WO2005058967A2 - Nouveau recepteur hybride anti-insuline/igf-i ou recepteur hybride anti-insuline/igf-i et anticorps igf-ir et applications - Google Patents

Nouveau recepteur hybride anti-insuline/igf-i ou recepteur hybride anti-insuline/igf-i et anticorps igf-ir et applications Download PDF

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WO2005058967A2
WO2005058967A2 PCT/IB2004/004360 IB2004004360W WO2005058967A2 WO 2005058967 A2 WO2005058967 A2 WO 2005058967A2 IB 2004004360 W IB2004004360 W IB 2004004360W WO 2005058967 A2 WO2005058967 A2 WO 2005058967A2
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antibody
hybrid
igf
cells
egfr
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WO2005058967A8 (fr
WO2005058967A3 (fr
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Liliane Goetsch
Nathalie Corvaia
Alain Duflos
Jean-François HAEUW
Olivier Leger
Alain Beck
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Pierre Fabre Medicament
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Publication of WO2005058967A8 publication Critical patent/WO2005058967A8/fr
Publication of WO2005058967A3 publication Critical patent/WO2005058967A3/fr
Priority to IL176202A priority Critical patent/IL176202A0/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6805—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a vinca alkaloid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to novel antibodies capable of binding specifically to i) the human Insulin/IGF-I hybrid receptor, isoform(s) A and/or B, (hereinafter referred as “hybrid-R” or sometimes as “hybrid-Rs", “hybrid-RsA” for isoform A or “hybrid-RsB” for isoform B) and/or ii) both the hybrid-R and insulin-like growth factor I receptor (hereinafter referred as "IGF-IR”) and/or capable of specifically inhibiting the tyrosine kinase activity of said hybrid-R or of both hybrid-R and IGF-IR, especially monoclonal antibodies of murine, chimeric and humanized origin, as well as the amino acid and nucleic acid sequences coding for these antibodies.
  • hybrid-R or sometimes as “hybrid-Rs", “hybrid-RsA” for isoform A or “hybrid-RsB” for isoform B
  • the invention likewise comprises the use of these antibodies as a medicament for the prophylactic and/or therapeutic treatment of cancers overexpressing, or with an abnormal activation of, hybrid-R or both hybrid-R and IGF-IR or any pathology connected with the overexpression, or an abnormal activation, of said receptor(s) as well as in processes or kits for diagnosis of illnesses connected with the overexpression, or an abnormal activation, of the hybrid-R or both hybrid-R and IGF-IR.
  • the invention finally comprises products and/or compositions comprising such antibodies in combination with anti-EGFR antibodies and/or compounds and/or anti-cancer agents or agents conjugated with toxins and their use for the prevention and/or the treatment of certain cancers.
  • IGF-IR insulin-like growth factor I receptor
  • IR insulin receptor
  • Sequence(s) coding for IGF-IR are registered under Accession Number NM_000875 in the NCBI Genbank. Another data, without limitation and incorporated herein by reference, describing IGF-IR sequence(s) is Ulrich et al., 1986, EMBO J., 5(10):2503-2512.
  • IGF-IR is a glycoprotein of molecular weight approximately 350,000.
  • IGF-IR binds its native ligands, preferably IGF1 and IGF2, with a very high affinity (Kd #1 nM) but is equally capable of binding to insulin with an affinity 100 to 1000 times less. Conversely, the IR binds insulin with a very high affinity although the IGFs only bind to the IR with a 100 times lower affinity.
  • the tyrosine kinase domain of IGF-IR and of IR has a very high sequence homology although the zones of weaker homology respectively concern the cysteine-rich region situated on the ⁇ -subunit and the C-terminal part of the -subunit.
  • the sequence differences observed in the ce-subunit are situated in the binding zone of the ligands and are therefore at the origin of the relative affinities of IGF-IR and of IR for the IGFs and insulin respectively.
  • the differences in the C-terminal part of the /3-subunit result in a divergence in the signalling pathways of the two receptors; IGF-IR mediating mitogenic, differentiation and antiapoptosis effects, while the activation of the IR principally involves effects at the level of the metabolic pathways (Baserga et al., Biochim. Biophys. Acta, 1332:F105-126, 1997; Baserga R., Exp. Cell. Res., 253:1-6, 1999).
  • the cytoplasmic tyrosine kinase proteins are activated by the binding of the ligand to the extracellular domain of the receptor.
  • the activation of the kinases in its turn involves the stimulation of different intra-cellular substrates, including IRS-1, IRS- 2, She and Grb 10 (Peruzzi F. et al., J. Cancer Res. Clin. Oncol, 125:166-173, 1999).
  • the two major substrates of IGF-IR are IRS and She which mediate, by the activation of numerous effectors downstream, the majority of the growth and differentiation effects connected with the attachment of the IGFs to this receptor (figure 2).
  • the availability of substrates can consequently dictate the final biological effect connected with the activation of the IGF-IR.
  • IRS-1 predominates, the cells tend to proliferate and to transform.
  • She dominates the cells tend to differentiate (Nalentinis B. et al, J. Biol.
  • the crucial discovery which has clearly demonstrated the major role played by IGF-IR in the transformation has been the demonstration that the R- cells, in which the gene coding for IGF-IR has been inactivated, are totally refractory to transformation by different agents which are usually capable of transforming the cells, such as the E5 protein of bovine papilloma virus, an overexpression of EGFR or of PDGFR, the T antigen of SN 40, activated ras or the combination of these two last factors (Sell C. et al., Proc. ⁇ atl. Acad. Sci., USA, 90:11217-11221, 1993; Sell C. et al., Mol. Cell. Biol, 14:3604-3612, 1994; Morrione A.
  • the E5 protein of bovine papilloma virus an overexpression of EGFR or of PDGFR
  • T antigen of SN 40 activated ras or the combination of these two last factors
  • IGF-IR is expressed in a great variety of tumors and of tumor lines and the IGFs amplify the tumor growth via their attachment to IGF-IR.
  • Other arguments in favor of the role of IGF-IR in carcinogenesis come from studies using murine monoclonal antibodies directed against the receptor or using negative dominants of IGF-IR.
  • murine monoclonal antibodies directed against IGF-IR inhibit the proliferation of numerous cell lines in culture and the growth of tumor cells in vivo (Arteaga C. et al, Cancer Res., 49:6237-6241, 1989; Li et al., Biochem. Biophys. Res. Com., 196:92-98, 1993; Zia F et al., J. Cell.
  • hybrid-R the capacity of 7C10 and h7C10 to recognize and/or inhibit the tyrosine kinase activity of hybrid-R or both hybrid-R and IGF-IR allow to avoid the escape of tumor consequent upon the expression, or the abnormal activation, of this hybrid-R.
  • Such an antibody could be an innovative therapeutic compound of a essential interest for the treatment of cancer.
  • Cancer pathologies are characterized by an uncontrolled cellular growth.
  • growth factors are specifically binding with their receptors and then transmit growth, transformation and/or survival signals to the tumoral cell.
  • the growth factor receprtors over-expression at the tumoral cell surface is largely described (Salomon DS 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 over-expression, or abnormal activation, leading to a direct perturbation of cellular growth regulation mechanisms can also affect the cell sensibility to induced apoptose by classical chemotherapies or radiotherapies.
  • IGF-IR is part of the tyrosine kinase receptors. It shows a high homology with the Insulin receptor (IR) which exist under two isoforms A and B. Sequences of IR, isoforms A and B, are registered under Accession Numbers X02160 and Ml 0051, respectively, in the NCBI Genbank. Other datas, without limitations, relating to IR are incorporated herein by references (Vinten et al, 1991, Proc. Natl Acad. Sci.
  • the IGF-IR and IR are tetrameric glycoproteins composed of two extracellular ⁇ - and two transmembrane ⁇ -subunits linked by disulfide bonds. Each ⁇ -subunit, containing the ligand-binding site is approximately 130- to 135-kDa, whereas each ⁇ - subunit containing the tyrosine kinase domain is approximately 90- to 95- kDa.
  • phosphorylated receptors recruit and phosphorylate docking proteins, including the insulin receptor substrate- 1 protein family (IRS1), Gabl and She (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.
  • IRS1 insulin receptor substrate- 1 protein family
  • Insulin binds with high affinity to the IR (100-fold higher than to the IGF-IR), whereas insulin-like growth factors (IGFl and IGF2) bind to the IGF-IR with 100-fold higher affinity than to the IR.
  • the human IR exists in two isoforms, IR-A and IR-B, generated by alternative splicing of the IR gene that either excludes or includes 12 amino acid residues encoded by a small exon (exon 11) at the carboxy-terminus of the IR ⁇ -subunit.
  • the relative abundance of IR isoforms is regulated by tissue specific and unknown factors (Moller et al, 1989, Mol.
  • IR-B is the predominant IR isoform in normal adult tissues (adipose tissue, liver and muscle) that are major target tissues for the metabolic effects of insulin (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.
  • the insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-R) (Soos et al, 1990, Biochem J., 270:383-390;
  • Hybrid-R has different functional characteristics.
  • Hybrid-RsB has reduced affinity for IGFl and especially for IGF2.
  • Hybrid-RsA has a high affinity for IGFl and bind also IGF2 and insulin at a physiological concentration range.
  • Hybrid-RsA up-regulates the IGF system by two different mechanisms i) binding (with high affinity) and activation by both IGFl and IGF2 (which do not occur with the Hybrid-RsB), ii) activation of the IGF-IR pathway after insulin binding.
  • Insulin binding to Hybrid-RsA phosphorylates the IGF-IR ⁇ -subunit and activates an IGF-IR-specific substrate (Crkll) so that Hybrid-RsA shifts insulin to IGF-IR signaling (Pandini et al, 2002).
  • Hybrid-R are more represented than IGF-IR (Bailyes et al, 1997).
  • Hybrid-RsA may also be overexpressed in a variety of human malignancies, including thyroid and breast cancers providing a selective growth advantage to malignant cells able to respond by a type IGF-IR signalisation following a stimulation by IGFl and/or IGF2 but also by insulin at physiological concentrations (Bailyes et al, 1997; Pandini et al, 1999, Clin.
  • the present invention allows to block the hybrid-R activity by generating a compound, and more particularly an antibody, of high affinity able to bind to said receptor and also to block its activation by its native ligands, preferably IGFl, IGF2 or Insulin.
  • the present invention also allows to jointly block the hybrid-R and IGF-IR activity by generating a compound, and more particularly an antibody, of high affinity able to bind to said two receptors and also to block their activation respectively their native ligands, preferably by IGFl, IGF2 or Insulin.
  • the present invention also deals with the use of an isolated antibody according to the present invention, or a fragment thereof, said antibody or fragment being able to bind to i) human hybrid-R, and/or to inhibit the binding of its native ligands, preferably IGFl, IGF2 and/or Insulin, and/or also able to specifically inhibit the tyrosine kinase activity of said hybrid-R, and/or ii) both human hybrid-R and human IGF-IR ,an ⁇ Vor to inhibit the binding of their native ligands, preferably IGFl and/or IGF2 and/or insulin, and/or also able to inhibit specifically the tyrosine kinase activity of said hybrid-R and IGF-IR.
  • said antibody is characterized in that it comprises the sequences of the 7C10 and h7C10 antibodies anti-IGF-IR, and fragment thereof, of the present invention, notably the antibodies anti-IGF-IR according to the present invention having a light chain comprising at least a CDR region selected in the group consisting in SEQ ID No. 2, 4 or 6 (or at least a CDR with at least 80% of homology after optimal alignment with SEQ ID No. 2, 4 or 6), and/or a heavy chain comprising at least a CDR region selected in the group consisting in SEQ ED No. 8, 10 or 12 (or at least a CDR with at least 80% of homology after optimal alignment with SEQ ID No. 8, 10 or 12).
  • said antibody is used for cancer therapy, more particularly breast cancer therapy.
  • breast tumoral cells specifically present on their surface IGF-IR but also a great number of Insulin receptor and, as a consequence, a great number of Hybrid-R (Frasca et al, 1999; Sciacca et al, 1999; Vella et al, 2001).
  • the object of the present invention is to be able to have available a murine monoclonal antibody, preferably a chimerized or humanized antibody, which will recognize hybrid-R or both hybrid-R and IGF-IR specifically and with great affinity. This antibody will interact little or not at all with the IR on insulin.
  • This antibody will be able to be active in vivo on all the types of tumors expressing, or with an abnormal activation of, hybrid-R or both hybrid-R and IGF-IR including estrogen-dependent tumors of the breast and tumors of the prostate, which is not the case for the anti-IGF-IR monoclonal antibodies (written MAb or MAB) currently available.
  • MAb anti-IGF-IR monoclonal antibodies
  • oIR3 which refers to the domain of IGF-IR, totally inhibits the growth of estrogen-dependent tumors of the breast (MCF-7) in vitro but is without effect on the corresponding model in vivo (Arteaga C. et al, J. Clin. Invest. 84:1418-1423, 1989).
  • the scFv-Fc fragment derived from the murine monoclonal 1H7 is only weakly active on the tumor of the breast MCF-7 and totally inactive on an androgen-independent tumor of the prostate (Li S.L. et al, Cancer Immunol. Immunother., 49:243-252, 2000). None of these known antibodies are described as being able to recognize, or inhibit the tyrosine kinase activity, of the hybrid-R.
  • the inventors have demonstrated a chimeric antibody (called C7C10) and two humanized antibodies respectively called h7C10 humanized form 1 and h7C10 humanized form 2, derivatives of the murine monoclonal antibody 7C10, recognising hybrid-R or both hybrid-R and IGF-IR and corresponding to all of the criteria stated above, that is to say to a nonrecognition of the receptor on the insulin, to an in vitro blockage of the IGFl and/or IGF2 proliferation induced but likewise to the in vivo inhibition of the growth of different tumors expressing, or with an abnormal activation of, hybrid-R or both hybrid-R and IGF-IR among which are an osteosarcoma, a non-small cell lung tumor and a pancreatic tumor BxPC3 but likewise and more particularly the estrogen-dependent tumor of the breast MCF-7 and an androgen- independent tumor of the prostate DU-145.
  • C7C10 chimeric antibody
  • two humanized antibodies respectively called h7C10 humanized
  • the intensity of inhibition of the tumor growth of MCF-7 cells in vivo by the antibody 7C10 is comparable, or even significantly superior, to that observed with tamoxifen, one of the reference compounds in the treatment of estrogen-dependent tumors of the breast.
  • these antibodies inhibit the phosphorylation of the tyrosine of the beta chain of IGF-IR and of IRS1, the first substrate of the receptor.
  • these antibodies cause the internalization of said receptor and its degradation contrary to what is usually observed with natural ligands which allow the rapid recycling of the receptor on the surface of the cells. It has been possible to characterize these antibodies by their peptidic and nucleic sequence, especially by the sequence of their regions determining their complementarity (CDR) for hybrid-R and/or IGF-IR.
  • CDR complementarity
  • the compound is preferably of IgGl isotype
  • other mechanisms of action involving effector functions such as for example ADCC and/or CDC, could explain the in vivo efficacy of the antobody of the present invention.
  • a subject of the present invention is an isolated antibody, or one of its functional fragments, said antibody or one of its said fragments being characterized in that it is capable of binding to the hybrid-R, isoform(s)
  • said isolated antibody comprising a light chain comprising at least one complementarity determining region CDR chosen from the CDRs of sequence SEQ ID Nos. 2, 4 and 6, and/or comprising a heavy chain comprising at least one CDR chosen from the CDRs of sequence SEQ ID Nos. 8, 10 and 12.
  • the terms “to bind” and “to attach” have the same meaning and are inter-changeable.
  • the terms polypeptides, polypeptide sequences, peptides and proteins attached to antibody compounds or to their sequence are interchangeable. It must be understood here that the invention does not relate to the antibodies in natural form, that is to say they are not in their natural environment but that they have been able to be isolated or obtained by purification from natural sources, or else obtained by genetic recombination, or by chemical synthesis, and that they can then contain unnatural amino acids as will be described further on.
  • CDR region or CDR it is intended to indicate the hypervariable regions of the heavy and light chains of the immunoglobulins as defined by Kabat et al.
  • 3 heavy chain CDRs and 3 light chain CDRs exist.
  • the te ⁇ n CDR or CDRs is used here in order to indicate, according to the case, one of these regions or several, or even the whole, of these regions which contain the majority of the amino acid residues responsible for the binding by affinity of the antibody for the antigen or the epitope which it recognizes.
  • percentage of identity between two nucleic acid or amino acid sequences in the sense of the present invention, it is intended to indicate a percentage of nucleotides or of identical amino acid residues between the two sequences to be compared, obtained after the best alignment (optimum alignment), this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length.
