HK1102600B - Novel anti-igf-ir antibodies and uses thereof - Google Patents

Abstract

The designated patent number for the application is CN101014625A.

Description

Novel anti-IGF-IR antibodies and uses thereof

The present invention relates to novel antibodies capable of binding specifically to the human insulin-like growth factor I receptor IGF-IR and/or capable of inhibiting specifically the tyrosine kinase activity of said IGF-IR, in particular monoclonal antibodies of murine, chimeric and humanized origin, as well as the amino acid and nucleic acid sequences coding for these antibodies. The invention also comprises the use of these antibodies as a medicament for the prophylactic and/or therapeutic treatment of cancers overexpressing IGR-IR or any disease associated with overexpression of said receptor, as well as the use of these antibodies in a method or kit for the diagnosis of a disease associated with overexpression of IGF-IR. Finally the invention comprises products and/or compositions comprising these antibodies in combination with anti-EGFR antibodies and/or anti-VEGF antibodies and/or antibodies against other growth factors involved in tumor progression or metastasis and/or compounds and/or anti-cancer agents or agents conjugated to 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 #1nM), 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, Bioehim. 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 (Valentis B.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.

Cancer pathology is characterized by uncontrolled cell growth. In several cancers, growth factors specifically bind to their receptors and then transmit growth, transformation and/or survival signals to tumor cells. Overexpression of growth factor receptors on the surface of tumor cells is extensively described (Salomon D.S.et al, Crit.Rev.Oncol.Hematol., 1995, 19: 183; Burrow S.et al, J.Surg.Oncol., 1998, 69: 21; Hakam A.et al, hum.Pathol., 1999, 30: 1128; Railo M.J.et al, Eur.J.cancer, 1994, 30: 307; Happerfield.L.C.et al, J.Pathol., 1997, 183: 412). This overexpression or aberrant activation leads to direct interference with cell growth regulation mechanisms and may also affect the sensitivity of cells to apoptosis induced by classical chemotherapy or radiotherapy.

During the last years, it has been shown that the respective humanization () Or chimeric (C225) antibodies target tumor cell surface overexpressed growth factor receptors such as EGFR or Her2/neu, resulting in significant inhibition of tumor growth in patients and in significant increase in efficacy of classical chemotherapy treatment (Carter p., Nature rev. cancer, 2001, 1 (2): 118; hortobagyi g.n., seminin. oncol., 2001, 28: 43; herbst r.s.et al, semin.oncol, 2002, 29: 27). In some preclinical studies, other receptors such as IGF-IR or VEGF-R (vascular endothelial growth factor receptor) have been identified as potential targets.

More specifically, IGF-IR is part of the tyrosine kinase receptor. It shows a high homology to the Insulin Receptor (IR), which exists in two isoforms a and B.

The sequences of IR isoforms a and B are registered in NCBI GenBank 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; Dumesicet 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 (dockingproteins), including the insulin receptor substrate-1 protein family (IRS1), Gab1 and Shc (Avrucch, 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 (Sasaoka et al, 1996, Endocrinology 137, 4427-4434; Nakae et al, 2001, endocr. Rev.22, 818-835; Dupont and LeRoith 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.

Human IR exists in two isoforms 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 isoforms is regulated by tissue-specific and unknown factors (Moller et al, 1989, mol. Endocrinol., 3, 1263-. IR-B is the predominant IR isoform in normal adult tissues (adipose tissue, liver and muscle), which are the major target tissues for insulin metabolism (Moller et al, 1989; Mosthaf et al, 1990). IR-A is the predominant isoform 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 Biological Chemistry, Vol.277, N.P.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 isoforms 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 (CrkII), 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.

The realization of such "therapeutic tools" capable of blocking both receptors simultaneously is of particular interest, since they would allow to avoid escape phenomena mediated by the expression or abnormal activation of IGF-IR and hybrid-R in the same tumor.

With regard to the increased interest in IGF-IR and more particularly monoclonal antibodies capable of binding to or inhibiting IGF-IR tyrosine kinase activity, the applicant has developed and characterized humanized monoclonal antibodies known as 7C10 or h7C10 (encoding F50035). International patent application PCT/FR 03/00178, which relates to such antibodies and their use, has been filed and is disclosed in publication number WO03/059951 at 24/7/2003. The contents of this patent application are incorporated herein by reference.

It is an object of the present invention to be able to have available other murine monoclonal antibodies, preferably chimeric or humanized antibodies, which will recognize IGF-IR specifically and with high affinity. These antibodies will interact with little or no IR. During the interaction between IGF1/IGF-IR and IGF2/IGF-IR, their attachment will inhibit IGF-IR expressing tumor growth in vitro by interacting primarily with activated signal transduction pathways. These antibodies are active in vivo against all tumors expressing IGF-IR, including estrogen-dependent tumors of the breast and prostate tumors.

The invention can also block hybrid-R and IGF-IR activity in combination by generating high affinity compounds, and more particularly antibodies, capable of binding to both receptors, and can also block their activation by IGF1, IGF2, or insulin.

