FIG. 41C: 11E1VH alignment with murine IGHJ 3x 01 germline genes;

FIG. 42A: 11E1VH alignment with human IGHV1-2 x 02 and IGHV1-46 x 01 germline genes;

FIG. 42B: 11E1VH alignment with human IGHJ4 x 03 germline genes;

FIG. 43: humanized 11E1VH with the mentioned mutations;

fig. 44A and 44B: c-Met phosphorylation assay in A549 cells. In the absence or presence of HGF, for 30 μ g/ml (FIG. 44A) or for determining EC₅₀Evaluation of 11E1 and 224G11 purified mabs over a dose range of values from 0.0015 to 30 μ G/ml (fig. 44B);

FIG. 45: combined use of 224G11Mab and Noviben in NSCLC NCI-H441 xenograft model in vivo

FIG. 46: in the NSCLC NCI-H441 xenograft model, 224G11Mab was used in combination with doxorubicin in vivo;

FIG. 47: in vivo combination of 224G11Mab and docetaxel in a NSCLC NCI-H441 xenograft model;

FIG. 48: in a NSCLC NCI-H441 xenograft model, 224G11Mab in combination with temozolomide in vivo;

fig. 49A, 49B, 49C, and 49D: the effect of anti-c-Met Mab on U87-MG spheroid growth;

fig. 50A and 50B: the in vitro activity of the chimeric and humanized forms of 224G11 in the phospho-c-Met assay;

FIG. 51: a Biacore analysis device;

FIG. 52: 224G11 activity in vivo on MDA-MB-231 cells co-implanted with MRC5 cells derived from human HGF in athymic nude mice;

FIG. 53: ELISA-based binding assay for Fc-cMet. anti-Fc-c-Met binding activity was measured in an ELISA-based assay, where anti-murine Fc binders were used to detect purified murine monoclonal antibodies 11E1, 224G11 and 227H1. Dose-dependent binding activity to plastic-coated recombinant Fc-cMet was measured at 450 nm;

FIG. 54: competition assay for HGF-cMet. In this ELISA-based assay, recombinant Fc-cMET remnant bound to plastic coated HGF in the presence of purified murine monoclonal antibodies 11E1, 224G11 and 227H1 was detected with an anti-murine Fc conjugate and measured at 450 nm;

FIG. 55: 227H 1-derived recombinant VH regions. The 227H1VH amino acid sequence was aligned with the selected human acceptor framework sequence (human receiving frame sequence) with only the mentioned amino acids found to be different from the murine 227H1VH sequence. The 227H1HZl, HZ2 and HZ3VH sequences correspond to the completed humanized version of the 227H1 murine VH region, with the remaining murine residues shown in bold. At HZ3, 10 residues (×) were automatically changed to their human counterparts. At HZ2, seven residues from the third group (3) were studied. In HZlVH, nine residues from the second group (2) were mutated to their human counterparts, and only six residues from the first group (1) were still murine;

FIG. 56: ELISA-based binding assays performed on Fc-cMet of recombinant 227H1 antibody. anti-Fc-cMet binding activity was measured in an ELISA-based assay, where anti-human Fc conjugates were used to detect chimeric and humanized 227H 1-derived recombinant antibodies. The dose-dependent binding activity of the humanized VH region derived 227H1 antibody to plastic coated recombinant Fc-cMet was measured at 450nm and then compared to those of the parental/reference chimeric antibodies;

FIG. 57: ELISA-based binding assay of Fc-cMet of recombinant 227H 1-derived antibodies. anti-Fc-cMet binding activity was measured in an ELISA-based assay, where anti-human Fc conjugates were used to detect chimeric and humanized 227H 1-derived recombinant antibodies. Dose-dependent binding activity of plastic coated recombinant Fc-cMet of humanized HZ4 VH-derived 227H1 antibody was measured at 450nm and then compared to those of the parental/reference chimeric antibodies;

FIG. 58: HGF-cMet competition assay for murine and recombinant antibodies of 227H1. In this ELISA-based assay, recombinant Fc-cMet residues bound to plastic coated HGF were detected with biotinylated irrelevant anti-cMet antibody in the presence of a different form of 227H1 antibody. Testing purified murine 227H1 monoclonal antibody, chimeric and HZ4VH derived humanized 227H1 derived recombinant antibody and comparing their ability to compete with HGF-cMet binding when measured at 450 nm;

FIG. 59: 227H1-HZ VH humanized variable domain sequences. Corresponding to amino acids that were actually changed to their human counterparts; | A Corresponding to the amino acids humanized during the completion of HZ3 to HZl; corresponding to the amino acids ultimately humanized in the 227H1-HZ VH sequence;

FIG. 60: 11E 1-derived recombinant VH regions. The 11E1VH amino acid sequence was aligned with the selected human acceptor framework sequence with only the mentioned amino acids found to be different from the murine 11E1VH sequence. The 11E1HZ VHl, VH2 and VH3 sequences correspond to the completed humanized version of the 11E1 murine VH region, with the remaining murine residues shown in bold. At HZ VH3, seven residues (×) were automatically changed to their human counterparts. Seven residues from the third group (3) were studied in HZ VH 2. At the HZ VHl, five residues from the second group (2) were mutated to their human counterparts, only five residues from the first group (1) were still murine;

FIG. 61: ELISA-based binding assay for Fc-cMet of recombinant 11E1 antibody. anti-Fc-cMet binding activity was measured in an ELISA-based assay, where anti-human Fc conjugates were used to detect chimeric and humanized 11E 1-derived recombinant antibodies. The dose-dependent binding activity of the humanized VH region derived 11E1 antibody to plastic coated recombinant Fc-cMet was measured at 450nm and then compared to those parental/reference chimeric antibodies;

FIG. 62: 11E 1-derived recombinant VL regions. The 11E1VL amino acid sequence was aligned with the selected human acceptor framework sequence with only the mentioned amino acids found to be different from the murine 11E1VL sequence. The 11E1HZ VLl, VL2 and VL3 sequences correspond to the completed humanized version of the 11E1 murine VL region, with the remaining murine residues shown in bold. At HZ VL3, ten residues (×) were automatically changed to their human counterparts. Eight residues from the third group (3) were studied at HZ VL 2. At HZ VLl, eight residues from the second group (2) were mutated to their human counterparts, and only four residues from the first group (1) were still murine;

FIG. 63: ELISA-based binding assay for Fc-cMet of recombinant 11E1 antibody. anti-Fc-cMet binding activity was measured in an ELISA-based assay, where anti-human Fc conjugates were used to detect chimeric and humanized 11E 1-derived recombinant antibodies. The dose-dependent binding activity of the humanized VL region derived 11E1 antibody to plastic coated recombinant Fc-cMet was measured at 450nm and then compared to those parental/reference chimeric antibodies;

FIG. 64: ELISA-based binding assay for Fc-cMet of recombinant 11E1 antibody. anti-Fc-cMet binding activity was measured in an ELISA-based assay, where anti-human Fc conjugates were used to detect chimeric and humanized 11E 1-derived recombinant antibodies. The dose-dependent binding activity of the single or dual humanised region derived 11E1 antibody to the plastic coated recombinant Fc-cMet was measured at 450nm and then compared to those of the parent/reference chimeric antibodies;

FIG. 65: 224G11VH region sequence. The 224G11VH amino acid sequence was aligned with the 227H1VH sequence (underlined non-homologous residues) and the selected human acceptor framework sequence, with only the amino acids mentioned which were found to be different from the murine 224G11VH sequence. The 224G11HZ VH0 sequence corresponds to the humanized version of the 224G11 murine VH region "227H 1/full IMGT" ("227H 1-based/full-IMGT"). Within this sequence, non-external-IMGT-CDR (no outside-IMGT-CDR) residues are still murine.

FIG. 66: ELISA-based binding assay of Fc-cMet to recombinant 224G11 antibody. anti-Fc-cMet binding activity was measured in an ELISA-based assay where anti-human Fc conjugates were used to detect chimeric and HZVH 0-derived humanized 224G 11-derived recombinant antibodies. The dose-dependent binding activity of the HZVH0 "fully IMGT" humanized VH region-derived 224G11 antibody to plastic-coated recombinant Fc-cMet was measured at 450nm and then compared to those parental/reference chimeric antibodies;

FIG. 67: 224G11 murine and recombinant antibodies were assayed for HGF-cMet competition. In this ELISA-based assay, recombinant Fc-cMet residues bound to plastic coated HGF were detected with biotinylated irrelevant anti-cMet antibody in the presence of a different form of the 224G11 antibody. Purified murine 224G11 monoclonal antibody, chimeric and HZVH0 derived humanized 224G11 derived recombinant antibodies were tested and compared for their ability to compete with HGF-cMet binding when measured at 450 nm;

FIG. 68: 224G11VL region sequence. The 224G11VL amino acid sequence was aligned with two selected human acceptor framework sequences, with only the amino acids mentioned found to be different from the murine 224G11VL sequence. The 224G11HZ VL3 sequence corresponds to the humanized version of the "shorter CDR 1" of the 224G11 murine VH region, while HZ VL6 corresponds to the "longer CDR 1" version, with the remaining murine residues shown in bold. For both basic humanized versions, the remaining murine residues were ranked for further humanization process, here corresponding to the amino acids that were humanized in the basic version, and 3,2 and 1 corresponding to the residue groups designed for the humanized version of the application;

FIG. 69: ELISA-based binding assay of Fc-cMet to recombinant 224G11 antibody. anti-Fc-cMet binding activity was measured in an ELISA-based assay where anti-human Fc conjugates were used to detect chimeric and humanized 22G 11-derived recombinant antibodies. The dose-dependent binding activity of humanized VL3 and VL6 region-derived 224G11 antibodies to plastic-coated recombinant Fc-cMet was measured at 450nm and then compared to those parental/reference chimeric antibodies;

FIG. 70: ELISA-based binding assay of Fc-cMet to recombinant 224G11 antibody. anti-Fc-cMet binding activity was measured in an ELISA-based assay where anti-human Fc conjugates were used to detect chimeric and humanized 224G 11-derived recombinant antibodies. The dose-dependent binding activity of the humanized VL region-derived 224G11 antibody to plastic-coated recombinant Fc-cMet was measured at 450nm and then compared to those parental/reference chimeric antibodies;

FIG. 71: 224G11 murine and recombinant antibodies were assayed for HGF-cMet competition. In this ELISA-based assay, recombinant Fc-cMet residues bound to plastic coated HGF in the presence of a different form of the 224G11 antibody were detected with biotinylated unrelated anti-cMet antibody. Purified murine 224G11 monoclonal antibody, chimeric and HZ VL4 derived humanized 224G11 derived recombinant antibodies were tested and compared for their ability to compete with HGF-cMet binding when measured at 450 nm;

FIG. 72: alignment of amino acid sequences of VL4 humanized 224G11VL region sequences. Corresponding to the amino acids that were actually changed to their human counterparts in the basic HZ VL6 version; | A Corresponding to amino acids that were humanized during the completion of HZ VL6 to HZ VL 4; corresponding to amino acids that are still murine in the 224G11-HZ VL4 sequence;

FIG. 73: ELISA-based binding assay of Fc-cMet to recombinant 224G11 antibody. anti-Fc-cMet binding activity was measured in an ELISA-based assay where anti-human Fc conjugates were used to detect chimeric and humanized 22G 11-derived recombinant antibodies. The dose-dependent binding activity of the plastic-coated recombinant Fc-cMet of the single or dual humanized domain derived 224G11 antibody was measured at 450nm and then compared to those of the parental/reference chimeric antibodies;

FIG. 74: 224G11 murine and recombinant antibodies were assayed for HGF-cMet competition. In this ELISA-based assay, recombinant Fc-cMet residues bound to plastic coated HGF in the presence of a different form of the 224G11 antibody were detected with biotinylated unrelated anti-cMet antibody. Purified murine 224G11 monoclonal antibody, chimeric and fully humanized 224G11 derived recombinant antibodies were tested and compared for their ability to compete with HGF-cMet binding when measured at 450 nm;

FIG. 75: ELISA-based binding assay of Fc-cMet to recombinant 224G11 antibody. anti-Fc-cMet binding activity was measured in an ELISA-based assay where anti-human Fc conjugates were used to detect chimeric and humanized 22G 11-derived recombinant antibodies. Dose-dependent binding activity of plastic-coated recombinant Fc-cMet of a single mutant of the fully humanized 224G11 antibody from VL4 was measured at 450nm and then compared to those of the parent/reference chimeric antibodies;

FIG. 76: ELISA-based binding assay of Fc-cMet to recombinant 224G11 antibody. anti-Fc-cMet binding activity was measured in an ELISA-based assay where anti-human Fc conjugates were used to detect chimeric and humanized 22G 11-derived recombinant antibodies. Dose-dependent binding activity of plastic-coated recombinant Fc-cMet of single and multiple mutants of VL 4-derived fully humanized 224G11 antibody was measured at 450nm and then compared to those of the parent/reference chimeric antibodies; and

FIG. 77: 224G11 murine and recombinant antibodies were assayed for HGF-cMet competition. In this ELISA-based assay, recombinant Fc-cMet residues bound to plastic coated HGF in the presence of a different form of the 224G11 antibody were detected with biotinylated unrelated anti-cMet antibody. Purified murine 224G11 monoclonal antibody, single or multiple mutants of chimeric and VL4 derived fully humanized 224G11 recombinant antibody were tested and compared for their ability to compete with HGF-cMet binding when measured at 450 nm;

Detailed Description

Example 1: production of anti-c-Met antibodies

To generate anti-c-Met antibodies, 8 week old BALB/c mice were immunized 3 to 5 times subcutaneously (20X 10) with CHO-transfected cell lines⁶Cell/dose/mouse) expressing c-Met on its cell membrane, or using a c-Met ectodomain fusion protein (10-15. mu.g/dose/mouse) (R)&D Systems, Catalog #358MT), or a fragment of the recombinant protein mixed with freonella adjuvant, 2 to 3 immunizations, the first immunization with complete freonella adjuvant and the subsequent immunization with incomplete freonella adjuvant. A mixed protocol was also performed in which mice received CHO-cMet cells and recombinant protein vaccination. Three before cell fusionDay, mice were given a bolus dose of recombinant protein or fragment by intraperitoneal or intravenous injection. Mouse spleens were then harvested and fused with SP2/0-Agl4 myeloma cells (ATCC) and HAT selected. Four fusions were performed. In general, for the preparation of monoclonal Antibodies or their functional fragments, in particular of murine origin, reference may be made to the techniques specifically mentioned in the handbook "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp.726,1988) or to the techniques for the preparation of hybridomas as described by Kohler and Milstein (Nature, 256: 495-497, 1975).