  • the comparisons of sequences between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having aligned them in an optimum manner, said comparison being able to be carried out by segment or by “comparison window”.
  • the optimum alignment of the sequences for the comparison can be carried out, in addition to manually, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App.
  • the percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences aligned in an optimum manner and in which the nucleic acid or amino acid sequence to be compared can comprise additions or deletions with respect to the reference sequence for an optimum alignment between these two sequences.
  • the percentage of identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the comparison window and by multiplying the result obtained by 100 in order to obtain the percentage of identity between these two sequences.
  • BLAST 2 sequences (Tatusova et al, "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250) available on the site http://www.ncbi.nlm.nih.gov/ gorf bl2.html, the parameters used being those given by default (in particular for the parameters "open gap penalty” : 5, and “extension gap penalty” : 2; the matrix chosen being, for example, the matrix "BLOSUM 62" proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program.
  • amino acid sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity with a reference amino acid sequence those having, with respect to the reference sequence, certain modifications, in particular a deletion, addition or substitution of at least one amino acid, a truncation or an elongation are preferred.
  • substitutions are preferred in which the substituted amino acids are replaced by "equivalent” amino acids.
  • the expression "equivalent amino acids” is aimed here at indicating any amino acid capable of being substituted with one of the amino acids of the base structure without, however, essentially modifying the biological activities of the corresponding antibodies and such as will be defined later, especially in the examples.
  • the antibodies according to the present invention are preferably specific monoclonal antibodies, especially of murine, chimeric or humanized origin, which can be obtained according to the standard methods well known to the person skilled in the art.
  • monoclonal antibodies or their functional fragments, especially of murine origin it is possible to refer to techniques which are described in particular in the manual "Antibodies” (Harlow and Lane, Antibodies: A
  • the monoclonal antibodies according to the invention can be obtained, for example, from an animal cell immunized against the hybrid-R or both against the hybrid-R and IGF-IR, or one of their fragments containing the epitope specifically recognized by said monoclonal antibodies according to the invention.
  • Said hybrid-R or both hybrid-R and IGF-IR, or one of their said fragments can especially be produced according to the usual working methods, by genetic recombination starting with a nucleic acid sequence contained in the cDNA sequence coding for the hybrid-R or IGF- IR or by peptide synthesis starting from a sequence of amino acids comprised in the peptide sequence of the hybrid-R or IGF-IR.
  • the monoclonal antibodies according to the invention can, for example, be purified on an affinity column on which the hybrid-R and/or IGF-IR or one of their fragments containing the epitope specifically recognized by said monoclonal antibodies according to the invention has previously been immobilized. More particularly, said monoclonal antibodies can be purified by chromatography on protein A and/or G, followed or not followed by ion-exchange chromatography aimed at eliminating the residual protein contaminants as well as the DNA and the LPS, in itself followed or not followed by exclusion chromatography on Sepharose gel in order to eliminate the potential aggregates due to the presence of dimers or of other multimers. In an even more preferred manner, the whole of these techniques can be used simultaneously or successively.
  • Chimeric or humanized antibodies are likewise included in antibodies according to the present invention.
  • chimeric antibody it is intended to indicate an antibody which contains a natural variable (light chain and heavy chain) region derived from an antibody of a given species in combination with the light chain and heavy chain constant regions of an antibody of a species heterologous to said given species.
  • the antibodies or their fragments of chimeric type according to the invention can be prepared by using the techniques of genetic recombination.
  • the chimeric antibody can be produced by cloning a recombinant DNA containing a promoter and a sequence coding for the variable region of a nonhuman, especially murine, monoclonal antibody according to the invention and a sequence coding for the constant region of human antibody.
  • a chimeric antibody of the invention encoded by such a recombinant gene will be, for example, a mouse-man chimera, the specificity of this antibody being determined by the variable region derived from the murine DNA and its isotype determined by the constant region derived from the human DNA.
  • the methods of preparation of chimeric antibodies it is possible, for example, to refer to the document Verhoeyn et al. (BioEssays, 8:74, 1988).
  • humanized antibody it is intended to indicate an antibody which contains
  • CDR regions derived from an antibody of nonhuman origin the other parts of the antibody molecule being derived from one (or from several) human antibodies.
  • some of the residues of the segments of the skeleton can be modified in order to conserve the affinity of the binding (Jones et al, Nature, 321:522- 525, 1986; Verhoeyen et al, Science, 239:1534-1536, 1988; Riechmann et al, Nature, 332:323-327, 1988).
  • the humanized antibodies according to the invention or their fragments can be prepared by techniques known to the person skilled in the art (such as, for example, those described in the documents Singer et al, J.
  • Such humanized antibodies according to the invention are preferred for their use in in vitro diagnostic methods, or in vivo prophylactic and/or therapeutic treatment.
  • an antibody fragment such as Fv, scFv (sc for single chain), Fab, F(ab') 2 , Fab', scFv-Fc fragments or diabodies, or any fragment of which the half- life time would have been increased by chemical modification, such as the addition of poly(alkylene) glycol such as poly(ethylene) glycol (“PEGylation") (pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab') 2 -PEG or Fab'-PEG) ("PEG” for Poly(Ethylene) Glycol), or by incorporation in a liposome, said fragments having at least one of the characteristic CDRs of sequence SEQ ID No.
  • PEGylation poly(ethylene) glycol
  • said functional fragments will be constituted or will comprise a partial sequence of the heavy or light variable chain of the antibody from which they are derived, said partial sequence being sufficient to retain the same specificity of binding as the antibody from which it is descended and a sufficient affinity, preferably at least equal to 1/100, in a more preferred manner to at least 1/10, of that of the antibody from which it is descended, with respect to the hybrid-R or both hybrid-R and IGF-IR.
  • Such a functional fragment will contain at the minimum 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the sequence of the antibody from which it is descended.
  • these functional fragments will be fragments of Fv, scFv, Fab,
  • antibody fragments of the invention can be obtained starting from antibodies such as described above by methods such as digestion by enzymes, such as pepsin or papain and/or by cleavage of the disulfide bridges by chemical reduction.
  • the antibody fragments comprised in the present invention can be obtained by techniques of genetic recombination likewise well known to the person skilled in the art or else by peptide synthesis by means of, for example, automatic peptide synthesizers such as those supplied by the company Applied Biosystems, etc.
  • the present invention concerns the antibody, or one of its functional fragments, as described above, said antibody being characterized in that it is also capable of binding the IGF-IR and inhibiting the binding of its native ligands, preferably designated herein as IGFl and/or IGF2, and/or capable of specifically inhibiting the tyrosine kinase activity of said IGF-IR.
  • the invention comprises the antibodies, or their functional fragments, according to the present invention, especially chimeric or humanized antibodies, obtained by genetic recombination or by chemical synthesis.
  • a subject of the invention is an antibody, or one of its functional fragments, according to the invention, characterized in that it comprises a heavy chain comprising at least one CDR chosen from the CDRs of sequence SEQ ID NO: 1
  • CDRH3 the third CDR of the heavy chain (CDRH3) has a greater size variability (greater diversity essentially due to the mechanisms of arrangement of the genes which give rise to it). It can be as short as 2 amino acids although the longest size known is 26. Functionally, CDRH3 plays a role in part in the determination of the specificity of the antibody (Segal et al, PNAS, 71 :4298- 4302, 1974; Amit et al, Science, 233:747-753, 1986; Chothia et al, J. Mol.
  • the present invention relates to an antibody or one of its functional fragments, according to the invention, characterized in that it comprises a heavy chain comprising at least two of the three CDRs or the three CDRs of sequence SEQ ID Nos. 8, 10 and 12, or at least two of three CDRs or three CDRs of sequence respectively having at least 80% identity after optimum alignment with the sequence SEQ ID Nos. 8, 10 and 12.
  • a subject of the invention is an antibody or one of its functional fragments, according to the invention, characterized in that it comprises a light chain comprising at least one CDR chosen from the CDRs of sequence SEQ ID Nos.
  • a subject of the invention is an antibody or one of its functional fragments according to the invention, characterized in that it comprises a light chain comprising at least two of the three CDRs or the three CDRs of sequence SEQ ID Nos. 2, 4 and 6, or at least two of three CDRs or three CDRs of sequence respectively having at least 80% identity after optimum alignment with the sequence SEQ ID Nos. 2, 4 and 6.
  • 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.
  • a subject of the present invention is an antibody or one of its functional fragments, according to the invention, characterized in that it does not attach or it does not attach in a significant manner to the human IR.
  • said functional fragments according to the present invention will be chosen from the fragments Fv, scFv, Fab, (Fab') , Fab', scFv-Fc, scFV-CH 3 or diabodies, or any functional fragment whose half-life would have been increased by a chemical modification, especially by PEGylation, or by incorporation in a liposome.
  • the invention relates to a murine hybridoma capable of secreting a monoclonal antibody according to the present invention, especially the hybridoma of murine origin such as deposited at the Centre National de Culture De Microorganisme (CNCM, National Center of Microorganism Culture)
  • the present invention relates to a murine antibody, or one of its functional fragments, according to the invention, characterized in that said antibody comprises a light chain of sequence comprising the amino acid sequence SEQ ID No. 54, or a sequence having at least 80% identity after optimum alignment with the sequence SEQ ID No. 54, or/and in that it comprises a heavy chain of sequence comprising the amino acid sequence SEQ ID No.
  • the present invention relates to a chimeric antibody, or one of its functional fragments, according to the invention, characterized in that said antibody moreover comprises the light chain and heavy chain constant regions derived from an antibody of a species heterologous to the mouse, especially man, and in a preferred manner in that the light chain and heavy chain constant regions derived from a human antibody are respectively the kappa or lambda and gamma- 1, gamma-2 or gamma-4 region.
  • 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 in which the skeleton segments FR1 to FR4 (such as defined below in examples 12 and 13, in tables 5 and 6) of said light chain and/or heavy chain are respectively derived from skeleton segments FR1 to FR4 of human antibody light chain and/or heavy chain.
  • the humanized antibody or one of its functional fragments, according to the present invention is characterized in that said humanized antibody comprises a light chain comprising the amino acid sequence SEQ
  • the humanized antibody, or one of its functional fragments, according to the invention is characterized in that said humanized antibody comprises a light chain comprising the amino acid sequence SEQ ID No. 65, and in that it comprises a heavy chain of sequence comprising the amino acid sequence SEQ ID No. 79 or 83, preferably SEQ ID No. 83.
  • the present invention relates to an isolated nucleic acid, characterized in that it is chosen from the following nucleic acids: a) a nucleic acid, DNA or RNA, coding for an antibody, or one of its functional fragments, according to the invention; b) a complementary nucleic acid of a nucleic acid such as defined in a); and c) a nucleic acid of at least 18 nucleotides capable of hybridizing under conditions of great stringency with at least one of the CDRs of nucleic acid sequence SEQ ID No. 1, 3, 5, 7, 9 or 11, or with a sequence having at least 80%, preferably 85%, 90%, 95% and 98%, identity after optimum alignment with the sequence SEQ ID No.
  • nucleic acid nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be employed indifferently in the present invention, it is intended to indicate a precise linkage of nucleotides, which are modified or unmodified, allowing a fragment or a region of a nucleic acid to be defined, containing or not containing unnatural nucleotides, and being able to correspond just as well to a double-stranded DNA, a single-stranded DNA as to the transcription products of said DNAs.
  • the present invention does not concern the nucleotide sequences in their natural chromosomal environment, that is to say in the natural state. It concerns sequences which have been isolated and/or purified, that is to say that they have been selected directly or indirectly, for example by copy, their environment having been at least partially modified. It is thus likewise intended to indicate here the isolated nucleic acids obtained by genetic recombination by means, for example, of host cells or obtained by chemical synthesis.
  • nucleic sequences having a percentage of identity of at least 80%, preferably 85%, 90%, 95% and 98% after optimum alignment with a preferred sequence, it is intended to indicate the nucleic sequences having, with respect to the reference nucleic sequence, certain modifications such as, in particular, a deletion, a truncation, an elongation, a chimeric fusion and/or a substitution, especially point substitution. It preferably concerns sequences in which the sequences code for the same amino acid sequences as the reference sequence, this being connected to the degeneracy of the genetic code, or complementary sequences which are capable of hybridizing specifically with the reference sequences, preferably under conditions of high stringency, especially such as defined below.
  • a hybridization under conditions of high stringency signifies that the temperature conditions and ionic strength conditions are chosen in such a way that they allow the maintenance of the hybridization between two fragments of complementary DNA.
  • conditions of high stringency of the hybridization step for the purposes of defining the polynucleotide fragments described above are advantageously the following.
  • the DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 42°C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 x SSC (1 x SSC corresponds to a 0.15 M NaCl + 0.015 M sodium citrate solution), 50% of formamide, 7% of sodium dodecyl sulfate (SDS), 10 x Denhardt's, 5% of dextran sulfate and 1% of salmon sperm DNA; (2) actual hybridization for 20 hours at a temperature dependent on the size of the probe (i.e.: 42°C, for a probe size > 100 nucleotides) followed by 2 washes of 20 minutes at 20°C in 2 x SSC + 2% of SDS, 1 wash of 20 minutes at 20°C in 0.1 x SSC + 0.1 % of SDS.
  • phosphate buffer 20 mM, pH 7.5
  • SSC sodium dodecyl sulfate
  • the last wash is carried out in 0.1 x SSC + 0.1% of SDS for 30 minutes at 60°C for a probe size > 100 nucleotides.
  • the hybridization conditions of high stringency described above for a polynucleotide of defined size can be adapted by the person skilled in the art for oligonucleotides of greater or smaller size, according to the teaching of Sambrook et al (1989, Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor).
  • the invention likewise relates to a vector comprising a nucleic acid according to the present invention.
  • the invention aims especially at cloning and/or expression vectors which contain a nucleotide sequence according to the invention.
  • the vectors according to the invention preferably contain elements which allow the expression and/or the secretion of the nucleotide sequences in a determined host cell.
  • the vector must therefore contain a promoter, signals of initiation and termination of translation, as well as appropriate regions of regulation of transcription. It must be able to be maintained in a stable manner in the host cell and can optionally have particular signals which specify the secretion of the translated protein. These different elements are chosen and optimized by the person skilled in the art as a function of the host cell used.
  • the nucleotide sequences according to the invention can be inserted into autonomous replication vectors in the chosen host, or be integrative vectors of the chosen host.
  • Such vectors are prepared by methods currently used by the person skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as lipofection, electroporation, thermal shock, or chemical methods.
  • the vectors according to the invention are, for example, vectors of plasmidic or viral origin. They are useful for transforming host cells in order to clone or to express the nucleotide sequences according to the invention.
  • the invention likewise comprises the host cells transformed by or comprising a vector according to the invention.
  • the host cell can be chosen from prokaryotic or eukaryotic systems, for example bacterial cells but likewise yeast cells or animal cells, in particular mammalian cells. It is likewise possible to use insect cells or plant cells.
  • the invention likewise concerns animals, except man, which comprise at least one cell transformed according to the invention.
  • a subject of the invention is a process for production of an antibody, or one of its functional fragments according to the invention, characterized in that it comprises the following stages: a) culture in a medium and appropriate culture conditions of a host cell according to the invention; and b) the recovery of said antibodies, or one of their functional fragments, thus produced starting from the culture medium or said cultured cells.
  • the cells transformed according to the invention can be used in processes for preparation of recombinant polypeptides according to the invention.
  • the processes for preparation of a polypeptide according to the invention in recombinant form characterized in that they employ a vector and/or a cell transformed by a vector according to the invention, are themselves comprised in the present invention.
  • a cell transformed by a vector according to the invention is cultured under conditions which allow the expression of said polypeptide and said recombinant peptide is recovered.
  • the host cell can be chosen from prokaryotic or eukaryotic systems.
  • nucleotide sequences according to the invention facilitating secretion in such a prokaryotic or eukaryotic system.
  • a vector according to the invention carrying such a sequence can therefore advantageously be used for the production of recombinant proteins, intended to be secreted.
  • the purification of these recombinant proteins of interest will be facilitated by the fact that they are present in the supernatant of the cell culture rather than in the interior of the host cells.
  • polypeptides according to the invention by chemical synthesis. Such a preparation process is likewise a subject of the invention.
  • the present invention concerns an antibody according to the invention such as described further above, characterized in that it is, moreover, capable of binding specifically to the human epidermal growth factor receptor EGFR and/or capable of specifically inhibiting the tyrosine kinase activity of said EGFR.
  • the growth factors are small proteins involved in the regulation of the proliferation and of the differentiation of normal cells. Some of these growth factors likewise play an important role in the initiation and the maintenance of cell transformation, being able to function as autocrine or paracrine factors.
