AU2003216748A1 - Novel anti-igf-ir antibodies and uses thereof - Google Patents

Abstract

An isolated antibody (Ab), and its functional fragments, that bind to human insulin-like growth factor-1 receptor (IGF-1R) and optionally: (i) inhibit natural binding of insulin-like growth factors (IGF)-1 and/or -2; and/or (ii) inhibit specifically tyrosine kinase activity of IGF-1R. An isolated antibody (Ab), and its functional fragments, that bind to human insulin-like growth factor-1 receptor (IGF-1R) and optionally inhibit natural binding of insulin-like growth factors (IGF)-1 and/or -2 and/or inhibit specifically tyrosine kinase activity of IGF-1R, is new. Ab comprises: (a) a light chain containing at least one complementarity-determining region (CDR) of one of 3 fully defined sequences comprising 16, 7 or 9 amino acids as given in the specification; or (b) a heavy chain containing at least one of the CDRs of one of 3 fully defined sequences comprising 6, 16 or 8 amino acids as given in the specification or sequences with 80% identity after optimal alignment. Independent claims are also included for the following: (1) murine hybridomas that secrete Ab; (2) an isolated nucleic acid (I) that: (a) is RNA or DNA and encodes Ab or its fragments; (b) is the complement of (a); or (c) has at least 18 nucleotides that can hybridize under stringent conditions with any of six sequences fully defined in the specification that encode the specified CDR, or with sequences having 80% identity after optimal alignment; (3) a vector containing (I); (4) a host cell containing the vector of (3); (5) a non-human transgenic animal containing at least one cell transfected with the vector of (3); (6) producing Ab or its fragments; (7) in vitro diagnosis of diseases induced by over- or under-expression of IGF-1R and or EGFR (epidermal growth factor receptor) by reaction with optionally labeled Ab; and (8) kit for performing method (7). ACTIVITY : Cytostatic; Antipsoriatic. The murine antibody 7C10, specific for IGF-1R, was tested (at 250 Microgram twice a week, intraperitoneally) against MCF-7 breast cancer cells, implanted in nude mice. After 37 days, the mean tumor volume was about 150 mm 3>; compared to about 800 mm 3> for controls and 300 mm 3> for animals treated with 10 Microgram tamoxifen. MECHANISM OF ACTION : Inhibition of signal transduction pathways mediated by IGF-1R, optionally also by epidermal growth factor (EGF) receptor, thus inhibiting growth and/or proliferation of tumor cells, especially where dependent on IGF, EGF, estrogens and/or HER2/neu.

Description

IN THE MATTER OF an Application for an Australian Patent corresponding to PCT Application PCT/FR3/00178 I, Michel PERNELLE, C/O CABINET REGIMBEAU, do solemnly and sincerely declare that I am conversant with the English and French languages and am a competent translator thereof, and that to the best of my knowledge and belief the following is a true and correct translation of the PCT Patent Application filed under No. PCT/FR3/00178 Date : June 25, 2004 Mic el PERNELLE For and on behalf of CABINET REGIMBEAU WO 03/059951 PCT/FRO3/00178 NOVEL ANTI-IGF-IR ANTIBODIES AND USES THEREOF The present invention relates to novel antibodies capable of binding specifically to the human insulin 5 like growth factor I receptor IGF-IR and/or capable of specifically inhibiting the tyrosine kinase activity of said IGF-IR receptor, especially monoclonal antibodies of murine, chimeric and humanized origin, as well as the amino acid and nucleic acid sequences coding for 10 these antibodies. The invention likewise comprises the use of these antibodies as a medicament for the prophylactic and/or therapeutic treatment of cancers overexpressing IGF-IR or any pathology connected with the overexpression of said receptor as well as in 15 processes or kits for diagnosis of illnesses connected with the overexpression of the IGF-IR receptor. The invention finally comprises products and/or compositions comprising such antibodies in combination with anti-EGFR antibodies and/or compounds and/or anti 20 cancer agents or agents conjugated with toxins and their use for the prevention and/or the treatment of certain cancers. The insulin-like growth factor I receptor called IGF-IR 25 is a receptor with tyrosine kinase activity having 70% homology with the insulin receptor IR. IGF-IR is a glycoprotein of molecular weight approximately 350,000. It is a hetero-tetrameric receptor of which each half -linked by disulfide bridges- is composed of an 30 extracellular a-subunit and of a transmembrane P subunit (see figure 1). IGF-IR binds IGF I and IGF II with a very high affinity (Kd #1 nM) but is equally capable of binding to insulin with an affinity 100 to 1000 times less. Conversely, the IR binds insulin with 35 a very high affinity although the IGFs only bind to the insulin receptor with a 100 times lower affinity. The tyrosine kinase domain of IGF-IR and of IR has a very high sequence homology although the zones of weaker - 2 homology respectively concern the cysteine-rich region situated on the a-subunit and the C-terminal part of the P-subunit. The sequence differences observed in the a-subunit are situated in the binding zone of the 5 ligands and are therefore at the origin of the relative affinities of IGF-IR and of IR for the IGFs and insulin respectively. The differences in the C-terminal part of the P-subunit result in a divergence in the signalling pathways of the two receptors; IGF-IR mediating 10 mitogenic, differentiation and antiapoptosis effects, while the activation of the IR principally involves effects at the level of the metabolic pathways (Baserga et al., Biochim. Biophys. Acta, 1332: F105-126, 1997; Baserga R., Exp. Cell. Res., 253:1-6, 1999). 15 The cytoplasmic tyrosine kinase proteins are activated by the binding of the ligand to the extracellular domain of the receptor. The activation of the kinases in its turn involves the stimulation of different 20 intra-cellular substrates, including IRS-1, IRS-2, Shc and Grb 10 (Peruzzi F. et al., J. Cancer Res. Clin. Oncol., 125:166-173, 1999). The two major substrates of IGF-IR are IRS and Shc which mediate, by the activation of numerous effectors downstream, the majority of the 25 growth and differentiation effects connected with the attachment of the IGFs to this receptor (figure 2). The availability of substrates can consequently dictate the final biological effect connected with the activation of the IGF-IR. When IRS-1 predominates, the cells tend 30 to proliferate and to transform. When Shc dominates, the cells tend to differentiate (Valentinis B. et al., J. Biol. Chem. 274:12423-12430, 1999). It seems that the route principally involved for the effects of protection against apoptosis is the phosphatidyl 35 inositol 3-kinases (PI 3-kinases) route (Prisco M. et al., Horm. Metab. Res., 31:80-89, 1999; Peruzzi F. et al., J. Cancer Res. Clin. Oncol., 125:166-173, 1999).

- 3 The role of the IGF system in carcinogenesis has become the subject of intensive research in the last ten years. This interest followed the discovery of the fact that in addition to its mitogenic and antiapoptosis 5 properties, IGF-IR seems to be required for the establishment and the maintenance of a transformed phenotype. In fact, it has been well established that an overexpression or a constitutive activation of IGF IR leads, in a great variety of cells, to a growth of 10 the cells independent of the support in media devoid of fetal calf serum, and to the formation of tumors in nude mice. This in itself is not a unique property since a great variety of products of overexpressed genes can transform cells, including a good number of 15 receptors of growth factors. However, the crucial discovery which has clearly demonstrated the major role played by IGF-IR in the transformation has been the demonstration that the R- cells, in which the gene coding for IGF-IR has been inactivated, are totally 20 refractory to transformation by different agents which are usually capable of transforming the cells, such as the E5 protein of bovine papilloma virus, an overexpression of EGFR or of PDGFR, the T antigen of SV 40, activated ras or the combination of these two last 25 factors (Sell C. et al., Proc. Natl. Acad. Sci., USA, 90: 11217-11221, 1993; Sell C. et al., Mol. Cell. Biol., 14:3604-3612, 1994; Morrione A. J., Virol., 69:5300-5303, 1995; Coppola D. et al., Mol. Cell. Biol., 14:4588-4595, 1994; DeAngelis T et al., J. Cell. 30 Physiol., 164:214-221, 1995). IGF-IR is expressed in a great variety of tumors and of tumor lines and the IGFs amplify the tumor growth via their attachment to IGF-IR. Other arguments in favor of 35 the role of IGF-IR in carcinogenesis come from studies using murine monoclonal antibodies directed against the receptor or using negative dominants of IGF-IR. In effect, murine monoclonal antibodies directed against IGF-IR inhibit the proliferation of numerous cell lines - 4 in culture and the growth of tumor cells in vivo (Arteaga C. et al., Cancer Res., 49:6237-6241, 1989; Li et al., Biochem. Biophys. Res. Com., 196:92-98, 1993; Zia F et al., J. Cell. Biol., 24:269-275, 1996; 5 Scotlandi K et al., Cancer Res., 58:4127-4131, 1998). It has likewise been shown in the works of Jiang et al. (Oncogene, 18:6071-6077, 1999) that a negative dominant of IGF-IR is capable of inhibiting tumor proliferation. 10 The object of the present invention is to be able to have available a murine monoclonal antibody, preferably a chimerized or humanized antibody, which will recognize IGF-IR specifically and with great affinity. This antibody will interact little or not at all with 15 the IR receptor on insulin. Its attachment will be able to inhibit in vitro the growth of tumors expressing IGF-IR by interacting principally with the signal transduction pathways activated during IGFl/IGF-IR and IGF2/IGF-IR interactions. This antibody will be able to 20 be active in vivo on all the types of tumors expressing IGF-IR including estrogen-dependent tumors of the breast and tumors of the prostate, which is not the case for the anti-IGF-IR monoclonal antibodies (written MAb or MAB) currently available. In effect, aIR3, which 25 refers to the domain of IGF-IR, totally inhibits the growth of estrogen-dependent tumors of the breast (MCF 7) in vitro but is without effect on the corresponding model in vivo (Arteaga C. et al., J. Clin. Invest. 84:1418-1423, 1989). In the same way, the scFv-Fc 30 fragment derived from the murine monoclonal 1H7 is only weakly active on the tumor of the breast MCF-7 and totally inactive on an androgen-independent tumor of the prostate (Li S. L. et al., Cancer Immunol. Immunother., 49:243-252, 2000). 35 In a surprising manner, the inventors have demonstrated a chimeric antibody (called C7C10) and two humanized antibodies respectively called h7C10 humanized form 1 and h7C10 humanized form 2, derivatives of the murine - 5 monoclonal antibody 7C10, recognising IGF-IR and corresponding to all of the criteria stated above, that is to say to a nonrecognition of the receptor on the insulin, to an in vitro blockage of the IGF1 and/or 5 IGF2 proliferation induced but likewise to the in vivo inhibition of the growth of different tumors expressing IGF-IR among which are an osteosarcoma and a non-small cell lung tumor but likewise and more particularly the estrogen-dependent tumor of the breast MCF-7 and an 10 androgen-independent tumor of the prostate DU-145. In the same way, and in a surprising manner, the intensity of inhibition of the tumor growth of MCF-7 cells in vivo by the antibody 7C10 is comparable, or even significantly superior, to that observed with 15 tamoxifen, one of the reference compounds in the treatment of estrogen-dependent tumors of the breast. Furthermore, it has been shown that these antibodies inhibit the phosphorylation of the tyrosine of the beta chain of IGF-IR and of IRS1, the first substrate of the 20 receptor. Moreover, it has likewise been established that these antibodies cause the internalization of said receptor and its degradation contrary to what is usually observed with natural ligands which allow the rapid recycling of the receptor on the surface of the 25 cells. It has been possible to characterize these antibodies by their peptidic and nucleic sequence, especially by the sequence of their regions determining their complementarity (CDR) for IGF-IR. 30 Thus, according to a first embodiment, a subject of the present invention is an isolated antibody, or one of its functional fragments, said antibody or one of its said fragments being capable of binding specifically to the human insulin-like growth factor I receptor and, if 35 necessary, preferably moreover capable of inhibiting the natural attachment of the ligands IGF1 and/or IGF2 of IGF-IR and/or capable of specifically inhibiting the tyrosine kinase activity of said IGF-IR receptor, characterized in that it comprises a light chain - 6 comprising at least one complementarity determining region CDR chosen from the CDRs of amino acid sequence SEQ ID Nos. 2, 4 or 6, or at least one CDR whose sequence has at least 80%, preferably 85%, 90%, 95% and 5 98% identity, after optimum alignment, with the sequence SEQ ID Nos. 2, 4 or 6, or in that it comprises a heavy chain comprising at least one CDR chosen from the CDRs of amino acid sequence SEQ ID Nos. 8, 10 and 12, or at least one CDR whose sequence has at least 10 80%, preferably 85%, 90%, 95% and 98% identity, after optimum alignment, with the sequence SEQ ID No. 8, 10 and 12. In the present description, the terms "to bind" and 15 "to attach" have the same meaning and are inter changeable. In the present description, the terms polypeptides, polypeptide sequences, peptides and proteins attached 20 to antibody compounds or to their sequence are interchangeable. It must be understood here that the invention does not relate to the antibodies in natural form, that is to 25 say they are not in their natural environment but that they have been able to be isolated or obtained by purification from natural sources, or else obtained by genetic recombination, or by chemical synthesis, and that they can then contain unnatural amino acids as 30 will be described further on. By CDR region or CDR, it is intended to indicate the hypervariable regions of the heavy and light chains of the immunoglobulins as defined by Kabat et al. (Kabat 35 et al., Sequences of proteins of immunological interest, 5th Ed., U.S. Department of Health and Human Services, NIH, 1991, and later editions). 3 heavy chain CDRs and 3 light chain CDRs exist. The term CDR or CDRs is used here in order to indicate, according to the - 7 case, one of these regions or several, or even the whole, of these regions which contain the majority of the amino acid residues responsible for the binding by affinity of the antibody for the antigen or the epitope 5 which it recognizes. By "percentage of identity" between two nucleic acid or amino acid sequences in the sense of the present invention, it is intended to indicate a percentage of 10 nucleotides or of identical amino acid residues between the two sequences to be compared, cbtained after the best alignment (optimum alignment), this percentage being purely statistical and the differences between the two sequences being distributed randomly and over 15 their entire length. The comparisons of sequences between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having aligned them in an optimum manner, said comparison being able to be carried out by segment or 20 by "comparison window". The optimum alignment of the sequences for the comparison can be carried out, in addition to manually, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2:482], by means of the local homology algorithm of 25 Neddleman and Wunsch (1970) [J. Mol. Biol. 48: 443], by means of the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444), by means of computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics 30 Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or else by BLAST N or BLAST P comparison software). The percentage of identity between two nucleic acid or 35 amino acid sequences is determined by comparing these two sequences aligned in an optimum manner and in which the nucleic acid or amino acid sequence to be compared can comprise additions or deletions with respect to the reference sequence for an optimum alignment between - 8 these two sequences. The percentage of identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by 5 dividing this number of identical positions by the total number of positions in the comparison window and by multiplying the result obtained by 100 in order to obtain the percentage of identity between these two sequences. 10 For example, it is possible to use the BLAST program, "BLAST 2 sequences" (Tatusova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247 15 250) available on the site http://www.ncbi.nlm.nih.gov/ gorf/bl2.html, the parameters used being those given by default (in particular for the parameters "open gap penalty" : 5, and "extension gap penalty" : 2; the matrix chosen being, for example, the matrix "BLOSUM 20 62" proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program. By amino acid sequence having at least 80%, preferably 25 85%, 90%, 95% and 98% identity with a reference amino acid sequence, those having, with respect to the reference -sequence, certain modifications, in particular a deletion, addition or substitution of at least one amino acid, a truncation or an elongation are 30 preferred. In the case of a substitution of one or more consecutive or nonconsecutive amino acid(s), the substitutions are preferred in which the substituted amino acids are replaced by "equivalent" amino acids. The expression "equivalent amino acids" is aimed here 35 at indicating any amino acid capable of being substituted with one of the amino acids of the base structure without, however, essentially modifying the biological activities of the corresponding antibodies - 9 and such as will be defined later, especially in the examples. These equivalent amino acids can be determined either 5 by relying on their structural homology with the amino acids which they replace, or on results of comparative trials of biological activity between the different antibodies capable of being carried out. 10 By way of example, mention is made of the possibilities of substitution capable of being carried out without resulting in a profound modification of the biological activity of the corresponding modified antibody. It is thus possible to replace leucine by valine or 15 isoleucine, aspartic acid by glutamic acid, glutamine by asparagine, arginine by lysine, etc., the reverse substitutions being naturally envisageable under the same conditions. 20 The antibodies according to the present invention are preferably specific monoclonal antibodies, especially of murine, chimeric or humanized origin, which can be obtained according to the standard methods well known to the person skilled in the art. 25 In general, for the preparation of monoclonal antibodies or their functional fragments, especially of murine origin, it is possible to refer to techniques which are described in particular in the manual 30 "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or to the technique of preparation from hybridomas described by Kohler and Milstein (Nature, 256:495-497, 1975). 35 The monoclonal antibodies according to the invention can be obtained, for example, from an animal cell immunized against the IGF-IR receptor, or one of its fragments containing the epitope specifically - 10 recognized by said monoclonal antibodies according to the invention. Said IGF-IR receptor, or one of its said fragments, can especially be produced according to the usual working methods, by genetic recombination 5 starting with a nucleic acid sequence contained in the cDNA sequence coding for the IGF-IR receptor or by peptide synthesis starting from a sequence of amino acids comprised in the peptide sequence of the IGF-IR receptor. 10 The monoclonal antibodies according to the invention can, for example, be purified on an affinity column on which the IGF-IR receptor or one of its fragments containing the epitope specifically recognized by said 15 monoclonal antibodies according to the invention has previously. been immobilized. More particularly, said monoclonal antibodies can be purified by chromatography on protein A and/or G, followed or not followed by ion exchange chromatography aimed at eliminating the 20 residual protein contaminants as well as the DNA and the LPS, in itself followed or not followed by exclusion chromatography on Sepharose gel in order to eliminate the potential aggregates due to the presence of dimers or of other multimers. In an even more 25 preferred manner, the whole of these techniques can be used simultaneously or successively. Chimeric or humanized antibodies are likewise included in antibodies according to the present invention. 30 By chimeric antibody, it is intended to indicate an antibody which contains a natural variable (light chain and heavy chain) region derived from an antibody of a given species in combination with the light chain and 35 heavy chain constant regions of an antibody of a species heterologous to said given species. The antibodies or their fragments of chimeric type according to the invention can be prepared by using the - 11 techniques of genetic recombination. For example, the chimeric antibody can be produced by cloning a recombinant DNA containing a promoter and a sequence coding for the variable region of a nonhuman, 5 especially murine, monoclonal antibody according to the invention and a sequence coding for the constant region of human antibody. A chimeric antibody of the invention encoded by such a recombinant gene will be, for example, a mouse-man chimera, the specificity of this 10 antibody being determined by the variable region derived from the murine DNA and its isotype determined by-the constant region derived from the human DNA. For the methods of preparation of chimeric antibodies, it is possible, for example, to refer to the document 15 Verhoeyn et al. (BioEssays, 8:74, 1988). By humanized antibody, it is intended to indicate an antibody which contains CDR regions derived from an antibody of nonhuman origin, the other parts of the 20 antibody molecule being derived from one (or from several) human antibodies. Moreover, some of the residues of the segments of the skeleton (called FR) can be modified in order to conserve the affinity of the binding (Jones et al., Nature, 321:522-525, 1986; 25 Verhoeyen et al., Science, 239:1534-1536, 1988; Riechmann et al., Nature, 332:323-327, 1988). The humanized antibodies according to the invention or their fragments can be prepared by techniques known to 30 the person skilled in the art (such as, for example, those described in the documents Singer et al., J. Immun. 150:2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10: 1-142, 1992; or Bebbington et al., Bio/Technology, 10:169-175, 1992). 35 Such humanized antibodies according to the invention are preferred for their use in in vitro diagnostic methods, or in vivo prophylactic and/or therapeutic treatment.

- 12 By functional fragment of an antibody according to the invention, it is intended to indicate in particular an antibody fragment, such as Fv, scFv (sc for single chain), Fab, F(ab') 2 , Fab', scFv-Fc fragments or 5 diabodies, or any fragment of which the half-life time would have been increased by chemical modification, such as the addition of poly(alkylene) glycol such as poly(ethylene) glycol ("PEGylation") (pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab') 2 -PEG 10 or Fab'-PEG) ("PEG" for Poly(Ethylene) Glycol), or by incorporation in a liposome, said fragments having at least one of the characteristic CDRs of sequence SEQ ID No. 2, 4, 6, 8, 10 or 12 according to the invention, and, especially, in that it is capable of exerting in a 15 general manner an even partial activity of the antibody from which it is descended, such as in particular the capacity to recognize and to bind to the IGF-IR receptor, and, if necessary, to inhibit the activity of the IGF-IR receptor. 20 Preferably, said functional fragments will be constituted or will comprise a partial sequence of the heavy or light variable chain of the antibody from which they are derived, said partial sequence being 25 sufficient to retain the same specificity of binding as the antibody from which it is descended and a sufficient affinity, preferably at least equal to 1/100, in a more preferred manner to at least 1/10, of that of the antibody from which it is descended, with 30 respect to the IGF-IR receptor. Such a functional fragment will contain at the minimum 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the sequence of the antibody 35 from which it is descended. Preferably, these functional fragments will be fragments of Fv, scFv, Fab, F(ab') 2 , F(ab'), scFv-Fc type or diabodies, which generally have the same - 13 specificity of binding as the antibody from which they are descended. According to the present invention, antibody fragments of the invention can be obtained starting from antibodies such as described above by 5 methods such as digestion by enzymes, such as pepsin or papain and/or by cleavage of the disulfide bridges by chemical reduction. In another manner, the antibody fragments comprised in the present invention can be obtained by techniques of genetic recombination 10 likewise well known to the person skilled in the art or else by peptide synthesis by means of, for example, automatic peptide synthesizers such as those supplied by the company Applied Biosystems, etc. 15 In a more preferred manner, the invention comprises the antibodies, or their functional fragments, according to the present invention, especially chimeric or humanized antibodies, obtained by genetic recombination or by chemical synthesis. 20 In a preferred embodiment, a subject of the invention is an antibody, or one of its functional fragments, according to the invention, characterized in that it comprises a heavy chain comprising at least one CDR of 25 sequence SEQ ID No. 12 or a sequence having at least 80% identity after optimum alignment with the sequence SEQ ID No. 12. Among the six short CDR sequences, the third CDR of the 30 heavy chain (CDRH3) has a greater size variability (greater diversity essentially due to the mechanisms of arrangement of the genes which give rise to it). It can be as short as 2 amino acids although the longest size known is 26. Functionally, CDRH3 plays a role in part 35 in thedetermination of the specificity of the antibody (Segal et al., PNAS, 71:4298-4302, 1.974; Amit et al., Science, 233:747-753, 1986; Chothia et al., J. Mol. Biol., 196:901-917, 1987; Chothia et al., Nature, 342:877-883, 1989; Caton et al., J. Immunol., 144:1965- - 14 1968, 1990; Sharon et al., PNAS, 87:4814-4817, 1990; Sharon et al., J. Immunol., 144:4863-4869, 1990; Kabat et al., J. Immunol., 147:1709-1719,.1991). 5 It is known that only a low percentage of the amino acids of the CDRs contribute to the construction of an antibody binding site, but these residues must be maintained in a very specific tridimensional conformation. 10 In a more preferred manner, the present invention relates to an antibody or one of its functional fragments, according to the invention, characterized in that it comprises a heavy chain comprising at least two 15 of the three CDRs or the three CDRs of sequence SEQ ID Nos. 8, 10 and 12, or at least two of three CDRs or three CDRs of sequence respectively having at least 80% identity after optimum alignment with the sequence SEQ ID No. 8, 10 and 12. 20 In a likewise preferred embodiment, a subject of the invention is an antibody or one of its functional fragments, according to the invention, characterized in that it comprises a light chain comprising at least one 25 CDR chosen from the CDRs of sequence SEQ ID No. 2, 4 or 6, or a CDR whose sequence has at least 80% identity after optimum alignment with the sequence SEQ ID No. 2, 4 or 6. 30 In a more preferred embodiment, a subject of the invention is an antibody or one of its functional fragments according to the invention, characterized in that it comprises a light chain comprising at least two of the three CDRs or the three CDRs of sequence SEQ ID 35 Nos. 2, 4 and 6, or at least two of three CDRs or three CDRs of sequence respectively having at least 80% identity after optimum alignment with the sequence SEQ ID No. 2, 4 and 6.