The resulting hybridomas were initially screened for c-Met recombinant protein by ELISA, and then screened for A549NSCLC, BxPC3 pancreatic, and U87-MG glioblastoma cell lines by FACS analysis (representative profiles are shown in FIG. 1) to ensure that the antibodies produced also recognize the native receptor on the tumor cells. In both experiments, positive responders were amplified, cloned, and a panel of hybridomas purified and screened for their ability to inhibit proliferation of cells in the BxPC3 model in vitro.

For this, 50000BxPC3 cells were plated in RPMI medium, 2mM l. glutamine, in SVF-free 96-well plates. After 24 hours of tiling, the antibody to be tested was added at a final concentration ranging from 0.0097 to 40 μ g/ml 60 minutes before the addition of 100ng/ml of hgf. After 3 days, the cells were treated with 0.5. mu. Ci of [ 2], [ 2]³H]Thymine was pulsed for 16 hours. Bound to DNA insoluble in trichloroacetic acid³H]The amount of thymine was quantified by liquid scintillation counting. The results are expressed as raw data to truly assess the intrinsic antagonistic effect of each Mab (fig. 2A and 2B).

The supernatants of the c-Met transfected cells were then analyzed by BRET to evaluate antibodies that inhibited cell proliferation by at least 50%. For this purpose, CHO stable cell lines expressing C-Met-Rluc or C-Met-Rluc and C-Met-K1100A-YFP were generated. One or two days prior to BRET experiments, cells were seeded in white 96-well plates containing DMEM-F12/FBS5% medium. To attach the cells to the plate, the cells were first cultured in 5% CO2 at 37 ℃. Cells were then starved overnight with 200 μ l of DMEM/well. Just prior to this experiment, DMEM was removed and cells were washed rapidly with PBS. Cells were incubated in PBS in the presence or absence of test antibody or reference compound for 10 minutes at 37 ℃ in a final volume of 50 μ l prior to addition of coelenterazine with or without HGF. After an additional 10 minutes of incubation at 37 ℃, the light emission obtained at 485nm and 530nm was started (initial) with a Mithras luminometer (Berthold) (1 s/wavelength/well, repeated 15 times).

BRET ratio was previously defined [ Angers et al, proc.natl.acad.sci.usa,2000, 97: 3684-3689] as follows: [ (530nm emission) - (485nm emission) X Cf ]/(485nm emission), where Cf corresponds to (530nm emission)/(485 nm emission) for cells expressing only Rluc fusion protein under the same conditions. Simplifying the equation indicates that BRET ratio corresponds to the 530/485nm ratio obtained when both partners are present, corrected for the 530/485nm ratio obtained when only the partner fused to r. For reasons of readability, the results are expressed in milliBRET units (mBU); mBU corresponds to the BRET ratio multiplied by 1000.

After this second in vitro test, 4 antibodies were selected that i) had no intrinsic activity as whole molecules in a functional test of proliferation, ii) significantly inhibited BxPC3 proliferation (fig. 2A and 2B) and iii) inhibited c-Met dimerization (fig. 3). Antibodies of these 3 IgGl κ isotypes are described as 11E1, 224G11, 223C4 and 227h1 in experiments, 5D5Mab, produced by Genentech and available at the ATCC, was added as a control for intrinsic antagonistic activity.

Figures 2A and 2B show that, in contrast to 5D5, 11El, 224G11, 223C4, and 227H1 did not have any agonist activity, whereas 5D5 induced a dose-dependent stimulatory effect on cell proliferation in the absence of ligand. Significant inhibition of cell proliferation was observed for all 4 selected antibodies. In this experiment 5D5 had no effect on HGF-induced cell proliferation.

When blocking dimerization was evaluated, it was observed that 224G11, 223C4, 11E1 and 227H1 had a significant effect on inhibiting dimerization, reaching 32, 55, 69 and 52%, respectively. The 5D5 antibody had no effect in this dimerization model compared to the basal signal in the respective experiments.

Example 2: protein recognition by anti-c-Met antibodies

To characterize the recognition pattern of the 3 selected antibodies, 3 ELISAs were established with the recombinant c-Met protein, its monomeric fragments (obtained by cleaving the recombinant c-Met-Fc protein and the recombinant SEMA domain).

The results presented in figure 4 show that 4 antibodies recognize dimeric and monomeric proteins. To accomplish these ELISAs, human dimeric c-Met protein (R)&D sytems, cat #358MT) was coated in PBS at a concentration of 0.7. mu.g/ml overnight at 4 ℃. After saturating the plates (Costar #3690) with 0.5% gelatin solution at 37 ℃ for 2 hours, hybridoma supernatants were incubated at 37 ℃ for 1 hour. Once washed with PBS, anti-mouse HRP antibody (Jackson ImmunoResearch, catalog # 115-. After 3 washes with PBS, peroxidase activity was revealed by the addition of 50. mu.l of TMB substrate (Uptima). The reaction was continued at room temperature for 5 minutes. The reaction was performed by adding 50. mu.l/well of 1M H₂SO₄The solution was stopped and read at 450nm on a plate reader. The same protocol was performed on the c-Met and SEMA domains of the monomers, but in this case the proteins were coated at 5 and 3. mu.g/ml, respectively.

The 5D5Mab introduced as a positive control recognized SEMA protein as expected. 224G11, 227H1 and 223C4 do not bind to the SEMA domain. 11E1 binds to SEMA.

To determine whether 11E1 and 5D5, both of which recognize the SEMA domain for competing overlapping epitopes, BIAcore analysis was performed. The BIAcore system, based on the surface plasmon resonance phenomenon, transmits data by monitoring binding events in real time. And then used to group antibodies in so-called "epitope mapping" experiments. Several antibodies that cannot bind to an antigenic molecule simultaneously are classified in the same group (same or adjacent binding sites). Conversely, when their respective binding sites are far enough to allow simultaneous binding of two antibodies, the latter are divided into two distinct groups. In such experiments, the antigen is usually used as a ligand (immobilized on a sensor chip) and the antibody without any label is used as the analyte (solution phase).

All the above experiments were performed on a BIAcore X instrument (GE Healthcare Europe GmbH). CM5 sensor chip (BIAcore) activated by murine anti-hexahistidine-tagged Mab (R & D System ref. mab050) was prepared using the amine coupling kit (BIAcore) according to the manufacturer's instructions. Electrophoresis buffer (HBS-EP) and regeneration buffer (Glycine, HCl) were from BIAcore. The recombinant soluble version of human HGF receptor produced as a chimeric molecule c-Met-Fc-hexahistidine tag comes from the R & D system (ref.358-MT-CF). The experiment was performed at 25 ℃ with a flow rate of 30. mu.l/min. A10. mu.g/ml solution of c-Met in running buffer was captured over a one minute period, typically injected into 270RU of c-Met soluble form on flow cell 2(fc 2). Flow cell 1(fcl) was used as a reference to examine non-specific binding between the antibody and the sensor chip matrix.

A continuous injection of the antibody to be tested was performed. The antibody was injected on the flow cells within 2 minutes. A second antibody (or the same) is then injected under the same conditions. A third injection with another antibody was made if no significant binding was observed. The sensor chip was then regenerated by a single 30s injection of regeneration buffer. Any antibody and c-Met-Fc were discarded at this stage.

Analysis of the results:

the ability of antibody "a" to block the binding of antibody "B" was calculated by the ratio BIA/C = (R2A/B/R1B) x 100: here R2A/B is a reaction equivalent to the binding of Mab "B" when it was injected after Mab "a", and RlB is equivalent to the binding of Mab "B" when it was first injected. A BIA/C of less than 20% indicates that A is capable of blocking the binding of B, and thus that A and B have adjacent binding sites.

Epitope mapping was performed with 2 mabs, 11E1 and 5D 5.

TABLE 3

The binding imaging of c-Met-Fc captured near 270RU by 10. mu.g/ml concentrations of Mab5D5 (first), 5D5 (second) and 11E1 (third) injected sequentially over 2 minutes all indicated that 5D5 and 11E1 bound clearly to two distant sites (FIG. 5A). This observation was confirmed by the interactive sequence of the antibodies (fig. 5B).

Table 3 summarizes the calculated ratios obtained with the different sequences of these 2 antibodies. Black values (over 75%) mean that Mab a does not block Mab B binding. Bold/italic values (below 20%) mean that the binding sites of the two antibodies (a and B) are identical or close enough not to bind simultaneously.

Example 3: effect of mabs on c-Met phosphorylation

To determine the activity of anti-c-Met antibodies on c-Met phosphorylation, a phosphor c-Met ELISA assay was arranged. Approximately 500000 a549 cells were seeded in each well of a 6-well plate containing F12K medium +10% FCS. Cells were starved 16 hours prior to addition of HGF (100ng/ml) and each antibody to be tested was added at a final concentration of 30 μ g/ml 15 minutes prior to ligand stimulation. 15 minutes after the addition, cold lysis buffer was added, the cells were scraped off and the cell lysate was collected and centrifuged at 13000rpm for 10 minutes at 4 ℃. Supernatants were quantitated using BCA kit (Pierce) and stored at-20 ℃. For ELISA assays, goat anti-c-Met antibody (R & D ref. af276) was used as capture antibody (coated overnight at 4 ℃) and 25 μ g protein from different cell lysates was added to each well in a 96-well plate after the saturation step with TBS-BSA5% buffer (1 hour at RT saturation). After incubation for 90 minutes at room temperature, the plate was washed four times and anti-phospho-c-Met antibody (rabbit anti-pY 1230-1234-cease1235 c-Met) was added. After another 1 hour incubation and four washes, anti-rabbit hrp (biosource) was added at room temperature for 1 hour, followed by luminol substrate, and then luminescence was evaluated with the Mithras device. The results in fig. 6B demonstrate and show that 11E1, 224G11, 223C4, and 227H1 inhibit C-Met phosphorylation by 68, 54, 80, and 65%, respectively, compared to 5D5Mab with a weaker inhibitory effect on C-Met phosphorylation (42%). In this experiment, a weak basal effect (less than 20%) was observed with 4 candidate antibodies (fig. 6A). As described in the various examples presented in this patent, this weak basal effect had no effect on the activity of the antibody in other in vitro and in vivo assays. 5D5 used as a control showed a significant basal effect in this experiment.

Example 4: replacement of radiolabeled HGF with anti-c-Met antibody

To determine whether anti-c-Met antibodies could replace HGF, binding experiments were scheduled. Briefly, protein a flash plates (FlashPlate) 96-well microplates (Perkin Elmer) were saturated with 0.5% gelatin (200 μ l/well, 2 hours at room temperature) in PBS before adding recombinant c-Met-Fc (R & D Systems) as a coating protein. 2000. mu.l of a 1. mu.g/ml solution of c-Met-Fc in PBS was added to each well. The plates were then incubated overnight at 4 ℃. Free residual protein A sites were further saturated with irrelevant hIgG (0.5. mu.g/well in PBS) for 2 hours at room temperature. After each step the plates were washed with PBS.

For competition assays, anti-C-Met monoclonal antibodies 11E1, 224G11, 223C4, 227H1 or HGF (R) at various concentrations ranging from 0.1pM to 1 μ M in the presence of PBS pH7.4 (R)&D Systems), 200pM [ 2], [ 2]¹²⁵I]Binding of HGF (specific activity 2,000Ci/mmol) to immobilized c-Met. The plates were incubated at room temperature for 6h and then counted using a Packard Top Count microplate scintillation counter. Non-specific binding was determined in the presence of 1 μ M HGF. Monoclonal antibody 9G4, which is not directed against c-Met but which recognizes specificallyColi protein, which was used as a murine IgG1 isotype control.

[¹²⁵I]-the total percentage of HGF specific binding is plotted on a semi-logarithmic graph as a function of ligand concentration. Inhibition of 50% (IC)₅₀) The concentration of each inhibitor required for radioligand binding was determined from the resulting sigmoidal competition curves (fig. 7A and 7B).