  • EGF epidermal growth factor
  • the inventors have, firstly, demonstrated that a coinhibition of the attachment of the IGFl and/or IGF2 and/or insulin to the hybrid-R or both the attachment of the IGFl and/or IGF2 and/or insulin to the hybrid-R with the attachement of the IGFl and/or IGF2 to the IGF-IR and of the attachment of the EGF to the EGFR allows a significant synergy of action of these two actions to be obtained against the in vivo tumor growth in nude mice carrying a tumor expressing these three receptors.
  • EGF and IGFl themselves act in synergy in the transformation of normal cells to cells with tumoral character and/or in the growth and/or the proliferation of tumor cells for certain tumors, especially for those overexpressing, or with an abnormal activation of, the three receptors EGFR,hybrid-R and IGF-IR and/or having an overactivation of the transduction signal mediated by these three receptors, in particular at the level of the tyrosine kinase activity of these receptors.
  • the invention concerns an antibody such as described further above, characterized in that it consists of a bispecific antibody comprising a second motif specifically inhibiting the attachment of the EGF to the EGFR and/or specifically inhibiting the tyrosine kinase activity of said EGFR.
  • second motif is intended to indicate above especially a sequence of amino acids comprising a fragment capable of specifically binding to EGFR, in particular a CDR region of a variable chain of an anti-EGFR antibody, or one of the fragments of this CDR region of sufficient length in order to exert this specific binding, or else several CDR regions of an anti-EGFR antibody.
  • the bispecific or bifunctional antibodies form a second generation of monoclonal antibodies in which two different variable regions are combined in the same molecule (Hollinger and Bohlen, 1999, Cancer and metastasis rev., 18:411-419). Their use has been demonstrated both in the diagnostic field and in the therapy field from their capacity to recruit new effector functions or to target several molecules on the surface of tumor cells.
  • These antibodies can be obtained by chemical methods (Glennie MJ et al, 1987, J. Immunol, 139:2367-2375; Repp R. et al, 1995, J. Hemat, 377-382) or somatic methods (Staerz U.D. and Bevan M.J., 1986, PNAS 83:1453-1457; Suresh M.R.
  • bispecific antibodies can be constructed as entire IgG, as bispecific Fab'2, as Fab'PEG or as diabodies or else as bispecific scFv but likewise as a tetravalent bispecific antibody or two attachment sites are present for each antigen targeted (Park et al, 2000, Mol. Immunol, 37(18): 1123-30) or its fragments as described further above.
  • the use of such bispecific antibodies has the advantage of reducing the toxicity of the treatment. This is because the use of a bispecific antibody allows the total quantity of circulating antibodies to be reduced and, consequently, the possible toxicity.
  • the bispecific antibody is a bivalent or tetravalent antibody.
  • the interest in using a tetravalent bispecific antibody is that it has a greater avidity in comparison with a bivalent antibody on account of the presence of three attachment sites for each target, respectively IGF-IR, hybrid-R and EGFR in the present invention.
  • said second motif is selected from the fragments Fv, Fab, F(ab') 2 , Fab', scFv, scFv-Fc, scFv-CH 3 and the diabodies, or any form whose half-life would have been increased like the pegylated fragments such as Fv-PEG, scFv-PEG, Fab-PEG, F(ab') 2 -PEG or Fab'-PEG.
  • said second anti-EGFR motif is descended from the mouse monoclonal antibody 225, its mouse-man chimeric derivative C225, or a humanized antibody derived from this antibody 225.
  • a subject of the invention is an antibody, or one of its functional fragments, according to the invention as a medicament, preferably a humanized antibody such as defined above.
  • Antibody for the remainder of the present description, must be understood as an i) anti-hybrid-R or ii) both anti-hybrid-R and anti- IGF-IR antibody as well as a bispecific anti-hybrid-R/EGFR or both anti-hybrid-R and anti-IGF-IR/EGFR antibody.
  • the invention likewise concerns a pharmaceutical composition
  • a pharmaceutical composition comprising by way of active principle a compound consisting of or comprising an antibody, or one of its functional fragments according to the invention, preferably mixed with an excipient and/or a pharmaceutically acceptable vehicle.
  • the present invention likewise concerns a pharmaceutical composition such as described further above which comprises a second compound chosen from the compounds capable of specifically inhibiting the attachment of the EGF to the human epidermal growth factor receptor EGFR and/or capable of specifically inhibiting the tyrosine kinase activity of said EGFR.
  • said second compound is chosen from the isolated anti-EGFR antibodies, or their functional fragments, capable of inhibiting by competition the attachment of the EGF to the EGFR.
  • said anti-EGFR antibody is chosen from the monoclonal, chimeric or humanized anti-EGFR antibodies, or their functional fragments.
  • said functional fragments of the anti-EGFR antibody are chosen from the fragments Fv, Fab, F(ab') 2 , Fab', scFv-Fc, scFv-CH 3 or diabodies, or any fragment whose half-life would have been increased, like pegylated fragments.
  • Said antibody can consist, in an even more preferred manner, of the mouse monoclonal antibody 225, its mouse-man chimeric derivative C225 (also called IMC-C225) or a humanized antibody derived from this antibody 225.
  • Another complementary embodiment of the invention consists in a composition such as described above which comprises, moreover, as a combination product for simultaneous, separate or sequential use, a cytotoxic/cytostatic agent and/or an inhibitor of the tyrosine kinase activity respectively of the receptors for IGFl and/or for EGF, a corticosteroid agent, an anti-emetic agent, a cancer -vaccine, an analgesic agent, an anti- vascular agent and/or an anti-proliferative agent.
  • a cytotoxic/cytostatic agent and/or an inhibitor of the tyrosine kinase activity respectively of the receptors for IGFl and/or for EGF a corticosteroid agent, an anti-emetic agent, a cancer -vaccine, an analgesic agent, an anti- vascular agent and/or an anti-proliferative agent.
  • “Separate use” is understood as meaning the administration, at the same time, of the two compounds of the composition according to the invention in distinct pharmaceutical forms.
  • “Sequential use” is understood as meaning the successive administration of the two compounds of the composition according to the invention, each in a distinct pharmaceutical form.
  • the composition according to the invention considerably increases the efficacy of the treatment of cancer.
  • the therapeutic effect of the anti-hybrid-R or both anti-hybrid-R and anti-IGF-IR antibody according to the invention is potentiated in an unexpected manner by the administration of a cytotoxic agent.
  • composition according to the invention concerns the possibility of using lower efficacious doses of active principle, which allows the risks of appearance of secondary effects to be avoided or to be reduced, in particular the effects of the cytotoxic agent.
  • this composition according to the invention would allow the expected therapeutic effect to be attained more rapidly.
  • said composition as a combination product according to the invention is characterized in that said cytotoxic/ cytostatic agent is chosen from the agents interacting with DNA, the antimetabolites, the topoisomerase I or II inhibitors, or else the spindle inhibitor or stabilizer agents or else any agent capable of being used in chemotherapy.
  • Such cytotoxic/cytostatic agents for each of the aforesaid classes of cytotoxic agents, are, for example, cited in the 2001 edition of VIDAL, on the page devoted to the compounds attached to the cancerology and hematology column "Cytotoxics", these cytotoxic compounds cited with reference to this document are cited here as preferred cytotoxic agents.
  • said composition as a combination product according to the invention is characterized in that said cytotoxic agent is coupled chemically to said antibody for simultaneous use.
  • said composition according to the invention is characterized in that said cytotoxic/cytostatic agent is chosen from the spindle inhibitor or stabilizer agents, preferably vinorelbine and/or vinflunine and/or vincristine.
  • Immunoliposomes are liposomes capable of vehicling compounds, such as cytotoxic and/or cytostatic agents, such as described above, and of addressing them to tumor cells by means of antibodies or of antibody fragments attached to their surface.
  • the antibodies or antibody fragments used are directed against antigens overexpressed at the surface of tumor cells and/or surface antigens the expression of which is restricted to tumor cells. They are preferably directed against tyrosine kinase receptors, and more particularly against the receptors for IGF-I, EGF or else VEGF.
  • a preferred antibody is a monoclonal or polyclonal, preferably monoclonal, or even humanized, antibody which will recognize the hybrid-R or both the hybrid-R and the IGF-IR specifically and with high affinity.
  • this antibody consists of the antibody which is the subject of the present invention.
  • the use of immunoliposomes for inhibiting tumor cell growth has been described in the literature.
  • Immunoliposomes combine the advantages of liposomes and of immunoconjugates. Liposomes in fact make it possible to encapsulate cytotoxic and/or cytostatic agents and thus to protect them against degradation. They also have the advantage of decreasing the toxicity of the vehiculed agents and of reducing the side effects that they induce. They may thus allow the use of agents which are much more toxic than the agents conventionally used in anticancer chemotherapies.
  • the conjugation of antibodies or of antibody fragments to the surface of liposomes has the advantage of thus providing a system for specific targeting and addressing of the cytotoxic agent encapsulated in the liposome.
  • the vehiculed agent since the vehiculed agent is not covalently coupled to the antibody or to the antibody fragment, it will be completely active as soon as it is introduced into the target cell
  • the antibodies or antibody fragments may be attached, without any limitation, covalently to the surface of the liposomes using conventional methods of bioconjugation.
  • the coupling of these antibodies or of the fragments will be carried out on the lipids or lipids carrying a PEG which have been inserted into the liposomal membrane. In the case of a PEG-lipid, the coupling will be carried out on the PEG in the distal position with respect to the lipid.
  • Liposomes careying PEG groups have the advantage of having longer half-lives than "naked" liposomes.
  • the thiol groups for this type of coupling may come from 2 sources.
  • Fab' or scFv fragments may be free cysteine residues introduced into a recombinant fragment of the antibody of interest, for example Fab' or scFv fragments with an additional cysteine residue, or released after enzymatic hydrolysis of the antibody of interest and controlled reduction, which is the case, for ex.ample, during the preparation of Fab' fragments from complete antibodies.
  • Complete antibodies can also be coupled, after controlled oxidation of the oligosaccharides carried by the heavy chains, to lipids or PEG-lipids exhibiting free amine or hydrazide groups.
  • immunoliposomes having antibodies anti-hybrid-R or both anti-hybrid-R and anti-IGR-IR, or fragments thereof, attached covalently to the surface of the liposomes, are comprised in the present invention.
  • Method for the treatment of cancer wherein such immunoliposomes are administrated to patient in need of such treatment, forms also part of the present invention.
  • said inhibitor of the tyrosine kinase activity of the receptors for IGFl and/or for EGF is selected from the group consisting of derived natural agents, dianilinophthalimides, pyrazolo- or pyrrolopyridopyrimidines or else quinazilines.
  • Such inhibitory agents are well known to the person skilled in the art and described in the literature (Ciardiello F., Drugs 2000, Suppl 1, 25-32).
  • inhibitors of EGFR can, without any limitation, consist of the anti-EGFR monoclonal antibodies C225 and 22Mab (ImClone Systems Incorporated), ABX-EGF (Abgenix/Cell Genesys), EMD-7200 (Merck KgaA) or the compounds ZD-1834, ZD- 1838 and ZD-1839 (AstraZeneca), PKI-166 (Novartis), PKI-166/CGP-75166 (Novartis), PTK 787 (Novartis), CP 701 (Cephalon), leflunomide (Pharmacia/Sugen), CI-1033 (Warner-Lambert Parke-Davis), CI-1033/PD 183, 805 (Warner-Lambert Parke-Davis), CL-387, 785 (Wyeth-Ayerst), BBR-1611 (Boehringer Mannheim GmbH/Roche), Naamidine A (Bristol-Myers Squibb), RC-39
  • the composition such as described above can likewise comprise another antibody compound directed against the extracellular domain of the HER2/neu receptor, as a combination product for simultaneous, separate or sequential use, intended for the prevention and for the treatment of cancer, especially the cancers overexpressing said HER2/neu receptor and the receptor IGF-IR and/or EGFR, such as especially cancer of the breast.
  • another antibody compound directed against the extracellular domain of the HER2/neu receptor as a combination product for simultaneous, separate or sequential use, intended for the prevention and for the treatment of cancer, especially the cancers overexpressing said HER2/neu receptor and the receptor IGF-IR and/or EGFR, such as especially cancer of the breast.
  • said anti-HER2/neu antibody of the composition according to the invention is the antibody called Trastuzumab (also called Herceptin).
  • the invention relates, in another aspect, to a composition characterized in that one, at least, of said antibodies, or one of their functional fragments, is conjugated with a cell toxin and/or a radioelement.
  • said toxin or said radioelement is capable of inhibiting at least one cell activity of cells expressing, or with an abnormal activation of, the hybrid-R or both the hybrid-R and the IGF-IR and/or EGFR, in a more preferred manner capable of preventing the growth or the proliferation of said cell, especially of totally inactivating said cell.
  • said toxin is an enterobacterial toxin, especially Pseudomonas exotoxin A.
  • the radio elements (or radioisotopes) preferably conjugated to the antibodies employed for the therapy are radioisotopes which emit gamma rays and preferably iodine 131 , yttrium 90 , gold 199 , palladium 100 , copper 67 , bismuth 217 and antimony 211 .
  • the radioisotopes which emit beta and alpha rays can likewise be used for the therapy.
  • toxin or radioelement conjugated to at least one antibody, or one of its functional fragments, according to the invention it is intended to indicate any means allowing said toxin or said radioelement to bind to said at least one antibody, especially by covalent coupling between the two compounds, with or without introduction of a linking molecule.
  • the agents allowing binding in a chemical (covalent), electrostatic or noncovalent manner of all or part of the components of the conjugate mention may particularly be made of benzoquinone, carbodiimide and more particularly EDC (1- ethyl-3- [3 -dimethylaminopropyl] -carbodiimide hydrochloride), dimaleimide, dithiobis- nitrobenzoic acid (DTNB), N-succinimidyl S-acetyl thio-acetate (SAT A), the bridging agents having one or more phenylazide groups reacting with the ultraviolets (UN.) and preferably ⁇ -[-4-(azidosalicylamino)butyl]-3 '-(2'-pyridyldithio)-propionamide (APDP), N-succinimid-yl 3-(2-pyridyldithio)propionate (SPDP), 6-hydrazino- nicotinamide (HY
  • Another form of coupling can consist in the use of a bifunctional ion chelator.
  • chelates derived from EDTA (ethylenediaminetetraacetic acid) or from DTPA (diethylenetriaminepentaacetic acid) which have been developed for binding metals, especially radioactive metals, and immunoglobulins.
  • DTPA and its derivatives can be substituted by different groups on the carbon chain in order to increase the stability and the rigidity of the ligand-metal complex (Krejcarek et al, 1977; Brechbiel et al, 1991; Gansow, 1991; US patent 4,831,175).
  • DTPA diethylenetriaminepentaacetic acid
  • said at least one antibody forming said conjugate according to the invention is chosen from its functional fragments, especially the fragments amputated of their Fc component such as the scFv fragments.
  • the present invention moreover comprises the use of the composition according to the invention for the preparation of a medicament. More particularly, according to another embodiment, the invention concerns the use of an antibody, or one of its functional fragments, and/or of a composition for the preparation of a medicament intended for the prevention or for the treatment of an illness induced by an overexpression and/or an abnormal activation of the hybrid-R and/or EGFR, and/or connected with a hyperactivation of the transduction pathway of the signal mediated by the interaction of the native ligands of these receptors, preferably of IGFl , IGF2 or insulin with hybrid-R, and/or of EGF with EGFR and/or HER2/neu.
  • the native ligands of these receptors preferably of IGFl , IGF2 or insulin with hybrid-R, and/or of EGF with EGFR and/or HER2/neu.
  • the invention concerns the use of an antibody, or one of its functional fragments, and/or of a composition for the preparation of a medicament intended for the prevention or for the treatment of an illness induced by an overexpression and/or an abnormal activation of both the hybrid-R and the IGF- IR and/or EGFR, and/or connected with a hyperactivation of the transduction pathway of the signal mediated by the interaction of the native ligands of these receptors, preferably of IGFl, IGF2 or insulin with hybrid-R, of IGFl or IGF2 with IGF-IR and/or of EGF with EGFR and/or HER2/neu.
  • a product or a composition for the preparation of a medicament intended for the prevention or for the treatment of a disease it is also comprised " a method of preventing or treatment of such disease comprising the administration of said product or composition in a patient in need of such treatment”.
  • said use according to the invention is characterized in that the administration of said medicament does not induce or induces only slightly secondary effects connected with inhibition of the IR, that is to say inhibition of the interaction of the IR with its natural ligands due to the presence of said medicament, especially by a competitive inhibition connected with the attachment of said medicament to the IR.