The invention also relates to the use of an isolated antibody or fragment thereof according to the invention, which antibody or fragment is capable of binding to i) human IGF-IR, and/or inhibiting the binding of its natural ligand, preferably IGF1 and/or IGF2, and/or is also capable of specifically inhibiting the tyrosine kinase activity of said IGF-IR, and/or which antibody or fragment is capable of binding to ii) hybrid-R, and/or inhibits the binding of its natural ligand, preferably IGF1, IGF2 and/or insulin, and/or is also capable of specifically inhibiting the tyrosine kinase activity of said hybrid-R.

According to another preferred embodiment, the antibody is used for cancer therapy, more particularly for breast cancer therapy.

Indeed, it is known that breast tumor cells present IGF-IR specifically on their surface, as well as a large number of insulin receptors, and therefore a large number of hybrid-R (Frasca et al, 1999; Sciac et al, 1999; Vella et al, 2001).

More specifically, the present invention relates to four different anti-IGF-IR monoclonal antibodies.

In a first aspect, the 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, if necessary, and preferably capable of inhibiting the natural attachment of the ligands IGF1 and/or IGF2 of IGF-IR and/or capable of specifically inhibiting the tyrosine kinase activity of said IGF-IR; characterized in that it comprises a light chain comprising at least one complementarity determining region CDR selected from the CDRs of amino acid sequences SEQ ID Nos.1, 2 and 3, or at least one CDR having at least 80%, preferably 85%, 90%, 95% and 98% identity in its sequence after optimal alignment with the sequences SEQ ID Nos.1, 2 and 3; or characterized in that it comprises a heavy chain comprising at least one complementarity determining region CDR selected from the CDRs of amino acid sequences SEQ ID Nos.4, 5 and 6, or at least one CDR having at least 80%, preferably 85%, 90%, 95% and 98% identity in its sequence after optimal alignment with the sequences SEQ ID Nos.4, 5 and 6.

In this specification and the corresponding examples, this antibody will be referred to as 13F 5.

In this specification, the terms "bonded" and "attached" have the same meaning and are interchangeable.

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.moi.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, 575Science Dr., Madison, Wis, or by BLAST N or BLAST P comparison Software).

The percent identity between two nucleic acid or amino acid sequences is determined by comparing the two sequences 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 2sequences-a new tools for matching proteins and nucleotides", FEMS Microbiol Lett.174: 247-250) may be used, which is available from the websites http:// www.ncbi.nlm.nih.gov/gorf/bl2.html, with the parameters using default values (especially for the parameters "open gap dependency": 5, and "extension gap dependency": 2; the matrix selected is, for example, the matrix suggested by the program, "BLOSUM 62"), by which the percent identity between the two sequences to be compared is calculated directly.

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. These equivalent amino acids can be determined by structural homology to the amino acids they replace, or by the results of a comparison of the biological activities of the different antibodies that can be performed.

By way of example, mention should be made of the possibility of substitutions which can be carried out without leading to deep modifications which correspondingly modify the biological activity of the antibody. Thus, it is naturally conceivable that leucine may be substituted with valine or isoleucine, aspartic acid with glutamic acid, glutamine with asparagine, arginine with lysine, and the like, and that reverse substitution may be performed under the same conditions.

In a second aspect, the 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, if necessary, preferably capable of inhibiting the natural attachment of the ligands IGF1 and/or IGF2 of IGF-IR and/or capable of specifically inhibiting the tyrosine kinase activity of said IGF-IR; characterized in that it comprises a light chain comprising at least one complementarity determining region CDR selected from the CDRs of amino acid sequences SEQ ID Nos.7, 8 and 9, or at least one CDR having at least 80%, preferably 85%, 90%, 95% and 98% identity in its sequence after optimal alignment with the sequences SEQ ID Nos.7, 8 and 9; or characterized in that it comprises a heavy chain comprising at least one complementarity determining region CDR selected from the CDRs of amino acid sequences SEQ ID Nos.10, 11 and 12, or at least one CDR having at least 80%, preferably 85%, 90%, 95% and 98% identity in its sequence after optimal alignment with the sequences SEQ ID Nos.10, 11 and 12.

In the following description, this antibody will be referred to as 12D 5.

In a third aspect, the 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, if necessary, preferably capable of inhibiting the natural attachment of the ligands IGF1 and/or IGF2 of IGF-IR and/or capable of specifically inhibiting the tyrosine kinase activity of said IGF-IR; characterized in that it comprises a light chain comprising at least one complementarity determining region CDR selected from the CDRs of amino acid sequences SEQ ID Nos.13, 14 and 15, or at least one CDR having at least 80%, preferably 85%, 90%, 95% and 98% identity in its sequence after optimal alignment with the sequences SEQ ID Nos.13, 14 and 15; or characterized in that it comprises a heavy chain comprising at least one complementarity determining region CDR selected from the CDRs of amino acid sequences SEQ ID Nos.16, 17 and 18, or at least one CDR having at least 80%, preferably 85%, 90%, 95% and 98% identity in its sequence after optimal alignment with the sequences SEQ ID Nos.16, 17 and 18.