- 15 In a more preferred manner, the antibody or one of its functional fragments according to the invention is characterized in that it comprises a heavy chain comprising the three CDRs of sequence SEQ ID Nos. 8, 10 5 and 12, or three CDRs of sequence respectively having at least 80% of identity after optimum alignment with the sequence SEQ ID No. 8, 10 and 12 and in that it moreover comprises a light chain comprising the three CDRs of sequence SEQ ID Nos. 2, 4 and 6, or three CDRs 10 of sequence respectively having at least 80% of identity after optimum alignment with the sequence SEQ ID No. 2, 4 and 6. According to another aspect, a subject of the present 15 invention is an antibody or one of its functional fragments, according to the invention, characterized in that it does not attach or it does not attach in a significant manner to the human insulin receptor IR. 20 In a preferred manner, said functional fragments according to the present invention will be chosen from the fragments Fv, scFv, Fab, (Fab') 2 , Fab', scFv-Fc or diabodies, or any functional fragment whose half-life would have been increased by a chemical modification, 25 especially by PEGylation, or by incorporation in a liposome. According to another aspect, the invention relates to a murine hybridoma capable of secreting a monoclonal 30 antibody according to the present invention, especially the hybridoma of murine origin such as deposited at the Centre National de Culture De Microorganisme (CNCM, National Center of Microorganism Culture) (Institut Pasteur, Paris, France) on September 19, 2001 under the 35 number 1-2717. The monoclonal antibody here called 7C10, or one of its functional fragments, characterized in that said antibody is secreted by the hybridoma deposited at the - 16 CNCM on September 19, 2001 under the number 1-2717 is, of course, part of the present invention. In a particular embodiment, the present invention 5 relates to a murine antibody, or one of its functional fragments, according to the invention, characterized in that said antibody comprises a light chain of sequence comprising the amino acid sequence SEQ ID No. 54, or a sequence having at least 80% identity after optimum 10 alignment with the sequence SEQ ID No. 54, or/and in that it comprises a heavy chain of sequence comprising the amino acid sequence SEQ ID No. 69, or a sequence having at least 80% identity after optimum alignment with the sequence SEQ ID No. 69. 15 According to a likewise particular aspect, the present invention relates to a chimeric antibody, or one of its functional fragments, according to the invention, characterized in that said antibody moreover comprises 20 the light chain and heavy chain constant regions derived from an antibody of a species heterologous to the mouse, especially man, and in a preferred manner in that the light chain and heavy chain constant regions derived from a human antibody are respectively the 25 kappa and gamma-1, gamma-2 or gamma-4 region. According to a likewise particular aspect, the present invention relates to a humanized antibody or one of its functional fragments, according to the invention, 30 characterized in that said antibody comprises a light chain and/or a heavy chain in which the skeleton segments FRI to FR4 (such as defined below in examples 12 and 13, in tables 5 and 6) of said light chain and/or heavy chain are respectively derived from 35 skeleton segments FRI to FR4 of human antibody light chain and/or heavy chain. According to a preferred embodiment, the humanized antibody or one of its functional fragments, according - 17 to the present invention is characterized in that said humanized antibody comprises a light chain comprising the amino acid sequence SEQ ID No. 61 or 65, or a sequence having at least 80% identity after optimum 5 alignment with the sequence SEQ ID No. 61 or 65, or/and in that it comprises a heavy chain comprising the amino acid sequence SEQ ID No. 75, 79 or 83, or a sequence having at least 80% identity after optimum alignment with the sequence SEQ ID No. 75, 79 or 83. 10 Preferably, the humanized antibody, or one of its functional fragments, according to the invention is characterized in that said humanized antibody comprises a light chain comprising the amino acid sequence SEQ ID 15 No. 65, and in that it comprises a heavy chain of sequence comprising the amino acid sequence SEQ ID No. 79 or 83, preferably SEQ ID No. 83. According to a novel aspect, the present invention 20 relates to an isolated nucleic acid, characterized in that it is chosen from the following nucleic acids: a) a nucleic acid, DNA or RNA, coding for an antibody, or one of its functional fragments, according to the invention; 25 b) a complementary nucleic acid of a nucleic acid such as defined in a); and c) a nucleic acid of at least 18 nucleotides capable of hybridizing under conditions of great stringency with at least one of the CDRs of 30 nucleic acid sequence SEQ ID No. 1, 3, 5, 7, 9 or 11, or with a sequence having at least 80%, preferably 85%, 90%, 95% and 98%, identity after optimum alignment with the sequence SEQ ID No. 1, 3, 5, 7, 9 or 11. 35 By nucleic acid, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be employed indifferently in the present invention, it is - 18 intended to indicate a precise linkage of nucleotides, which are modified or unmodified, allowing a fragment or a region of a nucleic acid to be defined, containing or not containing unnatural nucleotides, and being able 5 to correspond just as well to a double-stranded DNA, a single-stranded DNA as to the transcription products of said DNAs. It must also be understood here that the present 10 invention does not concern the nucleotide sequences in their natural chromosomal environment, that is to say in the natural state. It concerns sequences which have been isolated and/or purified, that is to say that they have been selected directly or indirectly, for example 15 by copy, their environment having been at least partially modified. It is thus likewise intended to indicate here the isolated nucleic acids obtained by genetic recombination by means, for example, of host cells or obtained by chemical synthesis. 20 By nucleic sequences having a percentage of identity of at least 80%, preferably 85%, 90%, 95% and 98%, after optimum alignment with a preferred sequence, it is intended to indicate the nucleic sequences having, with 25 respect to the reference nucleic sequence, certain modifications such as, in particular, a deletion, a truncation, an elongation, a chimeric fusion and/or a substitution, especially point substitution. It preferably concerns sequences in which the sequences 30 code for the same amino acid sequences as the reference sequence, this being connected to the degeneracy of the genetic code, or complementary sequences which are capable of hybridizing specifically with the reference sequences, preferably under conditions of high 35 stringency, especially such as defined below. A hybridization under conditions of high stringency signifies that the temperature conditions and ionic strength conditions are chosen in such a way that they - 19 allow the maintenance of the hybridization between two fragments of complementary DNA. By way of illustration, conditions of high stringency of the hybridization step for the purposes of defining the polynucleotide 5 fragments described above are advantageously the following. The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 420C for 3 hours in 10 phosphate buffer (20 mM, pH 7.5) containing 5 x SSC (1 x SSC corresponds to a 0.15 M NaCl + 0.015 M sodium citrate solution), 50% of formamide, 7% of sodium dodecyl sulfate (SDS), 10 x Denhardt's, 5% of dextran sulfate and 1% of salmon sperm DNA; (2) actual 15 hybridization for 20 hours at a temperature dependent on the size of the probe (i.e. : 420C, for a probe size > 100 nucleotides) followed by 2 washes of 20 minutes at 200C in 2 x SSC + 2% of SDS, 1 wash of 20 minutes at 200C in 0.1 x SSC + 0.1% of SDS. The last wash is 20 carried out in 0.1 x SSC + 0.1% of SDS for 30 minutes at 600C for a probe size > 100 nucleotides. The hybridization conditions of high stringency described above for a polynucleotide of defined size can be adapted by the person skilled in the art for 25 oligonucleotides of greater or smaller size, according to the teaching of Sambrook et al., (1989, Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor). 30 The invention likewise relates to a vector comprising a nucleic acid according to the present invention. The invention aims especially at cloning and/or expression vectors which contain a nucleotide sequence 35 according to the invention. The vectors according to the invention preferably contain elements which allow the expression and/or the secretion of the nucleotide sequences in a determined - 20 host cell. The vector must therefore contain a promoter, signals of initiation and termination of translation, as well as appropriate regions of regulation of transcription. It must be able to be 5 maintained in a stable manner in the host cell and can optionally have particular signals which specify the secretion of the translated protein. These different elements are chosen and optimized by the person skilled in the art as a function of the host cell used. To this 10 effect, the nucleotide sequences according to the invention can be inserted into autonomous replication vectors in the chosen host, or be integrative vectors of the chosen host. 15 Such vectors are prepared by methods currently used by the person skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as lipofection, electroporation, thermal shock, or chemical methods. 20 The vectors according to the invention are, for example, vectors of plasmidic or viral origin. They are useful for transforming host cells in order to clone or to express the nucleotide sequences according to the 25 invention. The invention likewise comprises the host cells transformed by or comprising a vector according to the invention. 30 The host cell can be chosen from prokaryotic or eukaryotic systems, for example bacterial cells but likewise yeast cells or animal cells, in particular mammalian cells. It is likewise possible to use insect 35 cells or plant cells. The invention likewise concerns animals, except man, which comprise at least one cell transformed according to the invention.

- 21 According to another aspect, a subject of the invention is a process for production of an antibody, or one of its functional fragments according to the invention, 5 characterized in that it comprises the following stages: a) culture in a medium and appropriate culture conditions of a host cell according to the invention; and 10 b) the recovery of said antibodies, or one of their functional fragments, thus produced starting from the culture medium or said cultured cells. The cells transformed according to the invention can be 15 used in processes for preparation of recombinant polypeptides according to the invention. The processes for preparation of a polypeptide according to the invention in recombinant form, characterized in that they employ a vector and/or a cell transformed by a 20 vector according to the invention, are themselves comprised in the present invention. Preferably, a cell transformed by a vector according to the invention is cultured under conditions which allow the expression of said polypeptide and said recombinant peptide is 25 recovered. As has been said, the host cell can be chosen from prokaryotic or eukaryotic systems. In particular, it is possible to identify nucleotide sequences according to 30 the invention, facilitating secretion in such a prokaryotic or eukaryotic system. A vector according to the invention carrying such a sequence can therefore advantageously be used for the production of recombinant proteins, intended to be secreted. In 35 effect, the purification of these recombinant proteins of interest will be facilitated by the fact that they are present in the supernatant of the cell culture rather than in the interior of the host cells.

- 22 It is likewise possible to prepare the polypeptides according to the invention by chemical synthesis. Such a preparation process is likewise a subject of the invention. The person skilled in the art knows the 5 processes of chemical synthesis, for example the techniques employing solid phases (see especially Steward et al., 1984, Solid phase peptide synthesis, Pierce Chem. Company, Rockford, 111, 2nd ed., (1984)) or techniques using partial solid phases, by 10 condensation of fragments or by a classical synthesis in solution. The polypeptides obtained by chemical synthesis and being able to contain corresponding .unnatural amino acids are likewise comprised in the invention. 15 The antibodies, or one of their functional fragments, capable of being obtained by a process according to the invention are likewise comprised in the present invention. 20 According to a second embodiment, the present invention concerns an antibody according to the invention such as described further above, characterized in that it is, moreover, capable of binding specifically to the human 25 epidermal growth factor receptor EGFR and/or capable of specifically inhibiting the tyrosine kinase activity of said EGFR receptor. In a general manner, the growth factors are small 30 proteins involved in the regulation of the proliferation and of the differentiation of normal cells. Some of these growth factors likewise play an important role in the initiation and the maintenance of cell transformation, being able to function as 35 autocrine or paracrine factors.. This is especially the case, in addition to the IGF1 described further above, for the epidermal growth factor EGF, which seems particularly involved in the appearance of the tumor - 23 phenotype, the progression of tumors and the generation of metastases. EGF and IGF1 exert their action through the 5 intermediary of their respective receptor here called EGFR and IGF-IR. It concerns in the two cases membrane receptors with tyrosine kinase activity whose overexpression is described in numerous cancers. It must, however, be noted that the interaction of these 10 two receptors is not clearly established and that the studies carried out by various teams in this connection give contradictory results as to the collaboration of these two receptors. 15 Studies carried out on prostate tumor cells show that the interruption of the autocrine loop EGF/EGFR by an anti-EGFR monoclonal antibody (here called "MAB" or "MAb") is manifested by a complete loss of the response of the DU145 cells to IGF1 (Connolly J.M. and Rose 20 D.P., Prostate, Apr. 24(4):167-75, 1994; Putz T. et al., Cancer Res., Jan. 1, 59(1):227-33, 1999). These results would suggest that a blockage of the receptor for the EGF would be sufficient in order to obtain a total inhibition of the transformation signals 25 generated by the activation of the two receptors (EGFR and IGF-IR). On the other hand, other studies (Pietrzkowski et al., Cell Growth Differ, Apr., 3(4):199-205, 1992; Coppola et al., Mol Cell Biol., Jul., 14(7):4588-95, 1994) have shown that an over 30 expression of EGFR necessitates the presence of a functional IGF-IR in order to exert its mitogenic and transformant potential, although IGF-IR does not necessitate, for its part, the presence of functional EGFR in order to mediate its action. This second series 35 of studies would be more in agreement with a strategy tending preferentially to block IGF-IR with the aim of simultaneously affecting the two receptors.

-24 In a surprising manner, the inventors have, firstly, demonstrated that a coinhibition of the attachment of the IGF1 and/or IGF2 to the IGF-IR receptor and of the attachment of. the EGF to the EGFR receptor allows a 5 significant synergy of action of these two actions to be obtained against the in vivo tumor growth in nude mice carrying a tumor expressing these two receptors. One of the more probable hypotheses which is able to explain this synergy of action is that the two growth 10 factors EGF and IGF1 (and/or IGF2) themselves act in synergy in the transformation of normal cells to cells with tumoral character and/or in the growth and/or the proliferation of tumor cells for certain tumors, especially for those overexpressing the two receptors 15 EGFR and IGF-IR and/or having an overactivation of the transduction signal mediated by these two receptors, in particular at the level of the tyrosine kinase activity of these receptors. 20 According to a preferred aspect of this embodiment, the invention concerns an antibody such as described further above, characterized in that it consists of a bispecific antibody comprising a second motif specifically inhibiting the attachment- of the EGF to 25 the EGFR and/or specifically inhibiting the tyrosine kinase activity of said EGFR receptor. The term "second motif" is intended to indicate above especially a sequence of amino acids comprising a 30 fragment capable of specifically binding to EGFR, in particular a CDR region of a variable chain of an anti EGFR antibody, or one of the fragments of this CDR region of sufficient length in order to exert this specific binding, or else several CDR regions of an 35 anti-EGFR antibody. The bispecific or bifunctional antibodies form a second generation of monoclonal antibodies in which two different variable regions are combined in the same - 25 molecule (Hollinger and Bohlen 1999 Cancer and metastasis rev. 18: 411-419). Their use has been demonstrated both in the diagnostic field and in the therapy field from their capacity to recruit new 5 effector functions or to target several molecules on the surface of tumor cells. These antibodies can be obtained by chemical methods (Glennie MJ et al. 1987 J. Immunol. 139, 2367-2375; Repp R. et al. 1995 J. Hemat. 377-382) or somatic methods (Staerz U.D. and Bevan M.J. 10 1986 PNAS 83, 1453-1457; Suresh M.R. et al. 1986 Method Enzymol. 121: 210-228) but likewise and preferentially by genetic engineering techniques which allow the heterodimerization to be forced and thus facilitate the process of purification of the antibody sought 15 (Merchand et al. 1998 Nature Biotech. 16:677-681). These bispecific antibodies can be constructed as entire IgG, as bispecific Fab'2, as Fab'PEG or as diabodies or else as bispecific scFv but likewise as a 20 tetravalent bispecific antibody or two attachment sites are present for each antigen targeted (Park et al. 2000 Mol. Immunol. 37 (18):1123-30) or its fragments as described further above. 25 In addition to an 'economic advantage from the fact that the production and the administration of a bispecific antibody are less onerous than the production of two specific antibodies, the use of such bispecific antibodies has the advantage of reducing the toxicity 30 of the treatment. This is because the use of a bispecific antibody allows the total quantity of circulating antibodies to be reduced and, consequently, the possible toxicity. 35 In a preferred embodiment of the invention,. the bispecific antibody is a bivalent or tetravalent antibody.

- 26 In practice, the interest in using a tetravalent bispecific antibody is that it has a greater avidity in comparison with a bivalent antibody on account of the presence of two attachment sites for each target, 5 respectively IGF-IR and EGFR in the present invention. In a similar manner to the selection of the functional fragments of the anti-IGF-IR antibody described above, said second motif is selected from the fragments Fv, 10 Fab, F(ab') 2 , Fab', scFv, scFv-Fc and the diabodies, or any form whose half-life would have been increased like the pegylated fragments such as Fv-PEG, scFv-PEG, Fab PEG, F(ab') 2 -PEG or Fab'-PEG. According to an even more preferred aspect of the invention, said second anti 15 EGFR motif is descended from the mouse monoclonal antibody 225, its mouse-man chimeric derivative C225, or a humanized antibody derived from this antibody 225. According to yet another aspect, a subject of. the 20 invention is an antibody, or one of its functional fragments, according to the invention as a medicament, preferably a humanized antibody such as defined above. Antibody, for the remainder of the present description, must be understood as an anti-IGF-IR antibody as well 25 as a bispecific anti-IGF-IR/EGFR antibody. The invention likewise concerns a pharmaceutical composition comprising by way of active principle a compound consisting of an antibody, or one of its 30 functional fragments according to the invention, preferably mixed with an excipient and/or a pharmaceutically acceptable vehicle. According to yet another embodiment, the present 35 invention likewise concerns a pharmaceutical composition such as described further above which comprises a second. compound chosen from the compounds capable of specifically inhibiting the attachment of the EGF to the human epidermal growth factor receptor - 27 EGFR and/or capable of specifically inhibiting the tyrosine kinase activity of said EGFR receptor. In a preferred aspect of the invention, said second 5 compound is chosen from the isolated anti-EGFR antibodies, or their functional fragments, capable of inhibiting by competition the attachment of the EGF to the EGFR. More particularly, said anti-EGFR antibody is chosen from the monoclonal, chimeric or humanized anti 10 EGFR antibodies, or their functional fragments. Even more particularly, said functional fragments of the anti-EGFR antibody are chosen from the fragments Fv, Fab, F(ab') 2 , Fab', scFv-Fc or diabodies, or any fragment whose half-life would have been increased, 15 like pegylated fragments. Said antibody can consist, in an even more preferred manner, of the mouse monoclonal antibody 225, its mouse-man chimeric derivative C225 (also called IMC-C225) or a humanized antibody derived from this antibody 225. 20 Another complementary embodiment of the invention consists in a composition such as described above which comprises, moreover, as a combination product for simultaneous, separate or sequential use, a 25 cytotoxic/cytostatic agent and/or an inhibitor of the tyrosine kinase activity respectively of the receptors for IGF-I and/or for EGF. "Simultaneous use" is understood as meaning the 30 administration of the two compounds of the composition according to the invention in a single and identical pharmaceutical form. "Separate use" is understood as meaning the 35 administration, at the same time, of the two compounds of the composition according to the invention in distinct pharmaceutical forms.

