HK1116201B - Novel anti-igf-ir antibodies and uses thereof - Google Patents

Description

Novel anti-IGF-IR antibodies and uses thereof

The present invention relates to novel antibodies capable of specifically binding to human insulin-like growth factor I receptor (IGF-IR) and/or capable of specifically inhibiting the tyrosine kinase activity of said IGF-IR, in particular monoclonal antibodies of murine, chimeric and humanized origin, as well as the amino and nucleic acid sequences coding for these antibodies. In a particular aspect, the invention relates to novel antibodies that are not only effective in inhibiting the binding of the ligand IGF1 to IGF-IR, but are also effective in inhibiting the binding of other ligands (e.g., IGF2) to the same receptor. The invention also comprises the use of these antibodies as a medicament for the prophylactic and/or therapeutic treatment of cancers overexpressing IGF-IR, stimulated by IGF1 and/or IGF2, or any disease associated with overexpression of said receptor, as well as methods or kits for diagnosing diseases associated with overexpression of IGF-IR and/or IGF-I/insulin hybrid receptor. The invention finally also comprises products and/or compositions comprising these antibodies together with, for example, anti-EGFR antibodies and/or compounds and/or anti-cancer agents or agents conjugated with toxins, and their use for the prevention and/or treatment of certain cancers.

The insulin-like growth factor I receptor, known as IGF-IR, is a well-described receptor with tyrosine kinase activity 70% homologous to the insulin receptor IR. IGF-IR is a glycoprotein with a molecular weight of about 350,000. It is a heterotetrameric receptor in which each half, connected by disulfide bridges, consists of an extracellular α -subunit and a transmembrane β -subunit. IGF-IR binds IGF1 and IGF2 with very high affinity (Kd #1 nM), but is also capable of binding insulin with 100-fold lower affinity. In contrast, IR binds insulin with very high affinity, whereas IGF binds to the insulin receptor with only 100-fold lower affinity. Although the cysteine-rich region and the C-terminal portion of the β -subunit, located in the α -subunit, have relatively low homology segments, the tyrosine kinase domains of IGF-IR and IR have very high sequence homology. The sequence differences observed in the alpha-subunit are located in the ligand binding segment and thus are at the origin of the relative affinities of IGF-IR and IR for IGF and insulin, respectively. The difference in the C-terminal portion of the β -subunit results in differentiation of the two receptor signaling pathways; IGF-IR mediates mitogenic, differentiating and anti-apoptotic effects, while IR activation is primarily involved in metabolic pathway level effects (Baserga et al, Biochim. Biophys. acta, 1332: F105-126, 1997; Baserga R., exp. cell. Res., 253: 1-6, 1999).

Binding of the ligand to the extracellular domain of the receptor activates the cytoplasmic tyrosine kinase protein. Kinase activation, in turn, is involved in stimulating different intracellular substrates, including IRS-1, IRS-2, Shc, and Grb 10(Peruzzi F.et. al., J.cancer Res. Clin. Oncol., 125: 166-. The two major substrates of IGF-IR are IRs and Shc, which mediate most of the growth and differentiation effects associated with IGF attachment to this receptor by activating a number of downstream effectors. Thus substrate availability may control the final biological effects associated with IGF-IR activation. When IRS-1 predominates, the cells tend to proliferate and transform. When Shc predominates, cells tend to differentiate (ValentinisB. et al., J.biol. chem.274: 12423-12430, 1999). The major pathway that appears to be involved in protection against apoptosis is the phosphatidylinositol 3-kinase (PI 3 kinase) pathway (Prisco M.et al, Horm. Metab.Res., 31: 80-89, 1999; Peruzzi F.et al, J.cancer Res.Clin. Oncol., 125: 166-.

The role of the IGF system in carcinogenesis has been the subject of much research in the last decade. This interest is after the discovery of the following facts: in addition to mitogenic and anti-apoptotic properties, IGF-IR appears to be required for establishment and maintenance of the transformed phenotype. Indeed, it has been established that overexpression or constitutive activation of IGF-IR in a large number of different cells leads to support-independent growth of the cells in fetal bovine serum-free medium and to tumor formation in nude mice. This is not in itself a unique property, since the products of a large number of different overexpressed genes can transform cells, including a large number of growth factor receptors. However, it has been clearly demonstrated that R-cells in which the IGF-IR-encoding gene has been inactivated are completely resistant to different factors which normally transform the cells, such as the E5 protein of bovine papilloma virus, the overexpression of EGFR or PDGFR, the T antigen of SV40, activated ras or a combination of the last two (Sell C.et al, Proc.Natl.Acad.Sci., USA, 90: 11217. 11221, 1993; Sell C.et al., mol.biol., 14: 3604. 3612, 1994; Morrione A.J., Virol., 69: 5300. 5303, 1995; Coppo D.et al., mol.cell.biol., 14: 4588. 4595, 1994; DeAngelis T.T.J., J.221.P.221.214).

IGF-IR is expressed in a number of different tumors and tumor lines, and IGF promotes tumor growth by its attachment to IGF-IR. Other arguments for the role of IGF-IR in carcinogenesis have come from studies using murine monoclonal antibodies directed against the receptor or using dominant negative mutants of IGF-IR. Indeed, murine monoclonal antibodies against IGF-IR inhibit proliferation of large numbers of cultured cell lines and growth of tumor cells in vivo (Aretag C.et al, Cancer Res., 49: 6237-. In analogy to this, it has also been shown in the work of Jiang et al (Oncogene, 18: 6071-6077, 1999) that dominant negative mutants of IGF-IR are capable of inhibiting tumor proliferation.

These antibodies capable of specifically binding IGF-IR have been described and several patent applications have been filed. By way of example, we may mention the patent application WO03/059951, filed by the applicant, in which a monoclonal antibody capable of binding IGF-IR (referred to as 7C10) is described. Other patent applications which may be mentioned are, for example, WO 02/053596(PFIZER INC. and ABGENIX INC.), WO 03/100008 (SCRING CORPORATION) or WO 03/106621(IMMUNOGEN INC.). All of these applications claim antibodies capable of specifically binding to IGF-IR and/or inhibiting its activity.

Although it is considered to be an obvious feature of each of these antibodies, it is clearly shown in the description of these applications that these antibodies are able to effectively inhibit the binding of two natural ligands to IGF-IR, in particular IGF 2. In vitro data such as inhibition of proliferation induced by IGF1 and IGF2 are shown in these applications and indicate that the antibodies can effectively inhibit IGF1 and IGF2 induced proliferation. However, since the major part of the antibody showed the ability to induce partial down-regulation of IGF-IR, it was not obvious that the anti-proliferative properties were directly linked to the displacement of IGF1 and IGF2 from IGF-IR.

In fact, for example, the data shown in WO 03/106621 relate only to IGF1 and not to the final inhibitory effect of IGF2 binding to IGF-IR (see examples C and FIG. 3 on pages 33-35, and example D and FIGS. 4-6 on pages 35-37).

The same observations were made in WO 02/053596 (see pages 78-79, example IV and FIG. 3, and page 82, example VII and FIG. 4).

Of all the presently described patent applications for the discovery of monoclonal or recombinant antibodies against human IGF-IR (hIGF-IR), only two (WO 2004/087756 and WO2005/005635) indeed show the efficacy of the antibodies (AK 1a and AK18, respectively) in inhibiting the binding of [125I ] IGF2 to hIFG-IR.

The assay method was identical in both cases, based on competitive binding to human colorectal adenocarcinoma (HT29) intact cells. Although the method is rational, there is no competition for positive controls, such as native hoigf-IR ligands (IGF1 and IGF2), which leads to unreliable competition binding data. On the other hand, it is shown that only incomplete competition (for [ 2]¹²⁵I]Inhibition of IGF2 binding to AK1a and AK18 antibodies is maximally 80%), even under high antibody concentration conditions, which makes it necessary to develop more effective antibodies to improve therapeutic efficacy in humans. A recent debate on the putative inhibitory effect on IGF 2-mediated responses by AK1a and AK18 is an example of the lack of inhibition showing functional IGF2 stimulatory signals mediated by hIGF-IR. Indeed, even if competition for IGF2 binding occurs, this must be correlated with the concomitant inhibition of downstream hIGF-IR signaling, and evidence must be enumerated to provide substantial evidence for the functional effects of the antibody.

It is an object of the present invention to provide useful murine monoclonal antibodies, preferably chimeric or humanized antibodies, which specifically recognize IGF-IR with high affinity and which are capable of inhibiting not only the binding of IGF1 to IGF-IR but also the binding of IGF2 to IGF-IR. The antibody interacts little or not at all with the IR. Its attachment will be able to inhibit the growth of IGF-IR overexpressing cell lines in vitro, mainly through interaction with signal transduction pathways activated during IGF1/IGF-IR and IGF2/IGF-IR interactions. The antibody will be active in vivo against all tumor types expressing IGF-IR, including estrogen dependent breast and prostate cancers, which is not the case with currently available anti-IGF-IR monoclonal antibodies (written as MAbs or MABs). Indeed, α IR3 (which refers to the IGF-IR domain) completely inhibited the in vitro growth of estrogen-dependent tumors of the breast (MCF-7), but had no effect on the corresponding in vivo model (Arteaga C.et al, J.Clin. invest.84: 1418-1423, 1989). Likewise, the scFv-Fc fragment derived from murine monoclonal 1H7 was only weakly active against tumors of mammary MCF-7 and completely inactive against androgen-independent prostate tumors (Li S.L.et al, Cancer Immunol.Immunother., 49: 243. sup. 252, 2000).

In a surprising manner, the inventors have generated a murine monoclonal antibody (designated I-3466) which recognizes IGF-IR and meets all the criteria mentioned above, that is to say does not recognize the receptor for insulin, blocks IGF1 and in particular IGF 2-induced proliferation in vitro and likewise inhibits the growth of different tumors expressing IGF-IR, osteosarcoma and prostate cancer, in vivo. Furthermore, these antibodies have been shown to inhibit IGF 1-and/or IGF-2-induced phosphorylation of tyrosine of the beta chain of IGF-IR on MCF-7 and HT29 cells. Furthermore, it was also equally established that these antibodies cause internalization of the receptor and its degradation, contrary to what is usually observed for natural ligands, which allow rapid recycling of the receptor on the cell surface. It has been possible to characterize these antibodies by their peptide and nucleic acid sequences, particularly by their sequence of the Complementarity Determining Regions (CDRs) directed against IGF-IR.

Thus, according to the first embodimentScheme, a subject of the present invention is an isolated antibody, or one of its functional fragments, capable of specifically binding to the human insulin-like growth factor I receptor and/or capable of specifically inhibiting the tyrosine kinase activity of the IGF-IR receptor, characterized in that it is capable of exhibiting an IC of less than 0.3nM, preferably less than 0.03nM₅₀Inhibits the natural attachment of its primary ligand IGF1 and is also capable of an IC of less than 0.3nM, preferably less than 0.1nM₅₀Inhibiting the natural attachment of its second ligand IGF 2.

More specifically, the invention relates to an isolated antibody, or a functional immunogenic fragment thereof, having binding affinity for the human insulin-like growth factor I receptor (IGF-IR), characterized in that it binds to said IGF-IR with an IC of less than 0.3nM, preferably less than 0.03nM₅₀Inhibits the binding of the natural binding partner IGF1 to the IGF-IR and it also has an IC of less than 0.3nM, preferably less than 0.1nM₅₀Inhibits the binding of the natural binding partner IGF2 to the IGF-IR.

Furthermore, the present invention relates to an isolated antibody, or a functional immunogenic fragment thereof, having tyrosine kinase inhibitory activity of the human insulin-like growth factor I receptor (IGF-IR), characterized in that it has an IC of less than 0.3nM, preferably less than 0.03nM, when bound to said IGF-IR₅₀Inhibits the binding of the natural binding partner IGF1 to the IGF-IR and it also has an IC of less than 0.3nM, preferably less than 0.1nM₅₀Inhibits the binding of the natural binding partner IGF2 to the IGF-IR.

In the present application, IC as explained in example 2₅₀Has been determined graphically.

With respect to the prior art, the Roche antibody (WO 2004/087756 and WO2005/005635) designated 18 has an average IC of 0.3nM against IGF1 and IGF2₅₀(see example 6 of WO2005/005635), i.e.beyond the IC obtained from antibody I-3466₅₀(see example 2).

In this specification, the terms "bonded" and "attached" have the same meaning and may be used interchangeably.

According to another embodiment of the invention, the antibody is also capable of binding to the IGF-I/insulin hybrid receptor.

In fact, IGF-IR shows high homology to Insulin Receptor (IR), which exists in two subtypes, A and B.

In NCBI GenBank, the sequences of IR subtypes a and B are registered as accession numbers X02160 and M10051, respectively. Other data relating to IR, without limitation, are incorporated herein by reference (Vinten et al, 1991, Proc. Natl. Acad. Sci. USA, 88: 249-252; Belfiore et al, 2002, The Journal of Biological Chemistry, 277: 39684-39695; Dumesic et al, 2004, The Journal of Endocrinology & Metabolism, 89 (7): 3561-3566).