In the following description, this antibody will be referred to as 2D 10.

Finally, in another aspect, the subject of the 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, if necessary, but preferably capable of inhibiting the natural attachment of the ligands IGF1 and/or IGF2 of IGF-IR and/or capable of specifically inhibiting the tyrosine kinase activity of said IGF-IR, characterized in that it consists of an antibody named 21E3 and is registered at CNCM as described below.

The antibodies according to the invention, namely 13F5, 12D5, 2D10 and 21E3, are preferably specific monoclonal antibodies, in particular of murine, chimeric or humanized origin, which can be obtained by the person skilled in the art according to well-known standard methods.

In general, for the preparation of monoclonal Antibodies or functional fragments thereof, in particular of murine origin, reference may be made to the techniques described in particular in the Manual "Antibodies" (Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp.726, 1988) or to the techniques described by Kohler and Milstein, prepared from hybridoma cells (Nature, 256: 495 497, 1975).

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

The monoclonal antibody according to the invention can be purified, for example, on an affinity column on which IGF-IR or one of its fragments comprising an epitope that can be specifically recognized by the monoclonal antibody according to the invention has been immobilized beforehand. More specifically, the monoclonal antibody can be purified by protein a and/or G chromatography, with or without ion exchange chromatography on residual protein contaminants and DNA and LPS, with or without exclusion chromatography on Sepharose gels to eliminate potential aggregates due to the presence of dimers or other multimers. In a more preferred manner, all of these techniques may be used simultaneously or sequentially.

Similarly, chimeric or humanized antibodies are also included in antibodies according to the invention.

Chimeric antibody, which means an antibody comprising native variable (light and heavy) regions derived from a given species, in combination with the light and heavy constant regions of an antibody of another species, which is heterologous to the given species.

The chimeric type antibody or a fragment thereof according to the present invention can be produced by using a gene recombination technique. For example, the chimeric antibody can be produced by cloning a recombinant DNA comprising a promoter and sequences encoding the variable region of a non-human, especially murine, monoclonal antibody according to the invention, as well as sequences encoding the constant region of a human antibody. The chimeric antibody of the present invention encoded by such a recombinant gene will be, for example, a mouse-human chimera, the specificity of which is determined by the variable region derived from mouse DNA and the isotype of which is determined by the constant region derived from human DNA. For a method for producing a chimeric antibody, reference may be made, for example, to Verhoeyn et al (BioEssays, 8: 74, 1988).

Humanized antibody means an antibody comprising CDR regions derived from a non-human antibody and the remainder of the antibody molecule is derived from one (or several) human antibodies. Furthermore, to preserve binding affinity, some residues of the backbone (known as FR) segment may be modified (Jones et al, Nature, 321: 522-S525, 1986; Verhoeyen et al, Science 239: 1534-S1536, 1988; Riechmann et al, Nature, 332: 323-S327, 1988).

Humanized antibodies or fragments thereof according to the invention may be prepared by techniques known to those skilled in the art (e.g., as described in the documents Singer et al, J.Immun.150: 2844-. These antibodies according to the invention are preferably used in vitro diagnostic methods, or in vivo prophylactic and/or therapeutic treatments.

A functional fragment of an antibody according to the invention is meant to refer in particular to 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 which should be able to increase the half-life by chemical modification, for example by addition of a poly (alkylene) glycol such as polyethylene glycol ("PEGylation", PEGylation ") (PEGylated fragments known as Fv-PEG, scFv-PEG, Fab-PEG, F (ab ') 2-PEG or Fab' -PEG) (" PEG "is polyethylene glycol), said fragment having at least one CDR which is characteristic of the sequence according to the invention SEQ ID Nos.1-6, 7-12 or 13-18, and being in particular characterized in that it is able to exert in a usual manner even part of the activity of the antibody from which it is derived, such as, inter alia, the ability to recognize and bind to IGF-IR and, if desired, the ability to inhibit IGF-IR activity.

Preferably, the functional fragment will consist of or comprise a partial sequence of the heavy or light variable chain of the antibody from which it is derived, said partial sequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably 1/100 being at least equal to the affinity of the antibody from which it is derived, and more preferably 1/10 in the case of 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 antibody sequence from which they are derived.

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

More specifically, the invention comprises an antibody or a functional fragment thereof according to the invention, in particular a chimeric or humanized antibody, obtained by genetic recombination or by chemical synthesis.

According to the first approach, the antibody will be defined by the heavy chain sequence.

In a first preferred way, the invention relates to an antibody or one of its functional fragments according to the invention, characterized in that it comprises a heavy chain comprising at least two of the three CDRs or three CDRs of sequence SEQ ID nos.4 to 6 or at least two of the three CDRs or three CDRs of sequence SEQ ID nos.4 to 6, respectively, having at least 80% identity after optimal alignment.