- 28 "Sequential use" is understood as meaning the successive administration of the two compounds of the composition according to the invention, each in a distinct pharmaceutical form. 5 In a general fashion, the composition according to the invention considerably increases the efficacy of the treatment of cancer. In other words, the therapeutic effect of the anti-IGF-IR antibody according to the 10 invention is potentiated in an unexpected manner by the administration of a cytotoxic agent. Another major subsequent advantage produced by a composition according to the invention concerns the possibility of using lower efficacious doses of active principle, 15 which allows the risks of appearance of secondary effects to be avoided or to be reduced, in particular the effects of the cytotoxic agent. In addition, this composition according to the 20 invention would allow the expected therapeutic effect to be attained more rapidly. In a particularly preferred embodiment, said composition as a combination product according to the 25 invention is characterized in that said cytotoxic/ cytostatic agent is chosen from the agents interacting with DNA, the antimetabolites, the topoisomerase I or II inhibitors, or else the spindle inhibitor or stabilizer agents or else any agent capable of being 30 used in chemotherapy. Such cytotoxic/cytostatic agents, for each of the aforesaid classes of cytotoxic agents, are, for example, cited in the 2001 edition of VIDAL, on the page devoted to the compounds attached to the cancerology and hematology column "Cytotoxics", these 35 cytotoxic compounds cited with reference to this document are cited here as preferred cytotoxic agents. In a particularly preferred embodiment, said composition as a combination product according to the - 29 invention is characterized in that said cytotoxic agent is coupled chemically to said antibody for simultaneous use. 5 In a particularly preferred embodiment, said composition according to the invention is characterized in that said cytotoxic/cytostatic agent is chosen from the spindle inhibitor or stabilizer agents, preferably vinorelbine and/or vinflunine and/or vincristine. 10 In order to facilitate the coupling between said cytotoxic agent and said antibody according to the invention, it is especially possible to introduce spacer molecules between the two compounds to be 15 coupled, such as poly(alkylene) glycols like polyethylene glycol, or else amino acids, or, in another embodiment, to use active derivatives of said cytotoxic agents into which would have been introduced functions capable of reacting with said antibody 20 according to the invention. These coupling techniques are well known to the person skilled in the art and will not be expanded upon in the present description. In another preferred embodiment, said inhibitor of the 25 tyrosine kinase activity of the receptors for IGF-I and/or for EGF is selected from the group consisting of derived natural agents, dianilinophthalimides, pyrazolo- or pyrrolopyridopyrimidines or else quinazilines. Such inhibitory agents are well known to 30 the person skilled in the art and described in the literature (Ciardiello F., Drugs 2000, Suppl. 1, 25 32). Other inhibitors of EGFR can, without any limitation, 35 consist of the anti-EGFR monoclonal antibodies C225 and 22Mab (ImClone Systems Incorporated), ABX-EGF (Abgenix/Cell Genesys), EMD-7200 (Merck KgaA) or the compounds ZD-1834, ZD-1838 and ZD-1839 (AstraZeneca), PKI-166 (Novartis), PKI-166/CGP-75166 (Novartis), PTK - 30 787 (Novartis), CP 701 (Cephalon), leflunomide (Pharmacia/Sugen), CI-1033 (Warner-Lambert Parke Davis), CI-1033/PD 183, 805 (Warner-Lambert Parke Davis), CL-387, 785 (Wyeth-Ayerst), BBR-1611 5 (Boehringer Mannheim GmbH/Roche), Naamidine A (Bristol Myers Squibb), RC-3940-II (Pharmacia), BIBX-1382 (Boehringer Ingelheim), OLX-103 (Merck & Co), VRCTC-310 (Ventech Research), EGF fusion toxin (Seragen Inc.), DAB-389 (Seragen/Lilgand), ZM-252808 (Imperial Cancer 10 Research Fund), RG-50864 (INSERM), LFM-A12 (Parker Hughes Cancer Center), WHI-P97 (Parker Hughes Cancer Center), GW-282974 (Glaxo), KT-8391 (Kyowa Hakko) or the "EGFR Vaccine" (York Medical/Centro de Immunologia Molecular). 15 According to yet another embodiment of the invention, the composition such as described above can likewise comprise another antibody compound directed against the extracellular domain of the HER2/neu receptor, as a 20 combination product for simultaneous, separate or sequential use, intended for the prevention and for the treatment of cancer, especially the cancers overexpressing said HER2/neu receptor and the receptor IGF-IR and/or EGFR, such as especially cancer of the 25 breast. Reference can be made especially to the publications of Albanell et al. (J. of the National Cancer Institute, 93(24):1830-1831, 2001) and of Lu et al. (J. of the 30 National Cancer Institute, 93(24):1852-1857, 2001) justifying the unexpected interest in combining an anti-HER2/neu antibody with an antiL-IGF-IR antibody according to the present invention. 35 In a particular manner, said anti-HER2/neu antibody of the composition according to the invention is the antibody called Trastuzumab (also called Herceptin).

- 31 The invention relates, in another aspect, to a composition characterized in that one, at least, of said antibodies, or one of their functional fragments, is conjugated with a cell toxin and/or a radioelement. 5 Preferably, said toxin or said radioelement is capable of inhibiting at least one cell activity of cells expressing the IGF-IR and/or EGFR receptor, in a more preferred manner capable of preventing the growth or 10 the proliferation of said cell, especially of totally inactivating said cell. Preferably also, said toxin is an enterobacterial toxin, especially Pseudomonas exotoxin A. 15 The radioelements (or radioisotopes) preferably conjugated to the antibodies employed for the therapy are radioisotopes which emit gamma rays and preferably 131 9100 67 iodine , yttrium 9 o, gold 1 99 , palladium' , copper, 20 bismuth 217 and antimony 211 . The radioisotopes which emit beta and alpha rays can likewise be used for the therapy. By toxin or radioelement conjugated to at least one 25 antibody, or one of its functional fragments, according to the invention, it is intended to indicate any means allowing said toxin or said radioelement to bind to said at least one antibody, especially by covalent coupling between the two compounds, with or without 30 introduction of a linking molecule. Among the agents allowing binding in a chemical (covalent), electrostatic or noncovalent manner of all or part of the components of the conjugate, mention may 35 particularly be made of benzoquinone, carbodiimide and more particularly EDC (l-ethyl-3-[3-dimethyl aminopropyl]-carbodiimide hydrochloride), dimaleimide, dithiobis-nitrobenzoic acid (DTNB), N-succinimidyl S acetyl thio-acetate (SATA), the bridging agents having - 32 one or more phenylazide groups reacting with the ultraviolets (U.V.) and preferably N-[-4 (azidosalicylamino)butyl]-3' -(2' -pyridyldithio) propionamide (APDP), N-succinimid-yl 3-(2 5 pyridyldithio)propionate (SPDP), 6-hydrazino nicotinamide (HYNIC). Another form of coupling, especially for the radioelements, can consist in the use of a bifunctional 10 ion chelator. Among these chelates, it is possible to mention the chelates derived from EDTA (ethylenediaminetetraacetic acid) or from DTPA (diethylenetriaminepentaacetic acid) 15 which have been developed for binding metals, especially radioactive metals, and immunoglobulins. Thus, DTPA and its derivatives can be substituted by different groups on the carbon chain in order to increase the stability and the rigidity of the ligand 20 metal complex (Krejcarek et al. (1977); Brechbiel et al. (1991); Gansow (1991); US patent 4 831 175). For example diethylenetriaminepentaacetic acid (DTPA) and its derivatives, which have been widely used in 25 medicine and in biology for a longtime either in their free form, or in the form of a complex with a metallic ion, have the remarkable characteristic of forming stable chelates with metallic ions and of being coupled with proteins of therapeutic or diagnostic interest 30 such as antibodies for the development of radioimmunoconjugates in cancer therapy (Meases et al., (1984); Gansow et al. (1990)). Likewise preferably, said at least one antibody forming 35 said conjugate according to the invention is chosen from its functional fragments, especially the fragments amputated of their Fc component such as the scFv fragments.

- 33 The present invention moreover comprises the use of the composition according to the invention for the preparation of a medicament. 5 More particularly, according to another embodiment, the invention concerns the use of an antibody, or one of its functional fragments, and/or of a composition for the preparation of a medicament intended for the prevention or for the treatment of an illness induced 10 by an overexpression and/or an abnormal activation of the IGF-IR and/or EGFR receptor, and/or connected with a hyperactivation of the transduction pathway of the signal mediated by the interaction of the 1-IGF1 or IGF2 with IGF-IR and/or of EGF with EGFR and/or 15 HER2/neu. Preferably, said use according to the invention is characterized in that the administration of said medicament does not induce or induces only slightly 20 secondary effects connected with inhibition of the insulin receptor IR, that is to say inhibition of the interaction of the IR receptor with its natural ligands due to the presence of said medicament, especially by a competitive inhibition connected with the attachment of 25 said medicament to the IR. The present invention moreover comprises the use of an antibody, or one of its functional fragments, preferably humanized, and/or of a composition according 30 to the invention for the preparation of a medicament intended to inhibit the transformation of normal cells into cells with tumoral character, preferably IGF dependent, especially IGF1- and/or IGF2-dependent and/or EGF-dependent and/or HER2/neu-dependent cells. 35 The present invention likewise relates to the use of an antibody, or one of its functional fragments, preferably humanized, and/or of a composition according to the invention for the preparation of a medicament - 34 intended to inhibit the growth and/or the proliferation of tumor cells, preferably IGF-dependent, especially IGF1- and/or IGF2-dependent and/or EGF-dependent and/or estrogen- dependent, and/or .HER2/neu-dependent cells. 5 In a general manner, a subject of the present invention is the use of an antibody, or one of its functional fragments, preferably humanized, and/or of a composition according to the invention, for the 10 preparation of a medicament intended for the prevention or for the treatment of cancer preferably expressing IGF-IR and/or EGFR, and/or of cancer preferably having a hyperactivation of the transduction pathway of the signal mediated by the interaction of IGF1 or IGF2 with 15 IGF-IR, such as, for example, the overexpression of IRS1 and/or of EGF with EGFR. The subject of the present invention is likewise the use of an antibody, or one of its functional fragments, 20 preferably humanized, and/or of a composition according to the invention, for the preparation of a medicament intended for the prevention or for the treatment of psoriasis, psoriasis whose epidermal hyperproliferation can be connected with the expression or the 25 overexpression of IGF-IR and/or EGFR, and/or with the hyperactivation of the transduction pathway of the signal mediated by the interaction of IGF-IR with its natural ligands (Wraight C.J. et al. Nat. Biotechnol., 2000, 18(5):521-526. Reversal of epidermal 30 hyperproliferation in psoriasis by insulin-like growth factor I receptor antisense oligonucleotides) and/or of EGFR with its natural ligands. Among the cancers which can be prevented and/or 35 treated, prostate cancer, osteosarcomas, lung cancer, breast cancer, endometrial cancer or colon cancer or any other cancer overexpressing IGF-IR is preferred.

- 35 According to yet another aspect, a subject of the present invention is a method of diagnosis, preferably in vitro, of illnesses connected with an overexpression or an underexpression, preferably an overexpression, of 5 the IGF-IR and/or EGFR receptor starting from a biological sample in which the abnormal presence of IGF-IR and/or EGFR receptor is suspected, characterized in that said biological sample is contacted with an antibody, or one of its functional fragments, according 10 to the invention, it being possible for said antibody to be, if necessary, labeled. Preferably, said illnesses connected with the overexpression of the IGF-IR and/or EGFR receptor in 15 said diagnosis method will be cancers. Said antibody, or one of its functional fragments, can be present in the form of an immunoconjugate or of a labeled antibody so as to obtain a detectable and/or 20 quantifiable signal. The antibodies labeled according to the invention or their functional fragments include, for example, antibodies called immunoconjugates which can be 25 conjugated, for example, with enzymes such as peroxidase, alkaline phosphatase, (u-D-galactosidase, glucose oxydase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase or glucose 6-phosphate dehydrogenase or by a molecule such 30 as biotin, digoxygenin or 5-bromodeoxyuridine. Fluorescent labels can be likewise conjugated to the antibodies or to their functional fragments according to the invention and especially include fluorescein and its derivatives, fluorochrome, rhodamine and its 35 derivatives, GFP (GFP for "Green Fluorescent Protein"), dansyl, umbelliferone etc. In such conjugates, the antibodies of :the invention or their functional fragments can be prepared by methods known to the person skilled in the art. They can be coupled to the - 36 enzymes or to the fluorescent labels directly or by the intermediary of a spacer group or of a linking group such as a polyaldehyde, like glutaraldehyde, ethylenediaminetetraacetic acid (EDTA), diethylene 5 triaminepentaacetic acid (DPTA), or in the presence of coupling agents such as those mentioned above for the therapeutic conjugates. The conjugates containing labels of fluorescein type can be prepared by reaction with an isothiocyanate. 10 Other conjugates can likewise include chemoluminescent labels such as luminol and the dioxetanes, bio luminescent labels such as luciferase and luciferin, or else radioactive labels such as iodine 123 , iodine 125 , 126 1377 99M 15 iodine , 2 6 iodine 3 3 , bromine , technetium 99m , indium"', indium 1 13 m, gallium 67 , gallium 68 , ruthenium 95 , ruthenium 97, ruthenium') , ruthenium 0 5 , mercury 1 7 , mercury 2 03 rhenium 9 9m, rhenium 1 01, rhenium0 5 , scandium 47 , tellurium 2 1m, tellurium 22m, telluriuml 25 m, thulium 1 65 , 20 thulium 1

,

67 thulium 1

'

68 , fluorine 18 , yttriuml 99 , iodine' 3 1 . The methods known to the person skilled in the art existing for coupling the therapeutic radioisotopes to the antibodies either directly or via a chelating agent such as EDTA, DTPA mentioned above can be used for the 25 radioelements which can be used in diagnosis. It is likewise possible to mention labeling with Na['112 5 ] by the chloramine T method [Hunter W.M. and Greenwood F.C. (1962) Nature 194:495] or else with technetium 9 9 m by the technique of Crockford et al. (US patent 4 424 200) or 30 attached via DTPA as described by Hnatowich (US patent 4 479 930). Thus, the antibodies, or their functional fragments, according to the invention can be employed in a process 35 for the detection and/or the quantification of an overexpression or of an underexpression, preferably an overexpression, of the IGF-IR and/or EGFR receptor in a biological sample, characterized in that it comprises the following steps: - 37 a) the contacting of the biological sample with an antibody, or one of its functional fragments, according to the invention; and b) the demonstration of the IGF-IR and/or 5 EGFR/antibody complex possibly formed. In a particular embodiment, the antibodies, or their functional fragments, according to the invention, can be employed in a process for the detection and/or the 10 quantification of the IGF-IR and/or EGFR receptor in a biological sample, for the monitoring of the efficacy of a prophylactic and/or therapeutic treatment of IGF and/or EGF-dependent cancer or else of psoriasis. 15 More generally, the antibodies, or their functional fragments, according to the invention can be advantageously employed in any situation where the expression of the IGF-IR and/or EGFR receptor must be observed in a qualitative and/or quantitative manner. 20 Preferably, the biological sample is formed by a biological fluid, such as serum, whole blood, cells, a tissue sample or biopsies of human origin. 25 Any procedure or conventional test can be employed in order to carry out such a detection and/or dosage. Said test can be a competition or sandwich test, or any test known to the person skilled in the art dependent on the formation of an immune complex of antibody-antigen 30 type. Following the applications according to the invention, the antibody or one of its functional fragments can be immobilized or labeled. This immobilization can be carried out on numerous supports known to the person skilled in the art. These supports 35 can especially include glass, polystyrene, poly propylene, polyethylene, dextran, nylon, or natural or modified cells. These supports can be either soluble or insoluble.

- 38 By way of example, a preferred method brings into play immunoenzymatic processes according to the ELISA technique, by immunofluorescence, or radio-immunoassay (RIA) technique or equivalent. 5 Thus, the present invention likewise comprises the kits or sets necessary for carrying out a method of diagnosis of illnesses induced by an overexpression or an underexpression of the IGF-IR and/or EGFR receptor 10 or for carrying out a process for the detection and/or the quantification of an overexpression or of an underexpression of the IGF-IR and/or EGFR receptor in a biological sample, preferably an overexpression of said receptor, characterized in that said kit or set 15 comprises the following elements: a) an antibody, or one of its functional fragments, according to the invention; b) optionally, the reagents for the formation of the medium favorable to the immunological reaction; 20 c) optionally, the reagents allowing the demonstration of IGF-IR and/or EGFR/antibody complexes produced by the immunological reaction. The invention moreover relates to the use of a 25 composition as a combination product according to the invention, for the preparation of a medicament intended for the prevention or for the treatment of cancer, especially cancers for which said cytotoxic agent or said anti-HER2/neu antibody is generally prescribed 30 and, especially, for which cancers the tumor cells express or overexpress the IGF-IR and/or EGFR receptor. A subject of the invention is likewise the use of an antibody according to the invention for the preparation 35 of a medicament intended for the specific targeting of a biologically active compound to cells expressing or overexpressing the IGF-IR and/or EGFR receptor.

- 39 It is intended here by biologically active compound to indicate any compound capable of modulating, especially of inhibiting, cell activity, in particular their growth, their proliferation, transcription or gene 5 translation. A subject of the invention is also an in vivo diagnostic reagent comprising an antibody according to the invention, or one of its functional fragments, 10 preferably labeled, especially radiolabeled, and its use in medical imaging, in particular for the detection of cancer connected with the expression or the overexpression by a cell of the IGF-IR and/or EGFR receptor. 15 The invention likewise relates to a composition as a combination product or to an anti-IGF-IR and/or EGFR/toxin conjugate or radioelement, according to the invention, as a medicament. 20 Preferably, said composition as a combination product or said conjugate according to the invention will be mixed with an excipient and/or a pharmaceutically acceptable vehicle. 25 In the present description, pharmaceutically acceptable vehicle is intended to indicate a compound or a combination of compounds entering into a pharmaceutical composition not provoking secondary reactions and which 30 allows, for example, facilitation of the administration of the active compound(s), an increase in its lifespan and/or in its efficacy in the body, an increase in its solubility in solution or else an improvement in its conservation. These pharmaceutically acceptable 35 vehicles are well known and will be adapted by the person skilled in the art as a function of the nature and of the mode of administration of the active compound(s) chosen.

- 40 Preferably, these compounds will be administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal, intraperitoneal or subcutaneous route, or by the oral route. In a more 5 preferred manner, the composition comprising the antibodies according to the invention will be administered several times, in a sequential manner. Their modes of administration, dosages and optimum 10 pharmaceutical forms can be determined according to the criteria generally taken into account in the establishment of a treatment adapted to a patient such as, for example, the age or the body weight of the patient, the seriousness of his/her general condition, 15 the tolerance to the treatment and the secondary effects noted. Other characteristics and advantages of the invention appear in the continuation of the description with the 20 examples and the figures whose legends are represented below. LEGENDS TO THE FIGURES 25 Figure 1: Schematic representation of IGF-IR. Figure 2: Scheme of the transduction of the signals mediated by IGF-IR during the attachment of IGFs. 30 Figures 3A, 3B and 3C: Recognition of native IGF-IR expressed on the surface of MCF-7 cells by the monoclonal antibody 7C10. For this experiment, the MCF-7 cells are incubated with 35 the 7C10 antibody or with a negative control antibody, then recovered with the aid of a fluorescent anti species secondary antibody. The labeling is read on a FACS. The first histogram (figure 3A) corresponds to the MCF-7 cells alone. In the second histogram (figure - 41 3B), the unshaded curve corresponds to the nonspecific labeling by a control isotype murine antibody. In the third histogram (figure 3C), the unshaded curve shows the recognition of IGF-IR by MAB 7C10. 5 Figures 4A, 4B and 4C: Labeling of Sf9 insect cells respectively expressing IGF-IR or IR. Figure 4A shows the labeling of nontransfected cells 10 alone (1) or cells labeled with control commercial monoclonal antibodies respectively recognizing IGF-IR (2) or IR (3). In figure 4B, Sf9 cells uniquely expressing IGF-IR are labeled with a IR3 (2) or anti IR(3), the peak (1) representing the single cells. In 15 figure 4C, Sf9 cells uniquely expressing IR are labeled with an anti-IR (3) or aIR3 (2), the peak (1) representing the single cells. Figure 5: Inhibitor effect of 7C10 antibody on the 20 proliferation of MCF-7 cells induced by IGF-I. The MCF-7 cells are incubated in the presence of increasing concentrations of IGF1 in the presence or in the absence of the MAB to be tested. The cell 25 proliferation is evaluated by following the incorporation of 3 H thymidine. The commercial antibody calR3 is used as a positive control of the experiment. The 7G3 is a murine anti-IGF-IR IgGl without activity on proliferation and used as a control isotype. 30 Figures 6A, 6B and 6C: - figure 6A: in vivo effect of the monoclonal antibody 7C10 on the growth of MCF-7 tumors established in nude mice; 35 - .figures 6B and 6C: figures respectively from publications of Arteaga et al. (J. Clin. Invest., 84, 1418-1423, 1989) and from Li et al. (Cancer Immunol. Immunother., 49, 243-252), and showing for figure 6B the effect of murine aIIR3 (likewise written aIR3) and - 42 for figure 6C the effect of a recombinant scFv-Fc derived from the 1H7 antibody on tumor growth. Figure 7: Comparative study of the effect of the MAb 5 7C10 and of tamoxifen on the growth in vivo of the tumor MCF-7. Figures 8A, 8B and 8C: Study of the antitumor activity of the murine antibody 7C10 in different xenograft 10 models of tumor cells in vivo. Figure SA shows the results obtained on an osteosarcoma model SK-ES-1, figure 8B concerns an androgen independent tumor of the prostate DU-145 and figure 8C 15 a model of non-small cell tumor of the lung A549. In these three models, the treatment was carried out twice per week i.p. at a rate of 250 pg/dose/mouse. The curves 7G3, EC2 and 9G4 correspond respectively to three murine IgGl used as an experiment control isotype 20 in each of the models. Figure 9: Study of the antitumor effect of the MAb 7C10 compared to navelbine (vinorelbine) as well as the synergy of the two compounds on the growth in vivo of 25 the line A549. Figure 10: Comparative activity of MAb cIR3, 7C10 and 1H7 on the IGF-2 proliferation induced by MCF-7 cells. 30 Figure 11: Comparison of the murine 7C10 and chimeric C7C10 MAb for the inhibition of the IGF1 proliferation of MCF-7 cells in vitro. The antibody 9G4 is a murine IgGl used as an experiment control isotype. 35 Figure 12: Comparative effect of the 7C10 and h7C10 MAb (humanized 1, written here 7H2HM) on the in vitro model of IGFl-induced proliferation of MCF-7 cells.