  • the present invention moreover comprises the use of an antibody, or one of its functional fragments, preferably humanized, and/or of a composition according to the invention for the preparation of a medicament intended to inhibit the transformation of normal cells into cells with tumoral character, preferably IGF-dependent, especially IGFl- and/or IGF2-dependent, and/or EGF-dependent and/or HER2/neu-dependent cells.
  • tumoral character preferably IGF-dependent, especially IGFl- and/or IGF2-dependent, and/or EGF-dependent and/or HER2/neu-dependent cells.
  • the present invention likewise relates to the use of an antibody, or one of its functional fragments, preferably humanized, and/or of a composition according to the invention for the preparation of a medicament intended to inhibit the growth and/or the proliferation of tumor cells, preferably IGF-dependent, especially IGFl- and/or IGF2- dependent, and/or EGF-dependent and/or estrogen- dependent, and/or HER2/neu- dependent cells.
  • IGF-dependent especially IGFl- and/or IGF2- dependent, and/or EGF-dependent and/or estrogen- dependent, and/or HER2/neu- dependent cells.
  • a subject of the present invention is the use of an antibody, or one of its functional fragments, preferably humanized, and/or of a composition according to the invention, for the preparation of a medicament intended for the prevention or for the treatment of cancer preferably expressing, or with an abnormal activation of, hybrid-R, or both hybrid-R and IGF-IR, and/or EGFR, and/or of cancer preferably having a hyperactivation of the transduction pathway of the signal mediated by the interaction between the hybrid-R and its native ligands, preferably IGFl;, IGF2 and insulin and/or IGF-IR and its native ligands, preferably IGFl and IGF2 or, for example, the overexpression of IRS1 and/or between EGFR with its native ligand, preferably EGF.
  • the subject of the present invention is likewise the use of an antibody, or one of its functional fragments, preferably humanized, and/or of a composition according to the invention, for the preparation of a medicament intended for the prevention or for the treatment of psoriasis, psoriasis whose epidermal hyperproliferation can be connected with the expression or the overexpression of the hybrid-R, or of both the hybrid-R and the IGF-IR, and/or EGFR, and/or with the hyperactivation of the transduction pathway of the signal mediated by the interaction of hybrid-R or both hybrid-R and IGF-IR respectively with their natural ligands (Wraight CJ. et al, Nat.
  • Biotechnol., 2000, 18(5):521-526 Reversal of epidermal hyperproliferation in psoriasis by insulin-like growth factor I receptor antisense oligonucleotides) and/or of EGFR with its natural ligands.
  • the cancers which can be prevented and/or treated with the antibody according to the invention and able to bind (or inhibit tyrosine kinase activity) the hybrid-R, breast cancer and thyroid cancer, or any other cancer overexpressing, or with an abnormal activation of hybrid-R is preferred.
  • both the hybrid-R and IGF-IR prostate cancer, osteosarcomas, lung cancer, breast cancer, endometrial cancer, pancreatic cancer, myeloid cancer, bladder cancer, thyroid cancer, melanoma or colorectal cancer or any other cancer overexpressing, or with an abnormal activation of both hybrid-R and IGF-IR is preferred.
  • a subject of the present invention is a method of diagnosis, preferably in vitro, of illnesses connected with an overexpression or an underexpression, preferably an overexpression, of the hybrid-R and/or EGFR starting from a biological sample in which the abnormal presence of hybrid-R and/or EGFR is suspected, characterized in that said biological sample is contacted with an antibody, or one of its functional fragments, according to the invention, it being possible for said antibody to be, if necessary, labeled.
  • a subject of the present invention is a method of diagnosis, preferably in vitro, of illnesses connected with an overexpression or an underexpression, preferably an overexpression, of both the hybrid-R and the IGF-IR, and/or EGFR starting from a biological sample in which the abnormal presence of both hybrid-R and IGF-IR, and/or EGFR is suspected, characterized in that said biological sample is contacted with an antibody, or one of its functional fragments, according to the invention, it being possible for said antibody to be, if necessary, labeled.
  • said illnesses connected with the overexpression, or with an abnormal activation of, of the hybrid-R or both the hybrid-R and the IGF-IR and/or EGFR in said diagnosis method will be cancers.
  • Said antibody, or one of its functional fragments can be present in the form of an immunoconjugate or of a labeled antibody so as to obtain a detectable and/or quantifiable signal.
  • the antibodies labeled according to the invention or their functional fragments include, for example, antibodies called immunoconjugates which can be conjugated, for example, with enzymes such as peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, glucose oxydase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase or glucose 6-phosphate dehydrogenase or by a molecule such as biotin, digoxygenin or 5-bromodeoxyuridine.
  • enzymes such as peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, glucose oxydase, glucose amylase, carbonic anhydrase, acety
  • Fluorescent labels can be likewise conjugated to the antibodies or to their functional fragments according to the invention and especially include fluorescein and its derivatives, fluorochrome, rhodamine and its derivatives, GFP (GFP for "Green Fluorescent Protein”), dansyl, umbelliferone etc.
  • the antibodies of the invention or their functional fragments can be prepared by methods known to the person skilled in the art.
  • conjugates containing labels of fluorescein type can be prepared by reaction with an isothiocyanate.
  • conjugates can likewise include chemoluminescent labels such as luminol and the dioxetanes, bio-luminescent labels such as luciferase and luciferin, or else radioactive labels such as iodine , iodine , iodine , iodine , bromine , technetium 99 “ 1 , indium 111 , indium 1131 ", gallium 67 , gallium 68 , ruthenium 95 , ruthenium 97 , ruthenium 103 , ruthenium 105 , mercury 107 , mercury 203 , rhenium 99 “ 1 , rhenium 101 , rhenium 105 , scandium 47 , tellurium 121 “ 1 , tellurium 122 “ 1 , tellurium 125 “ 1 , thulium 165 , thulium 167 , thulium 168 , fluorine 18 , yttrium 199
  • the antibodies, or their functional fragments, according to the invention can be employed in a process for the detection and/or the quantification of an overexpression or of an underexpression, preferably an overexpression, or with an abnormal activation of, of the IGF-IR and/or hybrid-R and/or EGFR in a biological sample, characterized in that it comprises the following steps: a) the contacting of the biological sample with an antibody, or one of its functional fragments, according to the invention; and b) the demonstration of the hybrid-R, or both hybrid-R and IGF-IR, and/or
  • the antibodies, or their functional fragments, according to the invention can be employed in a process for the detection and/or the quantification of the hybrid-R, or of both the hybrid-R and the IGF-IR, and/or EGFR in a biological sample, for the monitoring of the efficacy of a prophylactic and/or therapeutic treatment of IGF- and/or EGF-dependent cancer or else of psoriasis. More generally, the antibodies, or their functional fragments, according to the invention can be advantageously employed in any situation where the expression, or with an abnormal activation of, of the hybrid-R, or both hybrid-R and IGF-IR, and/or EGFR must be observed in a qualitative and/or quantitative manner.
  • the biological sample is formed by a biological fluid, such as serum, whole blood, cells, a tissue sample or biopsies of human origin.
  • a biological fluid such as serum, whole blood, cells, a tissue sample or biopsies of human origin.
  • Any procedure or conventional test can be employed in order to carry out such a detection and/or dosage.
  • Said test can be a competition or sandwich test, or any test known to the person skilled in the art dependent on the formation of an immune complex of antibody-antigen type.
  • the antibody or one of its functional fragments can be immobilized or labeled. This immobilization can be carried out on numerous supports known to the person skilled in the art. These supports can especially include glass, polystyrene, poly- propylene, polyethylene, dextran, nylon, or natural or modified cells. These supports can be either soluble or insoluble.
  • the invention also comprises the kits or sets necessary for carrying out a method of diagnosis of illnesses induced by an overexpression or an underexpression of both the hybrid-R and the IGF-IR, and/or EGFR or for carrying out a process for the detection and/or the quantification of an overexpression or of an underexpression of both the hybrid-R and the IGF-IR, and/or EGFR in a biological sample, preferably an overexpression, or with an abnormal activation of, of said receptor(s), characterized in that said kit or set comprises the following elements: a) an antibody, or one of its functional fragments, according to the invention; b) optionally, the reagents for the formation of the medium favorable to the immunological reaction; c) optionally, the reagents allowing the demonstration of hybrid-R/IGF-IR antibody and/or EGFR/antibody complexes produced by the immunological reaction.
  • the invention moreover 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 for the treatment of cancer, especially cancers for which said cytotoxic agent or said anti-HER2/neu antibody is generally prescribed and, especially, for which cancers the tumor cells express or overexpress the IGF-IR and/or EGFR.
  • a subject of the invention is likewise the use of an antibody according to the invention for the preparation of a medicament intended for the specific targeting of a biologically active compound to cells expressing or overexpressing the hybrid-R or both the hybrid-R and the IGF-IR and/or EGFR.
  • a subject of the invention is also an in vivo diagnostic reagent comprising an antibody according to the invention, or one of its functional fragments, preferably labeled, especially radiolabeled, and its use in medical imaging, in particular for the detection of cancer connected with the expression or the overexpression by a cell of the hybrid-R, or both the hybrid-R and the IGF-IR, and/or EGFR.
  • the invention likewise relates to a composition as a combination product or to an anti-hybrid-R, or both anti-hybrid-R and anti-IGF-IR, and/or EGFR/toxin conjugate or radioelement, according to the invention, as a medicament.
  • said composition as a combination product or said conjugate according to the invention will be mixed with an excipient and/or a pharmaceutically acceptable vehicle.
  • pharmaceutically acceptable vehicle is intended to indicate a compound or a combination of compounds entering into a pharmaceutical composition not provoking secondary reactions and which allows, for example, facilitation of the administration of the active compound(s), an increase in its lifespan and/or in its efficacy in the body, an increase in its solubility in solution or else an improvement in its conservation.
  • These pharmaceutically acceptable vehicles are well known and will be adapted by the person skilled in the art as a function of the nature and of the mode of administration of the active compound(s) chosen.
  • these compounds will be administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal, intraperitoneal or subcutaneous route, or by the oral route.
  • compositions comprising the antibodies according to the invention will be administered several times, in a sequential manner.
  • Their modes of administration, dosages and optimum pharmaceutical forms can be determined according to the criteria generally taken into account in the establishment of a treatment adapted to a patient such as, for example, the age or the body weight of the patient, the seriousness of his/her general condition, the tolerance to the treatment and the secondary effects noted.
  • the antibody, or fragments thereof could be use alone or in association with another antibody able to target another growth factor implied in the proliferation or dissemination of tumoral cells.
  • chemotherapeutic agent or another tyrosine kinase inhibitor in co-administration or in the form of an immuno-conjugate, said agent being chemical, biological and/or natural.
  • Fragments of said antibody could also be use in bispecific antibodies obtained by recombinant mechanisms or biochemical coupling, and then associating the specificity of the above described antibody with the specificity of other antibodies able to recognise other receptors involved in the proliferation, the angiogenese or any other mechanisms involved in the tumoral development.
  • cytotoxic and/or cytostatic active agent coupled to an addressing system, particularly to the antibodies 7C10, C7C10 or h7C10, or fragment thereof, according to the present invention capable of binding specifically to the hybrid-R or both the hybrid-R and the IGF-IR.
  • the present invention relates also to novel compounds comprising a cytotoxic and/or cytostatic active agent coupled to an addressing system. More particularly, the present invention relates to a compound comprising a Vinca alkaloid coupled to an antibody capable of binding specifically to the hybrid-R or both the hybrid-R and the
  • the invention also relates to the mode of coupling of the elements of said compound and also comprises the use of these compounds as a medicinal product for the prophylactic and/or therapeutic treatment of cancer, more particularly of cancers overexpressing hybrid-R and/or IGF-IR, or of any pathological condition associated with overexpression, or with an abnormal activation of, of said receptor(s).
  • chemotherapy represents one of the most effective means of combating cancer.
  • cytotoxic and/or cytostatic agents have been isolated or synthesized and make it possible to destroy or reduce, if not definitively, at least significantly, the tumor cells.
  • the toxic activity of these agents is not limited to tumor cells, and the non-tumor cells are also effected and can be destroyed. More particularly, side effects are observed on rapidly renewing cells, such as haematopoietic cells or cells of the epithelium, in particular of the mucous membranes.
  • the cells of the gastrointestinal tract are largely effected by the use of cytotoxic agents.
  • One of the aims of the present invention is also to be able to provide a compound which makes it possible to limit the side effects on normal cells while at the same time conserving a high cytotoxicity on tumor cells.
  • the applicant rather than developing new molecules, has sought to overcome the problem of toxicity of known molecules by limiting to tumor cells the access of said molecules. To do this, the applicant has developed an antibody-type addressing system for targeting only tumor cells.
  • One of the advantages of this approach is to be able to use known cytotoxic agents which are well defined in pharmacological and pharmacokinetic terms.
  • it is then possible to use strong cytotoxic agents which until now have been neglected in favour of cytotoxic agents which are less strong but which have a better therapeutic index (and therefore exhibit fewer side effects).
  • Another advantage lies in the use of an antibody, i.e. of a product of biological origin which does not add any toxicity to that of the cytotoxic agent.
  • the choice of the antibody makes it possible to accumulate with the action of the cytotoxic agent its own biological activity. The applicant has demonstrated that the use of a Vinca alkaloid coupled to an addressing device is of value in chemotherapy.
  • a subject of the present invention is a compound comprising at least one molecule of active agent coupled to an addressing system, said at least one molecule of active agent being a strong cytotoxic and/or cytostatic compound chosen from Vinca alkaloids, and said addressing system being a polyclonal or monoclonal antibody, which may be bispecific, or a functional fragment thereof, capable of targeting, preferably specifically, tumor cells.
  • a compound according to the invention is that the active agent is directly brought to the target cells by the antibody and, besides the fact that it does not degrade the other cells, its biological activity is not decreased.
  • One of the advantages associated with using antibodies as an addressing system is that it is possible to couple several active agents to them, thus increasing the efficacy of the compound. Specifically, since the compound is brought directly to the target cells, the fact that there are several active agents will not lead to an increase in side effects, but only to an increase in the desired in situ effect on the tumor cells.
  • targeting antibodies which can be used according to the invention, mention may be made, without any limitation, of the CeaVac antibodies directed against colorectal tumor cells, and the Y Theragyn/pemtumomab and OvaRex antibodies directed against ovarian tumor cells.
  • the present invention relates to a compound as described above, which comprises from 1 to 50 molecules of active agent, preferably from 1 to 10, and better still from 1 to 6.
  • the choice of the number of molecules of active agent depends, inter alia, on the molecular weight of each of the elements. For example, by way of indication, for an antibody of IgGl type with a molecular weight of 150 000 Da, it is preferred to couple from 4 to 6 molecules of vinblastine with a molecular weight of 900 Da (Petersen et al, Cancer Res., 1991, 51:2286). If the antibody is conjugated with too large an amount of cytotoxic agents, there is a risk that said agents will mask the recognition site for the antigen and decrease its activity.
  • the compound which is the subject of the invention is used as a medicinal product, and more particularly as an medicinal product intended for the treatment of cancer.
  • the present invention differs from the prior art not only in the sense that the choice of the antibody is aimed at targeting tumor cells as described above, but also in that said antibody exhibits an intrinsic activity on the tumor cells.
  • the compound as described above is also capable of inhibiting tumor cell proliferation and/or apoptotic function restoration by blocking transduction signals, the progression of cells in the cell cycle and/or membrane-bound receptor availability (phenomena of intemalization and of degradation of said receptor), or of reverting an apoptosis-resistant phenotype in the case of an antibody directed against the IGF-IR, insofar as it is widely described that overexpression of this receptor confers on tumor cells a means of withstanding apoptosis and in particular apoptosis induced by chemotherapy compounds (Beech D. J. et al, Oncology reports, 2001, 8:325-329; Grothe A. et al, J. Cancer Res.
  • Another mechanism of action of the compound as described above may be associated with the Fc portion of the antibody, if a whole antibody is used, and may consist of the setting up of effector mechanisms such as ADCC (antibody- dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity).
  • ADCC antibody- dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Avastin/Bevacizumab which acts on colorectal cancers by interfering with tumor angiogenesis
  • Rituxan/rituximab the activity of which is mainly related to the effector functions of the molecule, and in particular ADCC
  • Herceptin/trastuzumab which acts by inhibition of signal transduction and inhibition of cell progression in the cell cycle, and also, in large part, by initiating ADCC mechanisms.
  • Vinca alkaloids correspond to the family of natural compounds of which vinblastine, vincristine, anhydrovinblastine and leurosine, which are present in considerable amounts in plants, are demonstrative examples.
  • Vinca alkaloids should also be understood to mean all the derivatives present in small amounts, such as deoxyvinblastine or leurosidine, taken by way of non-limiting examples. It should also be understood to mean derivatives of natural structure but which are obtained by synthesis, such as, without any limitation, anhydrovinblastine.