In a second preferred manner, the invention relates to an antibody or one of its functional fragments according to the invention, characterized in that it comprises a heavy chain comprising at least two of the three CDRs or three CDRs of sequence SEQ ID nos.10 to 12, or at least two of the three CDRs or three CDRs of sequence SEQ ID nos.10 to 12, respectively, having at least 80% identity after optimal alignment.

In a third preferred way, the invention relates to an antibody or one of its functional fragments according to the invention, characterized in that it comprises a heavy chain comprising at least two of the three CDRs or three CDRs of sequence SEQ ID nos.16 to 18, or at least two of the three CDRs or three CDRs of sequence SEQ ID nos.16 to 18, respectively, having at least 80% identity after optimal alignment.

According to a second approach, the antibody will now be defined by the light chain sequence.

In a first embodiment, which is also preferred, the antibody according to the invention or one of its functional fragments is characterized in that it comprises a light chain comprising at least one CDR chosen from the CDRs of sequences SEQ ID nos.1 to 3 or a CDR whose sequence has at least 80% identity with sequences SEQ ID nos.1 to 3 after optimal alignment.

In a second embodiment, the antibody or one of its functional fragments according to the invention is characterized in that it comprises a light chain comprising at least one CDR chosen from the CDRs of sequences SEQ ID nos.7-9 or CDRs whose sequence has at least 80% identity with sequences SEQ ID nos.7-9 after optimal alignment.

In a third embodiment, the antibody or one of its functional fragments according to the invention is characterized in that it comprises a light chain comprising at least one CDR chosen from the CDRs of sequences SEQ ID nos.13 to 15 or a CDR whose sequence has at least 80% identity with sequences SEQ ID nos.13 to 15 after optimal alignment.

According to a third approach, antibodies will now be defined by a light chain sequence and a heavy chain sequence.

In a first preferred way, the antibody or one of its functional fragments according to the invention is characterized in that it comprises a heavy chain comprising the three CDRs of sequences SEQ ID nos.4 to 6 or having at least 80% identity after optimal alignment with sequences SEQ ID nos.4 to 6 and in that it further comprises a light chain comprising the three CDRs of sequences SEQ ID nos.1 to 3 or having at least 80% identity after optimal alignment with sequences SEQ ID nos.1 to 3.

In a second preferred manner, the antibody or one of its functional fragments according to the invention is characterized in that it comprises a heavy chain comprising the three CDRs of sequence SEQ ID nos.10 to 12 or having at least 80% identity after optimal alignment with sequence SEQ ID nos.10 to 12 and in that it further comprises a light chain comprising the three CDRs of sequence SEQ ID nos.7 to 9 or having at least 80% identity after optimal alignment with sequence SEQ ID nos.7 to 9.

In a third preferred way, the antibody or one of its functional fragments according to the invention is characterized in that it comprises a heavy chain comprising the three CDRs of sequences SEQ ID nos.16 to 18 or having at least 80% identity after optimal alignment with sequences SEQ ID nos.16 to 18 and in that it further comprises a light chain comprising the three CDRs of sequences SEQ ID nos.13 to 15 or having at least 80% identity after optimal alignment with sequences SEQ ID nos.13 to 15.

In another preferred embodiment, the antibody or one of its functional fragments, according to the invention and referred to as 13F5, is characterized in that it comprises a heavy chain having the sequence comprising the amino acid sequence SEQ ID No.20 and in that it further comprises a light chain having the sequence comprising the amino acid sequence SEQ ID No. 19.

In another preferred embodiment, the antibody according to the invention and referred to as 12D5, or one of its functional fragments, is characterized in that it comprises a heavy chain having the sequence comprising amino acid sequence SEQ ID No.22 or 23, and in that it further comprises a light chain having the sequence comprising amino acid sequence SEQ ID No. 21.

In another preferred embodiment, the antibody according to the invention and referred to as 2D10, or one of its functional fragments, is characterized in that it comprises a heavy chain having the sequence comprising amino acid sequence SEQ ID No.25 and in that it further comprises a light chain having the sequence comprising amino acid sequence SEQ ID No. 24.

Another possibility is, as part of the invention, an antibody wherein the three CDRs of the heavy chain are randomly selected from the CDRs of 13F5, 12D5 and 2D10, respectively, and wherein the three CDRs of the light chain are also randomly selected from the CDRs of 13F5, 12D5 and 2D10, respectively.

According to another aspect, a 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 the fragments Fv, scFv, Fab, (Fab ') 2, Fab', scFv-Fc or diabody, or any functional fragment which should increase the half-life by chemical modification, in particular by pegylation, or by incorporation into liposomes.

According to another aspect, the invention relates to a murine hybridoma capable of secreting a monoclonal antibody according to the invention, in particular a murine hybridoma deposited, for example, at Centre National De Culture De Microorganisme (CNCM, National Centre for microbial cultures) (Institut Pasteur, Paris, France).

The monoclonal antibody referred to herein as 13F5, or one of its functional fragments, is of course part of the present invention, characterized in that it is secreted by the hybridoma deposited at the CNCM with the number CNCM I-3193 at 3, 25 of 2004. The hybridoma is a murine hybridoma obtained by cell fusion (Sp20Agl4) of splenocytes from immunized mice and a myeloma cell line.