- 43 Figure 13: Effect of the 7C10 and h7C10 MAb (humanized 1, written here 7H2HM) on the transduction of the signal induced by IGF1. The first line of spots corresponds to the revelation, by an antiphospho 5 tyrosine antibody, of the phosphorylation of the immunoprecipitated P chain from the cells incubated in the presence of IGF1 alone or of IGF1 mixed with various antibodies to be tested. The 9G4 and the hIgGl are respectively the control isotypes of the forms 7C10 10 and h7C10 (likewise written 7H2HM). The second line of spots corresponds to the revelation of the P chain and shows that the quantity deposited in all of the wells is perfectly equivalent. 15 Figure 14: Sequence of the cDNA (SEQ ID No. 48), of its complementary strand (SEQ ID No. 50) and its translation into amino acids (SEQ ID No. 49), of the PCR fragment amplified from the mouse hybridoma 7C10 with the primers MKV-1 and MKC and which codes for the 20 3' end of the leader peptide and 7C10 VL. Figure 15: Sequence of the cDNA (SEQ ID No. 51), of its complementary strand (SEQ ID No. 53) and its translation into amino acids (SEQ ID No. 52), of the 25 PCR fragment amplified from the mouse hybridoma 7C10 with the primers MHV-12 and MHC-1, or MHV-8 and MHC-l and which codes for the 3' end of the leader peptide and 7C10 VH. 30 Figure 16: Recognition of the IGF-1 receptor by the chimeric antibody 7C10, likewise called C7C10 (supernatant of cos7-transfected cell culture). Figure 17: Comparison of the amino acid sequence of 35 mouse 7C10 VL (SEQ ID No. 54) with cells of other mouse antibodies having the greatest sequence homology. The numbering of the amino acids is that of Kabat et al. (1991). The residues in the framework regions - 44 (outside CDRs) which differ between 7C10 VL and Kabat mouse subgroup II (SEQ ID No. 57) are underlined. A dot indicates that the residue is identical at this position in comparison with the sequence of 7C10 VL. 5 DRB1-4.3 (SEQ ID No. 55) represents the sequence of the light chain of an anti-human mouse antibody MHC CLASS II B-Chain (access number in the Kabat databank is N011794). C94-5B11'CL (SEQ ID No. 56) represents the sequence of the light chain of a mouse antibody (access 10 number in the Kabat databank is P019314). Figure 18: Comparison of amino acid sequences of mouse 7C10 VL (SEQ ID No. 54) with cells of human light chains belonging to Kabat human subgroup II (SEQ ID No. 15, 60) and having the greatest sequence homology. The amino acid sequences are aligned and compared with that of mouse 7C10 VL. A dot indicates that the residue is identical at this position in comparison with the 20 sequence of 7C10 VL. GM607 (SEQ ID No. 58) represents the sequence of the kappa light chain secreted by the human lymphoblastoid line GM607 (Klobeck et al., Nucleic Acids Res., 12:6995-7006, 1984a and Klobeck et al., Nature, 309:73-76, 1984b, the access number in the 25 Kabat databank is N011606). DPK15/A19 (SEQ ID No. 59) represents the sequence of the human V germinal line kappa II. Figure 19: Comparison of amino acid sequences of 30 variable regions of the light chains (VL) of mouse 7C10 (SEQ ID No. 54), of human antibody GM 607 (SEQ ID No. 58) and of two versions of humanized 7C10 1 and 2 (SEQ ID Nos. 61 and 65). 35 The amino acid sequences are aligned and compared with that of mouse 7C10 VL. A dot indicates that the residue is identical at this position in comparison with the sequence of 7C10 VL. GM607 represents the sequence of the kappa light chain secreted by the human - 45 lymphoblastoid line GM607 (Klobeck et al., 1984a and 1984b, access number in the Kabat database: N011606). Figure 20: cDNA sequence (SEQ ID No. 62), its 5 complementary strand (SEQ ID No. 64) and its translation into amino acids (SEQ ID No. 63), of the gene constructed by de novo assembly coding for the leader peptide and the humanized version 1 of 7C10 VL. 10 Figure 21: cDNA sequence (SEQ ID No. 66), its complementary strand (SEQ ID No. 68) and its translation into amino acids (SEQ ID No. 67), of the gene constructed by de novo assembly coding for the leader peptide and the humanized version 2 of 7C10 VL. 15 Figure 22: Comparison of the amino acid sequences of mouse 7C10 VH (SEQ ID No. 69) with those of human mouse heavy chains belonging to Kabat mouse subgroup I(A) and having the greatest sequence homology. 20 The numbering of the amino acids is that of Kabat et al. (1991). The residues in the framework regions (outside CDRs) which differ between 7C10 VH and Kabat mouse subgroup I(A) (SEQ ID No. 71) are underlined. A 25 dot indicates that the residue is identical at this position in comparison with the sequence of mouse 7C10 VH. ANO3'CL (SEQ ID No. 70) represents the sequence of the heavy chain of a mouse antibody (access number in the Kabat databank: P001289). 30 Figure 23: Comparison of amino acid sequences of mouse 7C10 VH (SEQ ID No. 69) with those of human heavy chains belonging to the Kabat human subgroup II (SEQ ID No. 72) and having the greatest sequence homology. 35 The underlined residues are part of the canonical structures defined by Chothia et al. (1989). A dot indicates that the residue is identical at this position in comparison with the mouse 7C10 VH sequence.

- 46 Human VH FUR1'CL (SEQ ID No. 73) represents the sequence of the heavy chain of a human anti-lamin B antibody IgM/K of autoimmune origin (Mariette et al., Arthritis and Rheumatism, 36:1315-1324, 1993; access 5 number in Kabat: N020619). Human germline (SEQ ID No. 74) represents the sequence of the human germinal line 4.22 VH IV (Sanz et al., EMBO. J. 8:3741-3748, 1989). Figure 24: Comparison of the amino acid sequences of 10 the variable regions of the heavy chains (VH) of mouse 7C10 (SEQ ID No. 69) and of the three versions humanized by CDR-grafting humanized VH 1, 2 and 3 (respectively SEQ ID Nos. 75, 79 and 83). 15 The numbering of the residues corresponds to that of Kabat. The sequences are aligned and compared with that of mouse 7C10 VH. A dot indicates that the residue is identical at this position in comparison with the sequence of mouse 7C10 VH. 20 Figure 25: cDNA sequence (SEQ ID No. 76), its complementary strand (SEQ ID No. 78) and its translation into amino acids (SEQ ID No. 77), of the gene constructed by de novo assembly coding for the 25 leader peptide and the humanized version 1 of 7C10 VH. Figure 26: cDNA sequence (SEQ ID No. 80), its complementary strand (SEQ ID Nc. 82) and its translation into amino acids (SEQ ID No. 81), of the 30 gene constructed by de novo assembly coding for the leader peptide and the humanized version 2 of 7C10 VH. Figure 27: cDNA sequence (SEQ ID No. 84), its complementary strand (SEQ ID Nc. 86) and its 35 translation into amino acids (SEQ ID No. 85), of the gene constructed by de novo assembly coding for the leader peptide and the humanized version 3 of 7C10 VH.

- 47 Figure 28: Comparison of the recognition activity of the IGF-1 receptor by the chimeric antibody 7C10 (called "C7C10") and its humanized version 1 (7C10 hum 1) in ELISA. 5 Figure 29: Influence on the recognition activity of the IGF-l receptor of the humanized versions 1 and 2 of the light chain of the 7C10 antibody in ELISA. 10 Figure 30: Comparison of the recognition activity of the IGF-1 receptor by the chimeric antibody 7C10 and three humanized versions of the heavy chain (7C10 hum 1,. 2 and 3) in combination with humanized 7C10 VL 2 in ELISA. 15 Figure 31: Antitumor activity of the 7C10 antibody in an orthotopic model A549. Figures 32A, 32B, 32C and 32D: Study of the ADCC 20 observed at the level of A549 and MCF-7 cells cultured during 4 hours in the presence of the antibody 7H2HM (respectively figures 32C and 32D). The antibody h4D5 is used in parallel as an experiment positive control for the cells A549 and MCF-7 (respectively figures 32A 25 and 32B). Figures 33A, 33B and 33C: Effects of the antibodies 7C10 and 7H2HM on the cell cycle of the MCF-7 cells. 30 Figure 33A represents the proportion of MCF-7 cells in the GO/G1, S and G2/M phase in the absence of IGF1, expressed as a significant percentage of total MCF-7 cells observed. 35 Figure 33B represents the proportion of MCF-7 cells in the GO/G1, S and G2/M phase in the presence of IGF1, expressed as a percentage of total MCF-7 cells observed.

- 48 Figure 33C represents the proportion of MCF-7 cells in the S (U) and G2/M (0) phase, expressed as a percentage of total MCF-7 cells observed, in the presence of the compounds indicated in the figure compared with a 5 control sample in the absence of IGF1 ("0"). Figures 34A and 34B: Comparative effect of the antibodies 7C10 and 7H2HM on the growth of A549 cells in vitro (figure 34A) and on the growth of MCF-7 cells 10 in vivo (figure 34B). Figures 35A and 35B: Study of the synergy of the antibody 7H2HM combined with navelbine (NA) on the model A549 in vivo, compared with the control samples. 15 Figure 35A represents the development of the volume of the implanted tumor as a function of the treatment carried out starting from the commencement of the treatment and over approximately 50 days (figure 35A). Figure 35B represents in a particular manner the 20 results obtained for this development compared at approximately 48 days. In this figure, the results obtained with the antibody 7C10 have been introduced by way of comparison (the asterisks (*) correspond to the comparison control group/group (7C10 + Na) or control 25 group/group (7H2HM + Na) in a t-test). Figure 36: Study of the effect of the antibodies 7C10 and 7H2HM on apoptosis. 30 This figure represents the potentiation of the effect of doxorubicin by the antibodies 7C10 and 7H2HM (doxorubicin 2 pg/ml). Figures 37A to 37D: Demonstration by labeling in FACS 35 of the presence of EGFR and of IGF-IR on the surface of A549 cells. Figure 38: Effect of a coadministration of the MAB 7C10 and 225 on the in vivo growth of the tumor A549.

- 49 Figure 39: Effect of a coadministration of the MAB 7C10 and 225 on the survival of mice orthotopically implanted with A549 cells. 5 Figures 40A and 40B: Demonstration of the inhibition of tyrosine phosphorylation of the beta chain of IGF-IR and of IRS-1 by the MAB 7C10 and 7H2HM. 10 Figure 41: Demonstration of the induction of the internalization of IGF-IR by the MAB 7C10 and 7H2HM. Figures 42A to 42C: Demonstration of the degradation of IGF-IR by the MAB 7C10 and 7H2HM. 15 Example 1. Generation and selection of the murine monoclonal antibody (MAb) With the aim of generating MAb specifically directed 20 against IGF-IR and not recognizing the IR, a protocol comprising 6 screening stages was envisaged. It consisted in: - immunizing mice with recombinant IGF-IR, in order to generate hybridomas, 25 - screening the culture supernatants by ELISA on the recombinant protein which served for immunization, - testing all the supernatants of hybridomas positive by ELISA on the native receptor overexpressed on the surface of MCF-7 tumor cells, 30 - evaluating the supernatants of hybridomas positive in the two first screenings in terms of differential recognition of IGF-IR and of IR on insect cells infected with baculoviruses respectively expressing IGF-IR or IR, 35 - verifying that the antibodies selected at this stage were capable of inhibiting in vitro the induced IGF1 proliferation of the MCF-7 cells, - ensuring the in vivo activity, in nude mice, of the candidate retained in terms of impact on the growth - 50 of the tumor MCF-7. All of these different stages and results obtained will be briefly described below in example 1. 5 For the immunization stage, mice were injected twice, by the subcutaneous route, with 8 pg of recombinant IGF-IR. Three days before the fusion of the cells of the female rat with the cells of the murine myeloma 10 Sp2OAgl4, the mice were stimulated by an intravenous injection of 3 pg of the recombinant receptor. Fourteen days after the fusion, the supernatants of hybridomas were screened by ELISA, on plates sensitized by recombinant IGF-IR. The hybridomas whose supernatants 15 were found positive were conserved and amplified before being tested on the FACScan so as to verify that the antibodies produced were likewise capable of recognizing native IGF-IR. In order to do this, MCF-7 cells from an estrogen-dependent tumor of the breast 20 overexpressing IGF-IR were incubated with each of the culture supernatants produced by the hybridomas selected in ELISA. The native/MAb receptor complexes on the surface of the cell were revealed by a secondary anti-species antibody coupled to a fluorochrome. 25 Figures 3A to 3C show a histogram type obtained with the supernatant of the hybridoma 7C10 (figure 3C) compared with a cell labeling alone + secondary antibody (figure 3A) or with a labeling utilizing a control isotype (figure 3B). 30 At this stage of the selection, only the hybridomas secreting MAb at the same time recognizing the recombinant receptor and the native receptor were selected and cloned. The MAb secreted by these 35 hybridomas were produced and then purified before being tested on the FACScan, according to the method described above, on Sf9 insect cells expressing IGF-IR or IR in order to eliminate the hybridomas at the same time recognizing the two receptors. Figure 4A shows a - 51 total recovery of the histograms 1, 2, 3 respectively corresponding to the noninfected cells + secondary antibodies (1), to the noninfected cells labeled by aIR3 + secondary antibodies (2) and to the noninfected 5 cells labeled by an anti-IR antibody + secondary antibodies (3). This first result shows well the absence of IGF-IR and of IR detectable on the surface of these noninfected insect cells. Figure 4B shows a labeling of infected cells by a baculovirus expressing 10 IGF-IR. In this second figure, the aIR3, used as a positive control, labels well, as expected, the cells (peak 2), while the anti-IR (peak 3) is superimposed on the peak of single cells. Finally, in figure 4C, it is shown that the anti-IR labels well, as expected, the 15 Sf9 cells expressing the IR (peak 3), but in an unexpected manner, the aIR3 described in the literature as specific for IGF-IR seems likewise to recognize the IR (peak 2). 20 The results obtained in this third screening system are summarized in table 1 and show the generation of an MAb: 7C10, satisfying the criteria of recognition of the IGF-IR and of nonrecognition of the IR. The isotyping of the Mab 7C10 has shown that it involves an 25 IgGl. TABLE 1: Comparative reactivity of MAb 7C10 on Sf9 insect cells expressing IGF-IR or IR MFI (Mean fluorescence intensity) Noninfected IGFlR + IR + cells cells cells Cells 8 8 7 Anti-IR 4.6 9 91 Anti-IGF-IR (aIR3) 9 35 32 EC2 8 13 11 Anti-mouse FITC 4.3 9 13 UltraCulture medium 9 10 11 -52 15B9 7.5 25 77.8 9F5D 8 41 40 13G5 7.8 37 24 7C10 8.6 49 13 The two last screenings provided for the selection of the MAb consisted in verifying that the latter was very capable of inhibiting the cell proliferation induced by 5 the IGF-1 in vitro and in vivo on the cell line MCF-7. For the in vitro selection, the MCF-7 cells were inoculated, deprived of fetal calf serum, then incubated in the presence of increasing concentrations 10 of IGF-1 (from 1 to 50 ng/ml) in the presence or in the absence of the 7C10 antibody to be tested added to a final concentration of 10 pg/ml. In this experiment, the commercial aIR3 MAb was introduced as a positive control and the 7G3 MAb (isolated in parallel to the 15 7C10 and weakly recognizing the native receptor (MFI on the FACS of 50 compared with 200 for the MAb 7C10)) as a control isotype. The cell proliferation is estimated by following on the P counter the incorporation of tritiated thymidine by the cells. The results are 20 expressed as a proliferative index. The data presented in figure 5 show that IGF1 is capable of stimulating in a dose-dependent manner the proliferation of the MCF-7 cells. The MAb aIR3, used as a positive control, completely inhibits the proliferation of the MCF-7 25 cells induced by the IGF-1. In the same manner, the MAb 7C10 significantly inhibits the growth of the MCF-7 cells induced by IGF-1. Finally, the MAb 7G3 used as an isotype control turns out well, as expected, without effect on the tumor cell growth in vitro of the MCF-7 30 cell. The in vivo selection was carried out in an established tumor model. In order to do this, nude mice received a subcutaneous implant of slow-release estrogen, - 53 indispensable for the taking of the tumor in a murine model. Twenty-four hours after implantation of the estrogens, 5.106 MCF-7 cells are grafted onto the right flank of the mouse subcutaneously. Five days after this 5 cell graft, the tumors are measurable and batches of 6 mice are formed at random. The treatment of the mice is carried out twice per week, during 5 to 6 weeks, at the dose of 250 pg/dose/mouse. In the control group, the mice are treated in the same fashion with a murine 10 control isotype. The results presented in figure 6A show a very significant inhibition of the tumor growth induced by the antibody 7C10. This activity is particularly unexpected if reference is made to the data available concerning oIR3, always used as a 15 reference in the domain of the receptor for IGF1, and known for not having any activity in vivo on the growth of estrogen-dependent tumors (see figure 6B). In the same way, compared with the results obtained with the recombinant antibody scFv-Fc derived from the murine 20 MAb 1H7 (see figure 6C), the MAb 7C10 is much more efficacious in the in vivo inhibition of the growth of the MCF-7 cells. Example 2. Comparison of the effect of 7C10 and of 25 tamoxifen on the in vivo growth of the tumor MCF-7 With the aim of determining the effectiveness of the treatment by the antibody 7C10 in the context of 30 estrogen-dependent cancer of the breast, 7C10 was compared with the tamoxifen compound currently used for the treatment of mammary carcinoma in the context of developed forms with local and/or metastatic progression and in the context of the prevention of 35 recurrences (see VIDAL 2000, pages 1975-1976). In hormone-dependent cancers of the breast, a significant correlation exists between the expression of the receptors for estrogens (ER) and that of the - 54 IGF-IR (Surmacz E. et al., Breast Cancer Res. Treat., Feb., 47(3):255-267, 1998). Furthermore, it seems that the estrogens (E2) act in synergy with IGF1 (sometimes written IGF-I or IGFI) in order to stimulate cell 5 proliferation. It has in effect been shown that a treatment with E2 increases by approximately 10 times the mRNA level of IGF-IR as well as the expression level of the protein (Lee A.V. et al., Mol. Endocrinol., May, 13(5):787-796, 1999). This increase 10 is manifested by a significant increase in the phosphorylation of the IGF-IR. In addition, the E2 significantly stimulates the expression of IRS-1 ("IRS 1" for "Insulin Receptor Substrate-l") which is one of the substrates of the phosphorylated IGF-IR. 15 Tamoxifen has been widely used for many years in hormone therapy for the treatment of patients suffering from E2-dependent breast cancers (Forbes J.F., Semin. Oncol., Feb., 24 (1st Suppl. 1):S1-5-S1-19, 1997). This 20 molecule enters into competition with the estradiol and inhibits the attachment of this to its receptor (Jordan V.C., Breast Cancer Res. Treat., 31(1):41-52, 1994). It has in addition been demonstrated that tamoxifen is capable of inhibiting the IGF-IR-dependent 25 proliferation by inhibiting the expression of the receptor and its phosphorylation (Guvakova M.A. et al., Cancer Res., July 1, 57(13):2606-2610, 1997). These data as a whole seem to indicate that IGF-IR is an important mediator of the proliferation induced by the 30 E2/ER interaction. The long-term use of tamoxifen is associated with a significant increase in the risk of endometrial cancer (Fisher et al., J. of National Cancer Institute, 86, 35 7:527-537, 1994; VIDAL 2000, 1975-1976) and of collateral recurrence of E2-independent cancer of the breast (Li C.I. et al., J. Natl. Cancer Inst., July 4, 93(13):1008-1013, 2001). In this context, a comparison of the in vivo antitumor effect of the antibody 7C10 - 55 and of tamoxifen has been carried out on the MCF-7 model so as to determine the part of the activity connected with IGF-IR in the mediated ER proliferation. In order to do this, 7.106 MCF-7 cells were implanted 5 sc (subcutaneously) in nude mice, 24 hours after implantation in these same mice of a grain of estradiol with prolonged release (0.72 mg/tablet liberated over 60 days), indispensable for the establishment of any E2-dependent human tumor in this animal species. Five 10 days after this implantation, the tumors are measured and groups of 6 mice are formed. These groups are treated respectively with 1) the 7C10 antibody injected ip (intraperitoneally) at a rate of 250 pg/mouse, twice per week, 2) 10 pg of tamoxifen taken in PBS containing 15 3% of hydroxypropyl-cellulose (HPC) ip or 3) the solvent in which the tamoxifen is taken up (hydroxypropylcellulose). The tamoxifen is administered daily for 4 weeks except at the weekend. The mice treated with the MAb 7C10 likewise daily receive an 20 injection of PBS with 3% HPC. A study was previously carried out in order to verify that the solvent alone is without influence on the tumor growth. The results presented in figure 7 shown that the MAb 25 7C10 is capable of significantly inhibiting the growth of the tumor MCF-7 in vivo (the asterisks (*) correspond to the comparison control group/7C10 group in a t-test). In a surprising fashion, the antibody 7C10 seems to be significantly more efficacious than 30 tamoxifen for the inhibition of the tumor growth (the circles (0) correspond to the comparison tamoxifen group/7C10 group in a t-test) suggesting that this type of treatment by MAB might be substituted for treatment with tamoxifen. 35 Example 3. Demonstration of the antitumor activity of the MAb 7C10 in vivo on human tumors of different origins - 56 a) In vivo activity of the antibody 7C10 in three tumor models In order to generalize the activity of the 7C10 5 antibody to other tumors expressing the receptor for IGF1, 7C10 was tested in vivo in an androgen independent model of tumor of the prostate DU145 (likewise written DU-145), in an SKES-1 osteosarcoma model and in a model of non-small cell tumor of the 10 lung A549. The protocol is comparable to that described above for MCF-7 and the results presented in figures 8A to 8C show a significant activity of this MAB in the 3 tumor models. The activity observed in the model of tumor of the prostate is to be noted very particularly 15 inasmuch as the single chain scFv of the MAB 1H7 is without activity in an androgen-independent model of tumor of the prostate (Li et al., 2000). b) In vivo activity of the antibody 7C10 in an 20 orthotopic model A549 The conventional xenograft models as described above do not allow the study of drugs on metastatic dissemination. In effect, the tumors implanted s.c. 25 (subcutaneously) remain localized at the sight of injection and are therefore not really a reflection of the situation in man. In order to evaluate our antibody in a model closer to reality, the A549 cells were implanted in an intrapleural location. This model, 30 which is well described (Clin. Cancer Res. 2000 Jan; 6(1):297-304) allows a metastatic dissemination close to that observed in man to be observed, with mediastinal, pulmonary, cardiac and vertebral metas tases. In the study which was carried out, 106 A549 35 cells were injected intrapleurally into female nude mice. 7 days after implantation, the mice were divided into 2 batches of 22. One of these batches received a challenge dose of 500 pg/mouse and was then treated twice per week at a rate of 250 pg of 7C10/dose. The - 57 second batch was treated according to the same scheme with the control isotype 9G4. Figure 31 shows a significant extension of survival in the mice treated with the MAB 7C10 indicating that this antibody is 5 capable of having an action on metastatic dissemination. Example 4. Comparison of the MAb 7C10 with navelbine in vivo; effect of a 10 coadministration of the two treatments Navelbine is a chemotherapy compound indicated in non small cell cancer of the lung and in metastatic cancer of the breast. The comparative study of 7C10 and of 15 navelbine and the possible synergy between the two products was studied on the tumor model A549. For this study, 5.106 A549 cells were grafted subcutaneously on the right flank of the mouse. Five days after the cell graft, the tumors are measurable and the treatments 20 with MAb and/or navelbine are commenced. The MAb dose is always 250 pg/dose/mouse, twice per week, intra peritoneally. Concerning navelbine, it will be administered at the maximum dose tolerated by the mouse or 10 mg/kg, intraperitoneally. For this treatment 25 three injections will be carried out at intervals of 7 days. During the coadministrations, the two products are mixed before injection. The results presented in figure 9 show in a surprising 30 fashion that, in this model, the antibody 7C10 is as active as the conventional treatment with navelbine. A very significant synergy of the two products is likewise observed with five mice out of seven not having measurable tumors on day 72. 35 Example 5. Study of the in vitro inhibition of the IGF2-induced growth of the MCF-7 tumors As indicated above, IGF-IR is overexpressed by numerous - 58 tumors but it has furthermore been described that in a good part of the cancers of the breast and of the colon especially, the proliferation signal is given to this receptor via IGF2 (sometimes written IGF-II or IGFII). 5 It is therefore essential to ensure that the MAb 7C10 is likewise capable of inhibiting the IGF2 growth induced on the MCF-7 tumor in vitro. In order to do this, cells were inoculated into 96-well plates, deprived of fetal calf serum and stimulated by the 10 addition of 200 ng of IGF2 per ml, final concentration, of medium, in the presence and in the absence of the MAb to be tested introduced at a concentration of 10 pg/ml. The results presented in figure 10 show that IGF2, like IGF1, significantly stimulates the growth of 15 MCF-7 cells. The addition of a control isotype, 9G4, remains without effect on this stimulation. As already described by De L6on et al. (Growth Factors, 6:327-334, 1992), no effect is observed during the addition of the MAb aIR3. On the other hand, 7C10 totally inhibits the 20 growth induced by IGF2. Its activity is significantly better than that of 1H7. Example 6. Biological activity of the chimeric 7C10 (C7C10) and humanized (h7C10) antibodies 25 7C10 a) 7C10/C7C10 and 7C10/h7C10 comparison on the MCF-7 model in vitro 30 The chimeric form of the MAb 7C10 and the purified humanized form 1 (written here 7H2HM) were tested in vitro in the MCF-7 model as described above. The results presented respectively in figures 11 and 12 show that these two forms have perfectly preserved 35 their properties of inhibiting the IGFl-induced growth of the MCF-7 tumor. b) Comparative effect of the MAb 7C10 and h7C10 on the transduction of the signal induced by the - 59 attachment of IGF1 to its receptor The activity of the inhibition of the IGFI growth induced in vitro on the line MCF-7 ought to be the 5 translation of an inhibition of the transduction of the signal mediated by IGF1 during the attachment of the MAb 7C10 to the receptor. In order to verify this hypothesis, MCF-7 cells were incubated with or without IGF1, in the presence or in the absence of the 10 antibodies to be tested. After a short incubation time, the cells were lyzed, the P chain immunoprecipitated and the phosphorylation of this subunit estimated with the aid of an antiphosphotyrosine kinase antibody. The results presented in figure 13 show that the attachment 15 of the 7C10 or of the h7C10 significantly inhibits the phosphorylation of the B subunit of IGF-IR contrary to an irrelevant murine (9G4) or human antibody (written IgGl on the scheme). 20 c) Involvement of the 7H2HM antibody in the mechanisms of ADCC The inhibition of the transduction of the signal described above in paragraph b) is the principal 25 mechanism of action involved in the biological activity of the antibodies 7C10 and 7H2HM. It is, however, probable that during its administration in man, the antibody 7H2HM, of isotype IgGl, is capable of inducing cell lysis by a mechanism of ADCC type (Antibody 30 Dependent Cellular Cytotoxicity). In order to verify this point, NK (Natural Killer) cells coming from the peripheral blood of human donors are placed in the presence of A549 or MCF-7 cells previously incubated for 4 hours with 10 pg of 7H2HM antibody per 5.105 35 cells and labeled with 51 Cr -(50 pg). In this experiment, herceptin (written h4D5 on figures 32A and 32B) is used as an experiment positive control. Figures 32A to 32D show that, as expected, herceptin induces a significant ADCC on the two cells A549 and MCF-7 (see respectively - 60.