IGF-IR and IR are tetrameric glycoproteins consisting of two extracellular alpha-and two transmembrane beta-subunits linked by disulfide bonds. Each alpha-subunit containing the ligand binding site is approximately 130-135kDa, while each beta-subunit containing the tyrosine kinase domain is approximately 90-95 kDa. These receptors have over 50% overall amino acid sequence similarity and 84% similarity in the tyrosine kinase domain. Upon ligand binding, phosphorylated receptors recruit and phosphorylate docking proteins (docking proteins), including the insulin receptor substrate-1 protein family (IRS1), Gab1 and Shc (Avruch, 1998, mol.cell.biochem., 182, 31-48; Roth et al, 1988, Cold Spring Harbor Symp. Quant.biol.53, 537-543; White, 1998, mol.cell.biochem., 182, 3-11; Laviola et al, 1997, J.Clin.Invest.99, 830-837; Cheatham et al, 1995, Endocr.Rev.16, 117-142), leading to activation of different intracellular mediators. Although IR and IGF-IR similarly activate major signaling pathways, there are differences between the two receptors in recruitment of certain docking proteins and intracellular mediators (Sasaokaet., 1996, Endocrinology 137, 4427-4434; Nakae et al., 2001, Endocr. Rev.22, 818-835; Dupont and Le Roith 2001, Horm. Res.55, suppl.2, 22-26; Koval et al., 1998, biochem. J.330, 923-932). These differences are the basis for the major metabolic effects caused by IR activation, and for the major mitogenic, transforming and anti-apoptotic effects caused by IGF-IR activation (De Meyts et al, 1995, Ann.N.Y.Acad.Sci., 766, 388-S401; Singh et al, 2000, Prisco et al, 1999, Horm.Metab.Res.31, 80-89; ICido et al 2001, J.Clin.Endocrinol.Metab., 86, 972-979). Insulin binds to IR with high affinity (100-fold higher than to IGF-IR), whereas insulin-like growth factors (IGF1 and IGF2) bind to IGF-IR with 100-fold higher affinity than to IR.

There are two isoforms of human IR, IR- A and IR-B, which result from alternative splicing of the IR gene, excluding or including the 12 amino acid residues encoded by the small exon (exon 11) at the C-terminus of the IR α -subunit. The relative abundance of IR subtypes is regulated by tissue-specific and unknown factors (Moller et al, 1989, mol. Endocrinol., 3, 1263-. IR-B is the predominant IR subtype in normal adult tissues (adipose tissue, liver and muscle) that is the primary target tissue for insulin metabolism (Moller et al, 1989; Mosthaf et al, 1990). IR-A is the predominant subtype in fetal tissues and mediates fetal growth in response to IGF2 (FrascA et al, 1999, mol. cell. biol., 19, 3278-. Moreover, when cells are transformed and become malignant, dedifferentiation is often associated with increased relative abundance of IR-A (Pandini et al, 2002, The Journal of Biologic chemistry, Vol.277, N.degree.42, pp 39684-39695).

Based on their high homology, insulin and IGF-I half-receptors (consisting of one α -subunit and one β -subunit) can heterodimerize, leading to the formation of an insulin/IGF-I hybrid receptor (hybrid-R) (Soos et al, 1990, Biochemistry J., 270, 383-.

Both IR subtypes are capable of forming hybrids with IGF-IR. However, hybrid-R has different functional characteristics. hybrid-RsB has reduced affinity for IGF1 and especially for IGF 2. In contrast, hybrid-RsA has a high affinity for IGF1 and also binds IGF2 and insulin over a range of physiological concentrations. Expression of hybrid-RsA up-regulates the IGF system by two different mechanisms, i) binding (with high affinity) to and activation by IGF1 and IGF2 (which does not occur for hybrid-RsB), ii) activation of the IGF-IR pathway following insulin binding. Insulin binding to hybrid-RsA phosphorylates the IGF-IR β -subunit and activates IGF-IR-specific substrate (Crk II), such that hybrid-RsA switches insulin to IGF-IR signaling (Pandini et al, 2002).

In several tissues, such as the liver, spleen or placenta, hybrid-R is more representative than IGF-IR (Bailyes et al, 1997). When tumor tissue overexpresses or abnormally activates IGF-IR and IR-A (FrascA et al, 1999; Sciac et al, 1999, Oncogene 18, 2471-2479; VellA et al, 2001, mol. Pathol, 54, 121-124), hybrid-RsA can also be overexpressed in A variety of human malignancies, including thyroid and breast cancer, providing selective growth advantages to malignant cells that respond to the type of IGF-IR signaling following stimulation by physiological concentrations of IGF1 and/or IGF2, as well as insulin (Bailyes et al, 1997; Pandini et al, 1999, Clin. cancer Res, 5, 1935-1934; Belore et al, 1999, Biochimie (Paris)81, 403-407; FrascA et al, 1999, Sciac et al, VellA et al, 2001.

According to the invention, these antibodies are also characterized in that they are capable of binding to hybrid-R and, if desired, are additionally preferably capable of inhibiting the natural attachment of the ligands insulin, IGF1 and/or IGF-2 of hybrid-R and/or of specifically inhibiting the tyrosine kinase activity of said hybrid-R.

One subject of the present invention is an antibody, or one of its functional fragments, characterized in that it is also capable of 100% inhibition of the phosphorylation of the IGF-IR β -chain (preferably on HT29 cells) induced by IGF1 and/or IGF 2.

In another aspect, the antibody according to the invention, or one of its functional fragments, is characterized in that it does not exhibit any intrinsic antagonistic activity.

Furthermore, the antibody according to the invention, or one of its functional fragments, is characterized in that it is capable of inducing:

i) at least 30% of IGF-IR internalization on HT29 cells, and/or

ii) at least 85% of IGF-IR internalization on MCF-7 cells.

The antibody according to the invention, or one of its functional fragments, is characterized in that it is capable of inducing:

i) at least 50% degradation of IGF-IR on HT29 cells, and/or

ii) at least 65% degradation of IGF-IR on MCF-7 cells.

In another embodiment, the antibody according to the invention, or one of its functional fragments, is characterized in that it is capable of an IC at least equal to 1nM₅₀And preferably an IC of at least 0.7 and 0.5nM for IGF1 and IGF2 assays, respectively₅₀Inhibit the in vitro proliferation of MCF-7 cells induced by IGF1 and IGF 2.

The antibody according to the invention, or one of its functional fragments, is also characterized in that it is capable of inhibiting the growth of a tumor cell line in vivo.

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

In general, for the preparation of functional fragments of monoclonal Antibodies, in particular of murine origin, reference may be made to the techniques specifically described in the Manual "Antibodies (Antibodies)" (Harlow and Lane, Antibodies: Antibodies Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp.726, 1988), or to the techniques prepared from hybridomas as described by Kohler and Milstein (Nature, 256: 495-.

For example, the monoclonal antibody of the present invention can be obtained from an animal cell immunized with IGF-IR or a fragment thereof containing an epitope specifically recognized by the monoclonal antibody of the present invention. The IGF-IR or one of its fragments may be produced, inter alia, by genetic recombination methods starting with a nucleic acid sequence encoding IGF-IR contained in a cDNA sequence or by peptide synthesis methods starting with an amino acid sequence comprising an IGF-IR peptide sequence, according to conventional procedures.

For example, the monoclonal antibody of the invention may be purified on an affinity column to which IGF-IR or one of its fragments (which contains the epitope specifically recognized by the monoclonal antibody of the invention) has been previously immobilized. More specifically, the monoclonal antibodies can be purified by chromatography on proteins a and/or G, which may or may not be followed by ion exchange chromatography, with the aim of removing residual protein contaminants as well as DNA and LPS, which may or may not themselves be followed by Sepharose gel exclusion chromatography, in order to remove potential aggregates due to the presence of dimers or other multimers. In an even more preferred manner, all these techniques can be used simultaneously or sequentially.

Chimeric or humanized antibodies are also included within the scope of the antibodies of the invention.

By chimeric antibody is meant an antibody that contains the native variable (light and heavy) regions derived from an antibody of a particular species in combination with the light and heavy constant regions of an antibody of a heterologous species to the particular species.

The chimeric antibodies of the present invention or their fragments can be prepared by applying genetic recombination techniques. For example, chimeric antibodies can be produced by cloning sequences containing a promoter and a variable region according to the invention encoding a non-human, especially murine, monoclonal antibody. For example, the chimeric antibody of the present invention encoded by the recombinant gene will be a mouse-human chimera, the specificity of which is determined by the variable region derived from mouse DNA and its subtype is determined by the constant region derived from human DNA. For example, reference may be made to Verhoeyn et al (BioEssays, 8: 74, 1988) for methods for producing chimeric antibodies.

By humanized antibody is meant an antibody that contains CDR regions derived from an antibody of nonhuman origin, the remainder of the antibody molecule being derived from one (or from a plurality of) human antibodies. In addition, some residues of the backbone (referred to as FR) fragment may be modified so as to retain binding affinity (Jones et al, Nature, 321: 522-wall 525, 1986; Verhoeyen et al, Science, 239: 1534-wall 1536, 1988; Riechmann et al, Nature, 332: 323-wall 327, 1988).

Humanized antibodies of the invention or fragments thereof may be prepared by techniques well known to those skilled in the art (such as, for example, Singer et al, J.Immun., 150: 2844-2857, 1992; Mountain et al, Biotechnology. Gene. Eng.Rev., 10: 1-142, 1992; or Bebbington et al, Bio/Technology, 10: 169-175, 1992, as described in the following references). The use of these humanized antibodies of the invention is preferably used in vitro diagnostic methods, or in vivo prophylactic and/or therapeutic treatments. Other humanization methods known to the person skilled in the art are, for example, "CDR Grafting" (CDR Grafting) methods described by Protein Design Lab (PDL) in patent applications EP 0451261, EP 0682040, EP 09127, EP 0566647 or in U.S. Pat. No.5,530,101, U.S. Pat. No.6,180,370, U.S. Pat. No.5,585,089 and U.S. Pat. No.5,693,761. The following patent applications may also be mentioned: US5,639,641, US 6,054,297, US5,886,152 and US5,877,293. An antibody functional fragment according to the invention means in particular an antibody fragment such as Fv, scFv (sc for single chain), Fab, F (ab ') 2, Fab', scFv-Fc fragment or diabody (diabody), or any fragment whose half-life time is increased by chemical modification such as the addition of a poly (alkylene) glycol such as polyethylene glycol ("PEGylation") (a PEGylated fragment is called Fv-PEG, scFv-PEG, Fab-PEG, F (ab ') 2-PEG or Fab' -PEG) ("PEG" for polyethylene glycol), or by incorporation into liposomes, which fragment has at least one of the CDRs characteristic of the sequences SEQ ID No.1, 2, 3, 4, 5 or 6 according to the invention, and in particular it is capable of exerting even a part of the activity of the antibody from which it originates, such as in particular the ability to recognize and bind IGF-IR, if desired, and inhibiting the activity of IGF-IR.

Preferably, the functional fragment consists of or comprises a partial sequence of the heavy chain variable region or the light chain variable region of the antibody from which it is derived, said partial sequence being sufficient to maintain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least 1/100, more preferably at least 1/10 equal to the affinity of the antibody from which it is derived for IGF-IR.

Such functional fragments will comprise a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the sequence of the antibody from which they are derived.

Preferably, these functional fragments will be Fv, scFv, Fab, F (ab')₂F (ab'), scFv-Fc type or bifunctional antibody, which generally has the same binding specificity as the antibody from which it is derived. According to the present invention, the antibody fragment of the present invention can be obtained starting from an antibody (such as the above-mentioned antibody) by a method such as enzymatic digestion (such as pepsin or papain) and/or by a method such as chemical reduction cleavage of disulfide bridges. Alternatively, antibody fragments encompassed by the present invention may be obtained by genetic recombinant techniques also well known to those skilled in the art, or by peptide synthesis methods (e.g., automated peptide synthesizers, such as those provided by Applied Biosystems, Inc.), or the like.

In a more preferred manner, the invention comprises antibodies according to the invention or their functional fragments, in particular chimeric or humanized antibodies, obtained by genetic recombination or chemical synthesis.

More specifically, according to a preferred embodiment of the invention, said antibody is characterized in that it comprises a light chain comprising at least one Complementarity Determining Region (CDR) selected from the sequences SEQ ID nos. 1, 3 or 5 or at least one CDR having at least 80% identity with the sequences SEQ ID nos. 1, 3 or 5 after optimal alignment, or in that it comprises a heavy chain comprising at least one CDR selected from the CDRs of sequences SEQ ID nos. 2, 4 or 6 or at least one CDR having at least 80% identity with the sequences SEQ ID nos. 2, 4 or 6 after optimal alignment.

In the present specification, the terms polypeptide, polypeptide sequence, peptide and protein attached to an antibody compound or sequence thereof are interchangeable.