The monoclonal antibody referred to herein as 12D5, or one of its functional fragments, is of course part of the present invention, characterized in that it is secreted by the hybridoma deposited with the CNCM at 4,8, 2004 with the number CNCM I-3195. The hybridoma is a murine hybridoma obtained by fusing splenocytes from immunized mice with cells of a myeloma cell line (Sp20Agl 4).

The monoclonal antibody referred to herein as 2D10, or one of its functional fragments, is of course part of the present invention, characterized in that it is secreted by the hybridoma deposited with CNCM at 5/13 of 2004 with the number CNCM I-3214. The hybridoma is also a murine hybridoma obtained from the fusion of splenocytes from immunized mice with cells of a myeloma cell line (Sp20Agl 4).

The monoclonal antibody, herein designated 21E3, or one of its functional fragments, is of course part of the present invention, characterized in that it is secreted by the hybridoma deposited with the CNCM at 7/1/2004 with the number CNCM I-3249. The hybridoma was also a murine hybridoma obtained by cell fusion of immunized mouse splenocytes with a myeloma cell line (Sp20Agl 4).

According to an equally specific 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 light and heavy chain constant regions derived from antibodies of a species heterologous to the mouse, in particular human, and in a preferred manner 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.

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

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

Nucleic acids, nucleic sequences or nucleic acid sequences, polynucleotides, oligonucleotides, polynucleotide sequences, nucleotide sequences, these terms being used indifferently in the present invention, 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 capable of corresponding to double-stranded DAN, 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.

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 a host cell according to the invention in a culture medium and under suitable culture conditions; and

b) recovering said antibody or one of its functional fragments, produced starting from the culture medium or from said 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.) or using partially 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 as further described above, characterized in that it is also capable of specifically binding to the human epidermal growth factor receptor EGFR and/or is capable of specifically inhibiting the tyrosine kinase activity of said EGFR.

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

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.

In a particularly preferred embodiment, the composition, as a combination product according to the invention, is characterized in that the cytotoxic/cytostatic agent is selected from agents which interact with DNA, antimetabolites, topoisomerase I or II inhibitors, or spindle inhibitors or stabilizers, or any agents which can be used in chemotherapy. These cytotoxic/cytostatic agents, for each of the aforementioned types of cytotoxic agents, are cited, for example, in the VIDAL 2001 edition on the page of the cancerology and hematology column "cytotoxic agents" related compounds, the cytotoxic compounds cited herein by reference are cited as preferred cytotoxic agents.

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

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

In order 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 between the two compounds to be coupled, such as a poly (alkylene) glycol, such as polyethylene glycol, or an amino acid, or in another embodiment to use an active derivative of the cytotoxic agent, in which a function capable of reacting with the antibody according to the invention should have been introduced. These coupling techniques are well known to those skilled in the art and will not be detailed in this specification.

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

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 receptor IGF-IR, such as especially 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.

Preferably coupled to the antibody used for the therapeutic purposes of the radioactive element (or radioactivity)Isotope) is a gamma-ray 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 may 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 diseases induced by the overexpression and/or the aberrant activation of the IGF-I receptor and/or associated with an overactivation of the signal transduction pathway mediated by the interaction of 1-IGF1 or 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 tumor characteristics, preferably having IGF-dependence, in particular IGF 1-and/or IGF 2-dependence.

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 having IGF-dependence, in particular IGF 1-and/or IGF 2-dependence.

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 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, the epidermal hyperproliferation of which may be linked to the expression or overexpression of IGF-IR and/or to the overactivation of a signal transduction pathway mediated by the interaction of IGF-IR with its natural ligands (Wraight CJ.et al, Nat.Biotechnol., 2000, 18 (5): 521-526. reversal of epidermal hyperproliferation in psoriasis by an insulin-like growth factor I receptor antisense oligonucleotide). In another embodiment, an object 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 atherosclerosis.

Among the cancers that can be prevented and/or treated, prostate cancer, osteosarcoma, lung cancer, breast cancer, endometrial cancer or colon cancer or any other cancer that overexpresses IGF-IR is preferred.

According to another aspect, a subject of the present invention is a method for diagnosing, preferably in vitro, diseases associated with the overexpression or underexpression, preferably overexpression, of the IGF-I receptor, said IGF-I receptor being derived from a biological sample suspected of having an abnormal presence of the IGF-I receptor, characterized in that said biological sample is contacted with an antibody according to the invention, or one of its functional fragments, which antibody may be labeled, if necessary.

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

In another embodiment, the antibodies according to the invention can also be used for the treatment, prevention and/or diagnosis of diseases associated with not only IGF-IR overexpression but also hybrid-R overexpression.

More specifically, the antibody according to the invention is characterized in that it is also capable of binding to hybrid-R, isoforms a and/or B and of inhibiting the binding of its natural ligands, preferably IGF1 and/or IGF2 and/or insulin specified herein, and/or of specifically inhibiting the tyrosine kinase activity of said hybrid-R.