  • Vinca alkaloid should also be understood to mean all the compounds derived from these natural compounds by chemical or biochemical modification in one or more steps. These modifications may affect the "vindoline” component or the "velbanamine” component or both components simultaneously.
  • the Vinca alkaloids, as such, are known to those skilled in the art (Antitumor Bisindole Alkaloids from Catharanthus roseus (L.)).
  • the preferred derivatives according to the present invention are those which exhibit a pharmacological advantage established by virtue of cytotoxicity assays or activity assays on certain specific targets, such as tubulin, or which have demonstrated advantages in in vivo tests on animals.
  • cytotoxicity assays or activity assays on certain specific targets, such as tubulin, or which have demonstrated advantages in in vivo tests on animals.
  • the invention is therefore partly based on the choice of an original cytotoxic agent without any bias from the prior art.
  • a subject of the present invention is a compound as described above, in which said Vinca alkaloid is selected from vinblastine, deoxyvinblastine, deoxyleurosidine, vincristine, vindesine, vinorelbine, vinepidine, vinfosiltine, vinzolidine and vinflunine.
  • the subject of the invention has, more specifically, been demonstrated and exemplified using deoxyvinblastine and its 4'-S isomer, commonly known as deoxyleurosidine.
  • deoxyvinblastine and its 4'-S isomer commonly known as deoxyleurosidine.
  • the structure of each of these two compounds has been described for many years, but their pharmacological activity is considered to be moderate or weak (Neuss N. et al, Tetrahedron Letters, 1968, No.
  • the present invention therefore relates to a compound as described above, in which said Vinca alkaloid is (4'-R) deoxyvinblastine and/or (4'-S) deoxyleurosidine.
  • the greater activity of these two derivatives has been demonstrated against P388 murine leukaemia grafted intravenously on day 0.
  • the compound is administered intraperitoneally in a single dose on day 1.
  • the protocol for this test is described by
  • the in vivo activity of cytotoxic compounds is expressed by the T/C at a dose expressed in mg per kg.
  • the T/C corresponds to the ratio, multiplied by 100, of the median of the survival time of the treated animals to the median of the survival time of the control animals.
  • the present invention therefore relates to the use of (4'- R) deoxyvinblastine and/or (4'-S) deoxyleurosidine, collectively referred to as deoxyvinblastine in the remainder of the description, for treating cancer.
  • the present invention envisages the coupling of deoxyvinblastine to a compound of the monoclonal or polyclonal, preferably monoclonal, antibody type.
  • a preferred antibody making up the compound which is the subject of the present invention is a monoclonal or polyclonal, preferably monoclonal, antibody which will recognize the IGF-IR specifically and with high affinity, and which will have the ability to inhibit the growth of tumors, more particularly of tumors expressing the IGF-IR.
  • the cytoplasmic protein tyrosine kinases are activated by binding of the ligand to the extracellular domain of the receptor. Activation of the kinases leads, in turn, to stimulation of various intracellular substrates, including IRS-1, ISR-2, She and Grb 10
  • IRS-1 The two major substrates for the IGF-IR are IRS and She, which mediate, by activation of many downstream effectors, most of growth and differentiation effects associated with the binding of IGFs to this receptor. Substrate availability can, consequently, dictate the final biological effect associated with activation of the IGF-IR.
  • IRS-1 predominates, the cells tend to proliferate and to transform.
  • She dominates the cells tend to differentiate (Valentinis B. et al, J. Biol Chem., 274:12423-12430, 1999).
  • a subject of the present invention is a compound as described above (cytotoxic and/or cytostatic active agent coupled to an addressing system), comprising an antibody capable of recognizing the IGF-IR specifically and with high affinity. This antibody will interact little or not at all with the insulin receptor IR.
  • the scFv-Fc fragment derived from the murine monoclonal 1H7 is only weakly active on the MCF-7 breast tumor and completely inactive on an androgen-independent prostate tumor (Li S.L. et al, Cancer Immunol. Immunother., 49:243-252, 2000).
  • a subject of the present invention is a compound (cytotoxic and/or cytostatic active agent coupled to an addressing system) as described above, comprising an antibody, or one of its functional fragments, said antibody or one of its said fragments being capable of binding specifically to the human insulin-like growth factor-I receptor IGF-IR and, where appropriate, capable of inhibiting the natural binding of the IGF-IR ligands IGFl and/or IGF2, and/or capable of specifically inhibiting the tyrosine kinase activity of said IGF-IR.
  • a compound has a double advantage.
  • cytotoxic agent directly to tumor cells, more particularly tumor cells overexpressing, or with an abnormal activation of, the hybrid-R or both the hybrid-R and the IGF-IR, and thus to decrease the side effects in normal cells.
  • its mode of action is not limited to targeting.
  • the compound which is the subject of the present invention cumulates the action of the cytotoxic agent which makes it possible to destroy the tumor cells and the action of the antibody which will inhibit the growth of tumor cells, preferably of tumor cells expressing, or with an abnormal activation of, the hybrud-R or both the hybrid-R and the IGF-IR, by interacting with the signal transduction pathways, and will make it possible to decrease the resistance to apoptosis of cells overexpressing the receptor for IGFl and, consequently, to improve the activity of chemotherapy drugs, part of the mechanism of action of which lies in the induction of apoptosis.
  • the monoclonal antibody, or one of its functional fragments is the 7C10, a C7C10 or a h7C10, or fragment thereof, or their derived antibodies, as described in the first part of the present specification directed to the antibodies anti-hybrid-R or both anti-hybrid-R and anti-IGR-IR of the present invention.
  • the applicant filed a French patent application FR 03/08538 on July 11, 2003 for "Novel antitumor immunoconjugates". The content of this patent application is incorporated herein by way of reference.
  • Immunoliposomes containing such particular cytotoxic and/or cytostatic agents, such as described above,such as the vinca alkaloids, and of addressing them to tumor cells by means of antibodies or of antibody fragments attached to their surface are comprised in the present invention.
  • Method of treatement of cancer, particularly the preferred cancers cited above, comprising the administration of the present immunoliposomes forms also part of the present invention.
  • the antibodies or antibody fragments used are directed against antigens overexpressed at the surface of tumor cells and/or surface antigens the expression of which is restricted to tumor cells. They are preferably directed against tyrosine kinase receptors, and more particularly against the receptors for IGF-I, EGF or else VEGF.
  • a preferred antibody is a monoclonal or polyclonal, preferably monoclonal, or even humanized, antibody which will recognize the hybrid-R, or both the hybrid-R and the IGF-IR, specifically and with high affinity. Even more preferably, this antibody consists of the antibody anti-hybrid-R or both anti-hybrid-R and anti-IGR-IR which is the subject of the present invention described in the first part of the specification.
  • the monoclonal antibody as described above is also capable of binding specifically to the human epidermal growth factor receptor, EGFR, and/or capable of specifically inhibiting the tyrosine kinase activity of said EGFR.
  • the coupled monoclonal antibody consists of a bispecific antibody comprising a second unit which specifically inhibits the binding of EGF to the EGFR and/or which specifically inhibits the tyrosine kinase activity of said EGFR.
  • the bispecific antibody which can be used here for cytotoxic and/or cytostatic active agent coupled to an addressing system according to this invention is those as described in the first part of the presnt specification related to bispecific antibodies of the invention.
  • Another aspect of the invention concerns the mode of coupling between the antibody and the cytotoxic agent. Whatever the nature of the coupling, which may be direct or indirect, stable or labile, it should in no way impair the respective biological functions of the antibody and of the cytotoxic agent. It is clearly understood that any coupling satisfying this characteristic, and known to those skilled in the art, is included in the scope of the present patent application.
  • the coupling and more particularly the linkage used, must allow release of the deoxyvinblastine, in the 4- deacetylated or 3-acid, or 4-deacetylated and 3-acid, form, or in the form of one of these forms carrying all or part of said linkage used, in the target cells.
  • the coupling is chemical coupling. More particularly, said chemical coupling is composed of an anchorage on the Vinca alkaloid, an anchorage on the antibody and a linkage connecting these two anchorages.
  • linkage should be understood to mean any structure capable of providing a bond of whatever possible nature between the two elements of the compound, namely a chemical molecule and an antibody.
  • the anchorage on the Vinca alkaloid is effected on the acid function in the 3 -position after deacetylation of the 4-acetoxy group and demethylation of the ester function in the 3 -position of said Vinca alkaloid.
  • the anchorage on the Vinca alkaloid is effected on the acid function in the 3 -position directly by reaction on the ester function in the 3-position of said Vinca alkaloid.
  • the anchorage on the Vinca alkaloid is effected via an ester or thioester function on the hydroxyl function in the 3-position.
  • An additional embodiment consists in effecting the anchorage on the Vinca alkaloid via an amide function or an ester function or a hydrazide function on the acid function in the 4-position.
  • the anchorage on the antibody it should in no way denature the antibody, so as not to decrease its ability to recognize and interact with the tumor cells. To do this, it is preferable for the anchorage on the antibody to be effected on the oligosaccharides, the lysines and/or the aspartic acid and glutamic acid residues.
  • the Vinca alkaloid may also be coupled on the carboxylic functions of the antibody, carried by the aspartic acid and glutamic acid residues of the antibody.
  • an amine, hydrazide or hydrazine derivative of the Vinca alkaloid will be coupled on these residues in the presence of a compound of carbodiimide type, such as N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (or ED AC).
  • a compound of carbodiimide type such as N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (or ED AC).
  • the anchorage is effected on the oligosaccharides present on the asparagines (Asn) which are followed by a consensus sequence consisting of an amino acid and a serine or a threonine.
  • a preferred anchorage on the IgGl antibody used in the invention is on Asn297.
  • An additional embodiment consists in greatly increasing the density of the Vinca alkaloid in order to attain 10 to 50 mol per mole of antibody. Mention may be made of the coupling of a hemisuccinate derivative of the Vinca alkaloid on a lysine polymer
  • a hydrazide derivative of the Vinca alkaloid may be coupled on a dextran oxidized beforehand with meta-periodate.
  • the conjugate obtained is then coupled to the antibody via the lysine residues.
  • a hemisuccinate derivative of the Vinca alkaloid may be coupled on a dextran activated beforehand by controlled oxidation with meta-periodate and then substituted with a compound of diamide type. The conjugate obtained is then coupled on the lysine residues of the antibody.
  • the anchorage on the antibody is effected by reaction of an amine function, a hydrazine function, a hydrazide function or an acid function which has been activated. More particularly, the anchorage on the antibody is effected by reaction of an epoxide function or of a disulphide function, a sulphide function or an acid function which has been activated, with a nitrogen-containing residue or with a hydroxyl residue or with a thiol residue of said antibody. Mention may also be made, in a nonlimiting manner, of other linkages which may also be used to covalently attach the Vinca alkaloids to the antibodies or to their functional fragments (Garnett et al, Adv. Drug Deliv.
  • a preferred form of the invention uses a linkage which allows release of the deoxyvinblastine in the tumor cells.
  • a first means for achieving this consists in using a linkage connecting the two anchorages which consists of a peptide chain.
  • the linkage connecting the two anchorages consists of a linear or branched carbon-based chain.
  • one or more aromatic, ethylenic or acetylenic groups and also one or more ketone, amide, ester, hydrazide, hydrazone, amine, ether, sulphide or disulphide groups are included in the carbon chain in a distinct or combined manner.
  • the compound according to the invention is characterized in that the acid function in the 4-position of the Vinca alkaloid is coupled, via a hydrazide function, with an aldehyde residue of the antibody, generated beforehand.
  • the invention also relates to a pharmaceutical composition comprising, as active principle, a compound comprising or consisting of a Vinca alkaloid coupled to an antibody, or one of its functional fragments, according to the invention, to which a pharmaceutically acceptable excipient and/or vehicle is preferably added.
  • the present invention also comprises the use of the compound according to the invention for preparing a medicinal product.
  • the invention relates to the use of a compound as described above and/or of a composition comprising such a compound, for preparing a medicinal product intended for the prevention or treatment of cancers, in particular cancers induced by overexpression and/or activation, or with an abnormal activation of, of the hybrid-R, or both the hybrid-R and the IGF-IR, and/or EGFR which is abnormal, and/or associated with hyperactivation of the signal transduction pathway mediated by the interaction of IGFl, IGF2 or insulin with hybrid- R alone or in combination with the interaction of IGFl or IGF2 with IGF-IR and/or of EGF with EGFR.
  • the invention concerns a humanized anti-IGF-IR antibody, or antigen-binding fragment thereof, wherein said anti-IGF-IR antibody, or antigen-binding fragment, comprises at least one complementary determining region of non-human origin and at least one framework region having at least one human residue, wherein said antibody is characterized as: a) binding IGF-IR but not IR alone; and/or b) inhibiting the binding between a human IGF-IR and its native ligand, preferably designated herein as IGFl and/or IGF2, with an inhibition constant and/or IC 50 of less than 100 nM, preferably less than 50 nM; and/or c) specifically inhibiting the tyrosine kinase activity of said IGF-IR; and/or d) having a binding affinity
  • FR sequences of the light chain are linked with CDR1, CDR2, and CDR3 of sequences derived from a non-human source and comprising the amino acid sequences selected from the group consisting of SEQ ID Nos. 2, 4 and 6.
  • the present invention also deals with a humanized anti- hybrid-R antibody or antigen-binding fragment thereof, wherein said anti-hybrid-R antibody or antigen-binding fragment comprises at least one complementary determining region of non-human origin and at least one framework region having at least one human residue, wherein said antibody is characterized as: a) binding hybrid-R but not IR alone; and/or b) inhibiting the binding between a human hybrid-R and its native ligand, preferably designated herein as IGFl and/or IGF2 and/or insulin, with an inhibition constant and/or IC 50 of less than 100 nM, preferably less than 50 nM; and/or c) specifically inhibiting the tyrosine kinase activity of said hybrid-R; and/or d) having a binding affinity of 10 nM or less for said hybrid-R ; and/or e) down-regulating hybrid-R expression; and/or f) inhibiting in vivo tumor growth; wherein said antibody comprises
  • FR sequences of the light chain are linked with CDR1, CDR2, and CDR3 of sequences derived from a non-human source and comprising the amino acid sequences selected from the group consisting of SEQ ID Nos. 2, 4 and 6.
  • a humanized anti-IGF-IR and hybrid-R antibody or antigen-binding fragment thereof wherein said anti-IGF-IR and hybrid-R antibody or antigen-binding fragment comprises at least one complementary determining region of non-human origin and at least one framework region having at least one human residue, wherein said antibody is characterized as: a) binding IGF-IR and hybrid-R but not IR alone; and/or b) inhibiting the binding between a human IGF-IR and its native ligand, preferably designated herein as IGFl and/or IGF2, with an inhibition constant and/or IC 50 of less than 100 nM, preferably less than 50 nM, and also a human hybrid-R, and its natural ligand designated herein as IGFl and/or IGF2 and/or insulin, with an inhibition constant and/or IC 50 of less than 100 nM, preferably less than 50 nM; and/or c) specifically inhibiting the tyros
  • the invention also describes a method of modulating IGF-IR activity in IGF-IR- responsive mammalian cells comprising: contacting the cells with an antibody specific for said receptor, wherein said antibody comprises a light and a heavy chain, said light chain comprising at least one complementary determining region CDR chosen from the CDRs of sequence SEQ ID Nos. 2, 4 and 6 and said heavy chain comprising at least one complementary determining region CDR chosen from the CDRs of sequence SEQ ID Nos. 8, 10, and 12.
  • the invention describes a method of modulating hybrid-R activity in hybrid-R-responsive mammalian cells comprising: contacting the cells with an antibody specific for said receptor, wherein said antibody comprises a light and a heavy chain, said light chain comprising at least one complementary determining region CDR chosen from the CDRs of sequence SEQ ID Nos. 2, 4 and 6 and said heavy chain comprising at least one complementary determining region CDR chosen from the CDRs of sequence SEQ ID Nos. 8, 10, and 12.
  • an object of the present invention is a method of jointly modulating IGF-IR and hybrid-R activities in IGF-IR and hybrid-R-responsive mammalian cells comprising: contacting the cells with an antibody specific for said receptors, wherein said antibody comprises a light and a heavy chain, said light chain comprising at least one complementary determining region CDR chosen from the CDRs of sequence SEQ ID Nos. 2, 4 and 6 and said heavy chain comprising at least one complementary determining region CDR chosen from the CDRs of sequence SEQ ID Nos. 8, 10, and 12.
  • the methods above described, and wherein the antibody modulates IGF-IR activity is directed against, without limitation, mammalian cells selected from the group consisting of breast cancer cells, prostate cancer cells, colorectal cancer cells, lung cancer cells, bladder cancer cells, kidney cancer cells, thyroid cancer cells, osteosarcomas cells, pancreatic cancer cells, melanoma and myeloid cells.