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

By CDR regions or CDRs is meant the hypervariable regions of the heavy and light chains of an immunoglobulin, as defined by Kabat et al (Kabat et al, Sequences of proteins of immunological interest, 5th ed., u.s.department of Health and human services, NIH, 1991, and later). There are three heavy chain CDRs and three light chain CDRs. As used herein, the term CDR or CDRs is intended to indicate one of these regions, or several or even all of these regions, which comprise the majority of the amino acid residues responsible for binding by the affinity of the antibody for the antigen or its recognition epitope, as the case may be.

For the purposes of the present invention, a "percentage of identity" between two nucleic acid or amino acid sequences means the percentage of identical nucleotides or identical amino acid residues between the two sequences to be compared, obtained after optimal alignment (optimal alignment), which percentage is purely statistical and the differences between the two sequences are randomly distributed and cover their full length. Sequence comparison between two nucleic acid or amino acid sequences is usually carried out by comparing these sequences after they have been matched in an optimal manner, the comparison being able to be carried out by means of segments or by means of "comparison windows". In addition to being able to be performed manually, optimal alignments for comparing sequences can also be performed by Smith and Waterman (1981) [ ad. 482] by Neddleman and Wunsch (1970) [ j.mol.biol.48: 443] local homology algorithm, by Pearson and Lipman (1988) [ proc.natl.acad.sci.usa 85: 2444) similarity search methods, by computer Software implementation using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics computer Group, 575 Science Dr., Madison, Wis., or by BLAST N ORBLAST P comparison Software).

The percent identity between two nucleic acid or amino acid sequences is determined by comparing the two sequences that match in an optimal manner, and wherein the nucleic acid or amino acid sequence to be compared may contain additions or deletions to the reference sequence that best matches between the two sequences. Percent identity was calculated as follows: percent identity between two sequences is obtained by determining the number of identical positions of nucleotides or amino acid residues between the two sequences, by dividing the number of identical positions by the total number of positions in the "comparison window" and multiplying the result by 100.

For example, the BLAST program, "BLAST 2 sequences" (Tatusova et al, "BLAST 2 sequences-a new tools for matching protein and nucleotide sequences", FEMS Microbiol Lett.174: 247-250) may be used, which may be obtained from the website http: html, parameters using default values (especially for the parameters "open gap dependency": 5, and "extension gap dependency": 2; the matrix selected is, for example, the program proposed matrix "BLOSUM 62"), and the percentage of identity between the two sequences to be compared is calculated directly by the program.

By amino acid sequences having at least 80%, preferably 85%, 90%, 95% and 98% identity to the reference amino acid sequence, those sequences are preferred which have certain modifications relative to the reference sequence, in particular a deletion, addition or substitution, truncation or extension of at least one amino acid. In the case of substitution of one or more consecutive or non-consecutive amino acids, it is preferred to replace the amino acid being substituted with an "equivalent" amino acid. The expression "equivalent amino acid" means here any amino acid which can be replaced by one of the amino acids of the basic structure, which, however, does not substantially alter the biological activity of the corresponding antibody and which, for example, will be defined later, especially in the examples.

Some alternative embodiments of the invention provide anti-IGF-IR antibodies with other amino acid sequence modifications therein. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of the anti-IGF-IR antibody are prepared by introducing appropriate nucleotide changes in the nucleic acid of the anti-IGF-IR antibody or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequence of the anti-IGF-IR antibody. Any combination of deletions, insertions, and substitutions can be used to arrive at the final construct, so long as the final construct possesses the desired properties. Amino acid changes can also alter post-translational processing of the anti-IGF-IR antibodies, such as changing the number or position of glycosylation sites.

A useful method for identifying preferred mutagenic positions for certain residues or regions of anti-IGF-IR antibodies is known as "alanine scanning mutagenesis", e.g., Cunningham and Wells in Science, 244: 1081-. Here, a residue or set of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the IGF-IR antigen. Subsequently, those amino acid positions demonstrating functional sensitivity to the substitution are improved by further introducing other variations at the substitution positions. Thus, when the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a particular site, ala scanning or random mutagenesis is performed on the target codon or region and the expressed anti-IGF-IR antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino-and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an anti-IGF-IR antibody with an N-terminal methionyl residue or said antibody fused to a cytotoxic polypeptide. Other insertional variants of the anti-IGF-IR antibody molecule include fusions in the N-or C-terminus of the anti-IGF-IR antibody with an enzyme (e.g., ADEPT) or polypeptide that increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. At least one amino acid residue in these variants of the anti-IGF-IR antibody molecule is replaced with a different residue. The most interesting positions for substitution mutagenesis include the hypervariable regions, and furthermore FR conversion (FR alteration) is also under consideration. Conservative substitutions are shown in table 1 under the heading "preferred substitutions". If these substitutions result in a change in biological activity, then more changes (designated as "typical substitutions" in Table 1, or as further described with reference to the amino acid classes below) can be introduced and the product screened.

TABLE 1 amino acid substitutions

Initial residue	Exemplary replacement	Preferred alternatives
			Ala(A)Arg(R)Asn(N)Asp(D)Cys(C)Gln(Q)Glu(E)Gly(G)His(H)Ile(I)Leu(L)Lys(K)Met(M)Phe(F)Pro(P)Ser(S)Thr(T)Trp(W)Tyr(Y)Val(V)	val；leu；ilelys；gln；asngln；his; asp; lys; argglu; asnser; alaasn; gluasp; glnalaash; gln; lys; argleu; val; met; ala; phe; norleucine; ile; val; met; ala; phearg; gln; asnleu; phe; ilereu; val; ile; ala; tyraloghrerstyr; (ii) phetrp; phe; thr; a serile; leu; met; phe; ala; norleucine	vallysglngluserasnaspalaargleuileargleutyralathrsertyrpheleu

Significant changes in antibody biological properties can be achieved by selection substitutions that differ significantly in their effect on maintaining: (a) a polypeptide backbone structure in the replacement region, such as a sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain. The natural residues are divided into the following groups according to the properties of the common side chains:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidity: asp, glu;

(4) alkalinity: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions allow members of one of these classes to be swapped with another class.

Any cysteine residues not involved in maintaining the appropriate conformation of the anti-IGF-IR antibody can also be replaced, usually with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, a cysteine bond may be added to the antibody to improve its stability (especially in the case where the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitution variant comprises replacing one or more residues of a hypervariable region of a parent antibody (e.g. a humanized or human antibody). Typically, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they were produced. A convenient means for generating such replacement variants includes affinity maturation using phage display technology. Briefly, several hypervariable region sites (e.g., 6-7 sites) were mutated to generate all possible amino acid substitutions for each site. Thus, the antibody variants produced thereby are displayed in a monovalent fashion from filamentous phage particles as packaged within each particle in a fused form with the gene III product of M13. Phage display variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues which significantly contribute to antigen binding. Alternatively or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the point of contact between the antibody and human IGF-IR. These contact residues and adjacent residues are candidate substitutions according to the techniques detailed herein. Once such variants are generated, the collection of variants is screened as described herein and antibodies with superior properties in one or more relevant assays can be selected for further development.

Another type of amino acid variant of an antibody alters the original glycosylation pattern of the antibody. By altered is meant that one or more carbohydrate moieties are deleted from the antibody and/or one or more glycosylation sites are added that are not otherwise present in the antibody.

Glycosylation of antibodies is usually N-linked or O-linked. N-linked means that the carbohydrate moiety is attached to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatically attaching the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates potential glycosylation sites. O-linked glycosylation refers to the attachment of the sugar N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine and threonine, and 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to the antibody can be conveniently accomplished by altering the amino acid sequence such that the antibody comprises one or more of the above-described tripeptide sequences (directed to N-linked glycosylation sites). The alteration may also be achieved by adding or replacing one or more serine or threonine residues (to the O-linked glycosylation site) to the original antibody sequence.

Nucleic acid molecules encoding the amino acid sequence variants of the anti-IGF-IR antibodies are prepared by a variety of methods well known in the art. These methods include, but are not limited to, isolation from a natural source (in the case where natural amino acid sequence variants are present) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of a previously prepared variant or non-variant form of the anti-IGF-IR antibody.

In some cases, it may be desirable to improve the effector function of an antibody of the invention, e.g., to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) of the antibody and/or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or in addition, cysteine residues may be introduced in the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibody thus produced may have improved internalization capacity and/or enhanced complement-mediated cytocidal power and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, j.exp med., 176: 1191-1195 (1992) and shop, B.J.Immunol., 148: 2918-2922(1992). Hetero-bifunctional cross-linkers can also be used to prepare homodimeric antibodies with enhanced anti-tumor activity, such as Wolff et al, Cancer Research, 53: 2560, 2565 (1993). Alternatively, the antibody may be engineered to have a dual Fc region and may thus have enhanced complement hemolysis and ADCC capabilities. See Stevenson et al, Anti-Cancer Drug Design 3: 219-230(1989).

Likewise, to increase the serum half-life of the antibody, a salvage receptor binding epitope(s) may be incorporated into the antibody, particularly the antibody fragment, as described in U.S. Pat. No.5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an IgG molecule (e.g., IgG)₁、IgG₂、IgG₃Or IgG₄) Is responsible for increasing the in vivo serum half-life of the IgG molecule.

In a more preferred manner, the invention relates to an antibody according to the invention or a functional fragment thereof, characterized in that it comprises a heavy chain comprising at least two or three of the three CDRs of sequences SEQ ID nos. 2, 4 and 6 or of sequences having at least 80% identity after optimal alignment with sequences SEQ ID nos. 2, 4 and 6, respectively.

In a more preferred embodiment, the invention is directed to an antibody according to the invention or a functional fragment thereof, characterized in that it comprises a light chain comprising at least two or three of the three CDRs of sequences SEQ ID nos. 1, 3 and 5, or of sequences having at least 80% identity after optimal alignment with sequences SEQ ID nos. 1, 3 and 5, respectively.

In a more preferred manner, the antibody of the invention or a functional fragment thereof is characterized in that it comprises a heavy chain comprising the three CDRs of sequences SEQ ID nos. 2, 4 and 6, or of sequences having at least 80% identity, respectively, with sequences SEQ ID nos. 2, 4 and 6 after optimal alignment, and it further comprises a light chain comprising the three CDRs of sequences SEQ ID nos. 1, 3 and 5, or of sequences having at least 80% identity, respectively, with sequences SEQ ID nos. 1, 3 and 5 after optimal alignment.

According to the prior art, it is known that the maximum variability (length and composition) between 6 CDRs is observed for the 3 CDRs of the heavy chain and more specifically CDR-H3. Thus, it will be understood that preferred typical CDRs of an antibody of the invention are the 3 CDRs of the heavy chain, i.e. the CDRs encoded by the sequences SEQ ID nos. 2, 4 and 6 and more preferably the CDRs corresponding to CDR-H3 encoded by SEQ ID No. 6.

According to another aspect, one subject of the present invention is an antibody or one of its functional fragments according to the invention, characterized in that it is not attached or not attached in a significant way to the human insulin receptor IR.

In a preferred manner, said functional fragment according to the invention will be selected from Fv, scFv, Fab, F (ab')₂Fab', scFv-Fc or a fragment of a bifunctional antibody, or any functional fragment which has an increased half-life by chemical modification, in particular by PEGylation or by incorporation into liposomes.

According to another aspect, the invention relates to murine hybridomas capable of secreting the monoclonal antibodies of the invention, in particular murine hybridomas such as those deposited at the National Microorganism collection (CNCM, National Center of microbiology Culture) (institut tpasteur, Paris, France) at 23.6.2005 under accession No. I-3466 and derived from the fusion of Balb/c splenocytes of immunized mice with the Sp2O Ag14 myeloma cell line.

The monoclonal antibody, or one of its functional fragments, herein designated as I-3466, is characterized by the secretion of said antibody by the hybridoma deposited at CNCM under No. I-3466 on 23/6 of 2005, which hybridoma is, of course, part of the present invention.

In a particular embodiment, the invention relates to a murine antibody or a functional fragment thereof according to the invention, characterized in that said antibody comprises a light chain whose sequence comprises the amino acid sequence SEQ ID No.7 or a sequence having at least 80% identity after optimal alignment with the sequence SEQ ID No.7, or/and in that it comprises a heavy chain whose sequence comprises the amino acid sequence SEQ ID No.8 or a sequence having at least 80% identity after optimal alignment with the sequence SEQ ID No. 8.

According to a further particular aspect, the invention relates to a chimeric antibody or one of its functional fragments according to the invention, characterized in that said antibody also comprises the light and heavy chain constant regions derived from an antibody of a species heterologous to the mouse, in particular human, and preferably the light and heavy chain constant regions derived from a human antibody are the kappa and gamma-1, gamma-2 or gamma-4 regions, respectively.

In a particular embodiment corresponding to isotype IgG1, the complementary property of the antibody is to potentially exhibit effector functions such as ADCC (antibody dependent cellular cytotoxicity) and/or CDC (complement dependent cytotoxicity).