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.

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 includeChemiluminescent labels such as luminol, dioxetanes (dioxetanes), bioluminescent labels such as luciferase and luciferin, or radioactive labels such as iodine¹²³Iodine, iodine¹²⁵Iodine, iodine¹²⁶Bromine, 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 the IGF-I receptor in a biological sample, characterized in that it comprises the following steps:

a) contacting an antibody or functional fragment thereof according to the invention with a biological sample; 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 can be used in a method for detecting and/or quantifying the IGF-I receptor 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 the IGF-I receptor must 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.

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.

The invention therefore also comprises a kit or kit necessary for carrying out a method for the diagnosis of a disease induced by overexpression or underexpression of the IGF-I receptor or for carrying out a method for the detection and/or quantification of overexpression or underexpression of the IGF-I receptor in a biological sample, characterized in that said kit or kit comprises the following elements:

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

b) optionally reagents for forming a medium suitable for an immune response;

c) optionally reagents that allow the demonstration of the IGF-IR/antibody complex produced by the immune reaction.

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 the IGF-I receptor.

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 the IGF-I receptor.

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 the IGF-I receptor 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 appear in the continuation of the description, with the embodiments and the accompanying drawings, in which:

FIG. 1 shows the in vitro evaluation of anti-IGF-IR antibodies in the MCF-7 model,

FIGS. 2 and 3 represent the in vivo evaluation of anti-IGF-IR antibodies on DU145,

FIG. 4 shows that on intact cells expressing IGF-IR¹²⁵I]-a replacement for IGF-1,

FIG. 5 shows on immunocaptured HR-A [ alphA ], [ alphA ]¹²⁵I]-a replacement for IGF-1,

FIG. 6 shows on immunocaptured HR-B¹²⁵I]-a replacement for IGF-1,

FIG. 7 shows that on intact cells expressing IR-A¹²⁵I]-a replacement of the INS,

FIG. 8 shows that on intact cells expressing IR-B¹²⁵I]-replacement of INS.

Example 1: generation of anti-IGF-1R monoclonal antibodies

Hybridomas were generated by fusing spleen cells from BALB/c mice immunized with soluble α 2- β 2 heterotetrameric recombinant human IGF-1R (R & D System, Minneapolis, USA) with the SP2/0-Ag14 myeloma cell line. The resulting murine antibodies were first screened by ELISA and FACS analysis on MCF-7 cells. A final screen was then performed on Sf9-IGF-1r vs. Sf9-IR cells to eliminate antibodies recognizing IGF-1 and IR. Selected mabs (positive in ELISA and recognizing wild-type receptors on MCF-7 cells) were produced as ascites fluid and purified on protein a chromatography columns prior to in vitro and/or in vivo testing, as summarized in table 1.

Table 1: selection of anti-IGF-IR monoclonal antibodies

Example 2: in vitro Activity of anti-IGF-1R antibodies

Method of producing a composite material

MCF-7 cells from ATCC were routinely cultured in phenol red-free RPMI medium (Invitrogen Corporation, Scotland, UK), 10% FCS (Invitrogen Corporation), 1% L-glutamine (Invitrogen Corporation). 5X10 in serum-free medium⁴Density of individual cells/well MCF-7 cells were plated in 96-well tissue culture plates. After 24 hours, IGF1 was added in a dosage range of 1-50ng/ml to the medium in the absence or presence of a final concentration of 5. mu.g/ml of each antibody tested. After 3 days, 0.5. mu. Ci of [ 2], [ 2]³H]Cells were pulsed with thymidine (Amersham Biosciences AB, Uppsala, Sweden) for 16 hours. The amount of DNA incorporated in trichloroacetic acid-insoluble DNA by liquid scintillation counting³H]The amplitude of thymidine. Results are expressed as proliferation index (cpm of cells plus IGF1 plus antibody/cpm of cells plus antibody only).

Results

In vitro evaluation is the first screening of mabs (monoclonal antibodies) for mitogenic activity. For these experiments, the generated antibody prepared as ascites fluid was added to MCF-7 cells simultaneously with IGF1 and compared with a commercial α IR3Mab to select antibodies with at least the efficacy of the latter antibody. Positive mabs (5 mabs) described as (+) in Table 2, previously replied, had a proliferation index < 5 when cells were stimulated with the highest dose of IGF1(50 ng/ml). Figure 1 shows the in vitro activity of 4 of 6 potent in vitro inhibitors (2D10, 12D5, 12B1, 13F 5). 2F2 and 21E3 mabs have been identified as (+ -) -omega mabs (5 < proliferation index < 15 for the highest concentration of IGF 1), and 7G3 and 2B10 were identified as non-neutralizing antibodies (proliferation index > 15). It is interesting to note that 21E3 is the only Mab of IgG2 isotype.