As expected, the non-radiolabeled HGF is capable of completely substituting the [ 2], [ solution ] bound to the immobilized c-Met¹²⁵I]HGF, however control antibody 9G4 did not show any HGF blocking activity (fig. 7A and 7B). Monoclonal anti-C-Met antibodies 11E1, 224G11, 223C4 and 227H1 were able to have ICs at 20nM, 3nM, 2.7nM and 5.8nM, respectively₅₀Value inhibition [ alpha ]¹²⁵I]HGF binds to the cured c-Met. IC of the determined antibodies 224G11, 223C4 and 227H1₅₀IC of value and determined non-radiolabeled HGF₅₀Values (which were comprised between 3 and 5 nM) were compared, however antibody 11E1 showed a higher IC₅₀The value is obtained.

Example 5: inhibition of infiltration by anti-c-Met antibodies

To evaluate the inhibitory effect of anti-c-Met antibodies on the infiltration process, A549 cells were plated on BD BioCoat^TMMatrigel^TMInfiltration chamber (8 μm size (pre size) polycarbonate membrane over 6.5mm diameter hole). A459 cells were starved for 24 hours prior to performing the infiltration assay. 500000A 549 cells in chemotaxis buffer (DMEM medium, 0.1% BSA, 12mM Hepes) were then plated on a matrigel coated or not coated with the antibody to be tested (Mab final concentration 10. mu.g/ml) in the upper well of each chamber. Plates were incubated at 37 ℃ 5% CO₂After 1 hour incubation, the lower chamber was filled with growth medium containing 400ng/ml rhHGF or only with growth medium. The infiltration chamber was maintained at 37 ℃ with 5% CO₂Incubate for an additional 48 hours. At the end of this incubation time, the cells still on the upper surface of the filter were gently removed with a cotton swab and transferred to the lower surface of the filterThe cells were lysed, stained with CyQuant GR dye buffer (Invitrogen) and counted with a fluorescence reader Berthold Mithras LB 940. All conditions were tested in triplicate.

As expected, HGF induced significantly tumor cell infiltration compared to that observed with 10% FCS as a positive control (fig. 8). Murine IgG19G4 introduced as an isotype control had no significant effect on basal or HGF-induced infiltration when compared to cells plated without IgG. No agonist effect was noted for 11El, 224G11, 223C4, and 227H1 when added alone, whereas significant and comparable inhibition of HGF-induced infiltration was observed with 3 mabs.

Example 6: anti-c-Met antibodies inhibit wound healing

HGF stimulates migration. To determine whether anti-HGF antibodies inhibited migration, NCI-H441 cells were grown to high density and gapped with a P200 pipette tip. Cells were then stimulated to migrate through the gap with HGF (100ng/ml) in the presence or absence of 11E 1. Wells with only 11E1 were also evaluated. Six replicates of each test condition were evaluated and 3 independent experiments were performed. After overnight incubation, cells were visualized using an AxioVision Camera (Objective X4).

HGF induced significant migration resulting in complete closure of the gap within one night (fig. 9). 9G 4-unrelated IgG1 used as isotype control had no effect on cell migration. As expected, an agonist effect was observed when 5D5 was added alone, but significant inhibition of cell migration was observed with this antibody in the presence of HGF, with some gaps still open. When the Fab fragment of 5D5 was added alone, there was no agonist effect. However, in the presence of HGF, this fragment was observed to be inactive. As observed with isotype control 9G4, there was no agonist effect when MAb11E1 was added alone, but it was a full antagonist in the presence of HGF.

Example 7: scattering measurement

SK-HEP-1 cells were seeded at low density (1.10)⁴Cells/well) were seeded in 24-well plates of DMEM containing 10% FCS and HGF (100ng/ml) and test antibody (10 μ g/ml) were added simultaneously after 24 hours of growth. After 72 hours of incubation, the clones were fixed and used for 0.2% crystal violet staining in methanol and evaluation of scattered visible light was performed. Each experimental condition was performed in triplicate and 3 independent experiments were performed.

Addition of HGF to SK-HEP-1 cells induced significant cell scattering (fig. 10A and 10B). The 9G4 antibody introduced as an isotype control had no effect in the presence of either HGF alone or HGF. As expected, the 5D5 antibody alone showed significant agonist effect, but no inhibitory effect when added with HGF (fig. 10A). No agonist effect was observed with the addition of 11E1 (fig. 10A) or 224G11 (fig. 10B) alone. These antibodies demonstrated a very significant inhibitory effect in the presence of HGF (fig. 10A and 10B).

Example 8: three-dimensional luminal formation (tubulogenesis) assay

SK-HEP-1 cells were cultured at 1.10⁴Cells/well were seeded in 24-well plates of DMEM containing 10% FCS/matrigel (50/50) and incubated for 30 min before the addition of HGF (100ng/ml) and test antibody (10. mu.g/ml) simultaneously. After 7 days of incubation, cells were evaluated for the formation of visible luminal-like structures. Each test condition was subjected to 3 replicates and 3 independent experiments were performed.

Addition of HGF significantly induced SK-HEP-1 luminal-like structure formation (fig. 11). Antibody 9G4, introduced as an isotype control, was not effective either when added alone or when added in the presence of HGF. As expected, 5D5 showed significant agonist effect when added alone and no inhibitory effect when added with HGF. No agonist effect was observed with the addition of 11E1, 223C4, and 224G11 alone, while 11E1 and 223C4 demonstrated full inhibitory effect in the presence of HGF. 224G11Mab a partial but significant inhibition was observed.

Example 9: sphere forming

To evaluate the ability of anti-c-Met antibodies to inhibit tumor growth in vitro, U-87MG, human glioblastoma cell (ATCC # HTB-14) spheroids were generated in a model that was closer to the in vivo situation. Cells grown in monolayers were detached with trypsin-EDTA and resuspended in complete cell culture medium (DMEM) supplemented with 10% FBS. Spheroid formation was initiated by seeding 625 cells in DMEM-10% FCS into a single circular well at the bottom of a 96-well plate. To prevent cell adhesion to the substrate, the plates were pre-coated with poly (hydroxyethyl methacrylate) (polyHEMA) in 95% ethanol and allowed to air dry at room temperature. The plates were incubated at 37 ℃ in a humidified incubator with 5% CO2 under standard cell culture conditions. Purified monoclonal antibody (10. mu.g/ml) was added after 3 and 7 days of spheroid culture. Once cultured for 4 days, HGF (400ng/ml) was added. Spheroids were cultured for at least 10 days. The growth of the spheroids was then monitored by measuring the area of the spheroids with an automatic measurement module of axio vision software. Area in mum²And (4) expressing. 8-16 spheroids were evaluated per condition.

FIGS. 12A and 12B illustrate that no stimulatory effect was observed when HGF was added to the complete medium in the presence of 10% FCS. The 9G4 isotype control had no effect on spheroid growth as expected. Both 11E1 and 223C4 significantly reduced spheroid growth in the presence and absence of HGF. No effect was observed with the addition of the 5D5Fab fragment.

Example 10: in vivo activity of anti-c-Met Mab in U87MG xenograft model

Athymic mice of six to eight weeks of age were housed in sterile, filter-topped cages, housed under sterile conditions and treated according to French and European guidelines (French and European guideline). Selection U87-MG, glioblastoma cell line, expressing c-Met and the autocrine ligand HGF for in vivo evaluation. Mix 5x10⁶Cells were injected subcutaneously into mice. Then, six days after the cell transplantation, tumors (about 100 mm) were measured³) Animals with comparable tumor size were grouped into groups of 6 mice each and treated twice a week with 1 mg/dose of each antibody tested. Next, the mice were observed for changes in xenograft growth rate and body weight. Tumor volume was calculated by the formula: π (Pi)/6 × length × width × height.

Figure 13 summarizes the results obtained and demonstrates that all the antibodies tested significantly inhibited the in vivo growth of U87-MG cells. The use of an anti-IGF-lR antibody (IgG1) neutralized in group A demonstrated that the observed in vivo inhibition was specifically associated with modulation of the HGF-cMet axis.

Example 11: in vivo activity of anti-c-Met Mab in NCI-H441 xenograft model

NCI-H441 was derived from papillary lung adenocarcinoma, expresses high levels of c-Met, and demonstrates constitutive phosphorylation of c-Met RTK.

To determine whether this cell line expresses high levels of c-Met and is capable of producing HGF, quantitative RT-PCR and FACS or ELISA (Quantikine HGF; R)&D systems). For quantitative RT-PCR, total HGF or cMet transcript expression levels in cell lines were determined by using standard TaqMan^TMQuantitative PCR of the technique was used for evaluation. HGF or c-Met transcript levels were normalized to housekeeping gene ribosomal protein, large, P0(RPL0), and the results were expressed as normalized expression values (2-ddCT method).

The primer/probe set of RPL0 was: forward, 5'-gaaactctgcattctcgcttcctg-3' (SEQ ID No. 47); reverse, 5'-aggactcgtttgtacccgttga-3' (SEQ ID No. 48); and probe, 5'- (FAM) -tgcagattggctacccaactgttgca- (TAMRA) -3' (SEQ ID No. 49). Primer/probe set for HGF was forward, 5'-aacaatgcctctggttcc-3' (SEQ ID No. 50); reverse, 5'-cttgtagctgcgtcctttac-3' (SEQ ID No. 51); and probe, 5'- (FAM) -ccttcaatagcatgtcaagtggagtga- (TAMRA) -3' (SEQ ID No. 52). The primer/probe set for cMet is: forward, 5'-cattaaaggagacctcaccatagctaat-3' (SEQ ID No. 53); reverse, 5'-cctgatcgagaaaccacaacct-3' (SEQ ID No. 54); and probe, 5'- (FAM) -catgaagcgaccctctgatgtccca- (TAMRA) -3' (SEQ ID No. 55). The thermal cycling protocol consisted of melting at 50 ℃ for 2 minutes, 95 ℃ for 10 minutes, followed by 40 cycles of 95 ℃ for 15 seconds and 62 ℃ for 1 minute.

HGF mRNA was not found in NCI-H441 (FIG. 14), and HGF was not detected in NCI-H441 supernatant by ELISA. In these experiments, U87-MG, a glioblastoma cell line known as an autocrine cell line for HGF, was introduced as a positive control. RT-PCR analysis showed that significant levels of HGF mRNA were detected in U87-MG and HGF was detected at 1.9 ng/million cells in the supernatant of U87-MG cells. Both quantitative RT-PCRs and FACS analysis (fig. 15A and 15B) demonstrated that, as expected, NCI-H441 cells significantly over-expressed c-Met, and demonstrated that the expression was dramatically higher than that observed in U87-MG cells. In this experiment, the MCF-7 cell line was introduced as a negative control. In summary, NCI-H441 appears to be a non-autocrine constitutively activated cell line capable of HGF ligand-independent growth, where ligand-independent dimerization of c-met is the result of receptor overexpression.

Evaluation of the in vivo activity of anti-c-met antibodies on this non-autocrine cell line could deepen the understanding that they influence the potency of c-met dimerization.

Figure 16 demonstrates that 224G11, 11E1, and 227H1 significantly inhibited the in vivo growth of NCI-H441 suggesting that these antibodies, which are capable of inhibiting dimerization, can target ligand-independent inhibition of c-met in addition to ligand-dependent inhibition. As explained above in the present specification, due to the latter attribute, 224G11, 11E1, and 227H1 were shown to be different from the 5D5 one-armed (OA-5D5) anti-c-Met antibody.

Example 12: humanization procedure for CDR grafting of antibody 224G11

Humanization of the I-light chain variable region

Comparison of nucleotide sequence of 224G11VL with murine germline genes

As a starting step, the nucleotide sequence of 224G11VL was compared to the murine germline gene section in the IMGT database (http:// IMGT. cines. fr). .

The V regions of murine IGKV3-5 x 01 and IGKJ4 x 01 germline genes have been identified as having 99.31% sequence identity and the J regions as having 94.28% sequence identity, respectively. Regarding the identity obtained, it was decided to use the 224G11VL sequence directly for searching for human homologous sequences.

These alignments are shown in FIGS. 17A (V gene) and 17B (J gene).

Comparison of nucleotide sequence of 224G11VL with human germline genes

To identify the best human candidate sequences for CDR grafting, human germline genes showing the best identity with 224G11VL have been retrieved. For this purpose, the nucleotide sequence of 224G11VL was aligned with the portion of the human germline gene sequence in the IMGT database. For optimal selection, alignments between protein sequences were performed to retrieve better homologous sequences.

These two complementary approaches led to the identification of two possible accepted human V sequences for the murine 224G11VL CDR. The sequence identity of the human IGKV3-11 × 01 germline gene given by the nucleotide alignment was 75.99%, and the sequence identity of the human IGKV4-1 × 01 germline gene given by the protein sequence alignment was 67.30%. It is noteworthy that in both cases, the two closest germline genes and the analyzed sequences showed different CDR1 amino acid lengths (10 amino acids in 224G11 VL; 6 amino acids in IGKV3-11 x 01; 12 amino acids in IGKV4-1 x 01).

For the J region, the best homology score was first obtained from humans, with human IGKJ 3x 01 showing 80% sequence identity. But a higher number of consecutive identical nucleotides and better amino acid matches were found in the alignment with the human IGKJ4 × 02 germline gene (sequence identity 77.14%). Thus, IGKJ4 × 02 germline genes were selected as recipient human J regions of the murine 11E1VL CDRs.

The alignment is shown in FIGS. 18A (V region) and 18B (J region).