According to a further particular aspect, the present invention relates to a humanized antibody or one of its functional fragments according to the invention, characterized in that said antibody comprises a light chain and/or a heavy chain, wherein the framework fragments FR1-FR4 of said light and/or heavy chain are derived from the framework fragments FR1-FR4, respectively, of the light and/or heavy chain of a human antibody.

According to a preferred embodiment, the humanized antibody or a functional fragment thereof according to the invention is characterized in that said humanized antibody comprises a light chain comprising the amino acid sequence SEQ ID No.17 or a sequence having at least 80% identity after optimal alignment with the sequence SEQ ID No.17 or in that it comprises a heavy chain comprising the amino acid sequence SEQ ID No.18 or a sequence having at least 80% identity after optimal alignment with the sequence SEQ ID No. 18.

Preferably, the humanized antibody or a functional fragment thereof according to the invention is characterized in that said humanized antibody comprises a light chain comprising the amino acid sequence SEQ ID No.17 and in that it comprises a heavy chain sequence comprising the amino acid sequence SEQ ID No. 18.

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

a) nucleic acid, DNA or RNA encoding the antibody of the invention or one of its functional fragments;

b) nucleic acids complementary to the nucleic acids as defined under a);

c) a nucleic acid of at least 18 nucleotides capable of hybridising, under highly stringent conditions, to at least one CDR of the nucleic acid sequence SEQ ID No.9, 10, 11, 12, 13 or 14 or a sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity, after optimal alignment, to the sequence SEQ ID No.9, 10, 11, 12, 13 or 14;

d) a nucleic acid of at least 18 nucleotides capable of hybridizing under highly stringent conditions to at least the light chain of nucleic acid sequence SEQ ID No.15 and/or the heavy chain of nucleic acid sequence SEQ ID No.16 or a sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment to sequence SEQ ID No.15 or 16;

e) a nucleic acid of at least 18 nucleotides capable of hybridizing under highly stringent conditions with at least the light chain of nucleic acid sequence SEQ ID No.19 and/or the heavy chain of nucleic acid sequence SEQ ID No.20 or a sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with sequence SEQ ID No.19 or 20.

Nucleic acids, nucleic sequences or nucleic acid sequences, polynucleotides, oligonucleotides, polynucleotide sequences, nucleotide sequences, the terms of which will be used indifferently in the present description, are intended to mean precisely linked nucleotides, modified or not, which allow to define nucleic acid fragments or regions, comprising or not non-natural nucleotides, and which are capable of corresponding to double-stranded DNA, single-stranded DNA and transcripts of said DNA.

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

A nucleic acid sequence having a percentage of identity of at least 80%, preferably 85%, 90%, 95% and 98% to a preferred sequence after optimal alignment means a nucleic acid sequence which has certain modifications such as, in particular, deletions, truncations, extensions, chimeric fusions and/or substitutions, in particular point substitutions, relative to a reference nucleic acid sequence. Preferably, it relates to a sequence encoding an amino acid sequence identical to a reference sequence, which involves the degeneracy of the genetic code, or a complementary sequence capable of specifically hybridizing to a reference sequence, preferably under high stringency conditions, in particular as defined below.

Hybridization under conditions of high stringency means that the temperature conditions and ionic strength conditions are chosen such that they allow the maintenance of hybridization between two complementary DNA fragments. As an example, for the definition of the above polynucleotide fragment objective hybridization step of high stringency conditions, advantageously the following conditions.

DNA-DNA or DNA-RNA hybridization was performed in two steps: (1) prehybridization in phosphate buffer (20mM, pH 7.5) containing 5 XSSC (1 XSSC for 0.15M NaCl +0.015M sodium citrate solution), 50% formamide, 7% Sodium Dodecyl Sulfate (SDS), 10 XDenhardt's, 5% dextran sulfate, and 1% salmon sperm DNA at 42 ℃ for 3 hours; (2) the actual hybridization is carried out for 20 hours at a temperature which depends on the size of the probe (i.e.: 42 ℃ C. for a probe size > 100 nucleotides), followed by 2 washes in 2 XSSC + 2% SDS for 20 minutes at 20 ℃ and 1 wash in 0.1 XSSC + 0.1% SDS for 20 minutes. For probe size > 100 nucleotides at 60 degrees C, in 0.1x SSC + 0.1% SDS for 30 minutes of the final washing. The high stringency hybridization conditions described above for polynucleotides of defined size can be adapted by those skilled in the art for larger or smaller oligonucleotides according to the teachings of Sambrook et al (1989, Molecular cloning: a laboratory manual.2nd Ed. Cold Spring Harbor).

The invention likewise relates to vectors comprising a nucleic acid according to the invention.

The present invention is directed in particular to cloning and/or expression vectors comprising a nucleotide sequence according to the invention.

The vector according to the invention preferably comprises elements which allow the expression and/or secretion of the nucleotide sequence in a defined host cell. The vector must therefore contain a promoter, translation initiation and termination signals, and appropriate transcriptional regulatory regions. It must be able to be maintained in a stable manner in the host cell and optionally have specific signals specifying secretion of the translated protein. These various elements are selected and optimized by the person skilled in the art as a function of the host cell used. To this end, the nucleotide sequence according to the invention may be inserted into an autonomously replicating vector of the chosen host, or be an integrating vector of the chosen host.

These vectors are prepared by those skilled in the art by methods currently used, and the resulting clones can be introduced into an appropriate host by standard methods, such as lipofection, electroporation, heat shock, or chemical methods.

The vectors according to the invention are, for example, vectors of plasmid or viral origin. They can be used to transform host cells to clone or express the nucleotide sequences according to the invention.

The invention also encompasses a host cell transformed with or comprising a vector according to the invention.

The host cell may be selected from prokaryotic or eukaryotic systems, such as bacterial cells and yeast cells or animal cells, especially mammalian cells. Insect cells or plant cells may also be used.

The invention also relates to animals, except humans, comprising at least one cell transformed according to the invention.

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

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

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

Cells transformed according to the invention may be used in a method for producing a recombinant polypeptide according to the invention. The method for the recombinant production of a polypeptide according to the invention, which method is characterized in that it utilizes a vector according to the invention and/or a cell transformed by a vector, is itself encompassed by the invention. Preferably, the cells transformed with the vector according to the invention are cultured under conditions allowing the expression of the polypeptide and the recombinant peptide is recovered.

As said, the host cell may be selected from prokaryotic or eukaryotic systems. In particular, the nucleotide sequences according to the invention can be identified to facilitate secretion in these prokaryotic or eukaryotic systems. Thus, the vector according to the invention carrying such a sequence can advantageously be used for the production of recombinant proteins intended to be secreted. In this regard, the purification of these recombinant proteins of interest would benefit from the fact that: they are present in the supernatant of the cell culture rather than inside the host cell.

The polypeptides according to the invention can also be prepared by chemical synthesis. This preparation process is also subject of the present invention. Chemical synthesis methods are known to the person skilled in the art, for example using Solid phase techniques (see, inter alia, Steward et al, 1984, Solid phase peptide synthesis, Pierce chem. company, Rockford, 111, 2nd ed. (1984)) or using partial Solid phase techniques, by fragment condensation or by classical synthesis in solution. Polypeptides which are obtained by chemical synthesis and which are capable of comprising the corresponding unnatural amino acid are likewise encompassed by the invention.

The antibody or one of its functional fragments obtainable by the method according to the invention is likewise comprised in the invention.

According to a second embodiment, the invention relates to an antibody according to the invention, such as the antibody described above, characterized in that it is also capable of specifically binding to a receptor of the human tyrosine kinase family and/or of inhibiting the tyrosine kinase activity of this receptor.

In a first embodiment, such an antibody consists of a bispecific antibody comprising a second motif capable of specifically inhibiting the binding of EGF to the human Epidermal Growth Factor Receptor (EGFR) and/or inhibiting the tyrosine kinase activity of said EGFR. In a more preferred embodiment of the invention, the second anti-EGFR motif is produced by the murine monoclonal antibody 225, its chimeric derivative C225, or any humanized antibody derived from this antibody 225.

In a second embodiment, such an antibody consists of a bispecific antibody comprising a second motif capable of specifically inhibiting the activity modulated by the HER2/neu receptor and/or inhibiting the activity of said HER2/neu receptor tyrosine kinase. In a more preferred embodiment of the invention, the second anti-HER 2/neu motif is produced by the murine monoclonal antibody 4D5 or 2C4 or the humanized antibody Trastuzumab or Pertuzumab.

In a third embodiment, such an antibody consists of a bispecific antibody comprising a second motif capable of specifically inhibiting the binding of Hepatocyte Growth Factor (HGF) to the cMET receptor and/or specifically inhibiting the cMET receptor tyrosine kinase activity.

Finally, in a final embodiment, such an antibody consists of a bispecific antibody comprising a second motif capable of specifically inhibiting the binding of Macrophage Stimulating Protein (MSP) to a RON receptor and/or inhibiting the RON receptor tyrosine kinase activity.

According to another embodiment of the invention, it is also contemplated that the antibody according to the invention is capable of interacting with any kind of receptor involved in tumorigenesis, such as, for example and without limitation, VEGFR, FGF (fibroblast growth factor), PDGF (platelet-derived growth factor) or CXCR4 or 2 (chemokine receptor type 4 or 2).

Bispecific or bifunctional antibodies form a second generation monoclonal antibody in which two different variable regions are combined in the same molecule (Hollinger and Bohlen, 1999, Cancer and plasmasisis, rev. 18: 411-419). Their use has been demonstrated in the diagnostic field and in the therapeutic field, due to their ability to recruit the function of new effector factors 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-.

These bispecific antibodies can be constructed as intact IgG, bispecific Fab '2, Fab' PEG or bifunctional or other bispecific scFv, as well as the same tetravalent bispecific antibody or an antibody that presents two attachment sites for the respective antigen of interest (Park et al, 2000, mol.immunol., 37 (18): 1123-30) or fragments thereof as described above.

In addition to the economic benefits due to the labor-saving production and administration of bispecific antibodies over the production of two specific antibodies, the use of such bispecific antibodies has the advantage of reduced therapeutic toxicity. This is because the use of bispecific antibodies allows the total amount of circulating antibodies to be reduced and thus reduces the potential toxicity.

In a preferred embodiment of the invention, the bispecific antibody is a bivalent or tetravalent antibody.

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

According to yet another embodiment, the present invention also relates to a pharmaceutical composition as described above, comprising at least one second compound selected from compounds capable of specifically inhibiting the tyrosine kinase activity of IGF-IR, EGFR, HER2/neu, VEGFR, cMET and/or RON.

In a second preferred aspect of the invention, said second compound is selected from the group consisting of isolated anti-EGFR, anti-IGF-IR, anti-HER 2/neu, anti-VEGFR, anti-cMET and/or anti-RON antibodies or functional fragments thereof, capable of inhibiting proliferation and/or anti-apoptosis and/or angiogenesis and/or metastasis diffusion inducing activity mediated by said receptor.

According to another embodiment of the invention, the composition comprises at least one inhibitor of tyrosine kinase activity of IGF-IR, EGFR, HER2/neu, VEGFR, cMET and/or RON, as a composition for simultaneous, separate or sequential use.

In another preferred embodiment, the inhibitor of the tyrosine kinase activity of the receptor is selected from the group consisting of derivatized natural agents, dianilinophthalmides, pyrazolo-or pyrrolopyridopyrimidines or quinazolines. These inhibitors are well known to the person skilled in the art and are described in the literature (Ciardiello E, Drugs 2000, supply.1, 25-32).

A further supplementary embodiment of the present invention is a composition as described above, further comprising a cytotoxic/cytostatic agent and/or an inhibitor of the tyrosine kinase activity corresponding to IGF-I and/or EGF receptors, respectively, as a combination for simultaneous, separate or sequential use.

"simultaneous use" is understood to mean the administration of two compounds of a composition according to the invention in a single and the same pharmaceutical form.

"used separately" is understood to mean that the two compounds of the composition according to the invention are administered simultaneously, in different pharmaceutical forms.

"sequential use" is understood to mean that the two compounds of the composition according to the invention are each administered sequentially in different pharmaceutical forms.

In a common manner, the composition according to the invention significantly increases the efficacy of cancer treatment. In other words, the therapeutic effect of the anti-IGF-IR antibodies according to the invention is enhanced in an unexpected manner by the administration of cytotoxic agents. Another major corresponding advantage produced by the composition according to the invention relates to the possibility of using lower efficacy doses of the active ingredient, which allows to avoid or reduce the risk of secondary effects, in particular of cytotoxic agents, occurring.

In addition, such a composition according to the invention allows to obtain the desired therapeutic effect more quickly.

By "anti-cancer therapeutic agent" or "cytotoxic agent" is meant a substance that, when administered to a subject, treats or prevents the development of cancer in the subject's body. By way of non-limiting example of such an agent, it may be an alkylating agent, an antimetabolite, an antitumor antibiotic, a mitotic inhibitor, an inhibitor of chromatin function, an anti-angiogenic agent, an antiestrogen, an antiandrogen, or an immunomodulator.