figures 32A and 32B). 7H2HM is likewise capable of inducing an ADCC on the A549 cells (see figure 32C), but this phenomenon is of smaller amplitude on the MCF 7 cells (see figure 32D). 5 d) Effects of the antibodies 7C10 and 7H2HM on the cell cycle The inhibition of the cell growth observed in vitro on 10 the line MCF-7 should be manifested by an effect on the cell cycle. In order to reply to this question, 4.105 cells are inoculated into 6-well plates. 24 hours after inoculation, the calf serum is removed and IGF1 added in the presence or in the absence of the antibodies to 15 be tested. After incubation for 24 hours, the cells are recovered for the study of the cell cycle. Figure 33B demonstrates the effect of IGF1 on the entry into the cycle and the growth of the MCF-7 cells compared with the entry into the cycle and the growth of the MCF-7 20 cells in the absence of IGF1 (see figure 33A). After addition of the growth factor, a significant decrease in the GO/GI phase (from 88.2% to 56.3%) to the benefit of the S (from 7.8% to 31%) and G2/M (from 4% to 12.7%) phases is observed. During the addition of the 25 antibodies 7C10 and 7H2HM (see figure 33C), a significant inhibition of the entry into the cycle is observed. In it is to be noted that the murine antibody and its humanized homolog have a comparable activity on the cell cycle. The aIR3, introduced as a positive 30 control, seems slightly less active than the 7C10 and the 7H2HM in this test. The antibody 9G4 used as a control isotype is without effect on the cell cycle. e) Comparative activity in vivo of the antibodies 35 7C10 and 7H2HM on the model A549 In order to confirm the activity of the humanized antibody 7H2HM in vivo, the latter was compared with 7C10 in the model of non-small cell tumor of the lung - 61 A549. This experiment was carried out exactly as described above except for the dose of antibody which is 125 pg/dose twice per week in place of 250 pg/dose twice per week and that of the fact of the 5 nonavailability of great quantities of 7H2HM. The antibody 9G4 was used as an isotype control for 7C10 and an irrelevant human immunoglobulin of isotype IgGl (below called HIgGI) was used as a control for the humanized antibody 7H2HM. 10 Figure 34A shows that there are no significant differences between the 9G4 and HIgGl control curves. As expected, a significant inhibition of the tumor growth is observed with the murine antibody 7C10. 15 Concerning the humanized antibody 7H2HM, the activity observed is of exactly the same intensity as that observed with its murine counterpart. This data, in addition to the observations described above in vitro, indicates that the humanization has not modified the 20 properties of the antibody generated. On the other hand, in the xenograft models in the mouse, the activity of the humanized antibody seems to be integrally connected with a mechanism of inhibition of the transduction of the signal. In effect, if an ADCC 25 part was in play in the inhibition of the tumor growth in the Nude mouse, a difference ought to be observed between the activity of the murine and humanized antibodies. 30 An in vivo experiment was likewise carried out on the MCF-7 breast tumor model and shows that, as expected, the antibody 7H2HM is perfectly comparable with the murine antibody 7C10 for the inhibition of the growth of this tumor in vivo (figure 34B). 35 f) Demonstration of a synergy between the 7H2HM and navelbine The protocol described in example 4 was repeated with - 62 the aim of reproducing the results obtained with 7C10 with its humanized homolog: the antibody 7H2HM. The results presented in figures 35A and 35B show that, 5 as in the case of 7C10, a significant synergy is demonstrated between the humanized antibody 7H2HM and navelbine. g) Effect of the antibodies 7C10 and 7H2HM on the 10 apoptosis of MCF-7 cells in vitro As indicated above, IGF-IR is capable of confering protection against apoptosis when it is overexpressed on the surface of cells. Furthermore, it has been 15 demonstrated in these examples that the antibodies 7C10 and 7H2HM were capable of potentiating an active compound in chemotherapy. In order to test the power of the antibodies 7C10 and 7H2HM to induce apoptosis, and to explain in part their synergy potential with the 20 chemotherapy, experiments were conducted on the MCF-7 cells in the presence or in the absence of doxorubicin, a medicament known to induce the apoptosis of this cell line in vitro. In these experiments, the MCF-7 'cells are inoculated at 2.10 4 /cm 2 in Petri dishes and cultured 25 for 24 h in RPMI without phenol red supplemented with 10% of fetal calf serum (FCS). The cells are then washed twice with PBS and put back into culture in medium with 0% FCS. They are allowed an adaptation time of 10 minutes at 370C before the addition of the 30 antibodies at 10 pg/ml. After an extra 10 minutes at 370C, recombinant IGF-I (Sigma) is added to the culture medium to a final concentration of 50 ng/ml. The cells are left at 370C again for one hour in order to allow the attachment of the antibodies and of the IGF-I. 35 Finally, the doxorubicin (Sigma) is added to the culture medium at 2 pg/ml and the cells are incubated for 24 hours at 370C. The experiments have likewise been conducted with - 63 navelbine at a concentration of 10 pg/ml. The analysis of the cell viability is carried out by flow cytometric analysis after labeling with the 5 annexin V-FITC (20 minutes, 4 0 C) and DAPI (2 pg/ml) The percentage of dead cells considered is the labeled population Annexin + / DAPI +. The antibody 5C2 is used as a control isotype. 10 The results represented in figure 36 show that doxorubicin induces apoptosis in 8% of the MCF-7 cells. When the cells are treated conjointly with the antibody 7C10 and the doxorubicin a significant increase in cell death is observed. The same effect is shown with the 15 antibody 7H2HM. The same type of results was observed when the antibody is combined with navelbine. Example 7. Cloning strategy of genes coding for the variable regions of the heavy and light 20 chains of the monoclonal antibody (MAb) 7C10 The total RNA was extracted from 107 cells of hybridomas secreting the antibody 7C10 by using the TRI 25 REAGENTTM (according to the instructions given by the supplier, SIGMA, T9424). The first cDNA strand. was synthesized with the aid of the 'First strand cDNA synthesis' kit of Amersham-Pharmacia (#27-9621-01, according to the instructions given by the supplier) 30 For the two chains, the reaction was primed with the oligonucleotide Not I-d(T)18, comprised in the Kit. The cDNA:mRNA hybrid thus obtained was used for the amplification by PCR of the genes coding for the heavy 35 and light chains of the Mab 7C10. The PCR were carried out by using a combination of oligonucleotides specific for the heavy and light (Kappa) chains of mouse immunoglobulins. The primers corresponding to the 5' ends hybridize in the region corresponding to the - 64 signal peptides (Table 2 for heavy chains, Table 3 for light chains). These primers were compiled from a large number of mouse antibody sequences found in the databanks (Jones S.T. et al., Bio/Technology 9:88-89, 5 1991). The primers corresponding to the 3' ends hybridize in the constant regions of the heavy chains (CH1 domain of the subclass IgGl, not far from the V-C junction, MHC-1 primer Table 4) and light chains (Kappa domain not far from the V-C junction, MKC primer Table 10 4). TABLE 2: Oligonucleotide primers for the 5' region of the variable domains of the heavy chains of mouse immunoglobulin (MHV) ("MHV" for "Mouse Heavy Variable") 15 MHV-1: 5' ATGAAATGCAGCTGGGTCATSTTCTT 3' (SEQ ID No. 13) MHV-2: 5' ATGGGATGGAGCTRTATCATSYTCTT 3' (SEQ ID No. 14) MHV-3: 5' ATGAAGWTGTGGTTAAACTGGGTTTT 3' (SEQ ID No. 15) MHV-4: 5' ATGRACTTTGGGYTCAGCTTGRT 3' (SEQ ID No. 16) 20 MHV-5: 5' ATGGACTCCAGGCTCAATTTAGTTTT 3' (SEQ ID No. 17) MHV-6: 5' ATGGCTGTCYTRGSGCTRCTCTTCTG 3' (SEQ ID No. 18) MHV-7: 5' ATGGRATGGAGCKGGRTCTTTMTCTT 3' (SEQ ID No. 19) MHV-8: 5' ATGAGAGTGCTGATTCTTTTGTG 3' (SEQ ID No. 20) MHV-9: 5' ATGGMTTGGGTGTGGAMCTTGCTATT 3' (SEQ ID No. 21) 25 MHV-10: 5' ATGGGCAGACTTACATTCTCATTCCT 3' (SEQ ID No. 22) MHV-11: 5' ATGGATTTTGGGCTGATTTTTTTTATTG 3' (SEQ ID No. 23) MHV-12: 5' ATGATGGTGTTAAGTCTTCTGTACCT 3' (SEQ ID No. 24) NB KEY: R=A/G,Y=T/C,W=A/T,K=T/G,M=A/C,S=C/G. 30 TABLE 3: Oligonucleotide primers for the 5' region of the variable domains of kappa (light) chains of mouse immunoglobulin (MKV) ("MKV" for "Mouse Kappa Variable") MKV-1: 5' ATGAAGTTGCCTGTTAGGCTGTTGGTGCT 3' (SEQ ID No. 25) 35 MKV-2: 5' ATGGAGWCAGACACACTCCTGYTATGGGT 3' (SEQ ID No. 26) MKV-3: 5' ATGAGTGTGCTCACTCAGGTCCT 3' (SEQ ID No. 27) MKV-4: 5' ATGAGGRCCCCTGCTCAGWTTYTTGG 3' (SEQ ID No. 28) MKV-5: 5' ATGGATTTWCAGGTGCAGATTWTCAGCTT 3' (SEQ ID No. 29) MKV-5A: 5' ATGGATTTWCARGTGCAGATTWTCAGCTT 3' (SEQ ID No. 30) -65 MKV-6: 5' ATGAGGTKCYYTGYTSAGYTYCTGRG 3' (SEQ ID No. 31) MKV-7: 5' ATGGGCWTCAAGATGGAGTCACA 3' (SEQ ID No. 32) MKV-8: 5' ATGTGGGGAYCTKTTTYCMMTTTTTCAAT 3' (SEQ ID No. 33) MKV-9: 5' ATGGTRTCCWCASCTCAGTTCCTT 3' (SEQ ID No. 34) 5 MKV-10: 5' ATGTATATATGTTTGTTGTCTATTTC 3' (SEQ ID No. 35) MKV-11: 5' ATGGAAGCCCCAGCTCAGCTTCTCTT 3' (SEQ ID No. 36) MKV-12A: 5' ATGRAGTYWCAGACCCAGGTCTTYRT 3' (SEQ ID No. 37) MKV-12B: 5' ATGGAGACACATTCTCAGGTCTTTGT 3' (SEQ ID No. 38) MKV-13: 5' ATGGATTCACAGGCCCAGGTTCTTAT 3' (SEQ ID No. 39) 10 NB KEY: R=A/G,Y=T/C,W=A/T,K=T/G,M=A/C,S=C/G. TABLE 4: Oligonucleotide primers for the 3' ends of the mouse VH and VL genes 15 Light chain (MKC): 5' ACTGGATGGTGGGAAGATGG 3' (SEQ ID No. 40) Constant region of the mouse Kappa domain: 20 A D A A P T V S I F P P S S (SEQ ID No. 41) GCT GAT GCT GCA CCA ACT GTA TCC ATC TTC CCA CCA TCC AGT (SEQ ID No. 42) II I I I I l l I l l I l l I I I I l l (MKC) CC ATC TTC CCA CCA TCC AGT(SEQ ID No. 43) Heavy chain (MHC-1) 25 5' CCAGTGGATAGACAGATG 3' (SEQ ID No. 44) CH1 domain of mouse gamma-i (IgGl subclass): A K T T P P S V Y P L (SEQ ID No. 46) GCC AAA ACG ACA CCC CCA TCT GTC TAT CCA CTG (SEQ ID No. 45) 3 0 i l l I l i l l ll I I i l l (MHC-1) CCC CCA TCT GTC TAT CCA CTG (SEQ ID No. 47) Example 8. Sequences of immunoglobulins cloned from the mouse hybridoma 7C10 35 By following the amplification strategy described above, PCR products corresponding to the variable regions of the heavy (VH) and light (VL) chains were cloned by using the "pGEM®-T Easy Vector Systems" - 66 (Promega). For 7C10 VL, PCR products were obtained with the MKC primer in combination with the MKV1 and MKV2 primers. For 7C10 VH, PCR products were obtained with the MHC-1 primer in combination with the MHV8 and MHV12 5 primers. A thorough sequencing of the PCR products cloned in the pGem-T easy vectors revealed two different sequences for the light chain and one unique sequence for the heavy chain. 10 a) Variable region isolated from the oligo MKVl The DNA sequence obtained is characteristic of a variable region of functional Ig. This novel sequence is therefore presumed to be that coding for 7C10 VL. 15 The DNA (SEQ ID Nos. 48 and 50) and amino acid (SEQ ID No. 49) sequences of the cDNA coding for 7C10 VL are represented in figure 14. b) Variable region isolated from the oligo MKV2 20 The gene coding for this light chain comes from an aberrant mRNA transcript which is present in all the standard fusion partners derived from the original MOPC-21 tumor of which the mouse myeloma Sp2/Oagl4, 25 which was used in order to produce the 7C10 hybridoma, is part. This sequence contains an aberrant recombination between the V and J genes (deletion of four nucleotide bases involving a change in the reading frame) and a mutation of the invariable cysteine in 30 position 23 to tyrosine. These changes suggest that this light chain would be nonfunctional although nevertheless transcribed to messenger RNA. The DNA sequence of this pseudo light chain is not shown. 35 c) Variable region isolated from the oligos MHV8 and MHV12 The DNA sequences obtained with these two oligos are identical, apart from the sequence encoded by the oligo - 67 itself. This sequence is a novel sequence coding for a functional heavy chain presumed to be that of the monoclonal antibody 7C10. The DNA (SEQ ID Nos. 51 and 53) and amino acid (SEQ ID No. 52) sequences of the 5 cDNA coding for 7C10 VH are represented in figure 15. Example 9. Construction of chimeric mouse-man genes The chimeric antibody 7C10 was constructed so as to 10 have the mouse 7C10 regions VL and VH connected to the human constant regions kappa and gamma-l, respectively. Oligos were used in order to modify the 5' and 3' ends of the sequences flanking the DNA coding for 7C10 VL and VH in order to allow their cloning in vectors for 15 expression in mammalian cells. These vectors use the strong promoter HCMV in order effectively to transcribe the heavy and light chains of the chimeric antibody 7C10. On the other hand, these vectors likewise contain the replication origin of SV40 allowing an effective 20 replication of the DNA and, as a consequence, as a transitory expression of the proteins in cos cells. Example 10. Expression and evaluation of the recognition activity of the IGF-1 25 receptor of the chimeric antibody 7C10 The two plasmids containing the DNA coding for the chimeric 7C10 antibody were cotransfected in cos-7 cells (ATCC number CRL-1651) in order to study the 30 transitory expression of the recombinant antibody. After incubation for 72 hours, the culture medium was removed, centrifuged in order to eliminate the cell debris and analyzed by the ELISA technique for the production of human IgGl (see Example 16) and the 35 recognition of the receptor for IGF-1 (see Example 17). The ELISA tests for measurement of concentrations of human IgGl/Kappa showed that the expression of the chimeric antibody 7C10 in the cos-7 cells was between *- 68 300 and 500 ng/mm, which is comparable to the values obtained with the majority of antibodies. The ELISA tests for recognition of the receptor for 5 IGF-1 show that the chimeric antibody recognizes it specifically and with a good relative avidity (see figures 3A, 3B and 3C). This provides the functional proof that the good VH and VL of the 7C10 antibody have been identified. In addition, this chimeric form of 10 7C10 appears as being an indispensable tool in the evaluation of the affinity of the humanized forms. Example 11. Molecular modeling of the variable regions of the mouse antibody 7C10 15 In order to assist and to refine the humanization process by "CDR grafting", a molecular model of the VL and VH regions of the mouse antibody 7C10 was constructed. The model is based on the crystallographic 20 structure of the heavy chain 1AY1 and of the light chain 2PCP. Example 12. Process of humanization by CDR grafting of the variable region of the light 25 chain of the antibody 7C10 (7C10 VL) a) Comparison of the amino acid sequence of 7C10 VL with all the known mouse VL sequences 30 As a preliminary step to humanization by CDR grafting, the amino acid sequence of 7C10 VL was first compared with all the mouse VL sequences present in the databank of Kabat (Internet address: ftp://ftp.ebi.ac.uk/pub/ database/kabat/fastaformat/, last update of data dates 35 from 1999). 7C10 VL has thus been identified as belonging to the subgroup II of the Kappa light chains as defined by Kabat et al. (In Sequences of proteins of immunological interest ( 5 t h edn.), NIH publication No. 91-3242, US Department of Health and Human Services, - 69 Public Health Service, National Institutes of Health, Bethesda, 1991). The VL regions of monoclonal antibodies of mice having a sequence identity ranging up to 95% have been identified (DRB1-4.3 (SEQ ID No. 5 55): 95% and C94-5B11'CL (SEQ ID No. 56): 95%, see figure 17). In order to attempt to identify the out of the ordinary residues in the 7C10 VL sequence, the amino acid sequence of 7C10 VL (SEQ ID No. 54) was aligned with the consensus sequence of the subgroup II 10 of the mouse kappa chains (SEQ ID No. 57) as defined by Kabat (see figure 17). In the Kabat position number 3, the valine (V) normally present in the subgroup II of the Kappa light chains 15 according to Kabat (71%) is replaced by a leucine (L). A leucine in this position is not rare since it is found, for example, in DRB1-4.3 and C94-5B11'CL. According to the molecular model, this residue does not seem to play a particular role. Consequently, the 20 conservation of this residue in the humanized form will not be envisaged. In the Kabat position number 7, the threonine (T) normally present in the subgroup II of the Kappa light 25 chains according to Kabat (66%) is replaced by an iso leucine (I). An isoleucine in this position is relatively rare since it is only found 15 times among all the mouse VL sequences known and never among human VL sequences. The molecular model shows that this 30 residue (17) points toward the surface of the molecule but does not contact the CDRs (the residue of a CDR which is the closest would be the arginine in Kabat position number 42). In addition, it does not seem very probable that this residue 17 directly contacts the 35 antigen. Consequently, the conservation of this residue in the humanized form will not be envisaged, at any rate at first. In the Kabat position number 77, the arginine (R) - 70 normally present in the subgroup II of the Kappa light chains according to Kabat (95.5%) is replaced by a serine (S). A serine in this position is not rare. 5 b) Comparison of the amino acid sequence of 7C10 VL with all the known human VL sequences In order to identify the best human candidate for the "CDR grafting", the Kappa VL region of human origin 10 having the greatest homology possible with 7C10 VL was sought. To this end, the amino acid sequence of mouse kappa 7C10 VL was compared with all the human Kappa VL sequences present in the database of Kabat. Mouse 7C10 VL had the greatest sequence homology with the human 15 kappa VL regions of subgroup II as defined by Kabat et al. (1991). VH regions of monoclonal antibodies of human origin have been identified having a sequence identity ranging up to 75.9% (GM607 (SEQ ID No. 58), see figure 18) over the whole of the 112 amino acids 20 composing the variable region. A germinal line of human origin, DPK15/A19 (SEQ ID No. 59), having a sequence identity of 76% (see figure 18) was also identified, GM607 (Klobeck et al., 1984). GM607 was therefore chosen as a human sequence receptive of CDRs (according 25 to the definition of Kabat) of mouse 7C10 VL. By comparing the GM607 sequences with that of the consensus sequence of the human subgroup II (SEQ ID No. 60) (figure 18), no particular residue in the framework regions (Rch) could be identified, indicating by the 30 same fact that GM607 was a good candidate for CDR grafting. c) Humanized versions of 7C10 VL 35 The following stage in the humanization process consisted in joining the CDRs of mouse 7C10 VL to the framework regions (Rch) of the human light chain selected, GM607 (Klobeck et al., 19:34). At this stage of the process, the molecular model of the mouse Fv -71 regions of 7C10 is particularly useful in the choice of the mouse residues to be conserved as being able to play a role either in the maintenance of the tridimensional structure of the molecule (canonical 5 structure of the CDRs, VH/VL interface, etc.) or in the binding to the antigen. In the Rchs, each difference between the mouse (7C10 VL) and human (GM607) amino acids was examined scrupulously (see Table 5). In addition, the particular residues in the mouse sequence 10 7C10 VL which were identified (see example 12.a) were taken into account if needed. In the first version humanized by "CDR grafting" of 7C10 VL, human 1, a single change in the framework 15 regions (Rch) of GM607 was carried out. This change concerns the residue 2 (nomenclature of Kabat) situated in Rch 1. This residue enters in effect into the composition of the canonical structure of the CDR 1 of 7C10 VL and could therefore be critical for maintaining 20 this loop in its good conformation. The valine present in this position in the mouse 7C10 VL sequence is thus conserved in this same position in the humanized form (see Table 5 and figure 19 for the amino acid sequence (SEQ ID No. 61) and figure 20 for the DNA sequence (SEQ 25 ID Nos. 62 and 64) and the amino acid sequence comprising the peptide signal (SEQ ID No. 63). In the second version humanized by "CDR grafting" of 7C10 VL, human 2, no change in the Rchs of the human 30 light chain GM607 has been made. All the residues of the Rchs are thus of human origin including the residue 2 which has therefore been mutated in order to replace the valine present in mouse 7C10 VL by an isoleucine found in this same position in the human light chain 35 GM607 (see Table 5 and figure 19 for the amino acid sequence (SEQ ID No. 65) and figure 21 for the DNA sequence (SEQ ID Nos. 66 and 68) and the amino acid sequence comprising the peptide signal (SEQ ID No. 67)). This human form 2 is therefore totally humanized -72 (apart from, of course, CDRs themselves) since all the residues of the Rchs are those of the light chain of human origin, GM607. 5 TABLE 5: Alignment of the amino acid sequences leading to the design of the remodeled human 7C10 VL. regions Mouse Human Remodeled Remodeled Comments Kabat # FR light germinal GM human human or chain line 607 7C10 1 7C10 2 CDR 7C10 DPK15/A1 9 1 1 FR1 D D D D D 2 2 V* I* I* V* I* Cano L1 4 (16) Vernier zone 3 3 L V V V V 4 4 M M M M M Vernier zone 5 5 T T T T T 6 6 Q Q Q Q Q 7 7 I S S S S 8 8 P P P P P 9 9 L L L L L 10 10 S S S S S 11 11ii L L L L L 12 12 P P P P P 13 13 V V V V V 14 14 S T T T T 15i 15 L P P P P 16 16 G G G G G 17 17 D E E E E 18 18 Q P P P P 19 19 A A A A A 20 20 S S S S S 21 21 I I I I I -73 22 22 S S S S S 23 23 FR1 C C C C C 24 24 CDR1 R R R R R 25 25 I S* S* S* S* S* Cano L1 4(16) 26 26 S S S S S 27 27 Q Q Q Q Q 27A 28 S S S S S 27B 29 1I* L* L* i* i* Cano L1 4(16) 27C 30 V L -Li I 27D 31 H H H H H 27E 32 S S S S S 28 33 N N N N N 29 34 G G G G G 30 35 N Y Y n N 31 36 T N N t T 32 37 Y Y Y Y Y 33 38 L* L* L* L* L* Cano L1 4(16) 34 39 CDR1 Q D D q Q 35 40 FR2 W W W W W Vernier zone 36 41 Y Y Y Y Y VH/VL inter Vernier zone 37 42 L L L L L 38 43 Q Q Q Q Q VL/VH inter 39 44 K K K K K 40 45 P P P P P 41 46 G G G G G 42 47 Q Q Q Q Q 43 48 S S S S S 44 49 I P P P P P VL/VH inter