Example 3: in vivo Activity of anti-IGF-IR-1R antibodies

Method of producing a composite material

DU145 cells from ATCC were routinely cultured in DMEM medium (Invitrogen Corporation, Scotland, UK), 10% FCS (Invitrogen Corporation), 1% L-glutamine (Invitrogen Corporation). Two days prior to engraftment, cells were split into flasks so that they were in exponential growth phase. Two million DU145 in PBS were implanted into swiss nude mice. One day after implantation, animals were divided into 6 mice per group. Mice were treated with 200 μ g of each antibody to be tested by subcutaneous injection on the tumor contralateral side 3 times a week. The control group was treated with the murine isotype control (EC2) in the first screen or PBS was used for the subsequent screens, since no difference in tumor growth between these two groups of mice had been shown in the first experiment. Tumor volume was measured once a week and by the formula: pi/6 x length x width x height.

Results

Three in vivo experiments were performed to test a panel of mabs. Fig. 2 and 3 show that 13F5, 2D10, and 6E5 significantly inhibited the in vivo growth of DU145 cells. Statistical analysis (Mann and Whitney test) is shown in Table 2.

Second screening	D12	D20	D26	D30
					Mann-WhitnevPBS/2D10(Wilcoxon)	p＝0,0041	p＝0,0017	p＝0,0027	p＝0,0027
PBS/6E5Mann-Whitnev(Wilconon	p＝0,027	p＝0,013	p＝0,0092	p＝0,019
					PBS/12B1Mann-Whitney(Wilcoxon	p＝0,11	p＝0,11	p＝0,067	p＝0,11
PBS/2F2Mann-Whitney(Wilcoxon	p＝0,18	p＝0,067	p＝0,050	p＝0,14

Third screening	D12	D20	D26	D33
					PBS/16A12Mann-Whitnev(Wilcoxon	p＝0,063	p＝0,087	p＝0,19	p＝0,11

Table 2: statistical analysis of in vivo data

Example 4: evaluation of the ability of 2D10, 12D5, 13F5 to bind to IGF-IR and hybrid-R

The cells used in this study are listed thereafter:

-R +: r-fibroblasts stably transfected with IGF-I receptor (IGF-IR) cDNA

-R-/IR-A: r-fibroblasts stably transfected with insulin receptor isoform A (IR-A) cDNA

-R-/IR-B: r-fibroblasts stably transfected with insulin receptor isoform B (IR-B) cDNA

-R +/IR-A: r-fibroblasts stably co-transfected with IGF-I and insulin receptor isoform AcDNA and thus expressing hybrid receptor A (hybrid-RsA)

-R +/IR-B: r-fibroblasts stably co-transfected with IGF-I and insulin receptor isoform BcDNA, and thus expressing hybrid receptor A (hybrid-RsB)

Example 4-1: displacement analysis on IGF-IR by 2D10, 12D5, 13F5 and alpha IR-3 [ ¹²⁵ I]IGF1

In the absence or presence of increasing concentrations of a non-labeled ligand (IGF1, IGF2 or insulin) or antibody (2D10, 12D5, 13F5)¹²⁵I]IGF1(20,000cpm) bound to R + intact cells for 16 hours at 4 ℃. The results are plotted as a percentage of maximum specific binding and are shown in figure 4.

Both 2D10 and 13F5 efficiently and completely replaced IGF with sub-nanomolar affinity and were found to be compatible with the reference antibody, alpha IR-3 (IC)₅₀: 0.05nM) and in this example, 0.15 and 0.20nM IC, respectively₅₀. The affinity was higher than that of the native IGF-IR ligand IGF1 (2.2 nM in this example) and IGF2 (15 nM in this example).

Example 4-2: substitution analysis on hybrid-RsA by 2D10, 12D5, 13F5 and 47-9 [ ¹²⁵ I]IGF1

The hybrid-RsA from the R +/IR-A cell lysate was immunocaptured on Maxisorb plates coated with anti-IR antibody 83-7.

Then allowed in the absence or presence of increasing concentrations of non-labeled antibody (IGF1, IGF2 or insulin) or antibody (2D10, 12D5, 13F5, 47-9, 9G4)¹²⁵I]IGF1 (fig. 5) binds to the immunocaptured receptor. The results are plotted as a percentage of maximum specific binding and are shown in figure 5.

2D10 and 13F5 effectively and completely replaced the marker IGF1 with very similar subnanomolar affinities, in this example 0.2 and 0.35nM, respectively. By comparison, 47-9 produced an IC of 0.18nM₅₀Values (fig. 5).

These affinities were higher than those of the natural hybrid-RsA ligand IGF1 (2.0 nM in this example) and IGF2 (12 nM in this example).

Examples 4 to 3: substitution analysis on hybrid-RsB by 2D10, 12D5, 13F5 and 47-9 [ ¹²⁵ I]IGF1

hybrid-RsB from R +/IR-B cell lysates were immunocaptured on Maxisorb plates coated with 83-7 antibody.

Then allowed in the absence or presence of increasing concentrations of non-labeled antibody IGF1, IGF2, insulin or antibody (2D10, 12D5, 13F5, 47-9, 9G4)¹²⁵I]IGF1 (fig. 6) binds to the immunocaptured receptor. The results are plotted as a percentage of maximum binding.