Humanized version of 224G11VL

Considering the two possible accepted human V regions for the murine 224G11VL CDRs, humanized versions of the two 224G11VL regions will be described. The first corresponds to the initial experiment with a human framework (human frame) with a shorter CDR1 length (IGKV3-11 x 01), and the second with a longer CDR1 length (IGKV4-1 x 01).

a) Humanized version of 224G11VL based on IGKV3-11 x 01

The following humanization procedure consisted in the step of attaching the CDRs of the selected germline gene sequences IGKV3-11 x 01 and IGKJ4 x 02 and also murine 224G11VL to the framework of these germline gene sequences.

As depicted in fig. 19A, the residues in bold in the 224G11VL sequence correspond to the 25 amino acids found to be different between the 224G11VL region and the selected human framework (human FR, i.e., IGKV3-11 × 01 and IGKJ4 × 02).

Referring to (regarding) several criteria, such as the amino acid class changes between murine and human residues, their localization in the 3D structure of the variable region, which are known to be involved in the VH/VL interface, involved in antigen binding or CDR structure, 3 out of 25 different residues were identified for final mutation. The three most important defined residues and mutations in their human counterparts are murine M39 to human L, H40 to a and R84 to G. These residues scored as primary are shown in bold residues in the 224G11HZ1VL sequence, still murine, in FIG. 19A.

Of course, the residues to be tested are not limiting, but must be considered as preferred mutations.

With the aid of molecular models, further mutations can be identified. Residues rated as secondary, namely residues 15(L/P), 49(P/A), 67(L/R), 68(E/A), 93(P/S) and 99(V/F) may be mentioned, and mutations at these residues are also envisaged in another preferred embodiment.

Of course, the above-mentioned residues to be finally tested are not limiting, but must be considered as preferred mutations. In another preferred embodiment, all 16 other residues ranked tertiary among the 25 different amino acids can be reconsidered.

All of the above mutations will be tested individually or according to different combinations.

Figure 19A shows the humanized 224G11VL clearly identified by the above mutations performed based on IGKV3-11 x 01. The numbers under each proposed mutation correspond to the ranking of the mutation to be made.

b) Humanized version of 224G11VL based on IGKV4-1 x 01

The following humanization procedure consisted in ligating the CDRs of the selected germline gene sequences IGKV4-1 x 01 and IGKJ4 x 02 and murine 224G11VL to the framework of these germline gene sequences.

As depicted in fig. 19B, residues in bold in the 224G11VL sequence correspond to the 22 amino acids found to be different between the 224G11VL region and the selected human framework (human FR, i.e., IGKV4-1 × 01 and IGKJ4 × 02).

Referring to several criteria, such as its known involvement in the VH/VL interface, involvement in antigen binding or CDR structure, amino acid class changes between murine and human residues, positioning of this residue in the 3D structure of the variable region, 4 out of 22 different residues were identified for final mutation. The four most important defined residues and mutations in their human counterparts are: murine L4 was changed to human M, M39 to L, H40 to A and R84 to G. These residues ranked as primary are shown as bolded residues in the 224G11HZ2VL sequence still murine in fig. 19B.

Of course, the residues to be tested are not limiting, but must be considered as preferred mutations.

With the aid of molecular models, further mutations can be identified. Residues rated as secondary, i.e., residues 25(A/S), 66(N/T), 67(L/R) and 93(P/S), can be mentioned, and mutations at these residues are also contemplated in another preferred embodiment.

Of course, the above-mentioned residues to be finally tested are not limiting, but must be considered as preferred mutations. In another preferred embodiment, all 14 other residues ranked tertiary among 22 different amino acids can be reconsidered.

All of the above mentioned mutations will be tested either individually or according to different combinations.

Figure 19B shows that the mutations performed above identified a clear humanisation 224G11VL based on IGKV4-1 x 01. The numbers under each proposed mutation correspond to the ranking of the mutation to be made.

II-humanization of the heavy chain variable region

Comparison of nucleotide sequence of 224G11VH with murine germline genes

As a starting step, the nucleotide sequence of 224G11VH was compared to the murine germline gene portion of the IMGT database (http:// IMGT. cines. fr).

Murine IGHV1-18 x 01, IGHD2-4 x 01, and IGHJ 2x 01 germline genes have been identified with sequence identity 92.70% for the V region, 75.00% for the D region, and 89.36% for the J region. Regarding the identity obtained, it was decided to use the 224G11VH sequence directly to find human homologous sequences.

These alignments are shown in FIG. 20A (for the V gene), 20B (for the D gene) and 20C (for the J gene).

Comparison of nucleotide sequence of 224G11VH with human germline genes

To identify the best human candidate sequences for CDR grafting, human germline genes exhibiting the best identity with 224G11VH have been searched. For this purpose, the nucleotide sequence of 224G11VH was aligned with the human germline gene sequence portion of the IMGT database. For optimal selection, alignments between protein sequences were performed to retrieve better homologous sequences.

These two complementary approaches resulted in the identification of identical accepted human IGHV1-2 x 02V sequences for the murine 224G11VH CDRs, with 75.00% sequence identity at the nucleotide level and 64.30% at the protein level.

It is noted that the D region strictly belongs to the CDR3 region in the VH region. The humanization process is based on the CDR grafting (CDR-grafting). Analysis of the closest human D gene in this strategy is useless.

A search for homologous sequences in the J region identified the human IGHJ4 × 04 germline gene with 78.72% sequence identity.

Thus, human IGHV1-2 x 02V germline gene and human IGHJ4 x 01J germline gene were selected as the accepted human sequences for the murine 224G11VH CDRs.

The alignment is shown in FIGS. 21A (V region) and 21B (J region).

Humanized version of 224G11VH

The following humanization procedure consisted in ligating the CDRs of the selected germline gene sequences IGHV1-2 x 02 and IGHJ4 x 01 and murine 224G11VH to the framework of these germline gene sequences.

As depicted in fig. 22, the residues in bold in the 224G11VH sequence correspond to the 30 amino acids found to be different between the 224G11VH region and the selected human framework (human FR, i.e., IGHV1-2 × 02 and IGHJ4 × 01).

Referring to several criteria, such as its known involvement in the VH/VL interface, involvement in antigen binding or CDR structure, amino acid class changes between murine and human residues, positioning of this residue in the 3D structure of the variable region, 4 out of 30 different residues were identified for final mutation. The 4 most important defined residues and mutations in their human counterparts were murine D51 to human E, G55 to W, V80 to R and K82 to T. These residues ranked as primary are shown as bolded residues in the still murine 224G11HZVH sequence of fig. 22.

Of course, the residues to be tested mentioned above are not limiting, but must be considered as preferred mutations.

With the aid of molecular models, further mutations can be identified. Residues ranked as secondary, i.e., residues 25(T/A), 48(E/Q), 49(S/G), 53(I/M), 76(A/V), 78(L/M) and 90(D/E) may be mentioned, and mutations at these residues are also contemplated in another preferred embodiment.

Of course, the above-mentioned residues to be finally tested are not limiting, but must be considered as preferred mutations. In another preferred embodiment, all 19 other residues ranked tertiary among the 30 different amino acids can be reconsidered.

All of the above mutations will be tested individually or according to different combinations.

Figure 22 shows the above mutation clearly identified humanized 224G11 VH. The numbers under each proposed mutation correspond to the ranking of the mutation to be made.

Example 13: humanization procedure by CDR grafting of antibody 227H1

Humanization of the I-light chain variable region

Comparison of the nucleotide sequence of 227H1VL with that of a murine germline gene

As a starting step, the nucleotide sequence of 227H1VL was compared to the murine germline gene portion of the IMGT database (http:// IMGT. cines. fr). .

Murine IGKV3-5 x 01 and IGKJ4 x 01 germline genes have been identified as 96.90% sequence identity for the V region and 97.29% for the J region, respectively. Regarding the identity obtained, it was decided to use the 227H1VL sequence directly to find human homologous sequences.

These alignments are shown in FIGS. 23A (for the V gene) and 23B (for the J gene).

Comparison of the nucleotide sequence of 227H1VL with that of a human germline gene

To identify the human candidate sequences that are optimal for CDR grafting, human germline genes that exhibit the best identity with 227H1VL have been searched. For this purpose, the nucleotide sequence of 227H1VL was aligned with the human germline gene sequence portion of the IMGT database. For optimal selection, alignments between protein sequences were performed to retrieve better homologous sequences.

These two complementary approaches led to the identification of two possible accepted human V sequences for the murine 227H1VL CDR. The sequence identity of the human IGKV3-11 × 01 germline gene given by the nucleotide alignment was 74.91%, and the sequence identity of the human IGKV4-1 × 01 germline gene given by the protein sequence alignment was 64.00%. It is noteworthy that in both cases, the two closest germline genes and the analyzed sequences showed different CDR1 amino acid lengths (10 amino acids in 227H1 VL; 6 amino acids in IGKV3-11 x 01; 12 amino acids in IGKV4-1 x 01).

For the J region, the best homology score was first obtained from humans, with human IGKJ 3x 01 showing 78.38% sequence identity. But a higher number of consecutive identical nucleotides and better amino acid matches were found in the alignment of human IGKJ4 × 02 germline genes (sequence identity 75.68%). Therefore IGKJ4 × 02 germline genes were selected as the accepted human J-regions for the murine 227H1VL CDRs.

The alignment is shown in FIGS. 24A (V region) and 24B (J region).

Humanized version of 224G11VL

Considering the two possible accepted human V regions for the murine 227H1VL CDRs, two humanized versions of the 227H1VL region will be described. The first version corresponds to the initial trial of a human framework (human frame) with a shorter CDR1 length (IGKV3-11 x 01) and the second with a longer CDR1 length (IGKV4-1 x 01).

a) Humanized version of 227H1VL based on IGKV3-11 x 01

The following humanization procedure consisted in ligating the CDRs of the selected germline gene sequences IGKV3-11 x 01 and IGKJ4 x 02 and murine 227H1VL to the framework of these germline gene sequences.

As depicted in fig. 25A, the residues in bold in the 227H1VL sequence correspond to the 26 amino acids found to be different between the 227H1VL region and the selected human framework (human FR, i.e., IGKV3-11 × 01 and IGKJ4 × 02).

Referring to several criteria, such as its known participation in the VH/VL interface, participation in antigen binding or CDR structure, the amino acid class change between murine and human residues, the positioning of this residue in the 3D structure of the variable region, 3 out of 26 different residues were identified for final mutation. The 3 most important defined residues and mutations in their human counterparts are: murine I39 was changed to human L, H40 to A and R84 to G. These residues ranked as primary are shown as bolded residues in the 227H1HZ1VL sequence of fig. 25A, still murine.

Of course, the above-mentioned residues to be tested are not limitative, but must be considered as preferred mutations.

With the aid of molecular models, further mutations can be identified. Residues rated as secondary, namely residues 15(L/P), 25(V/A), 49(P/A), 67(L/R), 68(E/A), 93(P/S) and 99(S/F) may be mentioned, and mutations at these residues are also envisaged in another preferred embodiment.

Of course, the above-mentioned residues to be finally tested are not limiting, but must be considered as preferred mutations. In another preferred embodiment, all 16 other residues ranked tertiary among the 25 different amino acids can be reconsidered.

All of the above mutations will be tested separately or according to different combinations.

Figure 25A shows the performed IGKV3-11 x 01 based mutation identified a clear humanization of 227H1 VL. The numbers under each proposed mutation correspond to the ranking of the mutation to be made.

b) Humanized version of 227H1VL based on IGKV4-1 x 01

The following humanization procedure consisted in ligating the CDRs of the selected germline gene sequences IGKV4-1 x 01 and IGKJ4 x 02 and murine 227H1VL to the framework of these germline gene sequences.

As depicted in fig. 25B, the residues in bold in the 227H1VL sequence correspond to the 24 amino acids found to be different between the 227H1VL region and the selected human framework (human FR, i.e., IGKV4-1 × 01 and IGKJ4 × 02).

Referring to several criteria, such as its known involvement in the VH/VL interface, involvement in antigen binding or CDR structure, amino acid class changes between murine and human residues, positioning of this residue in the 3D structure of the variable region, 4 out of 24 different residues were identified for final mutation. The 4 most important defined residues and mutations in their human counterparts are: murine L4 was changed to human M, I39 to L, H40 to A and R84 to G. These residues ranked as primary are shown as bolded residues in the 227H1HZ2VL sequence, still murine in fig. 25B.

Of course, the above-mentioned residues to be tested are not limiting, but must be considered as preferred mutations.

With the aid of molecular models, further mutations can be identified. Residues rated as secondary, i.e., residues 25(A/S), 66(N/T), 67(L/R) and 93(P/S), can be mentioned, and mutations at these residues are also contemplated in another preferred embodiment.

Of course, the above-mentioned residues to be finally tested are not limiting, but must be considered as preferred mutations. In another preferred embodiment, all 16 other residues ranked tertiary among 22 different amino acids can be reconsidered.

All of the above mutations will be tested separately or according to different combinations.

Figure 25B shows the performed IGKV4-1 x 01 based mutation identified a clear humanization of 227H1 VL. The numbers under each proposed mutation correspond to the ranking of the mutation to be made.

II-humanization of the heavy chain variable region

Comparison of the nucleotide sequence of 227H1VH with that of a murine germline gene

As a starting step, the nucleotide sequence of 227H1VH was compared to the murine germline gene portion of the IMGT database (http:// IMGT. cines. fr).