(+)

- 74 45 50 K Q Q Q Q 46 51 L L L L L VL/VH inter Vernier zone 47 52 L L L L L Vernier zone 48 53 I I I I* I* Cano L2 1(7) Vernier zone 49 54 FR2 Y Y Y Y Y Vernier zone 50 55 CDR2 K L L k K 51 56 V* G* G* v* v* Cano L2 1(7) 52 57 I S* S* S* S* S* Cano L2 1(7) 53 58 N N N N N 54 59 R R R R R 55 60 L A A 1 L 56 61 CDR2 Y S S y Y 57 62 FR3 G G G G G 58 63 V V V V V 59 64 P P P P P 60 65 D D D D D 61 66 R R R R R 62 67 F F F F F 63 68 S S S S S 64 69 G* * G* G* G* Cano L2 1(7) Vernier zone 65 70 S S S S S 66 71 G G G G G Vernier zone 67 72 S S S S S 68 73 G G G G G Vernier - 75 zone 69 74 T T T T T Vernier zone 70 75 D D D D D 71 76 F* F* F* F* F* Cano L1 4(16) Vernier zone 72 77 T T T T T 73 78 L L L L L 74 79 K K K K K 75 80 I I I I I 76 81 S S S S S 77 82 S R R R R 78 83 V V V V V 79 84 E E E E E 80 85 A A A A A 81 86 E E E E E 82 87 D D D D D 83 88 L V V V V 84 89 G G G G G 85 90 V V V V V 86 91 Y Y Y Y y 87 92 I Y Y Y Y Y VL/VH inter 88 93 FR3 C C C C C 89 94 CDR3 F M M f F VL/VH inter 90 95 Q* Q* Q* * Q* Cano L3 1(9) 91 96 G A A g G VL/VH inter 92 97 S L L s S 93 98 I H Q h H 94 99 V T T v V 95 100 P* P* P* P* P* Cano L3 1(9) 96 101 I W Q w W VL/VH - 76 inter (+) 97 102 CDR3 T T T T 98 103 FR4 F F F F VL/VH inter (+) Vernier zone 99 104 G G G G 100 105 G Q Q Q 101 106 G G G G 102 107 T T T T 103 108 K K K K 104 109 L V V V 105 110 E E E E 106 111 I I I I 107 112 FR4 K K K K Legend: The first column (Kabat) indicates the position of the amino acid residue according to Kabat et al. (1991); the second column (#) indicates the position of 5 the amino acid residue in the regular sequence; the third column (FR or CDR) was made in order easily to identify the segments of the skeleton (FR1, FR2, FR3 and FR4) and the CDR segments (CDRI, CDR2 and CDR3) ("CDR" for "Complementarity-Determining Region") with 10 the three CDRs separating the four FRs; the fourth column (Mouse light chain 7C10) represents the amino acid sequence (SEQ ID No. 54) of the VL region of mouse antibody 7C10; the fifth column (Human germinal line DPK15/A19) represents the amino acid sequence (SEQ ID 15 No. 59) of the kappa II human V light chain of the germinal line; the sixth column (GM607) represents the amino acid sequence (SEQ ID No. 58) of the VL region of the human antibody GM607; the seventh and eighth columns (remodeled human 7C10 1 and 2) represent the 20 amino acid sequences of the humanized 1 and 2 antibody 7C10 VL (respectively SEQ ID Nos. 61 and 65). "*" - 77 indicates the parts of the canonical structure of the CDR loop such as defined by Chothia et al. (Nature, 342, 877-883, 1989). 5 Example 13. Process of humanization by CDR grafting of the variable region of the heavy chain of the antibody 7C10 (7C10 VH) a) Comparison of the amino acid sequence of 7C10 VH 10 with all of the known mouse VH sequences As a preliminary stage in humanization by CDR grafting, the amino acid sequence of 7C10 VH was first compared with all the mouse VH sequences present in the Kabat 15 databank (Internet address: ftp://ftp.ebi.ac.uk/ pub/database/kabat/fasta format/, last update of data dates from 1999). 7C10 VH has thus been identified as belonging to the subgroup I(A) of the heavy chains as defined by Kabat et al. (1991). VH regions of mouse 20 monoclonal antibodies having a sequence identity ranging up to 90.5% were identified (AN03'CL (SEQ ID No. 70), see figure 22). In order to attempt to identify the out of the ordinary residues in the sequence of 7C10 VH, we aligned the amino acid sequence 25 of 7C10 VH (SEQ ID No. 69) with the consensus sequence (SEQ ID No. 71) of the subgroup I(A) of the mouse heavy chains as defined by Kabat (see figure 22). Residue 17 (Kabat's numbering), Thr for the consensus 30 sequence of subgroup I(A) and Ser in 7C10 VH,. is located on the surface of the molecule with respect to the interface with the constant region. This residue does not seem to be important. 35 Residue 27 (Kabat's numbering), Asp for the consensus sequence of subgroup I(A) and Tyr in 7C10 VH, is a canonical residue for the CDR 1. Tyr in this position is not rare and is probably critical for maintaining CDR 1 in its good conformation.

- 78 Residue 84 (Kabat's numbering), Thr for the consensus sequence of the subgroup I(A) and Asn in 7C10 VH. Asn was found 93 times in mouse VH and 3 times in human VH. According to the molecular model, it is a surface 5 residue remote from the paratope. The numbering of the amino acids is that of Kabat et al. (1991). The residues in the framework regions (apart from CDRs) which differ between 7C10 VH and 10 Kabat mouse subgroup I(A) are underlined. ANO3'CL represents the sequence of the heavy chain of a mouse antibody (access number in the Kabat databank is P001289). 15 b) Comparison of the amino acid sequence of 7C10 VH with all of the known human VH sequences In order to identify the best human candidate for the "CDR grafting", the VH region of human origin having 20 the greatest possible homology with 7C10 VH was sought. To this end, the amino acid sequence of mouse 7C10 VH was compared with all the human VH sequences present in the Kabat databank. Mouse 7C10 VH had the greatest sequence homology with the human VH regions of the 25 subgroup II as defined by Kabat et al. (1991). VH regions of monoclonal antibodies of human origin were identified having a sequence identity ranging up to 67.3% (human VH FUR1'CL (SEQ ID No. 73, see figure 23) over the whole of the 98 amino acids encoded by the 30 variable gene (that is to say apart from CDR3 and region J). A germinal line of human origin, 4.22 VH IV (Sanz et al., 1989), having a sequence identity of 68.4%, according to the same criteria as for VH FUR1'CL, was also identified (human Germ-line (SEQ ID 35 No. 74), see figure 23). The sequence encoded by the germinal line 4.22 VH IV was chosen as a human sequence receptive of the CDRs (according to the definition of Kabat) of mouse 7C10 VH rather than VH FUR1'CL because in comparing the sequences of 4.22 VH IV and VH FUR1'CL - 79 with that of the consensus sequence of the human subgroup II (human Kabat sg II (SEQ ID No. 72), see figure 23 and table 6), no atypical residue in the framework regions (Rch) could be identified for 4.22 VH 5 IV although the presence of two atypical residues (Gln and Arg in positions 81 and 82A according to the nomenclature of Kabat, respectively) were identified in the sequence encoded by VH FUR1'CL. 10 c) Humanized versions of 7C10 VH The following stage in the humanization process consisted in joining the CDRs of mouse 7C10 VH to the framework regions (Rch) of the human germinal line 4.22 15 VH IV (Sanz et al., 1989). At this stage of the process, the molecular model of the mouse Fv regions of 7C10 is particularly useful in the choice of the mouse residues to be conserved as being able to play a role in the maintenance of the tridimensional structure of 20 the molecule (canonical structure of the CDRs, VH/VL interface, etc.) or in the binding to the antigen (belonging to the paratope). In the Rchs, each difference between the mouse (7C10 VH) and human (4.22 VH IV) amino acids was examined scrupulously (see Table 25 6). In addition, the particular residues in the mouse 7C10 VH sequence which had been identified (see Example 8.a) were taken into account if needed. In the first version of 7C10 VH humanized by "CDR 30 grafting", humanized 1, four changes in the framework regions (Rch) of 4.22 VH IV were carried out (see Table 6, figure 24 for the amino acid sequence (SEQ ID No. 75) and figure 25 for the DNA sequence (SEQ ID Nos. 76 and 78) and the amino acid sequence comprising the 35 peptide signal (SEQ ID No. 77)). These four changes concern: S Residue 30 (Kabat's nomenclature) situated in Rch 1. This residue enters in effect into the structural composition of the CDR1 of 7C10 VH (as - 80 defined by Chothia et al., 1989) and could therefore be critical for maintaining this loop in its correct conformation. The Thr present in this position in the mouse sequence 7C10 VH is 5 therefore conserved in this same position in the humanized form. Residue 48 (Kabat's nomenclature) situated in Rch 2. This residue is close to the CDRs, although according to the molecular model not in direct 10 contact with the latter, and could influence their ultimate conformation. The methionine present in this position in the mouse sequence 7C10 VH is therefore conserved in this same position in the humanized form 1. 15 * Residue 67 (Kabat's nomenclature) situated in Rch 3. This residue is close to the CDRs and according to the molecular model could contact Lysine 60 (Kabat's nomenclature) in the CDR 2. The isoleucine present in this position in mouse 20 sequence 7C10 VH is therefore conserved in this position in the humanized form 1. * Residue 71 (Kabat's nomenclature) situated in Rch 3. This residue is part of the canonical structure of the CDR 2 and should therefore be critical for 25 maintaining this loop in its correct conformation. The arginine present in this position in the mouse sequence 7C10 VH is therefore conserved in this position in the humanized form 1. 30 In the second version of 7C10 VH humanized by "CDR grafting", humanized 2, two changes in the framework regions (Rch) of 4.22 VH IV were carried out. These two changes concern the residues 30 and 71 (Kabat's nomenclature), already described in the humanized form 35 1 (see Table 6, figure 24 for the amino acid sequence (SEQ ID No..79) and figure 26 for the DNA sequence (SEQ ID Nos. 80 and 82) and the amino acid sequence comprising the peptide signal (SEQ ID No. 81)).

- 81 In the third form of 7C10 VH humanized by "CDR grafting", humanized 3, no change in the framework regions (Rch) of 4.22 VH IV was carried out. All the residues of the Rchs are therefore of human origin 5 including the residues 30, 48, 67 and 71 (Kabat's nomenclature) which have been conserved (see Table 6, figure 24 for the amino acid sequence (SEQ ID No. 83) and figure 27 for the DNA sequence (SEQ ID Nos. 84 and 86) and the amino acid sequence comprising the peptide 10 signal (SEQ ID No. 85)). This humanized form 3 is therefore totally humanized (apart, of course, from the CDRs themselves as defined by Kabat) since all the residues of the Rchs are those encoded by the VH gene of the germinal line 4.22 VH IV.

0 a.) I> 0 00 0.% VC a) V)- C) >C -F4-1V aI) 0N > C -- 4aW 0; 4PI / - 4 0 4 0 14 LLL-C' C 04-------------------------- 0) u) L Q O' 0 0 Q)' -c -NNcoN 0 000 O0 a) c5 cz ~ ~ ~ cu0 00 0 0 o PE 0. C,)~ - (yl ' C ~ I 4 ~ ( D r ~- 00 - N - N -1 -n 4, - m o 0 N'io 0 a N4 NA) NI -j C4 wN 2z y c 'n Ca) (A) V2 w na & IIT -.- > o= C, UI n ID f- w O C ' n - 0 a C )-r o r 0 a : L ' ,tv rl 10~~ "o .)' o o.a -r -r -t Lj a)) 42d C 0) ~ 0 0) >

-

> V) > >~ 0. Ln4 ~~E- u z'oc I -A I 40 04-4 H - (DI0 ~ -H 0lj : *(0 0 U2 (0H- 5 (. ~~- 424 L)0 SU) 0 -H- 0) ) r5 4-2 0) -c 00) 4- 0 ) 0 I 04 ) 4-)( q) 002 4 U U 0) U (1) 4 C r- 0) U) ajd 42J 4- 0) ( U) - 9 u )CO 0 (1) rT) u -H LI - U)~ _(-_ 0 4 () 4- (0 U) ~o-rH- ~0 0 4-) 04 0) c) -H -H W~ F0 (N 4 H 0- > ro > U - l 0Lr ~ 0-H 4 F- 0 U4 42 0) ) (0- (00 LH m) 0 0( 0 r 42 0 ~ ~ 01 U)~r1 ~ ~ ( 4--H >1 U) C U 0 U -H U) 4 H 0) 0 0 4J 0) -O F0-io -- -H -a~ (00 U U r4 (0 co (0 : 0 044 0i u 0 u ry)H -Hu- ) 0 (00 ~-4-4 42-0 - - UH 4-.: - u z -0 r: 0 0 0)0n1 0 - 00 > ro FL 0( 0 -H-) 0 ) O1) U ~ 0 ) ~ U Q 0 (0C 4

~

4 0 00 0 114N U)0 U H U) ]- C ) 0 : 0) 0) 44 U -H 1 U'3) 0 (J 4(0) )H a-H 0) C/) ~.i ~ )U) H - ~ ( U) UL) 44H L14 r- - 87 Example 14. Construction of the genes coding for the humanized versions 1 of 7C10 VL and VH by assembly of oligonucleotides 5 a) Principle The genes (leader peptide + variable regions VDJ for VH or VJ for VK) coding for the humanized variable regions were synthesized by solid-phase assembly on magnetic 10 beads coated with streptavidin. The genes coding for humanized 7C10 VH (445 base pairs) and humanized 7C10 VL (433 base pairs) are constructed by fusing two fragments of DNA owing to the presence of a KpnI restriction site present in the two sequences and 15 situated almost halfway along the gene (at 200 and 245 nucleotides with respect to the 5' end of the gene for VL and VH, respectively). The two fragments which are fused together are themselves assembled by an assembly technique which consists in using phosphorylated 20 oligonucleotides (approximately 30-35 mer) hybridized two by two (one oligo sense and the other antisense, with a homology of approximately 50%) in such a way that they overlap during elongation. A first oligonucleotide biotinylated in the 5' position is 25 attached to the magnetic beads and then the pairs of phosphorylated oligonucleotides are added one by one. The phosphodiester linkage between the juxtaposed phosphorylated oligonucleotides is produced by the enzyme T4 DNA ligase. 30 The genes thus synthesized de novo can be cloned directly (by digestion with restriction enzymes compatible with the expression vector chosen) or amplified by PCR in order to obtain more material as a 35 prelude to directional cloning by enzymatic digestion. The sequence of the gene thus constructed by de novo assembly is then verified by automatic sequencing of the DNA.