2D10 and 13F5 effectively and completely replaced the marker IGF1 with very similar subnanomolar affinities, in this exampleIn the examples, 0.04 and 0.15, respectively. By comparison, 47-9 was less potent with an IC of 0.40nM₅₀Values (fig. 6).

Examples 4 to 4: insulin receptor A (IR-A) viA 2D10, 12D5, 13F5 and MA-10 And substitution analysis on B (IR-B) isoform ¹²⁵ I]Insulin

In the absence or presence of increasing concentrations of a non-labeled ligand (IGF1, IGF2 or insulin) or antibody (2D10, 12D5, 13F5)¹²⁵I]Insulin (40,000cpm) binds to R-/IR-A or R-/IR-B intact cells for 16 hours at 4 ℃. The results are plotted as A percentage of maximum specific binding and are shown in FIGS. 7 and 8 for IR-A and IR-B, respectively.

None of 2D10, 12D5, or 13F5 replaced Insulin (IC) as compared to the reference antibody MA-10₅₀: 0.90 and 1.5nM correspond to IR-A (FIG. 7) and IR-B (FIG. 8), respectively).

Claims (21)

1. An isolated antibody or a functional fragment thereof, said antibody or said fragment thereof being capable of binding to the human insulin-like growth factor I receptor IGF-IR, characterized in that said antibody or functional fragment thereof comprises a light chain comprising at least said three complementarity determining region CDRs of sequences SEQ ID Nos.1, 2 and 3, and in that it comprises a heavy chain comprising at least said three CDRs of sequences SEQ ID Nos.4, 5 and 6.

2. The antibody of claim 1, designated 13F5 and characterized in that it comprises a heavy chain having the sequence comprising the amino acid sequence SEQ ID No.20 and in that it further comprises a light chain having the sequence comprising the amino acid sequence SEQ ID No. 19.

3. A murine hybridoma capable of secreting an antibody according to one of claims 1 or 2 and deposited with number I-3193 on 3/25 of 2004 at CNCM, Institut Pasteur, Paris.

4. An antibody secreted by the hybridoma of claim 3.

5. The antibody or functional fragment thereof of claim 1 or 2, characterized in that said antibody is a chimeric antibody and further comprises light and heavy chain constant regions from an antibody of a species heterologous to the mouse.

6. Antibody or functional fragment thereof according to claim 5, characterized in that the heterologous species is human.

7. A composition comprising, as an active ingredient, a compound consisting of an antibody or a functional fragment thereof as claimed in one of claims 1, 2, 4, 5 and 6.

8. Composition according to claim 7, characterized in that it further comprises, as a combination product for simultaneous or separate use, an antibody, a cytotoxic or cytostatic agent, and/or an inhibitor of the tyrosine kinase activity of each of the IGF-I and/or EGF receptors.

9. Composition according to claim 7, characterized in that it further comprises, as a combination product for sequential use, an antibody, a cytotoxic or cytostatic agent, and/or an inhibitor of the tyrosine kinase activity of each of the IGF-I and/or EGF receptors.

10. A composition according to one of claims 7, 8 and 9 as a medicament.

11. Use of an antibody or functional fragment thereof as claimed in one of claims 1, 2, 4, 5 and 6 and/or a composition according to any one of claims 7 to 10 for the preparation of a medicament intended for the prevention or treatment of diseases associated with overexpression and/or abnormal activation of the IGF-I receptor and/or diseases associated with overactivation of the signal transduction pathway mediated by the interaction of IGF1 or IGF2 with IGF-IR.

12. Use according to claim 11 for the preparation of a medicament intended to inhibit the transformation of normal cells into IGF-dependent cells characteristic of tumors.

13. The use of claim 12 for the preparation of a medicament, wherein the cells with tumor characteristics are IGF1 and/or IGF2 dependent cells.

14. Use according to claim 12 for the preparation of a medicament intended to inhibit the growth and/or proliferation of tumor cells, wherein said tumor cells are IGF-dependent cells.

15. The use of claim 14 for the preparation of a medicament, wherein the tumor cell is an IGF1 and/or IGF2 dependent cell.

16. Use according to claim 12 or 14, characterized in that the tumor cells are tumor IGF-dependent cells.

17. Use according to claim 12 or 14, characterized in that the tumor cells are tumor IGF1 and/or IGF2 dependent cells.

18. Use according to one of claims 11 to 15 for the preparation of a medicament intended for the prevention or treatment of cancer, wherein the cancer overexpresses IGF-IR.

19. Use according to claim 18, characterized in that the cancer is selected from prostate cancer, osteosarcoma, lung cancer, breast cancer, endometrial cancer or colon cancer.

20. Use of an antibody as claimed in any one of claims 1, 2, 4, 5 and 6 for the preparation of a medicament for the in vitro diagnosis of a disease induced by overexpression or underexpression of the IGF-I receptor from a biological sample suspected of having an abnormal presence of the IGF-I receptor.

21. Use according to claim 20, characterized in that the antibody is labeled.