Murine IGHV1-18 x 01, IGHD1-1 x 02 and IGHJ 2x 01 germline genes have been identified that are 92.70% sequence identity to the V region, 63.63% to the D region and 91.48% to the J region, respectively. Regarding the identity obtained, it was decided to use the 227H1VH sequence directly to find human homologous sequences.

These alignments are shown in FIG. 26A (for the V gene), 26B (for the D gene) and 26C (for the J gene).

Comparison of the nucleotide sequence of 227H1VH with that of a human germline gene

To identify the best human candidate sequences for CDR grafting, human germline genes exhibiting the best identity with 224G11VH have been searched. For this purpose, the nucleotide sequence of 227H1VH was aligned with the human germline gene sequence portion of the IMGT database. The accepted human IGHV1-2 x 02V sequence for the murine 224G11VH CDR was thereby identified with 72.92% sequence identity.

It is noted that the D region strictly belongs to the CDR3 region in the VH region. The humanization process is based on the CDR grafting method. In this strategy, it is not useful to analyze the closest human D gene.

A search for homologous sequences in the J region identified the human IGHJ4 × 04 germline gene with 78.72% sequence identity.

Thus, the human IGHV1-2 x 02V germline gene and the human IGHJ4 x 01J germline gene were selected as the accepted human sequences for the murine 227H1VH CDRs.

The alignment is shown in FIGS. 27A (V region) and 27B (J region).

To optimize selection, one skilled in the art can also align between protein sequences to aid in their selection.

227H1VH humanized version

The following humanization procedure consisted in ligating the CDRs of the selected germline gene sequences IGHV1-2 x 02 and IGHJ4 x 01 and murine 227H1VH to the framework of these germline gene sequences.

As depicted in figure 28, the residues in bold in the 227H1VH sequence correspond to the 32 amino acids found to be different between the 227H1VH region and the selected human framework (human FR, i.e., IGHV1-2 × 02 and IGHJ4 × 01).

Referring to several criteria, such as its known participation in the VH/VL interface, participation in antigen binding or CDR structure, the amino acid class change between murine and human residues, the positioning of this residue in the 3D structure of the variable region, 6 out of 32 different residues were identified for final mutation. The 6 most important defined residues and mutations in their human counterparts were murine L39 to human M, N40 to H, L55 to W, T66 to N, V80 to R and K82 to T. These residues ranked as primary are shown as bolded residues in the still murine 227H1HZVH sequence of fig. 28.

Of course, the above-mentioned residues to be tested are not limiting, but must be considered as preferred mutations.

With the aid of molecular models, further mutations can be identified. Residues rated as secondary, i.e., residues 48(K/Q), 49(T/G), 53(I/M), 76(A/V) and 78(L/M), can be mentioned, and mutations at these residues are also contemplated in another preferred embodiment.

Of course, the above-mentioned residues to be finally tested are not limiting, but must be considered as preferred mutations. In another preferred embodiment, all 21 other residues ranked tertiary among 30 different amino acids can be reconsidered.

All of the above mutations will be tested separately or according to different combinations.

Figure 28 shows the mutation identification of clear humanization of 227H1 VH. The numbers under each proposed mutation correspond to the ranking of the mutation to be made.

Example 14: humanization procedure by CDR grafting of antibody 223C4

Humanization of the I-light chain variable region

Comparison of nucleotide sequence of 223C4VL with murine germline genes

As a starting step, the nucleotide sequence of 223C4VL was compared to the murine germline gene portion of the IMGT database (http:// IMGT. cines. fr). .

Murine IGKV12-46 x 01 and IGKJ 2x 01 germline genes have been identified, respectively, as 99.64% for V-region sequence identity and 94.59% for J-region. Regarding the identity obtained, it was decided to use the 223C4VL sequence directly to find human homologous sequences.

These alignments are shown in FIGS. 29A (for the V gene) and 29B (for the J gene).

Comparison of nucleotide sequence of 223C4VL with human germline genes

To identify the best human candidate sequences for CDR grafting, human germline genes exhibiting the best identity with 223C4VL have been searched. For this purpose, the nucleotide sequence of 223C4VL was aligned with the human germline gene sequence portion of the IMGT database.

Human IGKV1-NL 1x 01 and IGKJ 2x 01 germline genes have been identified with sequence identity 78.49% for the V region and 81.08% for the J region. Thus, the human IGKV1-NL 1x 01 germline gene (V region) and IGKJ 2x 01 germline gene (J region) were selected as the accepted human sequences for the murine 223C4VL CDRs.

The alignment is shown in FIGS. 30A (V region) and 30B (J region).

To optimize selection, one skilled in the art can also perform alignments between protein sequences to aid in their selection.

Humanized versions of 223C4VL

The following humanization procedure consisted in ligating the CDRs of the selected germline gene sequences IGKV1-NL 1x 01 and IGKJ4 x 02 and murine 223C4VL to the framework of these germline gene sequences.

At this stage, a molecular model of the 223C4 murine Fv region can be developed and used to select murine residues for retention based on their role in maintaining the three-dimensional structure of the molecule or in antigen binding site and function. More specifically, 9 residues have been identified that will be the last to be mutated.

In the first step, the residues contained in the CDR anchors or structures will be determined. These residues are residue 66(R/N) and residue 68 (E/V).

In the second step, residues exposed to a solvent (solvant) and thus potentially immunogenic, are also assayed. These are residues 49(A/S), 51(K/Q), 69(S/D), 86(D/Q) and 92 (S/N).

Then, in a third step, residues comprised in the structure/folding of the variable region may also be mutated. These residues are 46(P/Q) and 96 (P/S).

Of course, the above-mentioned residues to be tested are not limitative, but must be considered as preferred mutations.

With the aid of molecular models, further mutations can be identified. Residues 9(S/A), 13(A/V), 17(D/E), 18(R/T), 54(L/V), 88(T/S), 90(T/K), 100(A/G) and 101(T/S) may be mentioned, and mutations at these residues are also envisaged in another preferred embodiment.

All of the above mutations will be tested separately or according to different combinations.

Figure 31 shows the mutation identification of a clear humanised 223C4 VL. The number under each proposed mutation corresponds to the rating of the mutation to be performed.

II-humanization of the heavy chain variable region

Comparison of nucleotide sequence of 223C4VH with murine germline genes

As a starting step, the nucleotide sequence of 223C4VH was compared to the murine germline gene portion of the IMGT database (http:// IMGT. cines. fr).

Murine IGHV1-18 x 01, IGHD6-3 x 01, and IGHJ6 x 01 germline genes have been identified that are 98.95% sequence identity for the V region, 72.72% for the D region, and 98.11% for the J region, respectively. Regarding the identity obtained, it was decided to use the 223C4VH sequence directly to find human homologous sequences.

These alignments are shown in FIG. 32A (for the V gene), 32B (for the D gene) and 32C (for the J gene).

Comparison of nucleotide sequence of 223C4VH with human germline genes

To identify the best human candidate sequences for CDR grafting, human germline genes exhibiting the best identity with 223C4VH have been searched. For this purpose, the nucleotide sequence of 223C4VH was aligned with the human germline gene sequence portion of the IMGT database.

Human IGHV1-2 x 02, IGHD1-26 x 01, and IGHJ6 x 01 germline genes were identified as 76.38% sequence identity for the V region, 75.00% for the D region, and 77.41% for the J region, respectively. Thus, the human IGHV1-2 x 02 germline gene (V region) and the human IGHJ6 x 01 germline gene (J region) were selected as the accepted human sequences for the murine 223C4VH CDRs.

The alignment is shown in FIGS. 33A (V region), 33B (for D region) and 33C (J region).

To optimize selection, one skilled in the art can also align between protein sequences to aid in their selection.

Humanized versions of 223C4VH

The following steps in the humanization process consisted in ligating the CDRs of the selected germline gene sequences IGHV1-2 x 02 and IGHJ6 x 01 and murine 223C4VH to the framework of these germline gene sequences.

At this stage of the process, a molecular model of the 223C4 murine Fv region can be developed and used to select murine residues for retention based on their role in maintaining the three-dimensional structure of the molecule or in antigen binding site and function. More specifically, 14 residues have been identified that are to be mutated last.

In the first step, the residues contained in the CDR anchors or structures will be determined. These residues are residues 40(H/D), 45(A/S), 55(W/D), 66(N/I) and 67 (Y/F).

In the second step, residues exposed to a solvent (solvant) and thus potentially immunogenic, are also assayed. They are residues 1(Q/E), 3(Q/L), 5(V/Q), 48(Q/M) and 80 (R/V).

Then, in a third step, residues comprised in the structure/folding of the variable region may also be mutated. These are residues 9(A/P), 13(K/V), 22(S/P) and 46 (P/H).

Of course, the above-mentioned residues to be tested are not limitative, but must be considered as preferred mutations.

With the aid of molecular models, further mutations can be identified. Residues 12(V/L), 21(V/I), 43(R/K), 49(G/S), 53(M/I), 68(A/N), 72(Q/K), 75(R/K), 76(V/A), 78(M/L), 82(T/K), 84(I/S), 92(S/R), 93(R/S), 95(R/T) and 97(D/E) may be mentioned, and mutations at these residues are also envisaged in another preferred embodiment.

All of the above mutations will be tested separately or according to different combinations.

Figure 34 shows the mutation identification of a clear humanised 223C4 VH. The numbers under each proposed mutation correspond to the ranking of the mutation to be made.

Example 15: mouse 224G11Mab alone or with chemotherapeutic agent novibenCombined use of antitumor Activity in an established xenograft NCI-H441 tumor model

Successful chemotherapeutic approaches rely in part on the balance between cellular response to apoptosis-inducing agents and the intracellular pro-apoptotic and anti-apoptotic pathways. The protective effect of activated c-Met on cell survival is documented. This effect results primarily from increased expression of anti-apoptotic Bcl-xl and Bcl-2 proteins caused by PI3-K mediated signaling, which in turn inhibits mitochondria-dependent apoptosis (caspase) 9). Indeed, it is conceivable that the HGF/c-Met system, which has a significant regulatory effect on the apoptotic process, can also affect the chemosensitivity of malignant cells. This hypothesis has passed through NovierCarry out the test, Novier BenIs a marketed chemotherapeutic agent for lung cancer treatment (Aapro et al, Crit. Rev. Oncol. Hematol.2001, 40: 251-grooved 263; Curran et al, Drugs aging.2002, 19: 695-grooved 697). The xenograft NCI-H441NSCLC model was used according to previous descriptions, this cell line for treatment of Novozymes (Kraus-Berthier et al, Clin. Cancer Res.,2000; 6: 297. sub.304) and targeted c-Met (Zou H.T.et al, Cancer Res).2007,67: 4408-.

Briefly, NCI-H441 cells from ATCC were routinely cultured in RPMI1640 medium, 10% FCS and 1% L-glutamine. Two days before implantation, cells were passaged to place them in exponential growth phase. Ten million NCI-H441 cells in PBS were implanted into 7-week-old Swiss nude mice. Three days after implantation, tumors were measured and animals with comparable tumor size were divided into 4 groups of 6 mice each. Mice were treated i.p. with a 2mg dose of 224G 11/mouse, then with 1mg antibody/mouse, twice a week, for 43 days. 9G4Mab was used as an isotype control.

Novier booki.p. injection at a dose of 8mg/kg, given on days 5, 12, 19 after cell injection. For 224G11 and NovierBoth are administered separately. In this experiment, both compounds were used at the optimal dose. Tumor volume was measured twice weekly and by the formula: p/6 × length × width × height.

FIG. 35 demonstrates 224G11 and Noveb when used alone as a single agent treatmentThe same is valid. A significant benefit was observed with the combination of the two therapies, which observed complete tumor regression in 3 out of 6 mice at day 63.

Example 16: C-Met inhibitors and angiogenesis

In addition to its direct role in regulating various tumor cell functions, activation of c-met is also involved in tumor angiogenesis. Endothelial cells express c-Met, and HGF stimulates the growth, infiltration and migration of endothelial cells (NakamuraY. et al., biochem. Biophys. Res., Commun.1995, 215: 483-488; Bussolino F. et al., J.cell biol.1992, 119: 629-641). It has been demonstrated that the synergistic regulation of HGF/c-Met growth, infiltration and migration in vascular endothelial cells leads to the formation of 3D capillary endothelial cell lumen-like structures in vitro (Rosen E.M.et al, supplement to Experientia1991, 59: 76-88).

To determine the possible effect of anti-c-Met Mab on HGF-induced angiogenesis, two sets of experiments were performed, which included i) evaluation of the effect of Mab on HUVEC proliferation, and ii) testing of Mab on HUVEC luminal-like structure formation.

For proliferation experiments, 7500 HUVECs were plated in each well of a 96-well plate previously coated with laminin. Cells were grown for 24 hours in EMB-2 assay medium supplemented with 0.5% FBS and heparin. Then, Mab to be tested (0.15 to 40. mu.g/ml) was added for 1 hour, followed by 20ng/ml of HGF. After further 24 hours, the cells were treated with 0.5. mu. Ci of³H]Thymine was pulsed. Incorporation of³H]The amount of thymine (d) is quantified by liquid scintillation counting. In this experiment, 9G4Mab was an irrelevant antibody used as an IgG1 isotype control.