Such compounds are, for example, those referred to on the page of the cited 2001 edition of VIDAL on "cytotoxins" in the cancerology and hematology columns, which cytotoxic compounds cited in this document are cited herein as preferred cytotoxic agents.

More specifically, the following agents are preferred in the present invention.

An "alkylating agent" refers to any substance that is capable of crosslinking or alkylating any molecule, preferably a nucleic acid (e.g., DNA) within a cell. Examples of alkylating agents include nitrogen mustards such as dichloromethyldiethylamine (mechlororethanamine), chlorambucol, melphalan, chlorodrate, pipobroman, prednimustine, disodium hydrogen phosphate (discotic-phosphate), or estramustine; oxaphosphorins such as cyclophosphamide, altretamine, trofosfamide, sulfonfamide or ifosfamide; aziridines (aziridines) or aziridines (imine-ethylenes) such as thiotepa, triethyleneamine (triethyleneamine) or altetramine; nitrosoureas such as carmustine (carmustine), streptozotocin, fotemustine (fotemustin), or lomustine (lomustine); alkyl sulfonates such as busulfan, busulfan or improsulfan; triazenes (triazenes) such as dacarbazine (dacarbazine); or platinum-based complexes such as cisplatin, oxliplatin, and carboplatin.

By "antimetabolite" is meant an agent that blocks cell growth and/or metabolism by interfering with certain activities, typically DNA synthesis. Examples of the antimetabolite include methotrexate, 5-fluorouracil, 2-deoxyfluorouridine (floxuridine), 5-fluorodeoxyuridine, capecitabine (capecitabine), cytarabine, fluoroadenosine (fludarabine), cytosine arabinoside, 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), chlorodeoxyadenosine (chlorodeoxyadenosine), 5-azacytidine, gemcitabine (gemcitabine), cladribine (cladribine), deoxyCorfuginin (deoxyoformycin), and pentostatin.

"antitumor antibiotic" refers to a compound that prevents or inhibits DNA, RNA and/or protein synthesis. Examples of antitumor antibiotics include doxorubicin, daunorubicin (daunorubicin), flavobilin (idarubicin), valrubicin, mitoxantrone (mitoxantrone), actinomycin D, mithramycin, plicamycin (plicamycin), mitomycin C, bleomycin, and procarbazine.

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

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

By "anti-angiogenic agent" is meant any drug, compound, substance, or agent that inhibits the growth of blood vessels. Exemplary anti-angiogenic agents include, but are not limited to, Razoxin (razoxin), marimastat (marimastat), batimastat (batimastat), prinomastat, tanomastat, ilomastat (ilomastat), CGS-27023A, halofuginone (halofuginon), COL-3, neovastat, BMS-275291, thalidomide (thalidoside), CDC 501, DMXAA, L-651582, batamine, endostatin (endostatin), SU5416, SU6668, interferon- α, EMD121974, interleukin-12, IM862, angiostatin, and vitaxin.

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

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

Immunomodulators are substances that stimulate the immune system.

Examples of immunomodulators include interferons, interleukins such as aldesukine (aldesleukin), OCT-43, denileukin diflox and interleukin-2, tumor necrosis factors such as tasonermine or other immunomodulators such as lentinan, cezopyran, roquinacre, pidotimod (pidotimod), imidure, thymopentin, poly I: C or levamisole together with 5-fluorouracil.

For more details, one skilled in the art may refer to the manual edited by "Association national standards des Engineers de Chimie therapeutics" and entitled "traite de Chimie therapeutics, vol.6, medical, analytical instruments and experimental dans Ie transaction producers, edition TEC & DOC, 2003".

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

In a particularly preferred embodiment, the composition of the invention is characterized in that the cytotoxic/cytostatic agent is chosen from spindle inhibitors or stabilizers, preferably vinorelbine (vinorelbine) and/or vinflunine (vinflunine) and/or vincristine.

To facilitate the coupling between the cytotoxic agent and the antibody according to the invention, it is possible in particular to introduce a spacer molecule, such as a polyalkylene glycol, for example polyethylene glycol, or another amino acid, between the two compounds to be coupled, or in another embodiment a derivative of the cytotoxic agent can be used, to which a function capable of reacting with the antibody according to the invention has been introduced. These coupling techniques are well known to those skilled in the art and will not be detailed in this specification.

Other EGFR inhibitors can be represented, without any limitation, by anti-EGFR monoclonal antibodies C225 and 22Mab (ImClone Systems incorporated), ABX-EGF (Abgenix/Cell Genesys), EMD-7200(Merck KgaA) or compounds ZD-1834, ZD-1838 and ZD-1839(AstraZeneca), PKI-166(Novartis), PKI-166/CGP-75166(Novartis), PTK 787(Novartis), CP 701(Cephalon), leflunomide (leflunomide) (Pharmacia/Sugen), CI-1033 (Warner-Lambert-Davids), CI-1033/PD, Gm805 (Warner-Lambert-Paravis), CL-387, 785 (Wyeth-Aegerst), R-Boehringer 1611 (Borne-Biotech), Gm805 (Warner-Lambert-David-II), and Sbyrex-39103 (Mb-R-Biocide), and Szel-R2 (Mb-R2, Mb-R2 (B-R2, Mb-R2, Bger-R2, Mb-R2, R-R2, and Szel (R-R2, R2, VRCTC-310(Ventech Research), EGF fusion toxin (Seragen Inc.), DAB-389(Seragen/Lilgand), ZM-252808(Imperial 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 "EGFR vaccine" (York Medical/Central de immunology Molecular).

According to another embodiment of the invention, the composition as described above may also comprise another anti-HER 2/neu receptor extracellular domain antibody compound as a combination product for simultaneous, separate or sequential use, intended for the prevention and treatment of cancer, in particular of cancers overexpressing said HER2/neu receptor and the receptors IGF-IR and/or EGFR, such as in particular breast cancer.

Reference may be made in particular to the publications of Albanell et al (J.of the National Cancer Institute, 93 (24): 1830-.

In a particular mode, said anti-HER 2/neu antibody according to the composition of the invention is an antibody known as Trastuzumab (also known as Herceptin).

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

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

Also preferably, the toxin is an enterobacterial toxin, especially pseudomonas exotoxin a.

The radioactive element (or radioisotope) preferably conjugated to the antibody used for the treatment is a gamma-emitting radioisotope, and preferably iodine¹³¹Yttrium, yttrium⁹⁰Gold, gold¹⁹⁹Palladium, palladium¹⁰⁰Copper, copper⁶⁷Bismuth, bismuth²¹⁷And antimony²¹¹. Radioisotopes that emit both beta and alpha radiation may also be used in therapy.

The toxin or radioactive element coupled to at least one antibody or one of its functional fragments according to the invention means any means allowing said toxin or said radioactive element to bind to said at least one antibody, in particular by covalent coupling between the two compounds, with or without the introduction of a linking molecule.

Among the reagents which allow to bind all or part of the components of the conjugate in a chemical (covalent), electrostatic or non-covalent manner, mention may be made in particular of benzoquinone, carbodiimides and more particularly EDC (1-ethyl-3- [ 3-dimethylaminopropyl ] -carbodiimide hydrochloride), bismaleimide, dithiodinitrobenzoic acid (DTNB), N-succinimidyl S-acetylthioacetate (SATA), bridging agents having one or more azidophenyl groups which are reactive with ultraviolet light (U.V.), and preferably N- [ -4- (azidosalicylamino) butyl ] -3 '- (2' -pyridyldithio) -propionamide (APDP), N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), 6-hydrazino-nicotinamide (HYNIC).

Another form of coupling, particularly for radioactive elements, may consist in using a bifunctional ion chelating agent.

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

For example, diethylenetriaminepentaacetic acid (DTPA) and its derivatives, which have been widely used in medicine and biology for a long time, either in free form or in the form of complexes with metal ions, have the remarkable property of forming stable chelates with metal ions and can be conjugated with proteins of therapeutic or diagnostic interest, such as antibodies, in order to develop radioimmunoconjugates for cancer therapy (Meases et al, 1984; Gansow et al, 1990).

Also preferably, the at least one antibody forming the conjugate according to the invention is selected from functional fragments thereof, in particular fragments truncated by its Fc part, such as scFv fragments.

The invention also comprises the use of a composition according to the invention for the preparation of a medicament.

More particularly, according to another embodiment, the invention relates to the use of one of the antibodies or functional fragments thereof, and/or of the composition for the preparation of a medicament intended for the prevention or treatment of a disease induced by overexpression and/or abnormal activation of IGF-IR, and/or associated with overactivation of the signal transduction pathway mediated by the interaction of IGF1 or IGF2 with IGF-IR.

According to yet another preferred embodiment, the invention relates to the use of an antibody or one of its functional fragments and/or a composition for the preparation of a medicament for the prevention or treatment of a disease caused by overexpression and/or abnormal activation of IGF-IR and/or associated with overactivation of the transduction pathway of signals mediated by the interaction of IGF2 with IGF-IR.

Preferably, said use according to the invention is characterized in that the administration of said drug does not induce or only induces slight secondary effects related to the inhibition of the insulin receptor IR, that is to say, due to the presence of said drug inhibiting the interaction of IR with its natural ligand, in particular by competitive inhibition linked to the attachment of said drug to the IR.

The invention also comprises the use of an antibody, preferably a humanized antibody or one of its functional fragments, and/or of a composition according to the invention for the preparation of a medicament intended to inhibit the transformation of normal cells into cells having the characteristics of a tumor, preferably cells that are IGF-dependent, in particular at least IGF 2-dependent.

The invention also relates to the use of an antibody, preferably a humanized antibody or one of its functional fragments, and/or a composition according to the invention for the preparation of a medicament intended to inhibit the growth and/or proliferation of tumor cells, preferably cells that are IGF-dependent, in particular at least IGF 2-dependent.

In a general manner, one subject of the present invention is the use of an antibody, preferably a humanized antibody or one of its functional fragments, and/or a composition according to the invention for the preparation of a medicament intended for the prevention or treatment of cancer, preferably expressing IGF-IR and/or preferably having an overactivation of the signal transduction pathway mediated by the interaction of IGF1 and/or IGF2 with IGF-IR, such as the overexpression of IRs 1.

A subject of the invention is also the use of an antibody, preferably a humanized antibody or one of its functional fragments, and/or a composition according to the invention, for the preparation of a medicament intended for the prevention or treatment of psoriasis whose epidermal hyperproliferation may be linked to the expression or overexpression of IGF-IR and/or to the overactivation of the signal transduction pathway mediated by the interaction of IGF-IR with its natural ligand (Wraight CJ.et al, Nat.Biotechnol., 2000, 18 (5): 521-526. epidermal hyperproliferation in psoriasis) and/or EGFR with its natural ligand.

The invention also relates to the use of an antibody or any functional fragment thereof (preferably humanized) and/or any composition comprising said antibody for the preparation of a medicament for the treatment or prevention of atherosclerosis.

Among the cancers that can be prevented and/or treated are preferably prostate cancer, osteosarcoma, lung cancer, breast cancer, endometrial cancer, colon cancer, multiple myeloma or ovarian cancer or any other cancer that overexpresses IGF-IR.

In a more preferred embodiment, due to the specific ability to displace IGF2, the invention relates to the use of an antibody or fragment thereof according to the invention for the prevention, diagnosis or treatment of colon cancer, since this cancer is known to be particularly related to IGF 2.

According to another aspect, a subject of the present invention is a method for the diagnosis of diseases, preferably in vitro, associated with the overexpression or underexpression, preferably overexpression, of IGF-IR, derived from a biological sample suspected of the abnormal presence of IGF-IR, characterized in that said biological sample is brought into contact with an antibody or one of its functional fragments according to the invention, which antibody, if necessary, can be labeled.

Preferably, the disease associated with IGF-IR overexpression in said diagnostic method is cancer.

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

One subject of the present invention is a method for in vitro diagnosis of a pathological condition characterized by an abnormal expression of IGF-IR with respect to the normal case, said method comprising contacting a biological sample suspected of containing IGF-IR with an antibody of the invention under conditions favouring the formation of an IGF-IR/antibody complex, and detecting said complex as an indication of the presence of said IGF-IR in said sample. In a preferred embodiment, the antibody is detectably labeled.

In a first aspect, the aberrant expression of IGF-IR is overexpression of IGF-IR. In a second aspect, the aberrant expression of IGF-IR is under-expression (overexpression) of IGF-IR.