  • the method above descirbed, wherein the antibody modulates hybrid-R activity is directed against, without limitation, mammalian cells selected from the group consisting of breast cancer cells or thyroid cancer cells. The same for the modulation of both hybrid-R and IGF-IR.
  • the invention also deals with method of decreasing IGF-IR activity in IGF-IR- responsive mammalian cells comprising: contacting the cells with an antibody specific for said receptor in an amount sufficient to decrease the activity of IGF-IR, wherein said antibody comprises a light and a heavy chain, said light chain comprising at least one complementary determining region CDR chosen from the CDRs of sequence SEQ ID Nos. 2, 4 and 6 and said heavy chain comprising at least one complementary determining region CDR chosen from the CDRs of sequence SEQ ID Nos. 8, 10, and 12.
  • the invention concerns, in a second embodiment, a method of decreasing hybrid-R activity in hybrid-R-responsive mammalian cells comprising: contacting the cells with an antibody specific for said receptor in an amount sufficient to decrease the activity of hybrid-R, wherein said antibody comprises a light and a heavy chain, said light chain comprising at least one complementary determining region CDR chosen from the CDRs of sequence SEQ ID Nos. 2, 4 and 6 and said heavy chain comprising at least one complementary determining region CDR chosen from the CDRs of sequence SEQ ID Nos. 8, 10, and 12.
  • a method of jointly decreasing IGF-IR and hybrid-R activities in IGF-IR and hybrid-R-responsive mammalian cells comprising: contacting the cells with an antibody specific for said receptors in an amount sufficient to decrease the activities of IGF-IR and hybrid-R, wherein said antibody comprises a light and a heavy chain, said light chain comprising at least one complementary determining region CDR chosen from the CDRs of sequence SEQ ID Nos. 2, 4 and 6 and said heavy chain comprising at least one complementary determining region CDR chosen from the CDRs of sequence SEQ ID Nos. 8, 10, and 12.
  • the method above mentionned, wherein the antibody decreases IGF-IR activity concerns mammalian cells selected from the group consisting of, without limitation, breast cancer cells, prostate cancer cells, colorectal cancer cells, lung cancer cells, bladder cancer cells, kidney cancer cells, thyroid cancer cells, osteosarcomas cells, pancreatic cancer cells, melanoma and myeloid cells.
  • the method above mentionned, wherein the antibody decreases hybrid-R activity concerns mammalian cells selected from the group consisting of, without limitation, breast cancer cells or thyroid cancer cells. The same for both hybrid-R and IGF-IR.
  • the invention also concerns a method of identifying an IGF-IR modulator comprising: a) contacting IGF-IR with an antibody according to the invention able to bind IGF-IR; b) contacting the complex of (a) with a compound library; c) identifying a compound which disrupts the complex of (a); and d) determining whether the compound exhibits agonist or antagonist activity at the IGF-IR, wherein this activity indicates identification of an IGF-IR modulator.
  • the invention consists in a method of identifying a hybrid-R modulator comprising: a) contacting hybrid-R with an antibody according to the invention able to bind hybrid-R; b) contacting the complex of (a) with a compound library; c) identifying a compound which disrupts the complex of (a); and d) determining whether the compound exhibits agonist or antagonist activity at the hybrid-R, wherein this activity indicates identification of a hybrid-R modulator.
  • a method of identifying an IGF-IR and hybrid-R modulator comprising the following stps is an object of the invention: a) contacting IGF-IR and hybrid-R with an antibody according to the invention able to bind IGF-IR and hybrid-R; b) contacting the complex of (a) with a compound library; c) identifying a compound which disrupts the complex of (a); and d) determining whether the compound exhibits agonist or antagonist activity at the IGF-IR and hybrid-R, wherein this activity indicates identification of an IGF-IR and hybrid-R modulator.
  • the present invention is also describing a method of identifying an IGF-IR modulator comprising: a) screening a library of peptide sequences, wherein said library of peptide sequences bind to IGF-IR, and wherein the library is derived from a peptide sequence comprising at least one sequence selected from the group consisting of SEQ ID Nos.
  • IGF-IR-responsive cell selected from the group consisting of cell lines displaying IGF-IR, such as, for example, MCF-7, T47D, BT20, ZR-75-1, MDA-MB-231 for breast cells, PC3 and DU145 for prostate cells, A549, A427 and SK- LU-1 for lung cells, HT29, Colo205 and CaCo-2 for colon cells, BC-PAP, FRO and ARO for thyroid cells, SK-OV-3 for ovarian cells, BxPC3, MiaPaCa-2 and LN36 for pancreas cells, SK-ES-1 for renal adrenal cancer sarcoma cells, Daoy, TE-671 and D283 Med for medulloblastoma cells, MM-1S and MM-1R for retinoblastoma multiple myeloma cells and SK-MEL-28 for determining whether the amino acid sequence isolated in (a) exhibits agonist or antagonist activity at IGF-IR-responsive cell selected from the group consist
  • a method of identifying a hybrid-R modulator comprising: a) screening a library of peptide sequences, wherein said library of peptide sequences bind to hybrid-R, and wherein the library is derived from a peptide sequence comprising at least one sequence, preferably 2, 3, 4, 5 and 6 different sequences, selected from the group consisting of SEQ ID Nos. 2, 4, 6, 8, 10 and 12; and b) determining whether the amino acid sequence isolated in (a) exhibits agonist or antagonist activity at hybrid-R-responsive cell selected from the group consisting of cell lines displaying hybrid-R, such as, for example, MDA-MB-231, MDA-MB-157,
  • MDA-MB-468, MDA-MB-453 and ZR-75 for breast cells and BC-PAP for thyroid cells wherein this activity indicates identification of an hybrid-R modulator.
  • it is also an object of the invention to develop a method of identifying an IGF-IR and hybrid-R modulator comprising: a) screening a library of peptide sequences, wherein said library of peptide sequences bind to IGF-IR and/or hybrid-R, and wherein the library is derived from a peptide sequence comprising at least one sequence, preferably 2, 3, 4, 5 and 6 different sequences, selected from the group consisting of SEQ ID Nos.
  • IGF-IR and/ir hybrid-R-responsive cell selected from the group consisting of cell lines displaying IGF-IR and/or hybrid-R, such as, for example, MCF- 7, T47D, BT20, ZR-75- 1, MDA-MB-231, MDA-MB-157, MDA-MB-468, MDA-MB- 453 and ZR-75 for breast cells, PC3 and DU145 for prostate cells, A549, A427 and SK- LU-1 for lung cells, HT29, Colo205 and CaCo-2 for colon cells, BC-PAP, FRO and ARO for thyroid cells, SK-OV-3 for ovarian cells, BxPC3, MiaPaCa-2 and LN36 for pancreas cells, SK-ES-1 for renal adrenal cancer sarcoma cells, Daoy, TE-671 and D283 Med for medulloblasto
  • IGF-IR and/ir hybrid-R-responsive cell selected from the group consisting of cell lines displaying IGF-IR
  • An object of the invention is A method of treating or preventing a medical condition in a subject, which medical condition is mediated by elevated expression and/or activation of IGFl, comprising administering a binding composition that specifically binds to IGF-IR comprising at least a member selected from the group consisting of: a) a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID No. 2, CDR-L2 defined by SEQ ID No. 4 and CDR-L3 defined by SEQ ID No. 6; and/or b) a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID No. 8, CDR-H2 defined by SEQ ID No. 10 and CDR-H3 defined by SEQ ID
  • an object of the invention is a method of treating or preventing a medical condition in a subject, which medical condition is mediated by elevated expression and/or activation of hybrid-R comprising administering a binding composition that specifically binds to hybrid-R comprising at least a member selected from the group consisting of: a) a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID No. 2, CDR-L2 defined by SEQ ID No. 4 and CDR-L3 defined by SEQ ID No. 6; and/or b) a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID No. 8, CDR-H2 defined by SEQ ID No. 10 and CDR-H3 defined by SEQ ID No. 12; to the subject.
  • a binding composition that specifically binds to hybrid-R comprising at least a member selected from the group consisting of: a) a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID No. 2, CDR-L2 defined by SEQ ID No. 4
  • an object of the invention is a method of treating or preventing a medical condition in a subject, which medical condition is mediated by elevated expression and/or activation of IGF-IR and hybrid-R comprising administering a binding composition that specifically binds to IGF-IR and hybrid-R comprising at least a member selected from the group consisting of: a) a light chain amino acid sequence which comprises CDR-L1 defined by SEQ
  • CDR-L2 defined by SEQ ID No. 4 and CDR-L3 defined by SEQ ID No. 6; and/or b) a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID No. 8, CDR-H2 defined by SEQ ID No. 10 and CDR-H3 defined by SEQ ID No. 12; to the subject.
  • Another object of the invention is a method of determining regression, progression or onset of a pathological disorder characterized by increased expression and/or activation of human IGF-IR relative to normal comprising incubating a sample obtained from a patient with said disorder with a detectable probe that is specific for said human IGF-IR under conditions favoring formation of a probe/IGF-IR complex, the presence of which is indicative of the regression, progression or onset of said pathological disorder in said patient.
  • another object of the invention is a method of determining regression, progression or onset of a pathological disorder characterized by increased expression and/or activation of human hybrid-R relative to normal comprising incubating a sample obtained from a patient with said disorder with a detectable probe that is specific for said human hybrid-R under conditions favoring formation of a probe/hybrid-R complex, the presence of which is indicative of the regression, progression or onset of said pathological disorder in said patient.
  • another aspect of the invention is a method of determining regression, progression or onset of a pathological disorder characterized by increased expression and/or activation of both human IGF-IR and hybrid-R relative to normal comprising incubating a sample obtained from a patient with said disorder with a detectable probe that is specific for said human IGF-IR and hybrid-R under conditions favoring formation of a probe/hybrid-R and probe.IGF-IR complex, the presence of which is indicative of the regression, progression or onset of said pathological disorder in said patient.
  • the probe is preferably an antibody, said antibody being preferably labeled with a radioactive label, a fluorescent label or an enzyme, and said antibody preferably comprising the antibody of the invention as described in the present specification.
  • Still another aspect of the invention is a method of following progress of a therapeutic regimen designed to alleviate a condition characterized by abnormal expression and/or activation of human IGF-IR relative to normal comprising: a) assaying a sample from a subject to determine level of expression and/or activation of said IGF-IR at a first time point; b) administering the antibody of the invention able to binf IGF-IR to said subject and assaying level of expression and/or activation of said IGF-IR at subsequent time points following administration of said antibody; and c) comparing said level of said IGF-IR at said subsequent time points to the level determined in (a) as a determination of effect of said therapeutic regimen, wherein a decrease in said level of expression and/or activation of IGF-IR subsequent to administration of said anti-IGF-IR antibody indicates a positive progression of the therapeutic regimen designed to alleviate said condition.
  • it is also a aspect of the invention to protect a method of following progress of a therapeutic regimen designed to alleviate a condition characterized by abnonnal expression and/or activation of human hybrid-R relative to normal comprising: a) assaying a sample from a subject to determine level of expression and/or activation of said hybrid-R at a first time point; b) administering the antibody of the invention able to bind hybrid-R to said subject and assaying level of expression and/or activation of said hybrid-R at subsequent time points following administration of said antibody; and c) comparing said level of said hybrid-R at said subsequent time points to the level determined in (a) as a determination of effect of said therapeutic regimen, wherein a decrease in said level of expression and/or activation of hybrid-R subsequent to administration of said anti-hybrid-R antibody indicates a positive progression of the therapeutic regimen designed to alleviate said condition.
  • the invention is claiming a method of following progress of a therapeutic regimen designed to alleviate a condition characterized by abnormal expression and/or activation of both human IGF-IR and hybrid-R relative to normal comprising: a) assaying a sample from a subject to determine level of expression and/or activation of said IGF-IR and hybrid-R at a first time point; b) administering the antibody of the invention able to bind both IGF-IR and hybrid-R to said subject and assaying level of expression and/or activation of said IGF- IR and hybrid-R at subsequent time points following administration of said antibody; and c) comparing said level of said IGF-IR and hybrid-R at said subsequent time points to the level determined in (a) as a determination of effect of said therapeutic regimen, wherein a decrease in said level of expression and/or activation of IGF-IR and hybrid-R subsequent to administration of said anti-IGF-IR and hybrid-R antibody indicates a positive progression of the therapeutic regimen designed to alleviate said condition.
  • the condition above mentioned comprises i) a tumor that expresses at least an
  • IGF-JR preferably breast cancer, prostate cancer, colorectal cancer, lung cancer, bladder cancer, kidney cancer, thyroid cancer, osteosarcomas, pancreatic cancer, melanoma and myeloid cancer; or ii) a tumor that expresses at least a hybrid-R, preferably breast cancer or thyroid cancer.
  • the methods above described can also include, before the administration of step b), the step consisting in measuring the concentration of a specific tumoral antigen in a body sample from the mammal, wherein an elevated concentration of said specific tumoral antigen above a reference range for said specific tumoral antigen indicates a increased risk for cancer.
  • Specific timoral antigen are described in example 43 hereinafter. Genetic markers can also be used (see example 43).
  • Methods above described, preferably methods of treating/preventing a medical condition or methods of following progress of a therapeutic regimen are comprising administering to said mammal the antibody of the invention in combination with an agent selected from the group consisting of a corticosteroid, anti-emetic, cancer vaccine, analgesic, anti-vascular agent, cytokines, non-specific immunostimulant and anti- proliferative agent.
  • an agent selected from the group consisting of a corticosteroid, anti-emetic, cancer vaccine, analgesic, anti-vascular agent, cytokines, non-specific immunostimulant and anti- proliferative agent.
  • said anti-emetic agent is selected from the group consisting of, without limitation, ondansetron hydrochloride, granisetron hydrochloride, metroclopramide, domperidone, haloperidol, cyclizine, lorazepam, prochlorperazine, dexamethasone, levomepromazine, or tropisetron.
  • said analgesic agent is selected from the group consisting of, without limitation, ibuprofen, naproxen, choline magnesium trisalicylate, or oxycodone.hydrochloride.
  • said anti-proliferative agent is selected from the group consisting of, without limitation, farnesyl protein transferase inhibitors, .alpha.v.beta.3 inhibitors, .alpha.v.beta.5 inhibitors, p53 inhibitors, VEGF inhibitors, VEGFR inhibitors, EGFR inhibitors and Her2neu inhibitors or heterodimers thereof, and PDGFR inhibitors.
  • Methods above described, preferably methods of treating/preventing a medical condition or methods of following progress of a therapeutic regimen are characterized in that said antibody comprises a heavy chain comprising the amino acid sequences of CDR-1, CDR-2, and CDR-3 wherein said heavy chain CDR's are selected from the group consisting of SEQ ID Nos.
  • a pharmaceutical composition for the treatment or prevention of a disorder in a mammal comprising an amount of a human anti-IGF-IR antibody that is effective in treating said disorder and a pharmaceutically acceptable carrier, wherein said disorder is selected from the group consisting of breast cancer, prostate cancer cells, colorectal cancer cells, lung cancer cells, bladder cancer cells, kidney cancer cells, thyroid cancer cells, osteosarcomas cells, pancreatic cancer cells, and myeloid cells, wherein said antibody comprises the antibody of the invention above described and able to bind IGF-IR.
  • Another embodiment is a pharmaceutical composition for the treatment or prevention of a disorder in a mammal comprising an amount of a human anti-hybrid-R antibody that is effective in treating said disorder and a pharmaceutically acceptable carrier, wherein said disorder is selected from the group consisting of breast cancer and thyroid cancer, wherein said antibody comprises the antibody of the invention able to bind hybrid-R.
  • Another embodiment is also a pharmaceutical composition for the treatment or prevention of a disorder in a mammal comprising an amount of a human anti-IGF-LR and hybrid-R antibody that is effective in treating said disorder and a pharmaceutically acceptable carrier, wherein said disorder is selected from the group consisting of breast cancer and thyroid cancer, wherein said antibody comprises the antibody of the invention able to bind IGF-IR and hybrid-R.
  • Said pharmaceutical composition(s) is further comprising an amount of anti- emetic, cancer vaccine, analgesic, anti-vascular agent, and anti-proliferative agent that, in combination with said antibody, is effective in treating said disorder.
  • the invention concerns, in another aspect, a method of detecting a binding partner for the antibody of the invention able to bind IGF-IR in a sample, the method comprising: a) incubating the antibody of the invention able to bind IGF-IR with a biological sample obtained from a patient with a cell proliferative disorder characterized by abnormal level of expression and/or activation of human IGF-IR under conditions sufficient to allow specific binding of the antibody to its binding partner, and b) detecting specific binding, wherein specific binding indicates the presence of a binding partner in the sample.