The results, presented as raw data in fig. 36, demonstrate that HGF is a potent inducer of HUVEC cell growth, as expected. Regardless of the dose determined, antibodies evaluated in the absence of HGF did not show proliferative activity of any agonist to HUVEC. In the presence of HGF, both 11E1 and 224G11Mab were observed to have significant dose-dependent inhibitory effects.

To evaluate the formation of HUVEC luminal-like structures, 25000 cells incubated with the test antibody for 30 minutes were plated in 48-well plates coated with matrigel. Then 50ng/ml HGF was added and the plates were incubated at 37 ℃. The medium was then collected and 5 μ M CMFDA was added 15 minutes before viewing with the microscope.

The results shown in fig. 37 demonstrate that HGF induces significant luminal-like structure formation as expected. The 9G4 antibody introduced as IgG1 isotype control did not have any effect on HGF-induced luminal-like structure formation, however both 11E1 and 224G11 significantly inhibited luminal-like structure formation.

Example 17: humanization procedure by CDR grafting of antibody 11E1

Humanization of the I-light chain variable region

Comparison of nucleotide sequence of 11E1VL with murine germline genes

As a starting step, the nucleotide sequence of 11E1VL was compared to the murine germline gene section of the IMGT database (http:// IMGT. cines. fr). .

Murine IGKV4-79 x 01 and IGKJ4 x 01 germline genes have been identified with 98.58% sequence identity to the V region and 97.22% to the J region, respectively. Regarding the identity obtained, it was decided to use the 11E1VL sequence directly to find human homologous sequences.

These alignments are shown in FIGS. 38A (for the V gene) and 38B (for the J gene).

Comparison of nucleotide sequence of 11E1VL with human germline genes

To identify the best human candidate sequences for CDR grafting, human germline genes exhibiting the best identity with 11E1VL have been searched. For this purpose, the nucleotide sequence of 11E1VL was aligned with the human germline gene sequence portion of the IMGT database.

Human IGKV3-7 x 02 and IGKV3D-7 x 01 have been identified, with both germline genes being 69.86% for V-region sequence identity. IGKV3-7 × 02 human germline genes are "ORFs" in the IMGT database, meaning that this sequence is found in the human genome, but there may be some recombination problems leading to the production of non-functional IGKV3-7 × 02 from natural antibodies. Therefore, IGKV3D-7 x 01 germline genes were selected as the accepted human V-regions for the murine 11E1 VLCDRs.

For the J region, the best homology score was first obtained from humans, with human IGKJ 3x 01 showing 78.38% sequence identity. However, a higher number of consecutive identical nucleotides and a better amino acid match (sequence identity of 75.68%) were found in the alignment of the human IGKJ4 × 02 germline genes. Thus IGKJ4 × 02 germline genes were selected as the accepted human J regions of the murine 11E1VL CDRs.

The alignment is shown in FIGS. 39A (V region) and 39B (J region).

To optimize selection, one skilled in the art can also align between protein sequences to aid in their selection.

Humanized versions of 11E1VL

The following humanization procedure consisted in ligating the CDRs of the selected germline gene sequences IGKV3D-7 x 01 and IGKJ4 x 02 and murine 11E1VL to the framework of these germline gene sequences.

As depicted in fig. 40, residues in bold in the 11E1VL sequence correspond to the 30 amino acids found to be different between the 11E1VL region and the selected human framework (human FR, i.e., IGKV3D-7 × 01 and IGKJ4 × 02).

Referring to several criteria, such as its known involvement in the VH/VL interface, involvement in antigen binding or CDR structure, amino acid class changes between murine and human residues, positioning of this residue in the 3D structure of the variable region, 4 out of 30 different residues were identified for final mutation. The 4 most important defined residues and mutations in their human counterparts are: murine L4 was changed to human M, Y40 to S, Y87 to F and T96 to P. These residues ranked as primary are shown as bolded residues in the still murine 11E1HZVL sequence of fig. 40.

Of course, the above-mentioned residues to be finally tested are not limiting, but must be considered as preferred mutations.

With the aid of molecular models, further mutations can be identified. Residues rated as secondary, i.e., residues 24(S/R), 53(W/L), 66(I/T), 67(L/R), 86(S/D), 95(Q/E), 99(A/F) or 121(E/D), can be mentioned, and mutations at these residues are also contemplated in another preferred embodiment.

Of course, the above-mentioned residues to be finally tested are not limiting, but must be considered as preferred mutations. In another preferred embodiment, all 18 other residues ranked tertiary among 30 different amino acids can be reconsidered.

All of the above mutations will be tested separately or according to different combinations.

FIG. 40 shows the performed above mutations identifying a clear humanised 11E1 VL. The numbers under each proposed mutation correspond to the ranking of the mutation to be made.

II-humanization of the heavy chain variable region

Comparison of the nucleotide sequence of 11E1VH with murine germline genes

As a starting step, the nucleotide sequence of 11E1VH was compared to the murine germline gene section of the IMGT database (http:// IMGT. cines. fr).

Murine IGHV1-7 x 01, IGHD4-l x 01, and IGHJ 3x 01 germline genes have been identified that are 94.10% sequence identity for the V region, 66.67% for the D region, and 100% for the J region, respectively. Regarding the identity obtained, it was decided to use the 11E1VH sequence directly to find human homologous sequences.

These alignments are shown in FIG. 41A (for the V gene), 41B (for the D gene) and 41C (for the J gene).

Comparison of the nucleotide sequence of 11E1VH with human germline genes

To identify the best human candidate sequences for CDR grafting, human germline genes exhibiting the best identity with 11E1VH have been searched. For this purpose, the nucleotide sequence of 11E1VH was aligned with the human germline gene sequence portion of the IMGT database. For optimal selection, alignments between protein sequences were performed to retrieve better homologous sequences.

These two complementary approaches led to the identification of two possible accepted human V sequences for the murine 11E1VH CDR. The nucleotide alignment gave 75.69% sequence identity for the human IGHV1-2 x 02 germline gene, while the protein sequence alignment gave 71.10% sequence identity for the human IGHV1-46 x 01 germline gene.

Notably, the D region strictly belongs to the CDR3 region in the VH region. The humanization process is based on the CDR grafting (CDR-grafting) method. Analysis of the closest human D gene in this strategy is useless.

Search for homologous sequences in the J region identified the human IGHJ4 × 03 germline gene with 80.85% sequence identity.

Human IGHV1-46 x 01V germline gene and human IGHJ4 x 03J germline gene were selected as the accepted human sequences for the murine 11E1VH CDRs, taking into account overall similarity and sequence alignment.

The alignment is shown in FIGS. 42A (V region) and 42B (J region).

Humanized versions of 11E1VH

The following humanization procedure was followed by the step of ligating the CDRs of the selected germline gene sequences IGHV1-46 x 01 and IGHJ4 x 03 and murine 11E1VH to the framework of these germline gene sequences.

As depicted in fig. 43, residues in bold in the 11E1VH sequence correspond to the 26 amino acids found to be different between the 11E1VH region and the selected human framework (human FR, i.e., IGHV1-46 × 01 and IGHJ4 × 03).

Referring to several criteria, such as its known involvement in the VH/VL interface, involvement in antigen binding or CDR structure, amino acid class changes between murine and human residues, positioning of this residue in the 3D structure of the variable region, 5 out of 26 different residues were identified for final mutation. The 5 most important defined residues and mutations in their human counterparts were murine N40 to human H, Y55 to I, D66 to S, a80 to R and K82 to T. These residues ranked as primary are shown as bolded residues in the still murine 11E1HZVH sequence of fig. 43.

Of course, the above-mentioned residues to be tested are not limiting, but must be considered as preferred mutations.

With the aid of molecular models, further mutations can be identified. Residues rated as secondary, namely residues 53(I/M), 71(L/F), 76(A/V), 78(L/M) and 87(A/V) may be mentioned, and mutations at these residues are also envisaged in another preferred embodiment.

Of course, the above-mentioned residues to be finally tested are not limiting, but must be considered as preferred mutations. In another preferred embodiment, all 16 other residues ranked tertiary among the 26 different amino acids can be reconsidered.

All of the above mutations will be tested separately or according to different combinations.

Figure 43 shows the mutation performed to identify a clear humanised 11E1 VH. The numbers under each proposed mutation correspond to the ranking of the mutation to be made.

Example 18: effect of purified Mab on c-met phosphorylation

In example 3, the effect of anti-c-Met mabs on phosphorylation was evaluated with dose supernatants of each hybridoma to be evaluated. Further experiments were performed with purified 11E1 and 224G11Mab, where the Mab was evaluated at a final concentration of 30. mu.g/ml (200nM) or dose range of 0.0015 to 30. mu.g/ml (0.01-200nM) to determine the IC of each antibody₅₀. The protocol used was the same as that described in example 3.

The results of 3 independent experiments are shown in fig. 44 and demonstrate that once (once) purified 11E1 and 224G11 showed no agonist effect when added alone to a549 cells, but in the presence of HGF, 87% and 75% antagonist effect, respectively. As expected, as agonistsThe 5D5Mab introduced for the positive control showed significant (58%) agonist effect when added alone, while there was only moderate antagonist effect (39%) in the presence of HGF. About EC₅₀Calculation of (11E 1) and IC of 224G11₅₀Are all of nanomolar scale.

Example 19: combined use of 224G11 and Noveba in NCI-H441 xenograft models in vivo

NCI-H441 cells from ATCC were routinely cultured in RPMI1640 medium, 10% FCS, 1% L-glutamine. Cells were passaged two days prior to implantation, leaving them in exponential growth phase at the time of implantation. Ten million NCI-H441 cells were implanted into athymic nude mice. Tumors were measured 5 days after implantation, and animals with comparable tumor sizes were divided into groups of 6 mice. i.p. treatment of mice with 2mg of 224G11 Mab/mouse followed by 1mg of antibody/mouse, twice a week up to 38 days, or 3 injections of noviben at 8mg/kg(D5, D12, D19). A third group for administration of the combination treatment is also included. Novier booki.p. injection administration. Tumor volume was measured twice weekly and by the formula: pi/6 × Length × Width × height, in NovienDuring the treatment, the animal weight was monitored daily. Statistical analysis was performed using either the t-test or the Mann-Whitney test at each measurement time. In this experiment, the mean tumor volume of the single modality treated group was for 224G11, noviben 41 days after the first injectionAnd Novier bookThe +224G11 decreased by 72%, 76% and 99.8%, respectively. On day 41, combination therapy significantly improved tumor growth compared to monotherapy treatment (on day 41, vs. noviban alone)P.ltoreq.0.041 compared to 224G11 alone and p.ltoreq.0.002) 4 of the 6 mice in the combination treatment group were tumor-free. The results are shown in FIG. 45.

These results were confirmed after 50 days of treatment end (D88), where 66% of the mice receiving the combination treatment still had no tumor.

Example 20: combined use of 224G11 and doxorubicin in vivo in the NCI-H441 xenograft model

NCI-H441 cells from ATCC were routinely cultured in RPMI1640 medium, 10% FCS, 1% L-glutamine. Cells were passaged two days prior to implantation, and allowed to grow exponentially at the time of implantation. Ten million NCI-H441 cells were implanted into athymic nude mice. Tumors were measured 5 days after implantation, and animals with comparable tumor sizes were grouped, 6 mice per group. Mice were treated i.p. with a drug loading dose of 2mg of 224G11 Mab/mouse and then with 1mg of antibody/mouse, two times a week, or with 5mg/kg4 injections of doxorubicin (D5, D12, D19, D26). A third group for administration of the combination therapy is also included. Doxorubicin was administered i.p. injection. Tumor volume was measured twice weekly and by the formula: pi/6 x length x width x height, and the animal weight was monitored daily during doxorubicin treatment. Statistical analysis was performed using either the t-test or the Mann-Whitney test at the time of each measurement. Both monotherapy and combination treatment showed significant antitumor activity (from D11 to D39p ≤ 0.002) compared to control group. The results are shown in FIG. 46.

Combination therapy also showed significant anti-tumor growth activity during periods D11 and D39 compared to monotherapy, suggesting that the use of anti-c-Met in combination with doxorubicin is beneficial.

Example 21: combined use of 224G11 and docetaxel in vivo in NCI-H441 xenograft model

NCI-H441 cells from ATCC were routinely cultured in RPMI1640 medium, 10% FCS, 1% L-glutamine. Cells were passaged two days prior to implantation, and allowed to grow exponentially at the time of implantation. Nine million NCI-H441 cells were implanted into athymic nude mice. Tumors were measured 5 days after implantation, and animals with comparable tumor sizes were grouped, 6 mice per group. i.p. mice were treated with a 2mg drug loading dose of 224G11 Mab/mouse, followed by 1mg antibody/mouse, twice a week, or 4 injections of docetaxel at 7.5mg/kg (D5, D12, D19, D26). A third group for administration of the combination therapy is also included. Docetaxel was administered i.p. injection. Tumor volume was measured twice weekly and by the formula: pi/6 x length x width x height, the animal weight was monitored daily during docetaxel treatment. Statistical analysis was performed using either the t-test or the Mann-Whitney test at the time of each measurement. Both monotherapy and combination treatment showed significant antitumor activity (from D11 to D35p ≦ 0.002) compared to the control group. The results are shown in FIG. 47.

Combination therapy also showed significant anti-tumor growth activity during D18 and D35 compared to single modality treatment, suggesting a benefit in the combined use of anti-c-Met and docetaxel.