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

Other conjugates may also include chemiluminescent labels such as luminol, dioxetanes (dioxetanes), bioluminescent labels such as luciferase and luciferin, or radioactive labels such as iodine¹²³Iodine, iodine¹²⁵Iodine, iodine¹²⁶Iodine, iodine¹³³Bromine, bromine⁷⁷Technetium, technetium^99mIndium, indium¹¹¹Indium, indium^113mGallium, gallium⁶⁷Gallium, gallium⁶⁸Ruthenium (II) and (III)⁹⁵Ruthenium (II) and (III)⁹⁷Ruthenium (II) and (III)¹⁰³Ruthenium (II) and (III)¹⁰⁵Mercury, mercury¹⁰⁷Mercury, mercury²⁰³Rhenium^99mRhenium¹⁰¹Rhenium¹⁰⁵Scandium (III)⁴⁷Tellurium^121mTellurium^122mTellurium^125mThulium, thulium¹⁶⁵Thulium, thulium¹⁶⁷Thulium, thulium¹⁶⁸Fluorine¹⁸Yttrium, yttrium¹⁹⁹Iodine, iodine¹³¹. The existing methods known to the skilled person for coupling therapeutic radioisotopes to antibodies, either directly or via chelating agents such as EDTA, DTPA mentioned above, can be used for the radioelements useful in diagnosis. Mention may also be made of the use of Na [ I ] by the chloramine T method¹²⁵]Marker [ Hunter w.m. and Greenwood f.c., 1962, Nature 194: 495]Also or alternatively with technetium by the technique of Crockford et al^99mMarkers (US patent 4,424,200), or attachment via DTPA as described by Hnatowich (US patent 4,479,930).

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

a) contacting a biological sample with an antibody of the invention or a functional fragment thereof; and

b) demonstrating the possible formation of IGF-IR/antibody complexes.

In a particular embodiment, the antibodies or functional fragments thereof according to the invention may be used in a method for detecting and/or quantifying IGF-IR in a biological sample, for monitoring the efficacy of a prophylactic and/or therapeutic treatment of IGF-dependent cancer or psoriasis or atherosclerosis.

More generally, the antibody or functional fragment thereof according to the invention can be advantageously used in any case where the expression of IGF-IR has to be observed in a qualitative and/or quantitative manner.

Preferably, the biological sample is formed from a biological fluid, such as human-derived serum, whole blood, cells, a tissue sample or a biopsy sample.

Another aspect of the invention is a diagnostic method for predicting the oncogenic potential of a prostate cell sample, comprising the steps of:

(a) providing a sample of human prostate tissue; and

(b) determining the presence of IGF-IR in a sample, said step comprising contacting said sample with an antibody of the invention under conditions conducive to the formation of an IGF-IR/antibody complex, wherein the presence of said complex is indicative of the carcinogenic potential of said cell in said tissue.

In another embodiment, the presence of said complex is indicative of the patient being at risk of developing or having an onset of a pathological condition characterized by said IGF-IR overexpression.

An object of the present invention is also a method of tracking the progress of a therapeutic regimen designed to alleviate a pathological disease characterized by an abnormal expression of IGF-IR expression, comprising the steps of:

(a) analyzing a sample from the subject to determine IGF-IR levels at a first time point;

(b) analyzing the sample at a second time point; and

(c) comparing the level at the second time point to the level determined in (a) as a determination of the effect of the treatment regimen, wherein a decrease in the level of IGF-IR in the sample can determine regression of the pathological disease in the patient, or an increase in the level of IGF-IR can determine progression of the pathological disease in the patient.

To carry out such detection and/or administration, any method or routine test may be used. The test may be a competition or sandwich test, or any test known to those skilled in the art that relies on the formation of antibody-antigen type immune complexes. After administration according to the invention, the antibody or one of its functional fragments may be immobilized or labeled. Immobilization can be carried out on a number of supports known to those skilled in the art. These supports may include, inter alia, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, or natural or modified cells. These supports may be soluble or insoluble.

By way of example, one preferred method uses an immunoenzymatic method according to the ELISA technique, by immunofluorescence or Radioimmunoassay (RIA) technique or equivalent.

Thus, the invention also comprises a kit or kit necessary for carrying out a diagnostic method for a disease induced by overexpression or underexpression of IGF-IR or for carrying out a method for detecting and/or quantifying overexpression or underexpression of IGF-IR in a biological sample, characterized in that said kit or kit comprises the following elements:

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

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

c) optionally, reagents that allow for the validation of the IGF-IR/antibody complex produced by the immune response.

The invention also relates to the use of a composition as a combination product according to the invention for the preparation of a medicament intended for the prevention or treatment of a cancer, in particular a cancer for which said cytotoxic agent or said anti-HER 2/neu antibody is generally prescribed, for which said tumor cells express or overexpress IGF-IR.

Another subject of the invention is the use of an antibody according to the invention for the preparation of a medicament intended to specifically target a biologically active compound to cells expressing or overexpressing IGF-IR.

Biologically active compound in this context means any compound capable of modulating, especially inhibiting, the activity of a cell, especially its growth, proliferation, transcription or gene translation.

Another subject of the invention is an in vivo diagnostic agent comprising an antibody according to the invention or one of its functional fragments, preferably labeled, in particular radiolabeled, and its use in medical imaging, in particular for detecting cancers associated with the expression or overexpression of IGF-IR by cells.

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

The composition according to the invention or the conjugate, preferably as a combination product, will be mixed with excipients and/or pharmaceutically acceptable carriers.

In the present specification, pharmaceutically acceptable carrier means a compound or combination of compounds that enters into a pharmaceutical composition without provoking secondary reactions and which allows, for example, to facilitate administration of the active compound, to increase its lifetime and/or in vivo efficacy, to increase its solubility in solution or to improve its preservation properties. Such pharmaceutically acceptable carriers are well known and will be adapted by those skilled in the art as a function of the nature and mode of administration of the active compound selected.

Preferably, the compounds are administered by systemic routes, in particular by intravenous, intramuscular, intradermal, intraperitoneal or subcutaneous routes, or by oral routes. In a more preferred manner, the composition comprising the antibody according to the invention will be administered several times in a sequential manner.

The mode of administration, dosage and optimal pharmaceutical form of the patient can be determined according to the criteria generally considered for determining the treatment to be applied to the patient, such as the age or weight of the patient, the severity of its general health, the tolerance to the treatment and the secondary effects mentioned.

Other features and advantages of the invention will appear in the following, continuous part of the description, together with the examples and the figures, the description of which follows.

Drawings

FIG. 1:monoclonal antibody I-3466 para¹²⁵I]Competition for binding of IGF-1 to IGF-IR.

As a function of ligand concentration, on a semi-logarithmic graph¹²⁵I]Specific binding (%) of IGF-1. The value of specific binding is the average of three experiments performed.

FIG. 2:monoclonal antibody I-3466 para¹²⁵I]Competition for binding of IGF-2 to IGF-IR.

As a function of ligand concentration, on a semi-logarithmic graph¹²⁵I]Specific binding (%) of IGF-2. The value of specific binding is the average of three experiments performed.

Fig. 3A and 3B:in vitro effects of I-3466 antibodies on IGF1 or IGF2 induced growth of MCF-7.

Fig. 4A and 4B:effect of I-3466Mab on IGF-IR β chain phosphorylation on MCF-7 cells induced by IGF1 (FIG. 4A) or IGF2 (FIG. 4B).

Fig. 5A and 5B:effect of I-3466Mab on IGF1 (fig. 5A) or IGF2 (fig. 5B) induced phosphorylation of IGF-IR β chain on HT29 cells.

FIGS. 6A-6C:i-3466 in vivo activity in DU145 (FIG. 6A), SK-ES-1 (FIG. 6B), HT29 (FIG. 6C), A549 (FIG. 6D) and MCF-7 (FIG. 6E) xenograft tumor models.

FIG. 7:comparison of the amino acid sequences of the light chain (VL) variable regions of mouse I-3466(SEQ ID No.7) and of the CDR-grafted I-3466 humanized (SEQ ID No. 20).

Symbol^*The (asterisks) indicate residues important for maintaining the conformation of the CDR loops, and the # (well) indicates conserved residues found at the VL/VH interface.

FIG. 8:comparison of the amino acid sequences of the heavy chain (VH) variable regions of mouse I-3466(SEQ ID No.8) and of the CDR-grafted I-3466 humanized (SEQ ID No. 21).

Symbol^*The (asterisks) indicate residues important for maintaining the conformation of the CDR loops, and the # (well) indicates conserved residues found at the VL/VH interface.

FIG. 9:SDS-PAGE analysis of purified I-3466 antibody. Legends are as follows:

m: a marker substance, a marker substance and a marker substance,

lane 1: I-3466A humanized variable region,

lane 2: i-3466 mouse variable region(s),

FIG. A: the SDS-PAGE was reduced to obtain a purified SDS-PAGE,

and B: non-reducing SDS-PAGE.

The constant regions of both mouse and humanized 3466 are human.

FIG. 10:schematic representation of a biosensor capture assay.

Human IgG1 Mab, an anti-constant Fc moiety, was covalently attached to the CM5 sensor surface. A limited amount of the Mab to be detected is immobilized and used to capture the analyte hIGF-IR-ECD. Using constants K for binding and dissociation rates, respectively_aAnd K_dTo characterize the binding of the Mab to the analyte. The equilibrium dissociation constant (KD) was calculated by the ratio of dissociation and association rate constants.

FIG. 11:graph of sensor results for binding and dissociation phases of IgG1 humanized I-3466/hIGF-IR-ECD complex for 5 different concentrations of hIGF-IR-ECD.

Example 1: generation and selection of murine monoclonal antibodies (MAbs)

To generate MAbs that are specific against IGF-IR and do not recognize IR, a protocol comprising 6 screening stages was performed.

It consists of the following stages:

immunizing mice with human recombinant IGF-IR, thereby producing hybridomas,

screening the culture supernatants by ELISA for recombinant proteins used for immunization,

all supernatants of positive hybridomas were tested by ELISA for natural receptors overexpressed on the surface of MCF-7 tumor cells,

-performing a binding assay to select antibodies specifically recognizing IGF-IR,

verifying that the antibodies selected at this stage are capable of inhibiting the proliferation of MCF-7 cells induced by IGF1 in vitro,

confirmation of the in vivo activity of the candidate retained according to the effect on the growth of the tumor MCF-7 in nude mice.

All these different phases and the results obtained will be briefly described in example 1.

For the immunization phase, mice were injected twice subcutaneously with 8 μ g of recombinant IGF-IR. Mice received 3 μ g of recombinant receptor by intravenous injection 3 days before splenocytes were fused with murine myeloma Sp2OAg14 cells. Supernatants from hybridomas were screened by ELISA on plates sensitized with recombinant IGF-IR 14 days after fusion. Hybridomas that remained positive for the supernatant were retained and amplified prior to testing by FACScan analysis, confirming that the antibodies produced were also able to recognize native IGF-IR.

Example 2: [¹²⁵I]-IGF1 and [ [ alpha ] ]¹²⁵I]Inhibition of IGF2 binding to human IGF-1 receptor preparation of Membrane lysates

NIH 3T3 cells stably transfected with human IGF-IR cDNA were cultured in DMEM supplemented with 10% fetal bovine serum. After detachment by scraping, serum-starved cells were further collected by centrifugation. The cell pellet was washed with phosphate buffer and resuspended in lysis buffer: 10mM Tris-HCl pH 7.5 buffer containing a protease inhibitor. Add about 1ml buffer to 25 × 10⁶In individual cells. The cells were further lysed by 3 freeze-thaw cycles followed by 30 Potter homogenizer treatments at 1,900 rpm. After sonication, nuclei and large cell debris were removed by centrifugation at 1,000g for 15 minutes at +4 ℃. Total cell membranes were obtained by centrifugation at 105,000g for 1 hour at +4 ℃. The membrane pellet was washed in lysis buffer and centrifuged at 105,000g for 1 hour at +4 ℃. The final pellet was resuspended in 50mM Tris-HCl buffer containing 150mM NaCl, 0.5% IGEPAL, 0.5% TritonX-100, 0.25% sodium deoxycholate and protease inhibitor and stirred overnight at +4 ℃. By centrifugation at 10,000g for 10 minutes at +4 deg.CThe soluble material is isolated from a soluble extract containing hIGF-IR. The protein concentration of the membrane lysate was measured by BCA (bicinchoninic) method, and IGF-IR was analyzed by Western blotting.

[¹²⁵I]-IGF1 and [ [ alpha ] ]¹²⁵I]Determination of IGF2 binding

Protein A FlashPlate was first coated with commercial monoclonal antibody 17-69(Neomarkers, Fremont, CA, USA), which has been shown to recognize the IGF-IR alpha subunit96 well microplates (Perkin Elmer, Boston, Mass., USA) to immobilize IGF-IR. 200 μ l of 20 μ g/ml antibody solution in PBS was added to each well and incubated overnight at +4 ℃. The buffer containing the residual 17-69 not bound to protein A was removed by aspiration. 200 μ l of 100 μ g/ml membrane lysate were added and incubated at room temperature for 2 hours to immobilize IGF-IR. The uncaptured proteins were removed by aspiration. For competition assays, 100pM of [ mu ] M was measured in a binding buffer containing 50mM Hepes pH7.6, 150mM NaCl, 0.05% Tween 20, 1% bovine serum albumin and 1mM PMSF in the presence of varying concentrations from 1pM to 1 μ M of monoclonal antibody I-3466 or ligands IGF1, IGF2 and insulin (Sigma, Saint-Quantin Fallavier, France)¹²⁵I]IGF1(Perkin Elmer, Boston, MA, USA) or¹²⁵I]Binding of IGF2(Amersham Biosciences, Saclay, France) to immobilized IGF-IR. Plates were incubated at room temperature for 2 hours and then counted on a Packard Top Count microplate scintillation counter. Non-specific binding was determined in the presence of 1 μ M IGF 1. Monoclonal antibody 9G4 (which is not directed against hIGF-IR, but specifically recognizes E.coli proteins) was used as a control mouse IgG1 subtype.