  • a second embodiment is a method of detecting a binding partner for the antibody of the invention able to bind hybrid-R in a sample, the method comprising: a) incubating the antibody of the invention able to bind hybrid-R with a biological sample obtained from a patient with a cell proliferative disorder characterized by abnormal level of expression and/or activation of human hybrid-R under conditions sufficient to allow specific binding of the antibody to its binding partner, and b) detecting specific binding, wherein specific binding indicates the presence of a binding partner in the sample.
  • a third embodiment of the invention is a method of detecting binding partners for the antibody of the invention able to bind IGF-IR and hybrid-R in a sample, the method comprising: a) incubating the antibody of the invention able to bind IGF-IR and hybrid-R with a biological sample obtained from a patient with a cell proliferative disorder characterized by abnormal level of expression and/or activation of both human IGF-IR and hybrid-R under conditions sufficient to allow specific binding of the antibody to its binding partner, and b) detecting specific binding, wherein specific binding indicates the presence of a binding partner in the sample.
  • the invention also deals with a method of purifying a hybrid receptor from a sample, the method comprising: a) incubating the antibody of the invention able to bind hybrid-R with a sample under conditions to allow specific binding of the antibody and the receptor, and b) separating the antibody from the sample and obtaining the purified receptor.
  • a second embodiment is a method of purifying a IGF-IR from a sample, the method comprising: a) incubating the antibody of the invention able to bind IGF-IR with a sample under conditions to allow specific binding of the antibody and the receptor, and b) separating the antibody from the sample and obtaining the purified receptor.
  • Figure 1 Schematic representation of IGF-IR.
  • Figure 2 Scheme of the transduction of the signals mediated by IGF-IR during the attachment of IGFs.
  • FIGS. 3A, 3B and 3C Recognition of native IGF-IR expressed on the surface of
  • MCF-7 cells by the monoclonal antibody 7C10.
  • the MCF-7 cells are incubated with the 7C10 antibody or with a negative control antibody, then recovered with the aid of a fluorescent anti-species secondary antibody.
  • the labeling is read on a FACS.
  • the first histogram corresponds to the MCF-7 cells alone.
  • the unshaded curve corresponds to the nonspecific labeling by a control isotype murine antibody.
  • the unshaded curve shows the recognition of IGF-IR by MAB 7C10.
  • Figures 4A, 4B and 4C Labeling of Sf9 insect cells respectively expressing IGF-IR or
  • Figure 4A shows the labeling of nontransfected cells alone (1) or cells labeled with control commercial monoclonal antibodies respectively recognizing IGF-IR (2) or IR
  • FIG. 4B Sf9 cells uniquely expressing IGF-IR are labeled with GTR3 (2) or anti-IR(3), the peak (1) representing the single cells.
  • FIG 4C Sf9 cells uniquely expressing IR are labeled with an anti-IR (3) or GJIR3 (2), the peak (1) representing the single cells.
  • Figure 5 Inhibitor effect of 7C10 antibody on the proliferation of MCF-7 cells induced by IGF-I. The MCF-7 cells are incubated in the presence of increasing concentrations of IGFl in the presence or in the absence of the MAB to be tested. The cell proliferation is evaluated by following the incorporation of 3 H thymidine. The commercial antibody ⁇ IR3 is used as a positive control of the experiment.
  • the 7G3 is a murine anti-IGF-IR IgGl without activity on proliferation and used as a control isotype.
  • Figures 6A, 6B and 6C - figure 6A: in vivo effect of the monoclonal antibody 7C10 on the growth of MCF-7 tumors established in nude mice; - figures 6B and 6C: figures respectively from publications of Arteaga et al. (J. Clin. Invest, 84, 1418-1423, 1989) and from Li et al. (Cancer Immunol. Immunother.,
  • Figure 7 Comparative study of the effect of the MAb 7C10 and of tamoxifen on the growth in vivo of the tumor MCF-7.
  • FIGS 8 A, 8B, 8C and 8D Study of the antitumor activity of the murine antibody
  • FIG. 7C10 in different xenograft models of tumor cells in vivo.
  • Figure 8 A shows the results obtained on an osteosarcoma model SK-ES-1
  • figure 8B concerns an androgen-independent tumor of the prostate DU-145
  • figure 8C a model of non-small cell tumor of the lung A549
  • figure 8D a model of pancreatic cancer BxPC3.
  • the treatment was carried out twice per week i.p. at a rate of 250 ⁇ g/dose/mouse.
  • the curves 7G3, EC2 and 9G4 correspond respectively to three murine IgGl used as an experiment control isotype in each of the models.
  • Figure 9 Study of the antitumor effect of the MAb 7C10 compared to navelbine (vinorelbine) as well as the synergy of the two compounds on the growth in vivo of the line A549.
  • Figure 10 Comparative activity of MAb oIR3, 7C10 and 1H7 on the IGF2 proliferation induced by MCF-7 cells.
  • Figure 11 Comparison of the murine 7C10 and chimeric C7C10 MAb for the inhibition of the IGFl proliferation of MCF-7 cells in vitro.
  • the antibody 9G4 is a murine IgGl used as an experiment control isotype.
  • Figure 12 Comparative effect of the 7C10 and h7C10 MAb (humanized 1, written here 7H2HM) on the in vitro model of IGFl -induced proliferation of MCF-7 cells.
  • Figure 13 Effect of the 7C10 and h7C10 MAb (humanized 1, written here 7H2HM) on the transduction of the signal induced by IGFl.
  • the first line of spots corresponds to the revelation, by an antiphospho-tyrosine antibody, of the phosphorylation of the immunoprecipitated ⁇ chain from the cells incubated in the presence of IGFl alone or of IGFl mixed with various antibodies to be tested.
  • the 9G4 and the hlgGl are respectively the control isotypes of the forms 7C10 and h7C10 (likewise written 7H2HM).
  • the second line of spots corresponds to the revelation of the ⁇ chain and shows that the quantity deposited in all of the wells is perfectly equivalent.
  • Figure 14 Sequence of the cDNA (SEQ ID No. 48), of its complementary strand (SEQ ID No. 50) and its translation into amino acids (SEQ ID No. 49), of the PCR fragment amplified from the mouse hybridoma 7C10 with the primers MKV-1 and MKC and which codes for the 3' end of the leader peptide and 7C10 VL.
  • Figure 15 Sequence of the cDNA (SEQ ID No. 51), of its complementary strand (SEQ ID No. 53) and its translation into amino acids (SEQ ID No.
  • Figure 17 Comparison of the amino acid sequence of mouse 7C10 VL (SEQ ID No.
  • the numbering of the amino acids is that of Kabat et al. (1991).
  • the residues in the framework regions (outside CDRs) which differ between 7C10 VL and Kabat mouse subgroup II (SEQ ID No. 57) are underlined.
  • a dot indicates that the residue is identical at this position in comparison with the sequence of 7C10 VL.
  • DRB 1-4.3 (SEQ ID No.
  • FIG. 55 represents the sequence of the light chain of an anti-human mouse antibody MHC CLASS II B-Chain (access number in the Kabat databank is N011794).
  • C94-5B11'CL represents the sequence of the light chain of a mouse antibody (access number in the Kabat databank is P019314).
  • Figure 18 Comparison of amino acid sequences of mouse 7C10 VL (SEQ ID No. 54) with cells of human light chains belonging to Kabat human subgroup II (SEQ ID No. 60) and having the greatest sequence homology. The amino acid sequences are aligned and compared with that of mouse 7C10 VL. A dot indicates that the residue is identical at this position in comparison with the sequence of 7C10 VL.
  • GM607 (SEQ ID No. 58) represents the sequence of the kappa light chain secreted by the human lymphoblastoid line GM607 (Klobeck et al, Nucleic Acids Res., 12:6995-7006, 1984a and Klobeck et al, Nature, 309:73-76, 1984b, the access number in the Kabat databank is N011606).
  • DPK15/A19 (SEQ ID No. 59) represents the sequence of the human V germinal line kappa II.
  • Figure 19 Comparison of amino acid sequences of variable regions of the light chains (VL) of mouse 7C10 (SEQ ID No. 54), of human antibody GM 607 (SEQ ID No. 58) and of two versions of humanized 7C10 1 and 2 (SEQ ID Nos. 61 and 65).
  • the amino acid sequences are aligned and compared with that of mouse 7C10 VL.
  • a dot indicates that the residue is identical at this position in comparison with the sequence of 7C10 VL.
  • GM607 represents the sequence of the kappa light chain secreted by the human lymphoblastoid line GM607 (Klobeck et al, 1984a and 1984b, access number in the Kabat database: N011606).
  • Figure 20 cDNA sequence (SEQ ID No. 62), its complementary strand (SEQ ID No. 64) and its translation into amino acids (SEQ ID No. 63), of the gene constructed by de novo assembly coding for the leader peptide and the humanized version 1 of 7C10 VL.
  • Figure 21 cDNA sequence (SEQ ID No. 66), its complementary strand (SEQ ID No.
  • the numbering of the amino acids is that of Kabat et al. (1991).
  • the residues in the framework regions (outside CDRs) which differ between 7C10 VH and Kabat mouse subgroup 1(A) (SEQ ID No. 71) are underlined. A dot indicates that the residue is identical at this position in comparison with the sequence of mouse 7C10 VH.
  • AN03'CL SEQ ID No. 70 represents the sequence of the heavy chain of a mouse antibody (access number in the Kabat databank: P001289).
  • Figure 23 Comparison of amino acid sequences of mouse 7C10 VH (SEQ ID No. 69) with those of human heavy chains belonging to the Kabat human subgroup II (SEQ LD No. 72) and having the greatest sequence homology.
  • the underlined residues are part of the canonical structures defined by Chothia et al. (1989). A dot indicates that the residue is identical at this position in comparison with the mouse 7C10 VH sequence.
  • Human VH FURl'CL (SEQ ID No. 73) represents the sequence of the heavy chain of a human anti-lamin B antibody IgM/K of autoimmune origin (Mariette et al, Arthritis and Rheumatism, 36:1315-1324, 1993; access number in Kabat: N020619).
  • Human germline (SEQ ID No. 74) represents the sequence of the human germinal line 4.22 VH IN (Sanz et al, EMBO. J. 8:3741-3748, 1989).
  • Figure 24 Comparison of the amino acid sequences of the variable regions of the heavy chains (VH) of mouse 7C10 (SEQ ID No. 69) and of the three versions humanized by CDR-grafting humanized VH 1, 2 and 3 (respectively SEQ ID Nos. 75, 79 and 83). The numbering of the residues corresponds to that of Kabat. The sequences are aligned and compared with that of mouse 7C10 VH. A dot indicates that the residue is identical at this position in comparison with the sequence of mouse 7C10 VH.
  • Figure 25 cDNA sequence (SEQ ID No. 76), its complementary strand (SEQ ID No. 78) and its translation into amino acids (SEQ ID No. 77), of the gene constructed by de novo assembly coding for the leader peptide and the humanized version 1 of 7C10 VH.
  • Figure 26 cDNA sequence (SEQ ID No. 80), its complementary strand (SEQ ID No. 82) and its translation into amino acids (SEQ ID No. 81), of the gene constructed by de novo assembly coding for the leader peptide and the humanized version 2 of 7C10 VH.
  • Figure 27 cDNA sequence (SEQ ID No. 84), its complementary strand (SEQ ID No. 86) and its translation into amino acids (SEQ ID No.
  • FIG. 85 Comparison of the recognition activity of the IGF-I receptor by the chimeric antibody 7C10 (called “C7C10”) and its humanized version 1 (7C10 hum 1) in ELISA.
  • Figure 29 Influence on the recognition activity of the IGF-I receptor of the humanized versions 1 and 2 of the light chain of the 7C10 antibody in ELISA.
  • Figure 30 Comparison of the recognition activity of the IGF-I receptor by the chimeric antibody 7C10 and three humanized versions of the heavy chain (7C10 hum 1, 2 and 3) in combination with humanized 7C1O VL 2 in ELISA.
  • Figure 31 Antitumor activity of the 7C10 antibody in an orthotopic model A549.
  • Figures 32 A, 32B, 32C and 32D Study of the ADCC observed at the level of A549 and MCF-7 cells cultured during 4 hours in the presence of the antibody 7H2HM (respectively figures 32C and 32D).
  • the antibody h4D5 is used in parallel as an experiment positive control for the cells A549 and MCF-7 (respectively figures 32A and 32B).
  • Figures 33A, 33B and 33C Effects of the antibodies 7C10 and 7H2HM on the cell cycle of the MCF-7 cells.
  • Figure 33 A represents the proportion of MCF-7 cells in the G0/G1, S and G2/M phase in the absence of IGFl, expressed as a significant percentage of total MCF-7 cells observed.
  • Figure 33B represents the proportion of MCF-7 cells in the G0/G1, S and G2/M phase in the presence of IGFl, expressed as a percentage of total MCF-7 cells observed.
  • Figure 33C represents the proportion of MCF-7 cells in the S ( ⁇ ) and G2/M (D) phase, expressed as a percentage of total MCF-7 cells observed, in the presence of the compounds indicated in the figure compared with a control sample in the absence of IGFl ("0").
  • Figures 34A and 34B Comparative effect of the antibodies 7C10 and 7H2HM on the growth of A549 cells in vitro (figure 34A) and on the growth of MCF-7 cells in vivo (figure 34B).
  • Figures 35 A and 35B Study of the synergy of the antibody 7H2HM combined with navelbine (NA) on the model A549 in vivo, compared with the control samples.
  • Figure 35A represents the development of the volume of the implanted tumor as a function of the treatment carried out starting from the commencement of the treatment and over approximately 50 days (figure 35A).
  • Figure 35B represents in a particular manner the results obtained for this development compared at approximately 48 days.
  • Figures 37A to 37D Demonstration by labeling in FACS of the presence of EGFR and of IGF-IR on the surface of A549 cells.
  • Figure 38 Effect of a coadministration of the MAB 7C10 and 225 on the in vivo growth of the tumor A549.
  • Figure 39 Effect of a coadministration of the MAB 7C10 and 225 on the survival of mice orthotopically implanted with A549 cells.
  • Figures 40 A and 40B Demonstration of the inhibition of tyrosine phosphorylation of the beta chain of IGF-IR and of IRS-1 by the MAB 7C10 and 7H2HM.
  • Figures 42A to 42C Demonstration of the degradation of IGF-IR by the MAB 7C10 and 7H2HM.
  • Figures 43 A and 43B Immuno-blotting with an anti-IGF-IR ⁇ -subunit and anti-IR ⁇ - subunit on filters containing cellular lysates obtained after immunoprecipitation and
  • Figure 44 Immunocapture of R+ cell lysates IGF-IR in Maxisorb plates coated with 17- 69 antibody and binding by 125 I-IGF1 in the absence or the presence of increasing concentrations of unlabeled ligand (IGF-I) or antibodies (7C10, h7C10, 1H7, 9G4).
  • IGF-I unlabeled ligand
  • Figure 45 Immunocapture of R-/IR-A cell lysates Hybrid-R A in Maxisorb plates coated with 83-7 antibody and binding by 125 I-IGF1 in the absence or the presence of increasing concentrations of unlabeled ligand (IGF-I) or antibodies (7C10, h7C10, 1H7, 9G4).
  • IGF-I unlabeled ligand
  • FIG. 46 Immunocapture of R-/IR-B cell lysates Hybrid-R B in Maxisorb plates coated with 83-7 antibody and binding by I-IGF1 in the absence or the presence of increasing concentrations of unlabeled ligand (IGF-I) or antibodies (7C10, h7C10, 1H7,
  • Figures 47 A and 47B Immuno-blotting analysis of antibody induced degradation of the
  • IGF-IR in A549 (A) and MCF-7 (B) cells IGF-IR in A549 (A) and MCF-7 (B) cells.
  • Figure 48 Immuno-blotting analysis of antibody degradation pathway of IGF-IR in MCF-7 cells.
  • Figure 49 Anti-tumoral activity of the murine antibody 7C10 co-administrated with an anti- VEGF antibody on mice orthopically implanted with A549 cells.
  • Figures 50 and 51 Comparison of the in vivo anti-tumoral activity of the 7C10 and h7C10 antibodies on the A549 (figure 50) and MCF-7 (figure 51) models.
  • Figures 52 and 53 Comparison of the anti-leukaemia activity of vinblastine and vincristine (figure 52) and of 4'R and 4'S deoxyvinblastines(figure 53).
  • Figure 54 In vivo antitumor activity of 4' R- and 4' S-deoxyvinblastines conjugated with IGR-IR antibodies on human tumors of various origins.
  • Figure 55 Schematic representation of the biosensor capturing assay.