Example 22: combined use of 224G11 and temozolomide in vivo in a U87MG xenograft model

U87-MG cells from ATCC were routinely cultured in DMEM medium, 10% FCS, 1% L-glutamine. Cells were passaged two days prior to implantation, and allowed to grow exponentially at the time of implantation. Five million U87-MG cells were implanted into athymic nude mice. Nineteen days after implantation, tumors were measured and animals with comparable tumor size were divided into groups of 6 mice. i.p. mice were treated with a 2mg drug loading dose of 224G11 Mab/mouse, followed by 1mg antibody/mouse, twice a week, or 3 injections of temozolomide at 5mg/kg (D19, D26, D33). A third group for administration of the combination therapy is also included. Temozolomide was administered i.p. injection. Tumor volume was measured twice weekly and by the formula: pi/6 x length x width x height, and the animal weight was monitored daily during temozolomide treatment. Statistical analysis was performed using either the t-test or the Mann-Whitney test at the time of each measurement. Both monotherapy and combination treatment showed significant antitumor activity compared to the control group (from D22 to D32p ≦ 0.002 (where control mice were euthanized for ethical reasons)). The results are shown in FIG. 48.

Combination therapy also showed significant anti-tumor growth activity (P ≦ 0.002, from 22 to 43 days (where control mice were euthanized for ethical reasons)), compared to monotherapy, which was temozolomide alone, and 224G11 alone from 29 to 53 days (the last day of treatment). Taken together, these data indicate that the combined use of anti-c-Met and temozolomide treatment is beneficial.

Example 23: sphere forming

As with the other mabs already described in example 9, we evaluated the ability of the 224G11Mab to inhibit tumor growth in vitro in the U87-MG spheroid model. For this purpose, U-87MG cells grown in monolayers were detached with trypsin-EDTA and resuspended in complete cell culture medium. Spheroid formation was initiated by seeding 625 cells in DMEM-2.5% FCS into a single well with a round bottom 96-well plate. To prevent cell adhesion to the substrate, the plates were pre-coated with poly (hydroxyethyl methacrylate) (polyHEMA) in 95% ethanol and allowed to air dry at room temperature. The plates were incubated at 37 ℃ in a humidified 5% CO2 (humidified incubator) under standard cell culture conditions. In a spherical bodyPurified monoclonal antibody (10. mu.g/ml) was added after 4 and 10 days of culture. Spheroids were cultured for 17 days. Then, growth of the spheroids was monitored by measuring the area of the spheroids with an automatic measurement module of axiovision software. Area in mum²And (4) expressing. 8-16 spheroids were evaluated per condition. After 10 days of culture and before the addition of antibody after 17 days of culture, the size of spheroids was measured.

Under these conditions, spheroids of the same species were obtained and no statistical difference was observed before the addition of antibody (fig. 49A).

As shown in fig. 49B-49D, the isotype control, 9G4 did not affect the growth of spheroids after 10 or 17 days of culture. Although the addition of 5D5 had no major effect on spheroid size, the addition of 224G11 and 11E1 significantly inhibited tumor growth.

Example 24: in vitro activity of chimeric and humanized forms of 224G11 in phosphorylation-c-Met assay

In functional assays, to compare the in vitro potency of murine, chimeric and humanized forms, culture supernatants from 224G11 hybridomas, and HEK293 transfected cells were dosed (dose) and tested as described in example 3. As already described in fig. 6B, the data summarized in fig. 50 show the expected results for unpurified murine antibody. Both chimeric and humanized unpurified antibodies showed comparable activity when added alone (fig. 50A) or incubated in the presence of HGF (fig. 50B).

Example 25: determination of the affinity constant (KD) of anti-c-Met antibodies by Biacore analysis

The binding affinity of the purified 11E1 and 224G11 antibodies was studied by BIAcore X, using as antigen a recombinant c-Met-extracellular region (ECD) fused to the human IgG1Fc region (R & D Systems). Since both the c-Met-Fc fusion protein and the antibody are bivalent compounds, Fab fragments (MW =50kDa) of Mab11E1 and 224G11 were generated by papain cleavage, purified and used in this assay to avoid affecting the affinity parameters. For this assay, an anti-histidine tag capture antibody was coated on a CM5 sensor chip. The running buffer was HBS-EP, the flow rate was 30. mu.l/min, and the assay was performed at 25 ℃. The soluble c-Met (ECD _ M1)2-Fc- (HHHHHHHHHH) 2 antigen was captured on the sensor chip (approximately 270 RU) and the test antibody was used as the analyte in solution. Regeneration of the sensor chip was performed using glycine, HCl ph1.5 buffer on two flow cells for half a minute.

The principle of this analysis is shown in fig. 51. The power generation parameters are summarized in table 4 below. They show that both 11El and 224G11 anti-c-Met antibodies bind the recombinant c-Met-Fc fusion protein with comparable affinity in the range of approximately 40 pM.

TABLE 4

Example 26: in vivo Activity of MDA-MB-231 of 224G11 and human HGF-derived MRC5 cell Co-transplanted athymic nude mouse

Both MDA-MB-213 and MRC5 cells from ATCC were cultured in DMEM medium, 10% FCS, 1% L-glutamine. Cells were passaged two days prior to implantation, and allowed to grow exponentially at the time of implantation. Five million MDA-MB-231 cells and 500000MRC5 cells were co-injected s.c. into athymic nude mice. Twelve days after implantation, tumors were measured and tumor size comparable animals were grouped, 6 mice per group. Mice were treated i.p. with a 2mg dose of 224G11 Mab/mouse, then with 1mg antibody/mouse, twice a week. Tumor volume was measured twice weekly and by the formula: pi/6 × length × width × height.

The results depicted in fig. 52 show that mice treated with 224G11 have a significant difference in median tumor growth compared to a group of controls.

Example 27: supplementary components for humanization of antibodies 227H1, 11E1 and 224G11

General procedure

Humanization of anti-c-Met antibodies, each chain was performed independently and sequentially, taking into account the amino acids analyzed for each variable region. The first attempt to evaluate the humanization process was an ELISA-based binding assay for recombinant Fc-cMet; the binding activity of the humanized antibody was compared to that of the recombinant chimeric antibody. In a second attempt, the ability of anti-c-Met antibodies to replace Fc-cMet binding to plastic coated recombinant HGF was evaluated; this competitive assay allows direct comparison of murine, chimeric and humanized versions of anti-c-Met antibodies.

FIGS. 53 and 54 illustrate the typical anti-c-Met binding activity of 227H1, 11E1, and 224G11 murine monoclonal antibodies.

FIG. 53 shows the anti-c-Met direct binding activity of the purified murine antibodies tested. In this assay, murine monoclonal anti-c-Met antibodies show a different but still dose-dependent anti-c-Met binding activity.

FIG. 54 shows the HGF-cMet binding competitive activity of purified murine antibodies. Competitive assays showed reliable differences between these anti-c-Met monoclonal antibodies, with moderate incomplete but reliable competitive activity for the 11E1 monoclonal antibody, while murine 224G11 and 227H1 showed a similar pattern of competitive activity with 100% of the maximal HGF binding substitution at high antibody concentrations. The 224G11 monoclonal antibody showed the best IC₅₀The value is obtained.

Notably, the direct binding activity of murine antibodies does not reflect their intrinsic HGF binding competitive properties.

These two assays were used to characterize recombinant chimeric and humanized versions of murine anti-c-Met antibodies. For this purpose, briefly, the anti-cMet variable regions, whether murine or humanized, were cloned into a line of LONZA' spcoplus expression vectors and recombinant IgG1/κ -derived antibodies were expressed in CHO cells. Expression culture supernatants were concentrated and extensively dialyzed against PBS and then adjusted to the dose of antibody concentration for expression and used directly to assess the corresponding anti-c-Met binding activity. Both direct binding and HGF competition assays were assessed to better characterize the recombinant chimeric or humanized versions.

Example 27-1: humanization of the 227H1 heavy chain variable region

To identify the human candidate sequences that are optimal for CDR grafting, human germline genes that exhibit the best identity with 227H1VH have been searched. Human IGHV1-2 x 02V germline genes and human IGHJ4 x 01J germline genes were selected as the accepted human sequences for the murine 227H1VH CDRs with the help of the IMGT database.

FIG. 55 shows an amino acid alignment of the murine 227H1VH region with a selected human framework. In the human FR band, only the amino acids found to be different from the 227H1 murine VH domain are depicted. Lines HZ3VH, HZ2VH and HZ1VH correspond to the humanized version of the implemented 227H1VH region, and the above ("variation located in" lines) mutations are clearly identified. The numbers under each proposed mutation correspond to the ranking of the mutation to be made.

In the first series of experiments, we constructed and analyzed the anti-c-Met binding activity of the three first humanized versions of the 227H1 murine VH domain when expressed in combination with the 227H1 chimeric light chain. The results obtained for the anti-c-Met direct binding assay are shown in figure 56. In this experiment, no difference in binding ability of the tested 227H 1-derived chimeric or partially humanized recombinant antibody was observed. Here, 26 of the 32 amino acids were found to be different in the murine 227H1VH region and in the selected human framework, and they were analyzed to find that the anti-c-Met binding activity of the 227H1 humanized VH region was unrelated to them when combined with a chimeric light chain.

In combination with site-specific mutation analysis of the last six murine residues in the humanized version of HZ1VH of region 227H1VH, we constructed a "fully IMGT humanized" version of the original HZ4VH and determined its anti-c-Met binding properties. The results of the direct binding assay are given in fig. 57, and the results of the HGF binding competition assay are given in fig. 58. It is noteworthy that both the recombinant chimeric and humanized 227H1 versions showed better competitive activity than the parental (parent) murine antibody.

However, in view of the experimental data obtained regarding the anti-c-Met binding properties of the 227H1VH region that was "fully IMGT" humanized, the resulting amino acid sequence depicted in figure 59 was selected and subjected to bioinformatic analysis to assess the level of "humanization" ("humaness") of the so-called 227H1-HZ VH humanized variable region.

For this purpose, a simple comparison of the framework sequences and the people database was made using the IMGT tool. Given the level of humanization we achieved in this process, 89 of the 89 framework-equivalent residues analyzed were found to be of reliable human origin. Only the CDR residues were found to differ, and if so, from the corresponding human germline genes and apparently at the hypervariable sites. Based on the IMGT coding system and homology analysis tools, we fully humanised the variable regions of murine antibodies for the first time.

Example 27-2: humanization of 11E1 monoclonal antibody

Humanization of the heavy chain variable region of I-11E1

To identify the best human candidate sequences for CDR grafting, human germline genes showing the best identity with the 11E1VH murine sequence have been searched. Human IGHV1-46 x 01V germline genes and human IGHJ4 x 03J germline genes were selected as accepted human sequences for the murine 11E1VH CDRs with the help of the IMGT database.

FIG. 60 shows an amino acid alignment of the murine 11E1VH region with a selected human framework. In the human FR band, only the amino acids found to be different from the 11E1 murine VH domain are depicted. Lines HZ VH3, HZ VH2, and HZ VH1 correspond to the humanized versions of the embodied 11E1VH region, and the above ("variation located in" lines) mutations are clearly identified. The numbers under each proposed mutation correspond to the ranking of the mutation to be made.

In the first series of experiments, we constructed and analyzed three first humanized versions of the 11E1 murine VH region when expressed in combination with a chimeric light chain of 11E 1. The results obtained for the anti-c-Met direct binding assay are shown in figure 61. In this experiment, similar binding capacity was observed for the tested chimeric or partially humanized recombinant antibodies derived from 11E 1. Here, 19 of the 24 amino acids were found to be different in the murine 11E1VH region and in the selected human framework, which were analyzed to find that the anti-c-Met binding activity of the 11E1 humanized VH region was independent of them when combined with the chimeric light chain.

Humanization of the light chain variable region of II-11E1

To identify the best human candidate sequences for CDR grafting, human germline genes showing the best identity with the 11E1VL murine sequence have been searched. Human IGKV3D-7 x 01V germline genes and human IGKJ4 x 01J germline genes were selected as accepted human sequences for the murine 11E1VL CDRs with the help of the IMGT database.

FIG. 62 shows an amino acid alignment of the murine 11E1VL region with a selected human framework. In the human FR band, only the amino acids found to be different from the 11E1 murine VL region are depicted. Lines HZ VL3, HZ VL2 and HZ VL1 correspond to the humanized versions of the 11E1VL region implemented, and the above ("changes in" lines) mutations are clearly identified. The numbers under each proposed mutation correspond to the ranking of the mutation to be made.

In the first series of experiments, we constructed and analyzed three first humanized versions of the 11E1 murine VL region when expressed in combination with the 11E1 chimeric heavy chain. The results obtained for the anti-c-Met direct binding assay are shown in figure 63. In this experiment, we observed similar binding capacity of the chimeric or partially humanized recombinant antibodies of 11E1 origin tested. Here, 26 of the 30 amino acids were found to be different in the murine 11E1VL region and the selected human framework, and they were analyzed to find that the anti-c-Met binding activity of the 11E1 humanized VL region was unrelated to them when combined with the chimeric heavy chain.

Humanization of III-11E1 antibodies

At this stage of humanization of the 11E1 monoclonal antibody, the humanized antibody sequence theoretically generated contained only 5 external CDR residues from the parental murine VH region (outside-CDR residue) and 4 external CDR residues from the parental murine VL sequence (see fig. 60, lines HZ VH1 and 62, lines HZ VL 1). The humanized version of 11El immediately characterizing the produced heavy and light chain combination was then determined. The results of the anti-c-Met direct binding assay are given in figure 64.