- 88 b) Experimental protocol of the de novo assembly technique Oligonucleotides phosphorylated in the 5' position or 5 biotinylated in the 5' position whose concentration was adjusted to 100 pM were ordered from MWG Biotech (see the sequences of the oligonucleotides used in Table 7 for the construction of humanized 7CLO VL, and Table 8 for the construction of humanized 7C10 VH). The 10 oligonucleotides were hybridized in pairs (an equimolar mixture, 500 pmol, of a sense oligo and of an antisense oligo in the buffer T4 DNA ligase is heated to 950C for 5 minutes and then allowed to cool on the bench to ambient temperature) according to a scheme described in 15 Table 9. The first biotinylated oligonucleotide is attached to magnetic beads coated with streptavidin (Dynabeads M-280 streptavidin, Dynal product No. 112-05). For 20 this, 500 pmol of the biotinylated oligonucleotide in a 15 mM NaCl solution are added to 50 pl of the decanted beads (use of a magnet holder) previously washed twice with 100 pl of TE 1X buffer (Tris-EDTA 100X buffer: 1 M Tris-HCi, pH 8, 0.1 M EDTA, Sigma T-9285). After 25 incubation at 370C for 15 min, the beads are washed twice with the wash buffer (10 mM Tris-HC1 pH 7.6, 10 mM EDTA and 50 mM NaCl) and the pairs of hybridized oligo-nucleotides are then added one by one. On each readdition of a pair of oligonucleotides, the mixture 30 is heated to 950C for 5 min and then allowed to cool on the bench to ambient temperature. Once ambient temperature is reached, 2 pl of 10 U/pl T4 DNA ligase (Biolabs) are added and the mixture is incubated for 20 min at 370C. The beads are then washed (wash buffer) 35 and the following pairs of oligonucleotides are then added in succession. The last unpaired oligo (antisense) is assembled in the following fashion. 5 pl of oligo (500 pmol) and 43 pl - 89 of T4 DNA ligase buffer are added to the decanted beads, then the mixture is heated to 950C for 5 min and allowed to cool on the bench to ambient temperature. Once ambient temperature is reached, 2 pl of T4 DNA 5 ligase are added and the mixture is incubated at 370C for 20 min. The beads are then washed twice with wash buffer and then twice with TE 1X buffer. The beads can then be conserved at 40C before 10 proceeding to the cloning and sequencing of the gene assembled de novo. TABLE 7: DNA sequence of oligonucleotides used for the construction of humanized 7C10 VL 1 by de novo assembly 15 LeaderMluI .biotin 5' -GTCAGAACGCGTGCCGCC (SEQ ID No. 87) 7CIOLresh.].sense 5' -ACCATGAAGTTGCCTGTTAGGCTGTTGGTGCT (SEQ ID No. 88) 7C 0Lresh. 2sense 5' -GATGTTCTGGTTTCCTGCTTCCAGCIAGTGATG (SEQ ID No. 89) 7ClOLresh. 3sense 5' -TTGTGATGACTCAGTCTCCA'CTCTCCCTGCCC (SEQ ID No. 90) 7ClOLresh. 4sense 5' -GTCACCCCTGGAGAGCCGGCCTCCATCTCCTG (SEQ ID No. 91) - 90 7C1 0Lresh.5sense 5'-CAGGTCTAGTCAGACCATTATACATAGTAATG (SEQ UID No. 92) 7Cl0Lresh.6sense 5'-GAAACACCTATTTGGAATGGTACCTGCAGA (SEQ ID No. 93) 7Cl0Lresh.7anti 5' -GGCAACTTCATGGTGGCGGCACGCGTTCTGAC (SEQ ID No. 94) 7Ci0Lresh. anti 5'-GAAACCAGAACATCAGCACCAACAGCCTAACA (SEQ ID No. 95) 7Cl0Lresh.9anti 5' -CTGAGTCATCACAACATCACTGCTGGAAGCAG (SEQ ID No. 96) 7Cl0Lresh. 10anti 5'-TCTCCAGGGGTGACGGGCAGGGAGAGTGGAGA (SEQ ID No. 97) 7Cl0Lresh. llanti 5'-TCTGACTAGACCTGCAGGAGATGGAGGCCGGC (SEQ ID No. 98) 7Cl0Lresh.12anti 5'-AAATAGGTGTTTCCATTACTATGTACAATGC (SEQ ID No. 99) 7Cl0Lresh.13sense 5'-CAGGGCAGTCTCCACAGCTCCTGATCTATAAA (SEQ ID No. 100) 7CI0Lresh. 14sense 5'-GTTTCTAATCGGCTTTATGGGGTCCCTGACAG (SEQ UL)D No. 101) 7Cl0Lresh.15sense 5' -GTTCAGTGGCAGTGGATCAGGCACAGATTTTA (SEQ ID No. 102) 7C10Lresh.16sense 5' -CACTGAAAATCAGCAGAGTGGAGGCTGAGGAT (SEQ ID No. 103) 7Clo0Lresh.17sense 5' -GTTGGGGTTTATTACTGCTTTCAAGGTTCACA (SEQ ID No. 104) 7C10Ltresh.18sense 5 '-TGTTCCGTGGACGTTCGGCCAAGGGACCAAGG (SEQ ID No. 105) 7C10Lresh.19sense 5' -TGGAAATCAAACGTGAGTGGATCCTCTGCG (SEQ ID No. 106) 7C10Lresh. KpnIREV 5'-TCTGCAGGTACCATTGC (SEQ I) No. 107) 7Cl0Lresh.Kpnlbiotin 5'-TGCAATGGTACCTGCAGAAGC (SEQ ID No. 108) 7C10Lresh.20anti 5' -AGACTGCCCTGGCTTCTGCAGGTACCAiTTGCA (SEQ ID No. 109) 7C10Lresh.21anti 5'-CGATTAGAAACTTTATAGATCAGGAGCTGTGG (SEQ ID No- 110) 7C10Lresh. 22anti 5'-TGCCACTGAACCTGTCAGGGACCCCATAAAGC (SEQ U) No. I 11) 7Cl0Lresh.23anti 5'-GATTTTCAGTGTAAAATCTGTGCCTGATCCAC (SEQ LD No. 112) 7C10Lresh.24anti 5' -TAAACCCCAACATCCTCAGCCTCCACTCTGCT (SEQ ID No. 113) 7C10Lresh.25anti 5'-TCCACGGAACATGTGAACCTTGAAAGCAGTAA (SEQ ID No. 114) 7Cl0Lresh.26anti 5' -TTTGATTTCCACCTTGGTCCCTTGGCCGAAC (SEQ ID No. 115) 7Cl0Lresh. BamHIantisense 5' -CGCAGAGGATCCACTCACG (SEQ D No. 116) TABLE 8: DNA sequence of oligonucleotides used for the construction of humanized 7C10 VH 1 by de novo assembly LeaderMl uI.biotin 5'-GTCAGAACGCGTGCCGCC (SEQ ID No. 117) 7C10Hresh. Sense 5' -ACCATGAAAGTGTTGAGTCTGTTGTACCTCTTGA (SEQ D No. 118) 7Cl0Hresh.2sense 5' -CAGCCATTCCTGGTATCCTGTCTCAGGTGCAGCT (SEQ ID No. 119) 7C10Hresh.3sense 5'-TCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCG (SEQ ID No. 120) 7Cl0Hresh. 4sense 5' -GAGACCCTGTCCCTCACCTGCACTGTCTCTGGT (SEQ ID No. 121) - 91 7C10Hresh.5sense 5'-TACTCCATCACCGGTGGTTATTTATGGAACTGG (SEQIDNo.122) 7ClOHresh.6sense 5' -ATACGGCAGCCCCCAGGGAAGGGACTGGAGTGG (SEQ ID No.123) 7Cl0Hresh. 7sense 5' -ATGGGGTATATCAGCTACGACGGTACCAATAAC (SEQ ID No.124) 7C10Hresh.8antisense 5' -TCAACACTTTCATGGTGGCGGCACGCGTTCTGAC (SEQ ID No. 125) 7Cl0Hresh. 9antisense. 5'-ATACCAGGAATGGCTGTCAAGAGGTACAACAGAC (SEQ ID No. 126) 7Cl0Hresh.10antisense 5' -TGGGCCCGACTCCTGAAGCTGCACCTGAGACAGG (SEQ ID No. 127) 7C10Hresh. llantisense 5'-TGAGGGACAGGGTCTCCGAAGGCTTCACCAGTCC (SEQ DD No. 128) 7Cl0Hresh.12antisense 5'-CCACCGGTGATGGAGTAACCAGAGACAGTGCAGG (SEQ ID No. 129) 7C10Hresh.13antisense 5' -CCCTGGGGGCTGCCGTATCCAGTTCCATAAATAA (SEQ D No. 130) 7Cl0Hresh.14antisense 5'-TAGCTGATATACCCCATCCACTCCAGTCCCTT (SEQ ID No. 131) 7C1OHresh.KpnIREV 5'-GTTATTGGTACCGTCG (SEQ ID No. 132) 7Cl0Hresh.KpnIbiotin 5' -TACGACGGTACCAATAACTAC (SEQ D) No. 133) 7Cl0Hresh.15sense 5' -AAACCCTCCCTCAAGGATCGAATCACCATATC (SEQ ID No. 134) 7C10Hresh.16sense 5'-ACGTGACACGT'CCAAGAACCAGTTC'CCCTGA (SEQ IDNo. 135) 7Cl0Hresh.17sense 5' -AGCTGAGCTCTGTGACCGCTGCGGACACTGCA (SEQ ID No. 136) 7C10Hresh.18sense 5'-GTGTATTACTGTGCGAGATACGGTAG(;GGTCTT (SEQ DNo. 137) 7C0 fresh. 19sense 5'-CTTTGACTACTGGGGCCAGGGAACCCTGGTCA (SEQ ID No. 138) 7C10Rresh.2Osense 5'-CCGTCTCCTCAGGTGAGTGGATCCTCTGCG (SEQ IDNo. 139) 7C10Hresh.2lantisense 5' -AGGGAGGGTTTGTAGTTATTGGTACCGTCGTA (SEQ ID No. 140) 7C10Hresh.22antisense 5' -ACGTGTCACGTGATATGGTGATTCGATCCTTG (SEQ ID No. 141) 7C10Hresh.23antisense 5'-AGAGCTCAGCTTCAGGGAGAACTGGTTCTTGG (SEQ ID No. 142) 7CO10Hresh.24antisense 5'-CAGTAATACACTGCAGTGTCCGCAGCGTCAC (SEQIDNo. 143) 7C10Hresh.25antisense 5'-AGTAGTCAAAGAAGACCCTACCGTATCTCGCA (SEQ IDNo. 144) 7Cl0Hresh.26antisense 5'-CTGAGGAGACGGTGACCAGGGTTCCCTGGCCCC (SEQ D)No. 145) 7C10Hresh.BamHIantisense 5'-CGCAGAGGATCCACTCAC (SEQ [DNo. 146) TABLE 9: Oligonucleotide - pairing protocol for the de novo assembly of genes coding for the humanized forms of 7C10 VH and VL 5 de novo assembly de novo assembly of the MlUI-KpnI fragment of the KpnT-BamHI fragment of 7C10 VL humanized 1 of 7C10 VL humanized 1 10 Biotinylated oligo leader Biotinylated oligo 7C10 L MIUI 7C10 VL KpnI Oligo pair 1 and 7 Oligo pair 13 and 20 Oligo pair 2 and 8 Oligo pair 14 and 21 15 Oligo pair 3 and 9 Oligo pair:c 15 and 22 - 92 Oligo pair 4 and 10 Oligo pair 16 and 23 Oligo pair 5 and 11 Oligo pair 17 and 24 Oligo pair 6 and 12 Oligo pair 18 and 25 Antisense oligo 7C10 Oligo pair 19 and 26 5 VL KpnI Antisense oligo 7C10 L BamHI de novo assembly de novo assembly of the MlUI-KpnI fragment of the KpnI-BamHI fragment 10 of 7C10 VL humanized 1 of 7C10 VL humanized 1 Biotinylated oligo leader Biotinylated oligo 7C10 H MlUI 7C10 VH KpnI Oligo pair 1. and 8 Oligo pair 15 and 21 15 Oligo pair 2 and 9 Oligo pair 16 and 22 Oligo pair 3 and 10 Oligo pair 17 and 23 Oligo pair 4 and 11 Oligo pair 18 and 24 Oligo pair 5 and 12 Oligo pair 19 and 25 Oligo pair 6 and 13 Oligo pair 20 and 26 20 Oligo pair 7 and 14 Antisense oligo 7C10 VH Antisense oligo 7C10 BamHI VH KpnI Example 15. Construction of the genes coding for the 25 humanized versions 2 of 7C10 VL and 7C10 VH and 3 of 7C10 VH by directed mutagenesis The humanized version 2 of 7C10 VH was obtained by 30 directed mutagenesis of the residues 48 and 67 (according to Kabat's nomenclature) of version 1. This directed mutagenesis was carried out with the aid of the system QuikChange TM Site-directed mutagenesis of Stratagene (kit #200518) according to the protocol 35 described by the manufacturer. The construction is carried out in two stages, first the residue 48 on version 1 was mutated with the aid of the pair of primers 7C10HhumanizedlQCM48 sense and antisense (see Table 10) and subsequently this version mutated at the -93 residue 48 was itself mutated at the residue 67 with the aid of the pair of primers 7C10HhumanizedlQCI67 sense and antisense (see Table 10). 5 The humanized version 3 of 7C10 VH was obtained by site-directed mutation of the residues 30 and 71 (according to Kabat's nomenclature) of version 2 likewise using the system QuikChange T M . This construction is carried out in two stages. At first, 10 the residue 30 on version 2 was mutated with the aid of the primers 7ClOHhumanizedQCT30 sense and antisense (see Table 10). Subsequently, this version mutated at the residue 30 was itself mutated at the residue 71 by using the pair of primers 7C10HhumanizedlV67QCR71 sense 15 and antisense (see Table 10) The humanized version 2 of 7C10 VL was obtained by site-directed mutation of the residue 2 (according to Kabat's nomenclature) of version 1 by using the system 20 QuikChange T M . The residue 2 on version 1 was mutated by using the pair of primers 7ClOLhumanizedlQCV2 sense and antisense (see Table 10). TABLE 10: List of the oligonucleotides used for the 25 directed mutagenesis by the stratagene QuikChanges system 7C10HhumanizedlQCT30. 5'-CTGGTTACTCCATCAGCGGTGGTTATTTATG (SEQ ID No. 147) sense 7Cl0HhumanizedlQCT30. 5'-CATAAATAACCACCGCTGATGGAGTAACCAG (SEQ ID No. 148) antisense 7C10HhumanizedlQCM48. 5'-GGGACTGGAGTGGATCGGGTATATCAGCTAC (SEQ ID No. 149) sense 7C10HhumanizedlQCM48. 5'-GTAGCTGATATACCCGATCCACTCCAGTCCC (SEQIDNo. 150) antisense 7CO10HhumanizedlQCI6 7 . 5'-TCCCTCAAGGATCGAGTCACCATATCACGTG (SEQ U) No. 151) sense 7C10HhumanizedlQCI67. 5'-CACGTGATATGGTGACTCGATCCTTGAGGGA (SEQ ID No. 152) antisense 7C0HhumanizedlV67QCR 7 1. -GATCGAGTCACCATATCAGTGGACACGTCCAAGAA (SEQ ID No. 153) sense CCAG 5' 7C10HhumanizedlV67QCR71. -CTGGTTCTTGGACGTGTCCACTGATATGGTGACTC (SEQ ID No. I54) antisense GATC 7C10LhumanizedlQCV2. 5'-GCTTCCAGCAGTGATATTGTGATGACTCAGT (SEQ ID No. 155) sense 7C1OLhumanizedlQCV2. I5'-ACTGAGTCATCACAATATCACTGCTGGAAGC (SEQ ID No. 156) antisense -94 Example 16. Transfection of the cos7 cells by electroporation The mammalian expression vectors containing the 5 chimeric or humanized versions of the heavy and light chains of the antibody 7C10 were tested in cos7 cells for the transitory expression of the recombinant antibodies 7C10. The DNA was introduced into the cos cells by electroporation with the aid of a BioRad 10 instrument (Gene Pulsar). The DNA (10 pg of each vector) is added to aliquots of 0.8 ml of cos cells at a concentration of 1 x 107 cells per ml in PBS buffer (without Ca++ and Mg++). A pulsation of 1900 volts and a capacity of 25 pF was delivered. The transfected cos 15 cells are then added to 8 ml of DMEM medium containing 5% of calf serum and incubated at 370C for 72 hours. The supernatant is then collected, centrifuged in order to eliminate the cell debris and tested by ELISA for the measurement of its concentration of recombinant 20 antibody 7C10 of IgGl/human Kappa type. Example 17. ELISA method for measuring the concentrations of recombinant antibody IgGl/human Kappa present in the 25 supernatant of the cos transfectants The supernatants produced by transitory expression in cos7 cells were tested for the presence of 7C10 antibody of IgGl/human Kappa type. For the detection of 30 the IgG1/human Kappa immunoglobulin, 96-well ELISA plates (Maxisorb, Nunc) were coated with a goat anti human IgG polyclonal antibody (specific for the gamma Fc fragment, Jackson Immuno-Research Laboratories Inc., #109-005-098). The supernatants of cos cells were 35 diluted in series and added to the coated wells. After incubation for one hour at 370C and washing, a goat anti-human light Kappa chain polyclonal antibody conjugated to peroxidase (HRP, Sigma, A-7164) was added. After incubation for 45 minutes at 370C and -95 washing, the TMB substrate (KPL #50--76-04) was added. After incubation for 10 minutes, the reaction was stopped by the addition of 1 M sulfuric acid and the optical density was read at 450 nm. A purified human 5 IgGl/human Kappa immunoglobulin (Sigma, 1-3889) of known concentration was used as a standard reference antibody. Example 18. ELISA method for determining the 10 recognition activity of 7C10 recombinant antibodies of human IgGl/Kappa type on the receptor for IGF-1 (IGF-IR) The cos7 culture supernatants were tested for their 15 capacity to recognize IGF-l R by an ELISA method. 96 well ELISA plates (Dynex Immulon 2HB) were coated with 100 pl per well of a solution of PBS containing 0.31 ng/pl of IGF-l R (Human Insulin-Like Growth Factor I soluble Receptor, R & D Systems, #391-GR) by 20 incubation for one night at 40C. After washing with PBS containing 0.05% Tween 20, the plates were saturated by the addition of a solution of PBS containing 0.5% gelatin solution and incubation at 37 0 C for 1 hour. After three washes with PBS, the samples of cos 25 supernatants to be tested, previously diluted in series in PBS containing 0.1% gelatin and 0.05% Tween 20, were added to the plates. After incubation at 370C for 1 hour followed by three washes (PBS containing 0.05% Tween 20), an anti-human IgG antibody (specific for the 30 Fc fragment) conjugated to peroxidase (HRP, Jackson Immuno-Research Laboratories Inc., #109-035-098) was added (dilution to 1/5000 in PBS containing 0.1% gelatin and 0.05% Tween 20). After incubation for 45 minutes at 370C and 3 washes (PBS containing 0.05% 35 Tween 20), the TMB substrate (KPL #50-76-04) was added. After incubation for 10 minutes, the reaction was stopped by addition of 1 M sulfuric acid and the optical density was read at 450 nm.

-96 Example 19. Determination of the recognition activity of IGF1-R by different versions of the humanized 7C10 antibody by "CDR grafting" 5 At first, we compared the recognition activity of humanized forms 1 of the heavy and light chains of 7C10 for the IGF-1 receptor with respect to the chimeric form. Figure 28 shows the results of an ELISA test of 10 recognition of the IGF-1R (see Example 18) from supernatants of the cos7 cells whose concentration of IgGl/human Kappa had been previously determined by ELISA (see Example 17). The titration curves of the four recombinant antibodies tested overlap perfectly 15 indicating that their relative affinities for IGF-IR are very similar. It is therefore concluded from this that the humanized form 1 of 7C10, composed of the humanized light chain 1 (1 single mouse residue present in the framework regions) in combination with the 20 humanized heavy chain 1 (4 mouse residues present in the framework regions), specifically recognizes the IGF-1 receptor and has an affinity very similar to that of the chimeric antibody (mouse variable regions). 25 Subsequently, we looked at the influence of the residue 2 (according to Kabat's nomenclature) of the humanized light chain of 7C10 (humanized version 1 versus humanized 2, see figure 19) on the recognition of the IGF-IR. Figure 29 shows the results of the ELISA test 30 for recognition of the IGF-IR (see Example 18) from supernatants of cos7 cells whose concentration of IgGl/human Kappa had been previously determined by ELISA (see Example 17). The two humanized versions 1 and 2 of the light.chain had been combined successively 35 with humanized 7C10 VH 1. The titration curves of the two combinations are superimposed indicating that the mutation of residue 2 of the light chain, which has been changed from one valine in the humanized version 1 to an isoleucine in the humanized form 2, apparently -97 has no influence on the relative affinity of recognition of the IGF1 receptor. The humanized form 2 of the light chain of 7C10 thus forms one version where no mouse residue (apart from CDRs) has been conserved. 5 This version, totally humanized, represents the preferred version of 7C10 VL. The totally humanized version of the 7C10 light chain (humanized version 2, see above) was tested in 10 combination with the three humanized versions of the heavy chain of 7C10. Figure 30 shows the results of the ELISA test for recognition of the IGF-1R from supernatants of cos7 cells whose concentration of IgGl/human Kappa had been previously determined by 15 ELISA (see Example 17). The titration curves are very similar and virtually overlap with the reference curve corresponding to the chimeric antibody, indicating that the three humanized versions 1, 2 and 3 of 7C10 VH give an identical relative affinity for IGF-1R when they are 20 combined with humanized 7C10 VL 2. Other ELISA tests conducted in parallel (results not shown) have however revealed that a point mutation of the residue 71 (Kabat's nomenclature) from an arginine (mouse) to a valine (human) involved a small loss of affinity of the 25 corresponding antibody for IGF-1R. It is thus reasonable to think that humanized 7C10 VH 2 has the same relative affinity for IGF-1R as humanized 7C10 VH 1. This humanized form 2 will therefore be preferred with respect to the form 1 since it only has two mouse 30 amino acids (residues 30 and 71, see figure 24). The humanized form 3 which does not have any mouse residue (apart from CDRs) will also be preferred since it only seems to involve a minimal loss of affinity. 35 In conclusion, it appears that two humanized forms of the antibody 7C10 according to the present invention are particularly preferred. A form constituted by the combination of humanized 7C10 VH 2 (2 conserved mouse residues) with humanized 7C10 VL 2 (no conserved mouse - 98 residue) and another form constituted by the combination of humanized 7C10 VH 3 (rno conserved mouse residue) with humanized 7C10 VL. 2 (no conserved mouse residue). This last form constitutes the ultimate 5 humanized version since no mouse residue is present at the same time in the heavy and light chains. Example 20. Expression of EGFR and of IGF-IR on the surface of A549 cells 10 The synergy of action obtained by the coadministration of two MABs directed respectively against IGF-IR and EGFR was studied in nude mice carrying a non-small cell lung tumor established by subcutaneous injection (s.c.) 15 of A549 cells (lung carcinoma cell line). At first, and in order to ensure the presence of the two receptors IGF-IR and EGFR on the surface of the A549 cell before injecting this into the mouse, 20 labeling for FACS reading of these cells was carried out with, respectively, the murine 7C10 anti-IGF-IR MAB (figure 37B) and the murine 225 anti-EGFR MAB (figure 37D). In order to do this, the cells were saturated for 30 min at 40C with a solution of PBS 10% FCS (fetal 25 calf serum), washed and then incubated for 30 min at 40C with the MAB of interest. After 3 new washes, the secondary anti-species antibody coupled to FITC (fluorescein isothiocyanate) is added. After incubation for 30 min, reading on the FACS (Fluorescence Activated 30 Cells Sorter) is carried out at 520 nm (excitation 488 nm). The results presented in figures 37A to 37D show that the A549 cells have on their surface a comparable 35 number of receptors for EGF and IGF1. In the two cases, the population is homogeneous with respect to the distribution of each of the receptors. The specificity of the labeling is confirmed by the use of an isotype control (figure 37C). These results validate the use of -99 the A549 cell as a model for the study of a synergy of action on two IGF-IR and EGFR receptors and for the study of a collaboration of these two receptors. 5 Example 21. Synergy of action of an anti-IGF-IR MAB and of an anti-EGFR MAB coadministered in vivo, in the nude mouse in the context of an antitumor treatment. 10 For this study, nude mice are grafted s.c. with 5.106 A549 cells. Five days after the cell graft, the tumors are measured and a homogeneous batch of mice in terms of tumor volume is formed. Starting from this batch, groups of '6 mice are generated at random. These mice 15 will be treated intraperitoneally (i.p.), twice per week with each of the MAB 7C10 and 225 individually at the dose of 250 pg/mouse or with the two MAB in coadministration. The MAB 9G4 is administered as an experiment isotype control. 20 The results presented in figure 38 show that each of the antibodies 7C10 and 225 administered alone is capable of inducing a significant decrease in the tumor growth in vivo. It can be noted that the two MAB tested 25 have a comparable activity on the growth of the tumor A549. In a surprising fashion with respect to the literature, a significant synergy is observed during simultaneous administration of the two MAB (p < or = 0.01 at each of the times of the kinetics in a t-test) 30 suggesting that a collaboration of the two receptors exists for the optimum growth of a tumor in vivo and that, contrary to the data in the literature, the blockage of one of the two axes does not suffice to totally inhibit the growth mediated by the second. 35 Example 22. Study of the antitumor activity of the murine antibodies 7C10 and 225 coadministered in mice orthotopically implanted with A549 cells.