Results

As a function of ligand concentration, on a semi-logarithmic graph¹²⁵I]-IGF1 and [ [ alpha ] ]¹²⁵I]-percentage of specific binding of IGF2 plotted. From the resulting sigmoidal competition curves (FIGS. 1 and 2) graphically determined at 50% (IC)₅₀) Inhibiting radioactivity in a sampleThe concentration of each inhibitor required for body binding.

The IGF1 and IGF2 ligand effectively replace [ 2]¹²⁵I]Binding of IGF1 to immobilized hIGF-IR, whereas insulin and the subtype control antibody 9G4 do not inhibit at a concentration of less than 500nM¹²⁵I]Binding of IGF1 (FIG. 1). Monoclonal antibody I-3466 can have an IC of 0.021nM₅₀(which compares IC determined for non-radiolabeled IGF1₅₀Inhibition by 30 times lower¹²⁵I]Binding of IGF1 (FIG. 1).

Further, the antibody I-3466 exhibits¹²⁵I]Strong binding inhibitory activity of IGF2 on immobilized hIGF-IR (FIG. 2). In this case, IC derived from the competition curve₅₀The value was approximately 0.1 nM. This IGF 2-blocking activity of I-3466 approximates the inhibitory activity induced by IGF2 (IC)₅₀0.06 nM). This is slightly less than the inhibitory activity (IC) of IGF1₅₀0.03nM) and significantly greater than the inhibitory activity (IC) of insulin₅₀200 nM). As expected, the control antibody 9G4 did not show any IGF2 blocking activity.

Example 3: in vitro Effect of I-3466 antibodies on IGF1 or IGF2 induced growth of MCF-7

As noted above, a number of tumor cells overexpress IGF-IR, and it has been reported that in breast and colon cancers, the amount is very significant, with the receptor being provided with a proliferative signal by IGF2 (sometimes written as IGF-2, IGF-II or IGF II I). It must therefore be ensured that MAb I-3466 is able to inhibit IGF1 and IGF2 induced growth of MCF-7 cells in vitro as well. To this end, 200. mu.l of cells in serum-free medium (phenol red-free RPMI medium plus L-glutamine) were incubated at 5X 10⁴Individual cells/well were plated in 96-well plates. 24 hours after inoculation, a final concentration of 50. mu.g/ml (6.6nM) of IGF1 or IGF2 (final concentration of 100ng/ml (13.2nM)) was added to MCF-7 cells in the presence or absence of I-3466 or murine non-neutralizing anti-IGF-IR antibody (7G3) (used as a subtype control) and incubated for an additional 52 hours.

Antibodies were detected at final concentrations ranging from 10. mu.g/ml (66nM) to 0.0097. mu.g/ml (0.065 nM). Subsequently, the [ 2] of 0.25. mu. Ci is used³H]Thymidine pulsed cells for 16 hours and quantification of DNA incorporation by liquid scintillation counting³H]The amount of thymidine. IGF-1 and IGF-2 significantly stimulated the growth of MCF-7 cells (Table 2).

No significant inhibition was observed when cells were treated with increasing doses of subtype control antibody.

In contrast, when cells were incubated with increasing doses of the I-3466 antibody, significant dose-dependent inhibition of proliferation induced by IGF1 (90%) and IGF2 (84%) was observed, with IC₅₀0.7nM and 0.5nM, respectively (Table 2 and FIGS. 3A-3B).

TABLE 2

In vitro Effect of I-3466 antibodies on IGF1(A) or IGF2(B) -induced growth of MCF-7

Example 4: i-3466 inhibits IGF1 and IGF 2-induced phosphorylation of the IGF-IR beta chain

In 20ml phenol red free RPMI at 5X 10⁴Individual cell/cm²(75cm²Plate of (1), COSTAR) MCF7 or HT29 cells were cultured for 24 hours in RPMI mixed with 5mM glutamine, penicillin/streptomycin (100U/100. mu.g/ml, respectively) and 10% fetal bovine serum. After washing three times in PBS, cells were incubated for 12 hours in phenol red free medium (RPMI) containing no fetal bovine serum mixed with 5mM glutamine, penicillin/streptomycin, 0.5. mu.g/ml bovine serum albumin (Sigma A-8022) and 5. mu.g/ml transferrin (Sigma T8158).

For activation, the cells were first incubated with the blocking antibody to be detected (10. mu.g/ml) at 37 ℃ for 15 minutes, followed by addition of IGF1 or IGF2 for an additional 2 minutes. The reaction was stopped by aspirating the incubation medium and the plate was placed on ice. Cells were lysed by adding 0.5ml lysis buffer (50mM tris-HCl pH 7.5, 150mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate) mixed with protease inhibitors (one tablet per 50ml, Boehringer Ref: 1697498) and phosphatase inhibitors (Calbiochem Ref: 524625 (1/100)). The cells were scraped and the suspension recovered and placed on a mixer for 1.5 hours at 4 ℃. The solution was centrifuged at 12,000rpm for 10 minutes (4 ℃) and the protein concentration of the supernatant was quantified by BCA.

Mu.g of cell lysate protein was mixed with anti-IGF-IR (Santa cruz Ref: sc-713) for immunoprecipitation and placed on a mixer and kept at 4 ℃ for 1.5 hours. The immunoprecipitates were recovered by addition of protein A-agarose (Boehringer Ref: 1134515) and placed on a mixer overnight at 4 ℃. Agarose beads were washed twice with 1ml lysis buffer, twice with wash buffer 1(50mM tris-HCl pH 7.5; 500mM NaCl; 0.1% Nonidet P40; 0.05% sodium deoxycholate (Boehringer 1332597) mixed with protease inhibitors and phosphatase inhibitors) and once with wash buffer 2(50mM tris-HCl; 0.1% Nonidet P40; 0.05% sodium deoxycholate (Boehringer Ref: 1332597) mixed with protease inhibitors and phosphatase inhibitors (1/100)). The immunoprecipitates were resuspended in Laemmli buffer and heated to 100 ℃ for 5 minutes. The supernatants were analyzed by polyacrylamide SDS gel (8% Novex EC6015) electrophoresis. Proteins were transferred to nitrocellulose membranes and immunoblotted with either an anti-phosphotyrosine antibody conjugated to HRP (BD transduction LabsPY20) or an anti-IGF-IR beta chain antibody (Santa Cruz Ref: sc 713) followed by an anti-rabbit antibody conjugated to HRP. Blots were visualized by chemiluminescence (Amersham RPN2209) followed by autoradiography on Kodak X-mat AR film.

FIGS. 4A and 4B show MCF-7 cells that were unstimulated (lane 1, panels A and B) or stimulated with 50ng/ml IGF1 (lane 2, panel A) or 100ng/ml IGF2 (lane 2, panel B) alone. As expected, no basal level stimulation of IGF-IR was observed in MCF-7 cells, whereas significant phosphorylation of the IGF-IR beta chain was observed when MCF-7 cells were incubated with IGF1 or IGF 2. No stimulation was observed when cells were treated with IgG1 subtype control (lane 3, panels A and B) or I-3466 antibody alone (lane 4, panels A and B), indicating that I-3466 exhibited no agonistic effect on IGF-IR. When I-3466 was added with IGF1 or IGF2, complete inhibition of ligand-induced phosphorylation was observed (lane 5, panels A and B). The 9G4 antibody used as isotype control had no effect on IGF1 or IGF2 induced phosphorylation (lane 6, panels a and B).

The same inhibitory activity of I-3466 was observed on HT29 cells stimulated with IGF1 or IGF2 (FIGS. 5A and 5B). These results are consistent with the results described in example 2, example 2 showing that I-3466 is capable of displacing IGF1 and IGF2 from IGF-IR.

Example 5: internalization and degradation studies of IGF-IR

Internalization and degradation were analyzed by FACS analysis. Internalization studies were performed using a murine biotinylated anti-IGF-IR monoclonal antibody (Mab) (hereinafter referred to as 12B1 Mab) and binding to a different epitope from the antigen recognized by the I-3466 antibody. 9G4 Mab was introduced as a subtype control. Both antibodies were prepared in our laboratory. Treatment of confluent MCF-7 cells with trypsin and 1X 10 from each cell suspension⁶Individual cells were seeded in FACS buffer on 96-well plates. Plates were incubated with IGF1(50ng/ml) or with 30. mu.g/ml I-3466, 9G4, mIgG1 for 4 hours at 37 ℃.

Cells incubated with FACS buffer alone were used to determine basal levels of IGF-IR expression.

The cells were then washed twice and 20. mu.g/ml biotinylated 12B1 MAb was added to the plate. After incubation at 4 ℃ for 30 min to avoid receptor internalization, cells were washed 3 times at 4 ℃ and purified by addition of streptavidin Alexa Fluor488 conjugates (Molecular Probes Europe BV, Leiden, Netherlands) were stained. For the degradation assay, biotinylated 12B1 and streptavidin Alexa Fluor were usedAn additional cell permeability treatment step was added prior to staining the cells with the 488 conjugate.

Table 3 shows that I-3466 leads to down-regulation of IGF-IR on MCF-7 and HT29 cells after 4 hours of incubation. When cells were incubated with 9G4 Mab (used as a subtype control), no downregulation was observed.

TABLE 3

Internalization/degradation assay for IGF-IR using I-3466 antibody

A-internalization assay

Cell line ID	Examined Mab	MFI 12B1-mIgG1	% internalization
				MCF-7	PBS9G4I-3466	24722631	86
HT29	PBS9G4I-3466	696342	33

B-degradation test

Cell line ID	Examined Mab	MFI 12B1-mIgG1	% degradation
				MCF-7	PBS9G4I-3466	11210537	65
HT29	PBS9G4I-3466	344823	52

Example 6: the I-3466Mab is heterogeneous to DU145, SK-ES-1, HT29, A549 and MCF-7 Antitumor effect of transplanted tumor model

To explore the activity of the I-3466 antibody on tumor growth in vivo, 5 xenograft tumor models were applied: androgen-independent DU145 prostateCarcinoma, osteosarcoma SK-ES-1, colon carcinoma HT29, non-small cell lung carcinoma A549 and breast cancer MCF-7. For this purpose, 2X 10 are used for DU-145, SK-ES-1, HT29, A549 and MCF-7, respectively⁶、5×10⁷、5×10⁶、10×10⁶And 5X 10⁶Individual cells were injected subcutaneously into 6-8 week old female athymic nude mice. For DU-145 and SK-ES-1, mice were treated with 200. mu.g of unpurified antibody 24 hours after cell injection.

Treatment was repeated twice a week. For HT29, when tumor volume reached 49-59mm³Treatment was started and mice were injected intraperitoneally 3 times a week with 0.5mg of purified antibody.

For A549, the initial tumor volume is between 38-43mm³For the MCF-7 test, the initial tumor volume at the beginning of the treatment was between 42 and 59mm³. Tumor volume was evaluated once or twice a week and calculated by the following formula: π/6 × Length × Width × height.

FIGS. 6A-6E show partial results performed with non-purified antibodies, which indicate that I-3466 was able to significantly inhibit tumor growth in vivo for the 5 test cell lines.

Example 7: cloning, preparation and characterization of humanized I-3466 IgG1 antibody

The chemically synthesized variable regions of the light and heavy chains of humanized antibody I-3466 were PCR amplified and cloned into antibody expression vectors carrying human light chain kappa and heavy chain IgG1 constant regions, respectively. The PCR primers contained 36 nucleotides, 15 for IN-Fusion (IN-Fusion) annealing to the overlapping cloning region of the vector and 21 for annealing to the variable region. The exact nucleotide sequence is shown below:

for light chain variable region cloning:

IF-humanized I-3466-LCK-F (SEQ ID No.21)

[5’-ACAGATGCCAGATGCGACATTGTGATGACCCAGTCC]，

IF-humanized I-3466-LCK-R (SEQ ID No.22)

[5’-TGCAGCCACCGTACGCTTGATCTCCACCTTGGTGCC]，

For heavy chain variable region cloning:

IF-humanized I-3466-HCG1-F (SEQ ID No.23)

[5’-ACAGGTGTCCACTCGGAGGTGCAGCTGGTGGAGTCT]，

IF-humanized I-3466-HCG1-R (SEQ ID No.24)

[5’-GCCCTTGGTGGATGCGGAGGAGACTGTCACCAGGGT]

The vector was linearized by digesting the vector with BmtI and FspI. The In-Fusion reaction (In-Fusion reaction) was carried out according to the protocol recommended by the vendor. The resulting clones were verified by sequencing. Human embryonic kidney 293 cells were transfected with light and heavy chain plasmid DNA. The medium containing the secreted antibody was collected and concentrated. The antibody was affinity purified using a protein a/G column and concentrated, and dialyzed in PBS. Protein concentration was determined by OD280nm and purity was analyzed by reducing and non-reducing SDS-PAGE (fig. 9).