In this experiment, similar binding capacity was observed for the chimeric or humanized recombinant antibodies of 11E1 origin tested. Analysis of HGF binding competition properties and site-directed mutation analysis of the effects of the remaining 9 murine residues were performed independently or in combination in the VH1/VL1 "pre-humanized" version of this selected 11E1 monoclonal antibody.

Examples 27 to 3: humanization of the 224G11 monoclonal antibody

Humanization of the I-224G11 heavy chain variable region

To identify the best human candidate sequences for CDR grafting, human germline genes showing the best identity with the 224G11VH murine sequence have been searched.

Given the high sequence homology of the 224G11 and 227H1VH region sequences, and as has been confirmed by tools using the IMGT database, the same human IGHV1-2 x 02V germline gene and human IGHJ4 x 01J germline gene were selected as the accepted human sequences for the murine 224G11VH CDRs.

Based on this high homology, it was decided to directly transfer (transfer) the humanization information obtained from humanization of the 227H1VH region (see example 27), and we then designed a "fully IMGT" humanized version as depicted in figure 65, which represents an amino acid alignment of the murine 227H1 and 224G11VH regions with the selected human framework. In the human FR row, only the amino acids found to be different from the 224G11 murine VH region are described. Line HZVH0 corresponds to the "complete IMGT" humanized version of region 224G11VH, as obtained from the "complete IMGT" 227H1-HZVH region.

A humanized version of the "complete IMGT" of the murine VH region of 224G11 has then been constructed and analyzed for anti-c-Met binding activity when expressed in combination with a 224G11 chimeric light chain. The results obtained for the anti-c-Met direct binding assay are shown in fig. 66, while fig. 67 shows the HGF binding competition assay. In view of the experimental data obtained for the anti-c-Met binding properties of the region 224G11VH that was "fully IMGT" humanized, the resulting amino acid sequences depicted in FIG. 65 were selected and bioinformatically analyzed to assess the "humanization" level of the so-called 224G11-HZ VH0 region.

In view of the humanization strategy used herein, it was necessary to perform humanization analysis of the 224G11HZ VH0 sequence with reference to example 27. As already described for the humanization of the 227H1VH region, we demonstrated the reliability of the IMGT coding system and homology analysis tools, and also demonstrated the possibility of inter-antibody transfer of the humanization strategy under the constraints of their intrinsic homology.

Humanization of the light chain variable region of II-224G11

To identify the best human candidate sequences for CDR grafting, human germline genes showing the best identity with the 224G11VL murine sequence have been searched. With the help of the IMGT database, two possible accepted human V regions for the murine 224G11VL CDR were identified. For region 224G11VL, two humanization strategies were planned. The first corresponded to the initial trial of a human framework with a shorter CDR1 length (IGKV3-11 x 01) and the second with a longer CDR1 length (IGKV4-1 x 01).

FIG. 68 shows an amino acid alignment of the murine 224G11VL region with two selected human frameworks. In the shorter and longer lines of Hu-FR, only the amino acids found to be different from the murine VL region of 224G11 are described. The HZ VL3, line HZ VL6 corresponds to the basic humanized version of the 224G11VL region, and the mutations described above ("level" lines) are clearly identified. The numbers under each proposed mutation correspond to the order in which the mutation is to be made, whether the basic "shorter" or "longer" CDR1 framework is selected.

In a first set of experiments, two basic humanized versions of the murine VL region of 224G11 were constructed and analyzed for anti-c-Met binding activity when expressed in combination with the 224G11 chimeric heavy chain. The results obtained for the anti-c-Met direct binding assay are shown in figure 69. In this experiment, a similar anti-c-Met binding capacity was observed for the chimeric and HZ VL6 ("longer CDR 1")) versions, whereas little binding was detected for the HZ VL3 ("shorter CDR 1") recombinant 224G 11-derived antibodies.

In a second set of experiments, we constructed and analyzed the anti-c-Met binding activity of a humanized version of the implemented HZVL 6-derived 224G11VH region when expressed in combination with the 224G11 chimeric heavy chain. Two additional humanized forms were analyzed; in the HZ VL5 version, the third set of seven residues (grade 3) was humanized, and in the HZ VL4 version only the first set of four remaining residues (residues rated at grade 1) remained murine. The results obtained for the anti-c-Met direct binding assay are shown in figure 70. In this experiment, no difference in binding capacity was observed between the tested 224G 11-derived chimeric or partially humanized recombinant antibodies. Here, 18 of the 22 amino acids were found to be different in the murine 224G11VL region and the selected "longer CDR 1" human framework, and analysis of them revealed that the anti-c-Met binding activity of the 224G11 humanized VL region was independent of them when combined with the chimeric heavy chain.

The humanized version of HZ VL4 of region 224G11VL was then tested in an HGF binding competition assay. As shown in fig. 71, the results obtained demonstrate that the competitive activities of murine and recombinant chimeric and HZ VL4 humanized 224G 11-derived antibodies are similar.

At this stage of humanization of the 224G11VL region, the resulting sequence contained only 4 external CDR residues from the murine parent sequence. As shown in fig. 72, the 4 § labeled residues L4, M39, H40 and R84.

Based on the IMGT coding system and homology analysis tools, we demonstrated that human frameworks that show structural differences in CDR length remain appropriate in the humanization process. It was then decided to characterize the resulting humanized versions of the heavy and light chains of the 224G11 antibody. Site-directed mutagenesis analysis was then performed on the effect of the remaining 4 murine residues when expressed in combination with the VH0 humanized version of the heavy chain.

Humanization of III-224G11 antibodies

In the first series of experiments, we constructed and analyzed a fully humanized version of the 224G11 antibody for anti-c-Met binding activity. This recombinant version includes VH and VL regions, respectively, that are both humanized for VH0 and VL 4. The results obtained for the anti-c-Met direct binding assay are shown in figure 73. In this experiment, the anti-c-Met binding activity of fully human 224G11 was found to be similar to the "single-chain" humanized and chimeric recombinant 224G11 version.

The fully humanized version of region 224G11VL was then tested in an HGF binding competition assay. The results obtained are shown in fig. 74, which demonstrates that the competitive activities of the parental murine and recombinant chimeric and fully humanized 224G11 derived antibodies are similar.

At this stage of humanization of the 224G11 antibody, the resulting sequence contained only 4 external CDR residues from the murine parent light chain variable region sequence. We then analyzed single variants of site directed mutations in the VL4 humanized VL region when expressed in combination with a humanized version of VH0 heavy chain. For the direct binding assay we identified possible related residues in 4 experiments, i.e. M39 and H40, as shown in figure 75.

It was decided to analyze multiple mutants of the hZ VL4 humanized 224G11VL region when expressed in combination with the HZ VH0 humanized 224G11VH region. For the direct binding assay as shown in figure 76 and for the HGF binding competition assay as shown in figure 77, the polyamino acid mutants of the VL4 region were analyzed to identify the best humanised combinations. On the basis of the single mutant analysis, a focus was made on the double or triple mutants which could show the best anti-c-Met activity. The VH0/VL4-2x mutant corresponded to a humanized VH domain of HZ VH0224G11 expressed with a humanized VL domain of HZ VL4224G11 (with double L4M/R84G mutation). The VH0/VL4-3x mutant corresponded to the HZ VH0224G11 humanized VH domain expressed with the HZ VL4224G11 humanized VL domain (with three L4M/M39L/R84G mutations).

In view of the experimental data obtained for the anti-c-Met binding properties of the fully humanized 224G11 antibody, bioinformatic analysis was then performed on both the heavy and light chain variable region sequences to assess the level of "humanization" of the VH0/VL4-2x and VH0/VL4-3x best humanized versions. "complete IMGT" humanization of the VH0224G11VH region has been previously demonstrated. With respect to the level of humanization of VL4-2x and-3 x224G11 humanized VL region versions, they contained only murine residues M39 and/or H40. These two possible key residues are located at the end of the CDR1, and M39 is the N-terminal CDR anchor (anchor). Given the problem of CDR length we are faced with in humanizing the 224G11VL region, and given those positions that are part of the Kabat definition of the VL CDR1, the level of humanization of the fully humanized 224G11 antibody should exhibit strongly induced immunogenicity due to having the fewest conserved murine residues.

Claims (28)

1. An isolated antibody or one of its functional bivalent fragments, characterized in that said antibody or functional bivalent fragment thereof comprises a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences SEQ ID nos. 7, 8 and 9, respectively; the light chain comprises CDR-L1, CDR-L2 and CDR-L3 having the amino acid sequences of SEQ ID Nos. 15, 16 and 17, respectively.

2. The antibody or one of its functional bivalent fragments according to claim 1, characterized in that said antibody or functional bivalent fragment thereof comprises a heavy chain of amino acid sequence SEQ ID No.20 and a light chain of amino acid sequence SEQ ID No. 23.

3. A murine hybridoma capable of secreting the antibody according to claim 1, characterized in that said hybridoma is the murine hybridoma deposited at the institute of pasteur, paris CNCM at 6/7/2007 under accession number I-3786.

4. Antibody or one of its functional bivalent fragments according to any one of claims 1 and 2, characterized in that it is a monoclonal antibody.

5. The antibody or one of its functional bivalent fragments according to claim 4, characterized in that said antibody is a chimeric antibody, wherein the light and heavy chain constant regions are derived from an antibody of a species heterologous to the mouse.

6. Chimeric antibody or one of its functional bivalent fragments according to claim 5, characterized in that said heterologous species is human.

7. Humanized antibody or one of its functional bivalent fragments according to claim 6, characterized in that the light and heavy chain constant regions derived from human antibodies are the light chain kappa region and the heavy chain gamma-1, gamma-2 or gamma-4 region, respectively.

8. The antibody of any one of claims 1,2 and 4-7, wherein the antibody is capable of inhibiting ligand-dependent and ligand-independent c-Met activation.

9. The antibody of claim 8, wherein the antibody is capable of specifically binding to c-Met.

10. The antibody of claim 9, wherein the antibody is capable of inhibiting tumor cell proliferation by at least 50% of at least one tumor type.

11. The antibody of claim 10, wherein the antibody is capable of inhibiting c-Met dimerization.

12. The antibody of any one of claims 1,2, and 4-11, wherein the antibody is bivalent.

13. An isolated nucleic acid, characterized in that it is selected from the group consisting of:

a) nucleic acid encoding the antibody according to any one of claims 1,2 and 4 to 12 or one of its functional bivalent fragments;

b) DNA nucleic acid comprising

A nucleic acid sequence comprising the sequences SEQ ID No.43 and SEQ ID No. 46;

c) RNA nucleic acids corresponding to the nucleic acids defined in b); and

d) nucleic acids complementary to the nucleic acids defined in a) and b).

14. A vector comprising the DNA nucleic acid of parts a) and b) of claim 13.

15. A host cell comprising the vector of claim 14.

16. Method for producing an antibody according to one of claims 1,2 and 4 to 12 or one of its functional bivalent fragments, characterized in that it comprises the following phases:

a) culturing the cell of claim 14 in a culture medium and under suitable culture conditions; and

b) harvesting said antibody or one of its functional bivalent fragments produced from the culture medium or said cultured cells.

17. Use of an antibody or one of its functional bivalent fragments according to claims 1,2 and 4 to 12 or obtained by the process of claim 16 for the preparation of a medicament for the prevention or treatment of malignancies associated with c-Met activation.

18. A composition comprising as active principle a compound consisting of one of the antibodies or of a functional bivalent fragment thereof according to one of claims 1,2 and 4 to 12, or obtained by the method according to claim 16.

19. Composition according to claim 17, characterized in that it further comprises, as a combination product, one preparation for simultaneous, separate or sequential use, wherein said preparation is an anti-tumor antibody.

20. Composition according to claim 18 or 19, characterized in that it further comprises at least one preparation as a combined product for simultaneous, separate or sequential use, wherein said preparation is a cytotoxic/cytostatic agent.

21. The composition of claim 20, wherein said cytotoxic/cytostatic agent is chemically conjugated to said antibody or said divalent functional fragment for simultaneous use.

22. The composition of claim 21, wherein said cytotoxic/cytostatic agent is selected from the group consisting of alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, antiangiogenic agents, antiestrogens, antiandrogens, and immunomodulators.

23. The composition of claim 22, wherein said cytotoxic/cytostatic agent is a mitotic inhibitor.

24. Composition according to claim 18 or 19, characterized in that at least one of said antibodies or one of its functional bivalent fragments is bound to a cytotoxin and/or a radioactive element.

25. Use of a composition according to any one of claims 18 to 23 for the manufacture of a medicament for the prevention or treatment of malignancies associated with c-Met activation.

26. Use of an antibody or one of its functional bivalent fragments according to one of claims 1,2 and 4 to 12, or obtained by a method according to claim 16, or a composition according to one of claims 18 to 24, for the preparation of a medicament for the prevention or treatment of a malignant tumor.

27. Use according to claim 26, characterized in that said malignant tumour is selected from the group consisting of prostate cancer, osteosarcoma, lung cancer, breast cancer, endometrial cancer, glioblastoma and colon cancer.

28. Use according to claim 25, 26 or 27, characterized in that the malignancy is a cMet activation-associated malignancy selected from HGF-dependent and/or independent malignancies.