- 100 The use of orthotopic models for the evaluation of the antitumor activity presents a particular interest with respect to the process of metastatic dissemination of a 5 tumor. In order to evaluate the antitumor activity of an antibody mixture directed respectively against

IGF

IR and EGFR, 106 A549 cells (non-small cell lung cancer) were implanted in the intrapleural cavity of nude mice. It is to be noted that the consequence of this type of 10 tumor implantation is a metastatic dissemination similar to that observed in man and leads to the death of the animals. Figure 39 shows that the administration of the antibodies 225 and 7C10 alone allows a comparable and a significant gain in survival to be 15 observed. In a surprising fashion, the coadministration of these two antibodies increases in a considerable fashion the survival of the animals suggesting that this treatment could have an impact on the metastatic dissemination of the tumor cells. 20 Example 23. 7C10 and 7H2HM inhibit the phosphorylation of the tyrosine of the chain of IGF-IR and of IRS-I 25 MCF7 cells are cultured for 24 hours at 5.104 cells/cm 2 (75 cm 2 plates, COSTAR) in 20 ml of RPMI without phenol red, mixed with 5 mM of glutamine, penicillin/ streptomycin (respectively 100 U/100 pg/ml) and 10% of fetal calf serum. After three washes in PBS, the cells 30 were incubated for 12 hours in medium (RPMI) without phenol red, devoid of fetal calf serum and mixed with 5 mM of glutamine, penicillin/streptomycin, bovine serum albumin at 0.5 pg/ml (Sigma A-8022) and transferrin at 5 pg/ml (Sigma T8158). 35 For activation, the cells were first incubated. at 37 0 C for 2 minutes with blocking antibodies (10 pg/ml) and then IGF-I (Sigma 13769, 50 ng/ml) was added for two additional minutes. The reaction was stopped by - 101 aspiration of the incubation medium and the plates were laid on ice. The cells were solubilized by addition of 0.5 ml of lysis buffer (50 mM tris-HC1l pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate), mixed 5 with protease inhibitors (1 tablet per 50 ml, Boehringer Ref.: 1697 498), and phosphatase inhibitors (Calbiochem Ref.: 524625 (1/100)). The cells were scraped off and the suspension was recovered and placed on a shaker at 40C for 1.5 hours. The solutions were 10 centrifuged at 12,000 rpm for ten minutes (4oC) and the protein concentrations of the supernatants were quantified by BCA. 500 pg of proteins of the cell lysate were mixed with 15 the anti-IGF-IR . (Santa cruz Ref.: sc-713) for immunoprecipitation and incubated on the shaker at 40C for 1.5 hours. The immunoprecipitates were recovered by addition of protein A-agarose (Boehringer Ref.: 1 134 515) and incubated all night on the shaker at 40C. For 20 the immunoprecipitation of IRS-1, anti-IRS-1 antibodies coupled to agarose beads (Santa cruz Ref.: 559Ac) were used. The agarose beads were washed twice with 1 ml of lysis buffer, twice with a wash buffer 1 (50 mM tris HCl pH 7.5; 500 mM NaCl; 0.1% Nonidet P40; 0.05% sodium 25 deoxycholate (Boehringer 1 332 597), mixed with protease inhibitors and phosphatase inhibitors) and once with a wash buffer 2 (50 mM tris-HCl; 0.1% Nonidet P40; 0.05% sodium deoxycholate (Boehringer Ref.: 1 332 597), mixed with protease inhibitors and phosphatase 30 inhibitors 1/100). The immunoprecipitates were resuspended in a Laemmli buffer, heated to 1000C for 5 minutes.. The supernatants were analyzed by electrophoresis on polyacrylamide SDS gel (8% Novex EC6015). The proteins were transferred to a 35 nitrocellulose membrane followed by either an immunoblot with anti-phosphotyrosine antibodies conjugated to HRP (upstate Biotechnology 4G10) or beta anti-chain of IGF-IR or anti-IRS-i (Santa Cruz Ref.: sc 8038) followed by an anti-rabbit antibody conjugated to - 102 HRP. The imprints were revealed by chemiluminescence (Amersham RPN 2209) followed by autoradiography on Kodak X-mat AR films. 5 Figure 40A represents MCF7 cells nonstimulated (0) or stimulated either with IGF-1 (50 ng/ml) alone (0+IGF-1) or combined with monoclonal or humanized anti-IGF-IR antibodies (10 pg/ml) 7C10, 1H7, 7H2HM. The antibodies 9G4 or hIgGl are murine or human immunoglobulins of 10 isotype IgGl used as an experiment negative control. The beta chains of the IGF-IR were immunoprecipitated and blotted with phosphorylated anti-tyrosine antibodies. The results obtained show that the monoclonal or humanized anti-IGF-IR 7C10, 1H7 and 7H2HM 15 antibodies inhibit the phosphorylation of the tyrosine of the beta chain of the IGF-IR. Figure 40B represents MCF7 cells nonstimulated (0) or stimulated either with IGF-1 (50 ng/ml) alone (0+IGF-1) 20 or combined with monoclonal or humanized anti-IGF-IR antibodies (10 pg/ml) 7C10, 1H7, 7H2HM. As described above, the antibodies 9G4 or hIgGl are murine or human immunoglobulins of isotype IgGl used as an experiment negative control. The IRS-1 was immunoprecipitated and 25 blotted with phosphorylated anti-tyrosine antibodies. The. results obtained show that the monoclonal antibodies 7C10, 7H2HM and 1H7 inhibit the phosphorylation of the tyrosine of the IRS-1. 30 Example 24. 7C10 and 7H2HM induces the internalization of the IGF-IR MCF7 and A549 cells were suspended to 1.107 cells/ml in PBS with 10% of fetal calf serum (FACS buffer). 1.106 35 cells were incubated for 30 minutes at 37'C with the monoclonal antibodies at 10 pg/ml (7C10, 7G3, 9G4) or at 20 pg/ml for 7H2HM. After washing, the cells were labeled at 40C for 30 minutes with a biotinylated anti IGF-IR (monoclonal antibody 12B1) and finally incubated - 103 at 4 0 C for 30 minutes with a conjugate of streptavidin 488 alexa Fluor®. The cells were analyzed by FACScan (Becton-Dickinson,. Enembogegem, Belgium) with the Cellquest software after elimination of debris. 5 Figure 41 shows the A549 cells without coloration ( 1 st peak), the A549 cells incubated with 7C10 or 7H2HM ( 2 nd peak) and the A549 cells incubated with an irrelevant mouse or rat IgGl ( 3 rd peak). A decrease by two of the 10 surface expression of the IGF-IR by the cells is seen when the cells have been previously incubated with 7C10 or 7H2HM. Example 25. 7C10 and 7H2HM induce the degradation of 15 the IGF-IR MCF-7 cells were cultured for 24 hours at 10.104 cells/cm 2 (75 cm 2 , Costar) in 15 ml of complete medium. Next, the cultures were washed three times with PBS and 20 incubated for 12 hours with medium devoid of serum. Next, the cells were incubated with cycloheximide at 25 pg/ml alone or with 10 pg/ml of monoclonal antibody 7C10, 9G4, 7G3 or of IGF-I (50 ng/ml). In certain experiments, before incubation with the monoclonal 25 antibodies, the cells were treated for 1 hour at 370C with MG-132 (10 pM, Calbiochem 474791) in order to inhibit the proteasome activities. After incubation, the cells were washed and solubilized by addition of a lysis buffer. 20 pg of proteins were analyzed by 30 electrophoresis on polyacrylamide gel at 8% of SDS and transferred to a nitrocellulose membrane followed by a beta anti-chain immunoblot of the IGF-IR such as described further above. 35 The analysis by Western-blot (figure 42A) of the integrity of the IGF-IR shows that 7C10 and 7H2HM induce the degradation of the receptor while the natural ligand does not cause any degradation of the latter. No degradation of the receptor is observed with -104 the 9G4, an irrelevant antibody used as an isotype control. Figure 42B demonstrates, and with respect thereto, that the degradation is inhibited by a proteasome inhibitor MG132 (incubation period of 2 5 hours). Comparable results were obtained with the humanized antibody 7H2HM (figure 42C).

Claims (54)

1. An isolated antibody, or one of its functional 5 fragments, said antibody or one of its said fragments being capable of binding to the human insulin-like growth factor I receptor IGF-IR and, if necessary, inhibiting the natural attachment of its ligands IGF1 and/or IGF2 and/or capable of specifically inhibiting 10 the tyrosine kinase activity of said IGF-IR receptor, characterized in that it comprises a light chain comprising at least one complementarity determining region CDR chosen from the CDRs of sequence SEQ ID No. 2, 4 or 6, or at least one CDR whose sequence has at 15 least 80% identity after optimum alignment with the sequence SEQ ID No. 2, 4 or 6, or in that it comprises a heavy chain comprising at least one CDR chosen from the CDRs of sequence SEQ ID Nos. 8, 10 and 12, or at least one CDR whose sequence has at least 80% identity 20 after optimum alignment with the sequence SEQ ID No. 8, 10 and 12.

2. The antibody, or one of its functional fragments, as claimed in claim 1, characterized in that it 25 comprises a heavy chain comprising at least one CDR of sequence SEQ ID No. 12 or a sequence having at least 80% identity after optimum alignment with the sequence SEQ ID No. 12. 30

3. The antibody, or one of its functional fragments, as claimed in claim 1 or 2, characterized in that it comprises a heavy chain comprising at least two of the three CDRs or the three CDRs of sequence SEQ ID Nos. 8, 10 and 12, or at least two of three CDRs or three CDRs 35 of sequence respectively having at least 80% identity after optimum alignment with the sequence SEQ ID No. 8, 10 and 12. -106

4. The antibody, or one of its functional fragments, as claimed in one of claims 1 to 3, characterized in that it comprises a light chain comprising at least one CDR chosen from the CDRs of sequence SEQ ID No. 2, 4 or 5 6, or a CDR whose sequence has at least 80% identity after optimum alignment with the sequence SEQ ID No. 2, 4 or 6.

5. The antibody, or one of its functional fragments, 10 as claimed in one of claims 1 to 4, characterized in that it comprises a light chain comprising at least two of the three CDRs or the three CDRs of sequence SEQ ID Nos. 2, 4 and 6, or at least two of three CDRs or three CDRs of sequence respectively having at least 80% 15 identity after optimum alignment with the sequence SEQ ID No. 2, 4 and 6.

6. The antibody, or one of its functional fragments, as claimed in one of claims 1 to 5, characterized in 20 that it does not attach in a significant manner to the human insulin receptor IR.

7. The antibody as claimed in one of claims 1 to 6, characterized in that said functional fragment is 25 chosen from the fragments Fv, Fab, F(ab') 2 , Fab', scFv, scFv-Fc and the diabodies, or any fragment whose half life would have been increased such as pegylated fragments. 30

8. A murine hybridoma capable of secreting an antibody as claimed in one of claims 1 to 6.

9. The murine hybridoma as claimed in claim 8 deposited at the CNCM, Institut Pasteur, Paris, on 35 September 19, 2001 under the number 1-2717.

10. An antibody, or one of its functional fragments, characterized in that said antibody is secreted by the hybridoma as claimed in claim 9. - 107

11. The antibody, or one of its functional fragments, as claimed in one of claims 1 to 7, characterized in that said antibody comprises a light chain of sequence 5 comprising the amino acid sequence SEQ ID No. 54, or a sequence having at least 80% identity after optimum alignment with the sequence SEQ ID No. 54, or/and in that it comprises a heavy chain of sequence comprising the amino acid sequence SEQ ID No. 59, or a sequence 10 having at least 80% identity after optimum alignment with the sequence SEQ ID No. 69.

12. The antibody or one of its functional fragments, as claimed in claim 11, characterized in that said 15 antibody is a chimeric antibody and moreover comprises the light chain and heavy chain constant regions derived from an antibody of a species heterologous to the mouse. 20

13. The chimeric antibody, or one of its functional fragments, as claimed in claim 12, characterized in that said heterologous species is man.

14. The chimeric antibody, or one of its functional 25 fragments, as claimed in claim 13, characterized in that the light chain and heavy chain constant regions derived from a human antibody are respectively the kappa and gamma-1, gamma-2 or gamma-4 region. 30

15. The antibody or one of its functional fragments, as claimed in one of claims 1 to 7, characterized in that said antibody is a humanized antibody and comprises a light chain and/or a heavy chain in which the skeleton segments FR1 to FR4 of said light chain 35 and/or heavy chain are respectively derived from skeleton segments FR1 to FR4 of human antibody light chain and/or heavy chain.

16. The humanized antibody, or one of its functional - 108 fragments, as claimed in claim 15, characterized in that said antibody comprises a light chain comprising the amino acid sequence SEQ ID No. 61 or 65, or a sequence having at least 80% identity after optimum 5 alignment with the sequence SEQ ID No. 61 or 65, or/and in that it comprises a heavy chain comprising the amino acid sequence SEQ ID No. 75, 79 or 83, or a sequence having at least 80% identity after optimum alignment with the sequence SEQ ID No. 75, 79 or 83. 10

17. The humanized antibody, or one of its functional fragments, as claimed in claim 15 or 16, characterized in that said antibody comprises a light chain comprising the amino acid sequence SEQ ID No. 65, and 15 in that it comprises a heavy chain of sequence comprising the amino acid sequence SEQ ID No. 79 or 83, preferably SEQ ID No. 83.

18. An isolated nucleic acid, characterized in that it 20 is chosen from the following nucleic acids: a) a nucleic acid, DNA or RNA, coding for an antibody, or one of its functional fragments, as claimed in one of claims 1 to 7 and 10 to 17; b) a complementary nucleic acid of a nucleic acid 25 such as defined in a); and c) a nucleic acid of at least 18 nucleotides capable of hybridizing under conditions of great stringency with at least one of the CDRs of sequence SEQ ID No. 1, 3, 5, 7, 9 or 11, or with a sequence 30 having at least 80% identity after optimum alignment with the sequence SEQ ID No. 1, 3, 5, 7, 9 or 11.

19. A vector comprising a nucleic acid as claimed in claim 18. 35

20. A host cell comprising a vector as claimed in claim 19.

21. A transgenic animal with the exception of man - 109 comprising at least one cell transformed by a vector as claimed in claim 19.

22. A process for production of an antibody, or one of 5 its functional fragments, as claimed in one of claims 1 to 7 and 10 to 17, characterized in that it comprises the following stages: a) culture in a medium and appropriate culture conditions of a cell as claimed in claim 20; and 10 b) the recovery of said antibodies, or one of their functional fragments, thus produced starting from the culture medium or said cultured cells.

23. An antibody, or one of its functional fragments, 15 capable of being obtained by a process as claimed in claim 22.

24. The antibody, or one of its functional fragments, as claimed in any one of claims 1 to 7, 10 to 17 and 20 23, characterized in that it is, moreover, capable of attaching specifically to the human epidermal growth factor receptor and/or capable of specifically inhibiting the tyrosine kinase activity of said EGFR receptor. 25

25. The antibody as claimed in claim 24, characterized in that it consists of a bispecific antibody and in that it comprises a second motif specifically inhibiting the attachment of the EGF to the human 30 epidermal growth factor receptor EGFR and/or specifically inhibiting the tyrosine kinase activity of said EGFR receptor.

26. The antibody as claimed in claim 25, characterized 35 in that it is bivalent or tetravalent.

27. The antibody as claimed in one of claims 25 or 26, characterized in that said second motif is selected from the fragments Fv, Fab, F(ab') 2 , Fab', Fab'PEG, -110 scFv, scFv-Fc and the diabodies, or any form whose half-life would have been increased.

28. The antibody as claimed in any one of claims 25 to 5 27, characterized in that said second anti-EGFR motif is descended from the mouse monoclonal antibody 225, its mouse-man chimeric derivative C225, or a humanized antibody derived from this antibody 225. 10

29. The antibody, or one of its functional fragments, as claimed in one of claims 1 to 7, 10 to 17 and 23 to 27 as a medicament.

30. A composition comprising by way of active 15 principle a compound consisting of an antibody, or one of its functional fragments, as claimed in one of claims 1 to 7, 10 to 17 and 23 to 29.

31. The composition as claimed in claim 30, 20 characterized in that it comprises a second compound chosen from the compounds capable of specifically inhibiting the attachment of the EGF to the human epidermal growth factor receptor EGFR and/or capable of specifically inhibiting the tyrosine kinase activity of 25 said EGFR receptor.

32. The composition as claimed in claim 31, characterized in that said second compound is chosen from the isolated anti-EGFR antibodies, or their 30 functional fragments, capable of inhibiting by competition the attachment of the EGF to the EGFR.

33. The composition as claimed in claim 32, characterized in that said anti-EGFR antibody is chosen 35 from the monoclonal, chimeric or humanized anti-EGFR antibodies, or their functional fragments.

34. The composition as claimed in either of claims 32 or 33, characterized in that said functional fragments - 111 of the anti-EGFR antibody are chosen from the fragments Fv, Fab, F(ab') 2 , Fab', scFv-Fc and the diabodies, or any fragment whose half-life would have been increased, like pegylated fragments. 5

35. The composition as claimed in one of claims 32 to 34, characterized in that said anti-EGFR antibody is the mouse monoclonal antibody 225, its mouse-man chimeric derivative C225, or a humanized antibody 10 derived from this antibody 225.

36. The composition as claimed in any one of claims 30 to 35, characterized in that it comprises, moreover, as a combination product for simultaneous, separate or 15 sequential use, a cytotoxic/cytostatic agent and/or an inhibitor of the tyrosine kinase activity respectively of the receptors for IGF-I and/or for EGF.

37. The composition as claimed in claim 36, 20 characterized in that said cytotoxic/cytostatic agent is chosen from the agents interacting with DNA, the antimetabolites, the topoisomerase I or II inhibitors, or the spindle inhibitor or stabilizer agents or else any agent capable of being used in chemotherapy. 25

38. The composition as claimed in claim 36 or 37, characterized in that said cytotoxic/cytostatic agent is coupled chemically to at least one of the elements of said composition for simultaneous use. 30

39. The composition as claimed in claim 37 or 38, characterized in that said cytotoxic/cytostatic agent is chosen from the spindle inhibitor or stabilizer agents, preferably vinorelbine, vinflunine or 35 vincristine.

40. The composition as claimed in one of claims 36 to 39, characterized in that said inhibitor of the tyrosine kinase activity respectively of the receptors - 112 for IGF-I and/or for EGF is selected from the group consisting of derived natural agents, dianilino phthalimides, pyrazolo- or pyrrolopyridopyrimidines. or else quinazilines. 5

41. The composition as claimed in any one of claims 30 to 40, characterized in that it comprises, moreover, another antibody compound directed against the extracellular domain of the HER2/neu receptor, as a 10 combination product for simultaneous, separate or sequential use intended for the prevention and for the treatment of cancer.

42. The composition as claimed in claim 41, 15 characterized in that said antibody directed against the extramembrane domain of the HER2/neu receptor is Trastuzumab, or one of its functional fragments.

43. The composition as claimed in any one of claims 30 20 to 42, characterized in that one, at least, of said antibodies, or one of its functional fragments, is conjugated with a cell toxin and/or a radioelement.

44. The composition as claimed in one of claims 30 to 25 43 as a medicament.

45. The use of an antibody, or one of its functional fragments, as claimed in one of claims 1 to 7, 10 to 17 and 23 to 29 and/or of a composition as claimed in any 30 one of claims 30 to 44 for the preparation of a medicament intended for the prevention or for the treatment of an illness connected with an overexpression and/or an abnormal activation of the IGF-IR and/or EGFR receptor, and/or connected with a 35 hyperactivation of the transduction pathway of the signal mediated by the interaction of IGF1 or IGF2 with IGF-IR and/or of EGF with EGFR.

46. The use as claimed in claim 45, characterized in - 113 that the administration of said medicament does not induce or only slightly induces secondary effects connected with inhibition of the insulin receptor IR. 5

47. The use as claimed in claim 45 or 46 for the preparation of a medicament intended to inhibit the transformation of normal cells into cells with tumoral character, preferably IGF-dependent, especially IGF1 and/or IGF2-dependent and/or EGF-dependent and/or 10 HER2/neu-dependent cells.

48. The use as claimed in any one of claims of 45 to 47 for the preparation of a medicament intended to inhibit the growth and/or the proliferation of tumor 15 cells, preferably IGF-dependent, especially IGF1 and/or IGF2-dependent and/or EGF-dependent and/or HER2/neu-dependent cells.

49. The use as claimed in one of claims of 45 to 48 20 for the preparation of a medicament intended for the prevention or for the treatment of cancer.

50. The use as claimed in claim 49, characterized in that said cancer is a cancer chosen from prostate 25 cancer, osteosarcomas, lung cancer, breast cancer, endometrial cancer or colon cancer.

51. The use as claimed in one of claims of 45 to 48 for the preparation of a medicament intended for the 30 prevention or for the treatment of psoriasis.

52. A method of in vitro diagnosis of illnesses induced by an overexpression or an underexpression of the IGF-IR and/or EGFR receptor starting from a 35 biological sample in which the abnormal presence of IGF-IR and/or EGFR receptor is suspected, characterized in that said biological sample is contacted with an antibody as claimed in one of claims 1 to 7, 10 to 17 and 23 to 29, it being possible for said antibody to - 114 be, if necessary, labeled.

53. A kit or set for carrying out a method of diagnosis of illnesses induced by an overexpression or 5 an underexpression of the IGF-IR and/or EGFR receptor or for carrying out a process for the detection and/or the quantification of an overexpression or of an underexpression of the IGF-IR and/or EGFR receptor in a biological sample, preferably an overexpression of said 10 receptor, characterized in that said kit or set comprises the following elements: a) an antibody, or one of its functional fragments, as claimed in one of claims 1 to 7, 10 to 17 and 23 to 29; 15 b) optionally, the reagents for the formation of the medium favorable to the immunological reaction; c) optionally, the reagents allowing the demonstration of IGF-IR/antibody and/or EGFR/antibody complexes produced by the immunological reaction. 20

54. The use of an antibody, or one of its functional fragments, as claimed in one of claims 1 to 7, 10 to 17 and 23 to 29, for the preparation of a medicament intended for the specific targeting of a biologically 25 active compound to cells expressing or overexpressing the IGF-IR and/or EGFR receptor.