Example 8: BIACore analysis of humanized I-3466 IgG1 antibody

Apparatus and materials

The BIAcore T100 instrument, CM5 biosensor chip, HBS-EP buffer, acetate buffer (pH 5), glycine-HCl buffer (pH 1.5), amine coupling kit were all from BIAcore (Upsala, Sweden). Anti-human IgG Fc was from Jackson ImmunoResearch laboratories Inc. (West Grove, USA), and the soluble human insulin-like growth factor-1 receptor (hIGF-IR) extracellular domain (ECD) was from R & D Systems (Minneapolis, USA).

Biacore assay

All experiments were performed at 25 ℃ at a flow rate of 40. mu.l/min. To prepare the BIAcore assay (see FIG. 10), an anti-human IgG-Fc antibody (50. mu.g/ml in acetate buffer, pH 5) was immobilized on a carboxymethyl dextran sensor chip (CM5) using an amine coupling method as described in the instructions. 11042 and 11111 Resonance Units (RU) of anti-IgG Fc antibodies were attached to Flow Cells (FC) 1 and 3, respectively. The purified Mab to be tested was diluted in 0.5% P20, HBS-EP buffer at a concentration of 5. mu.g/ml and injected onto FC3 to achieve 500-1000 RU. FC1 was used as a reference cell. Specific signals (specific signals) correspond to the difference in signals obtained at FC2 versus FC 1. Analytes (soluble hIGF-IR, apparent molecular weight 365kDa) dissolved in 0.5% P20, HBS-EP buffer were injected at 5 different concentrations (100, 50, 25, 12.5 and 6.25nM) during 90 seconds. These concentrations were prepared from stock solutions in 0.5% P20, HBS-EP buffer. During 10 minutes, the dissociation phase of the analyte was monitored. Working buffer (Running buffer) was also injected under the same conditions as a dual control. After each cycle (antibody + hIGF-IR injection), both flow cells were regenerated by injection of 20-45. mu.l glycine-HCl buffer (pH 1.5). Such regeneration is sufficient to remove all captured Mab and Mab/hIGF-IR complex on the sensor chip.

Results

By the association and dissociation rate constants k, respectively_aAnd k_dMab humanized I-3466 was characterized for binding to the analyte hIGF-IR-ECD. The equilibrium dissociation constant (KD) was calculated by the ratio of the dissociation and association rate constants. The results are given in table 4 below. A graph of sensor results corresponding to different analyte concentrations is shown in fig. 11.

TABLE 4

IGF-IRAbs	ka(1/Ms)	kd(1/s)	KD(M)
				IgG1 HI3466	1.84E+05	6.29E-05	3.42E-10

Sequence listing

<110> Pieeerfahole pharmaceutical Co

Liliyana, Geqi

Natales Kelvya

<120> novel anti-IGF-IR antibodies and uses thereof

<130>D23693

<150>FR 05/07829

<151>2005-07-22

<150>US 60/701,622

<151>2005-07-22

<160>24

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Claims (46)

1. An isolated antibody or a functional fragment thereof, characterized in that it comprises a heavy chain comprising the three CDRs of sequences SEQ ID nos. 2, 4 and 6 and in that it further comprises a light chain comprising the three CDRs of sequences SEQ ID nos. 1, 3 and 5.

2. The antibody or one of its functional fragments according to claim 1, which has binding affinity for the human insulin-like growth factor I receptor (IGF-IR), characterized in that it inhibits the binding of the natural binding partner IGF1 to said IGF-IR with an IC50 of less than 0.3nM and that it also inhibits the binding of the natural binding partner IGF2 to said IGF-IR with an IC50 of less than 0.3nM when binding to said IGF-IR.

3. The isolated antibody or one of its functional fragments according to claim 1, which has human insulin-like growth factor I receptor (IGF-IR) tyrosine kinase inhibitory activity, characterized in that, upon binding to said IGF-IR, it inhibits the binding of the natural binding partner IGF1 to said IGF-IR with an IC50 of less than 0.3nM and it also inhibits the binding of the natural binding partner IGF2 to said IGF-IR with an IC50 of less than 0.3 nM.

4. The antibody or one of its functional fragments according to claim 1, characterized in that it is also capable of 100% inhibition of IGF1 and/or IGF2 induced phosphorylation of the IGF-IR β chain.

5. The antibody or one of its functional fragments according to claim 1, characterized in that it is devoid of any intrinsic agonist activity.

6. The antibody or one of its functional fragments according to claim 1, characterized in that it is capable of inducing, by the same FACS analysis method:

i) at least 30% of IGF-IR internalization on HT29 cells, and/or

ii) at least 85% of IGF-IR internalization on MCF-7 cells.

7. The antibody or one of its functional fragments according to claim 1, characterized in that it is capable of inducing, by the same FACS analysis method:

i) at least 50% degradation of IGF-IR on HT29 cells, and/or

ii) at least 65% degradation of IGF-IR on MCF-7 cells.

8. The antibody or one of its functional fragments, according to claim 1, characterized in that it is capable of inhibiting IGF1 and IGF 2-induced proliferation of MCF-7 cells in vitro with an IC50 directed at least equal to 1 and 0.5nM, respectively, against IGF1 and IGF 2.

9. Antibody or one of its functional fragments, according to claim 1, characterized in that it does not attach in a significant way to the human Insulin Receptor (IR).

10. Antibody or one of its functional fragments, according to claim 1, characterized in that said functional fragment is selected from the group consisting of fragments Fv, Fab, F (ab')₂Fab', scFv-Fc and bifunctional antibodies.

11. The antibody or a functional fragment thereof according to claim 1, characterized in that said antibody comprises a light chain comprising the amino acid sequence SEQ ID No.7 and it comprises a heavy chain comprising the amino acid sequence SEQ ID No. 8.

12. The antibody or one of its functional fragments, according to claim 1, characterized in that said antibody is a chimeric antibody and further comprises light and heavy chain constant regions derived from an antibody of a species heterologous to the mouse.

13. Chimeric antibody or one of its functional fragments, according to claim 12, characterized in that said heterologous species is human.

14. Chimeric antibody or one of its functional fragments, according to claim 13, characterized in that the light and heavy chain constant regions derived from human antibodies are the kappa and gamma-1, gamma-2 or gamma-4 regions, respectively.

15. Humanized antibody or a functional fragment thereof according to claim 14, characterized in that it comprises a light chain comprising the amino acid sequence SEQ ID No.17 and it comprises a heavy chain comprising the amino acid sequence SEQ ID No. 18.

16. A murine hybridoma capable of secreting the antibody of claim 1 deposited with the CNCM of the institute for pasteur in paris at 23 days 6/2005 with numbers 1-3466.

17. An antibody or a functional fragment thereof, characterized in that said antibody is secreted by a hybridoma cell according to claim 16.

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

a) a nucleic acid encoding the antibody of claim 1 or a functional fragment thereof;

b) nucleic acids complementary to the nucleic acids as defined under a).

19. A vector comprising the nucleic acid of claim 18.

20. A host cell comprising the vector of claim 19.

21. Method for producing the antibody of claim 1 or one of its functional fragments, characterized in that it comprises the following phases:

a) culturing the cell of claim 20 in a culture medium and under suitable culture conditions; and

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

22. A composition comprising as an active ingredient a compound consisting of the antibody of claim 1 or one of its functional fragments.

23. Composition according to claim 22, characterized in that it comprises at least a second compound chosen from compounds capable of specifically inhibiting the tyrosine kinase activity of IGF-IR, EGFR, HER2/neu, cMET and/or RON.

24. Composition according to claim 23, characterized in that said second compound is selected from the group consisting of isolated anti-EGFR, -IGF-IR, -HER2/neu, -cMET and/or-RON antibodies or functional fragments thereof, capable of inhibiting the proliferative and/or anti-apoptotic and/or angiogenic and/or metastatic fringing induction activity mediated by said receptor.

25. Composition according to claim 23, characterized in that it comprises at least one inhibitor of tyrosine kinase activity of IR, IGF-IR, EGFR, HER2/neu, cMET and/or RON as a combined product for simultaneous, separate or sequential use.

26. A composition according to claim 25, characterised in that the inhibitor of tyrosine kinase activity is selected from the group consisting of diphenylamino phthalimides, pyrazolo-or pyrrolopyridopyrimidines or quinazolines.

27. Composition according to claim 23, characterized in that it further comprises a cytotoxic/cytostatic agent as a combined product for simultaneous, separate or sequential use.

28. The composition according to claim 27, characterized in that said cytotoxic/cytostatic agent is chosen from agents that interact with DNA, antimetabolites, topoisomerase I or II inhibitors, or spindle inhibitors or stabilizers or any other agent that can be used in chemotherapy.

29. The composition according to claim 27, characterized in that said cytotoxic/cytostatic agent is chemically coupled to at least one component of said composition for simultaneous application.

30. Composition according to claim 27, characterized in that the cytotoxic/cytostatic agent is chosen from spindle inhibitors or stabilizers.

31. Composition according to claim 30, characterized in that said cytotoxic/cytostatic agent is chosen from vinorelbine, vinflunine or vincristine.

32. Composition according to claim 23, characterized in that at least one of said antibodies or one of its functional fragments is conjugated with a cytotoxin and/or a radioactive element.

33. Use of an antibody or one of its functional fragments according to claim 1 for the preparation of a medicament, characterized in that said medicament is used for the prevention or treatment of a cancer selected from prostate cancer, osteosarcoma, non-small cell lung cancer, breast cancer or colon cancer.

34. Use according to claim 33, characterized in that the cancer is colon cancer.

35. Use of an antibody of claim 1 for the preparation of a composition for use in a method for in vitro diagnosis of a pathological condition characterized by abnormal expression of IGF-IR relative to the normal, said method comprising contacting a biological sample suspected of containing IGF-IR with said composition under conditions conducive to the formation of an IGF-IR/antibody complex, and detecting said complex as an indication of the presence of said IGF-IR in said sample.

36. The use according to claim 35, wherein the antibody is detectably labeled.

37. The use according to claim 35, wherein the aberrant expression of IGF-IR is overexpression of IGF-IR.

38. The use according to claim 35, wherein the aberrant expression of IGF-IR is under-expression of IGF-IR.

39. Use of an antibody of claim 1 for the preparation of a composition for use in an in vitro diagnostic method for predicting the oncogenic potential of a prostate cell sample, said method comprising the steps of:

(a) providing a sample of human lung or breast tissue; and

(b) determining whether IGF-IR is present in a sample, said step comprising contacting said sample with said composition under conditions conducive to the formation of an IGF-IR/antibody complex, wherein the presence of said complex is indicative of the oncogenic potential of said cell in said tissue.

40. Use according to claim 39, wherein the presence of said complex is indicative of a patient at risk of developing or developing a pathological condition characterized by overexpression of said IGF-IR.

41. Use of an antibody of claim 1 for the preparation of a composition for use in a method of tracking the progress of a treatment regimen designed to alleviate a pathological condition characterized by abnormal IGF-IR expression, said method comprising the steps of:

(a) analyzing a sample from the subject to determine IGF-IR levels at a first time point;

(b) analyzing the sample at a second time point; and

(c) comparing said level at said second time point with the level determined in (a) as a determination of the effect of said treatment regimen, wherein a decrease in the level of IGF-IR in said sample determines regression of said pathological condition in said subject, or an increase in the level of IGF-IR determines progression of said pathological condition in said subject.

42. A kit or a combination thereof for carrying out a method for diagnosing a disease caused by overexpression or underexpression of IGF-IR or for carrying out a method for detecting and/or quantifying overexpression or underexpression of IGF-IR in a biological sample, characterized in that it comprises the following components:

a) the antibody of claim 1 or a functional fragment thereof;

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

c) optionally, reagents allowing the validation of the IGF-IR/antibody complex produced by the immune reaction.

43. Use of the antibody of claim 1 or one of its functional fragments for the preparation of a medicament intended for the specific targeting of a biologically active compound to cells expressing or overexpressing IGF-IR.

44. A method for modulating IGF-IR activity in IGF-IR responsive mammalian cells for non-therapeutic purposes, comprising: contacting a cell with the antibody of claim 1 under conditions sufficient to modulate the IGF-IR activity relative to a non-IGF-IR reactive cell.

45. A method for reducing IGF-IR activity in an IGF-IR reactive mammalian cell for a non-therapeutic purpose relative to a normal cell, comprising: contacting a cell with the antibody of claim 1.

46. A method of purifying IGF-IR from a sample, the method comprising: a) incubating the antibody of claim 1 with a sample under conditions that allow specific binding of the antibody to the receptor, and b) isolating the antibody from the sample and obtaining the purified receptor.