  • a Mab directed against the constant Fc portion of either mouse or human IgGl were covalently attached onto a CM5 sensor surface.
  • a limited amount (400 RU) of Mab to be tested were immobilized and used to capture the analyte hIGF-IR.
  • the binding of Mab to the analyte is described by the association and dissociation rate constants k a and k , respectively.
  • Figures 56 A, 56B and 56C Determination of h7C10 ICso in the IGFl induced proliferative assay. Results are expressed as proliferative indexes in panel A. Panel B shows an example of IC 50 calculation and panel C summarizes the IC 50 data obtained with 5 different batches of h7C 10 antibody.
  • 9G4 and 7G3 Mabs are respectively a irrelevant IgGl antibody and an non neutralizing anti- IGF-IR antibody (IgGl isotype).
  • Figure 58 staining of either tumoral or normal tissues from lung and breast cancer patients. Comparison with tissues from normal regions.
  • Figure 59 h7C10 down regulation of IGF-IR in vivo.
  • Figures 60A, 60B, 60C Determination of h7C10 IC 50 in the IGF2 induced proliferative assay. Results are expressed as proliferative indexes in panel A. Panel B shows an example of IC 50 calculation and panel C summarizes the IC 50 data obtained with 5 different batches of h7C 10 antibody.
  • Example 1 Generation and selection of the murine monoclonal antibody (MAb) With the aim of generating MAb specifically directed against IGF-IR and not recognizing the IR, a protocol comprising 6 screening stages was envisaged. It consisted in: - immunizing mice with recombinant IGF-IR, in order to generate hybridomas, - screening the culture supematants by ELISA on the recombinant protein which served for immunization, - testing all the supematants of hybridomas positive by ELISA on the native receptor overexpressed on the surface of MCF-7 tumor cells, - evaluating the supematants of hybridomas positive in the two first screenings in terms of differential recognition of IGF-IR and of IR on insect cells infected with baculoviruses respectively expressing IGF-IR or IR, - verifying that the antibodies selected at this stage were capable of inhibiting in vitro the induced IGFl proliferation of the MCF-7 cells, - ensuring the in vivo
  • mice were injected twice, by the subcutaneous route, with 8 ⁇ g of recombinant IGF-IR.
  • the mice were stimulated by an intravenous injection of 3 ⁇ g of the recombinant receptor.
  • the supematants of hybridomas were screened by ELISA, on plates sensitized by recombinant IGF-IR.
  • the hybridomas whose supematants were found positive were conserved and amplified before being tested on the FACScan so as to verify that the antibodies produced were likewise capable of recognizing native IGF-IR.
  • MCF-7 cells from an estrogen-dependent tumor of the breast overexpressing IGF- IR were incubated with each of the culture supematants produced by the hybridomas selected in ELISA.
  • the native/MAb receptor complexes on the surface of the cell were revealed by a secondary anti-species antibody coupled to a fluorochrome.
  • Figures 3 A to 3C show a histogram type obtained with the supernatant of the hybridoma 7C10 (figure 3C) compared with a cell labeling alone + secondary antibody (figure 3 A) or with a labeling utilizing a control isotype (figure 3B).
  • the hybridomas secreting MAb at this stage of the selection, only the hybridomas secreting MAb at the same time recognizing the recombinant receptor and the native receptor were selected and cloned.
  • the MAb secreted by these hybridomas were produced and then purified before being tested on the FACScan, according to the method described above, on Sf9 insect cells expressing IGF-IR or IR in order to eliminate the hybridomas at the same time recognizing the two receptors.
  • Figure 4A shows a total recovery of the histograms 1, 2, 3 respectively corresponding to the noninfected cells + secondary antibodies (1), to the noninfected cells labeled by oIR3 + secondary antibodies (2) and to the noninfected cells labeled by an anti-IR antibody + secondary antibodies (3).
  • This first result shows well the absence of IGF-IR and of IR detectable on the surface of these noninfected insect cells.
  • Figure 4B shows a labeling of infected cells by a baculovirus expressing IGF-IR.
  • the o R3, used as a positive control labels well, as expected, the cells (peak 2), while the anti-IR (peak 3) is superimposed on the peak of single cells.
  • the two last screenings provided for the selection of the MAb consisted in verifying that the latter was very capable of inhibiting the cell proliferation induced by the IGFl in vitro and in vivo on the cell line MCF-7.
  • the MCF-7 cells were inoculated, deprived of fetal calf serum, then incubated in the presence of increasing concentrations of IGFl (from 1 to 50 ng/ml) in the presence or in the absence of the 7C10 antibody to be tested added to a final concentration of 10 g/ml.
  • the commercial ⁇ IR3 MAb was introduced as a positive control and the 7G3 MAb (isolated in parallel to the 7C10 and weakly recognizing the native receptor (MFI on the FACS of 50 compared with 200 for the MAb 7C10)) as a control isotype.
  • the cell proliferation is estimated by following on the ⁇ counter the incorporation of tritiated thymidine by the cells.
  • the results are expressed as a proliferative index.
  • the data presented in figure 5 show that IGFl is capable of stimulating in a dose-dependent manner the proliferation of the MCF-7 cells.
  • the MAb 7C10 significantly inhibits the growth of the MCF-7 cells induced by IGF-I.
  • the MAb 7G3 used as an isotype control turns out well, as expected, without effect on the tumor cell growth in vitro of the MCF-7 cell.
  • nude mice received a subcutaneous implant of slow-release estrogen, indispensable for the taking of the tumor in a murine model. Twenty-four hours after implantation of the estrogens, 5.10 6 MCF-7 cells are grafted onto the right flank of the mouse subcutaneously. Five days after this cell graft, the tumors are measurable and batches of 6 mice are formed at random.
  • mice The treatment of the mice is carried out twice per week, during 5 to 6 weeks, at the dose of 250 ⁇ g/dose/mouse.
  • the mice are treated in the same fashion with a murine control isotype.
  • the results presented in figure 6A show a very significant inhibition of the tumor growth induced by the antibody 7C10. This activity is particularly unexpected if reference is made to the data available concerning o R3, always used as a reference in the domain of the receptor for IGFl, and known for not having any activity in vivo on the growth of estrogen-dependent tumors (see figure 6B).
  • the MAb 7C10 is much more efficacious in the in vivo inhibition of the growth of the MCF-7 cells.
  • This difference of activity could be relaty, whithout any limitation, to some particular properties of 7C10 and h7C10 antibodies which recognize the hybrid-R, isoform(s) A and/or B, (see example 26).
  • Another non exclusive hypothesis could be the recognition by these antibodies of the atypical IGF-IR as described in the paper of Siddle et al. (Siddle et al, 1994, Horn Res. 41(suppl 2):56- 65).
  • K D Equilibrium dissociation constants (K D ) of a series of 7C10 and h7C10 antibodies directed against the extracellular domain of human insulin-like growth factor- 1 receptor (hlGF-IR) were calculated by the ratio between dissociation and association rate constants, as defined by surface plasmon resonance using a BIAcore X instrument. Capture of the investigated antibodies was used to favour a proper presentation of their antigen-binding site ( Figure 55). Therefore, a mixture of goat anti- human IgG Fc and rabbit anti-mouse IgG Fc polyclonal antibodies were covalently linked on both flowcells (FCl and FC2) of a CM5 sensorchip.
  • FC2 being a reference cell to evaluate non specific interactions of the analyte with the matrix.
  • the analyte corresponding to the extracellular domain of hIGFl-R, was tested at 5 different concentrations ranging from 12.5 to 200 nM at 25°C at a flow rate of 30 ⁇ l/min.
  • a mean K D of 0.86 ⁇ 0.09 nM was obtained for 3 different batches of A2CHM Mab. These values were closed to that obtained for the mouse Mab 7C10 (K D : 0.86 ⁇ 0.14 nM) Table 2.
  • 7C10 in the context of estrogen-dependent cancer of the breast, 7C10 was compared with the tamoxifen compound currently used for the treatment of mammary carcinoma in the context of developed forms with local and/or metastatic progression and in the context of the prevention of recurrences (see VIDAL 2000, pages 1975-1976).
  • ER receptors for estrogens
  • IGF-IR IGF-IR
  • tamoxifen The long-term use of tamoxifen is associated with a significant increase in the risk of endometrial cancer (Fisher et al, J. of National Cancer Institute, 86, 7:527-537, 1994; VIDAL 2000, 1975-1976) and of collateral recurrence of E2-independent cancer of the breast (Li CL et al, J. Natl Cancer Inst, July 4, 93(13):1008-1013, 2001).
  • a comparison of the in vivo antitumor effect of the antibody 7C10 and of tamoxifen has been carried out on the MCF-7 model so as to determine the part of the activity connected with IGF-IR in the mediated ER proliferation.
  • MCF-7 cells were implanted sc (subcutaneously) in nude mice, 24 hours after implantation in these same mice of a grain of estradiol with prolonged release (0.72 mg/tablet liberated over 60 days), indispensable for the establishment of any E2- dependent human tumor in this animal species. Five days after this implantation, the tumors are measured and groups of 6 mice are formed.
  • HPC hydroxypropyl-cellulose
  • the tamoxifen is administered daily for 4 weeks except at the weekend.
  • the mice treated with the MAb 7C10 likewise daily receive an injection of PBS with 3% HPC. A study was previously carried out in order to verify that the solvent alone is without influence on the tumor growth.
  • the MAb 7C10 is capable of significantly inhibiting the growth of the tumor MCF-7 in vivo (the asterisks (*) conespond to the comparison control group/7C10 group in a t-test).
  • the antibody 7C10 seems to be significantly more efficacious than tamoxifen for the inhibition of the tumor growth (the circles (°) correspond to the comparison tamoxifen group/7C10 group in a t-test) suggesting that this type of treatment by MAB might be substituted for treatment with tamoxifen.
  • Example 3 Demonstration of the antitumor activity of the MAb 7C10 in vivo on human tumors of different origins a) In vivo activity of the antibody 7C10 in 4 tumor models In order to generalize the activity of the 7C10 antibody to other tumors expressing the receptor for IGFl, 7C10 was tested in vivo in an androgen-independent model of tumor of the prostate DU145 (likewise written DU-145), in an SKES-1 osteosarcoma model, in a model of non-small cell tumor of the lung A549 and in a model of pancreatic cancer BxPC3.
  • the protocol is comparable to that described above for MCF-7 and the results presented in figures 8A to 8D show a significant activity of this MAB in the 4 tumor models.
  • the activity observed in the model of tumor of the prostate is to be noted very particularly inasmuch as the single chain scFv of the MAB 1H7 is without activity in an androgen-independent model of tumor of the prostate (Li et al, 2000).
  • the conventional xenograft models as described above do not allow the study of drugs on metastatic dissemination. In effect, the tumors implanted s.c. (subcutaneously) remain localized at the sight of injection and are therefore not really a reflection of the situation in man.
  • Example 4 Comparison of the MAb 7C10 with navelbine in vivo; effect of a coadministration of the two treatments Navelbine is a chemotherapy compound indicated in non-small cell cancer of the lung and in metastatic cancer of the breast.
  • the comparative study of 7C10 and of navelbine and the possible synergy between the two products was studied on the tumor model A549.
  • 5.10 6 A549 cells were grafted subcutaneously on the right flank of the mouse. Five days after the cell graft, the tumors are measurable and the treatments with MAb and/or navelbine are commenced.
  • the MAb dose is always 250 ⁇ g/dose/mouse, twice per week, intra-peritoneally.
  • navelbine it will be administered at the maximum dose tolerated by the mouse or 10 mg lcg, intraperitoneally. For this treatment three injections will be carried out at intervals of 7 days. During the coadministrations, the two products are mixed before injection. The results presented in figure 9 show in a surprising fashion that, in this model, the antibody 7C10 is as active as the conventional treatment with navelbine. A very significant synergy of the two products is likewise observed with five mice out of seven not having measurable tumors on day 72.
  • Example 5 Study of the in vitro inhibition of the IGF2-induced growth of the MCF-7 tumors As indicated above, IGF-IR is overexpressed by numerous tumors but it has furthermore been described that in a good part of the cancers of the breast and of the colon especially, the proliferation signal is given to this receptor via IGF2 (sometimes written IGF-II or IGFII). It is therefore essential to ensure that the MAb 7C10 is likewise capable of inhibiting the IGF2 growth induced on the MCF-7 tumor in vitro.
  • IGF2 sometimes written IGF-II or IGFII
  • Example 6 Biological activity of the chimeric 7C10 (C7C10) and humanized (h7C10) antibodies 7C10 a) 7C 10/C7C 10 and 7C 10/h7C 10 comparison on the MCF-7 model in -vitro The chimeric form of the MAb 7C10 and the purified humanized form 1
  • NK Natural Killer cells coming from the peripheral blood of human donors are placed in the presence of A549 or MCF-7 cells previously incubated for 4 hours with 10 ⁇ g of 7H2HM antibody per 5.10 5 cells and labeled with 51 Cr (50 ⁇ g).
  • herceptin (written h4D5 on figures 32A and 32B) is used as an experiment positive control.
  • Figures 32 A to 32D show that, as expected, herceptin induces a significant ADCC on the two cells A549 and MCF-7 (see respectively figures 32A and 32B).
  • 7H2HM is likewise capable of inducing an ADCC on the A549 cells (see figure 32C), but this phenomenon is of smaller amplitude on the MCF-7 cells (see figure 32D).
  • other effector functions such as, for example, CDC. d) Effects of the antibodies 7C10 and 7H2HM on the cell cycle The inhibition of the cell growth observed in vitro on the line MCF-7 should be manifested by an effect on the cell cycle.
  • 4.10 5 cells are inoculated into 6-well plates. 24 hours after inoculation, the calf serum is removed and IGFl added in the presence or in the absence of the antibodies to be tested.
  • Figure 33B demonstrates the effect of IGFl on the entry into the cycle and the growth of the MCF-7 cells compared with the entry into the cycle and the growth of the MCF-7 cells in the absence of IGFl (see figure 33 A).
  • a significant decrease in the G0/G1 phase from 88.2% to 56.3%) to the benefit of the S (from 7.8% to 31%) and G2/M (from 4% to 12.7%) phases is observed.
  • the antibodies 7C10 and 7H2HM see figure 33C
  • a significant inhibition of the entry into the cycle is observed.
  • the murine antibody and its humanized homolog have a comparable activity on the cell cycle.
  • the antibody 9G4 used as a control isotype is without effect on the cell cycle.
  • Comparative activity in vivo of the antibodies 7C10 and 7H2HM on the model A549 In order to confirm the activity of the humanized antibody 7H2HM in vivo, the latter was compared with 7C10 in the model of non-small cell tumor of the lung A549.
  • the MCF-7 cells are inoculated at 2.10 4 /cm 2 in Petri dishes and cultured for 24 h in RPMI without phenol red supplemented with 10% of fetal calf serum (FCS). The cells are then washed twice with PBS and put back into culture in medium with 0% FCS. They are allowed an adaptation time of 10 minutes at 37°C before the addition of the antibodies at 10 ⁇ g/ml. After an extra 10 minutes at 37°C, recombinant IGFl (Sigma) is added to the culture medium to a final concentration of 50 ng/ml. The cells are left at 37°C again for one hour in order to allow the attachment of the antibodies and of the IGF-I.
  • FCS fetal calf serum
  • the doxorubicin (Sigma) is added to the culture medium at 2 ⁇ g/ml and the cells are incubated for 24 hours at 37°C
  • the experiments have likewise been conducted with navelbine at a concentration of 10 ⁇ g/ml.
  • the analysis of the cell viability is carried out by flow cytometric analysis after labeling with the annexin V-FITC (20 minutes, 4°C) and DAPI (2 ⁇ g/ml). The percentage of dead cells considered is the labeled population Annexin + / DAPI +.
  • the antibody 5C2 is used as a control isotype.
  • the results represented in figure 36 show that doxorubicin induces apoptosis in 8% of the MCF-7 cells. When the cells are treated conjointly with the antibody 7C10 and the doxorubicin a significant increase in cell death is observed. The same effect is shown with the antibody 7H2HM. The same type of results was observed when the antibody is combined with navelbine.
  • Example 7 Cloning strategy of genes coding for the variable regions of the heavy and light chains of the monoclonal antibody (MAb) 7C10
  • the total RNA was extracted from 10 7 cells of hybridomas secreting the antibody 7C10 by using the TRI REAGENTTM (according to the instructions given by the supplier, SIGMA, T9424).
  • the first cDNA strand was synthesized with the aid of the 'First strand cDNA synthesis' kit of Amersham-Pharmacia (#27-9621-01, according to the instructions given by the supplier).
  • the reaction was primed with the oligonucleotide Not I-d(T)18, comprised in the Kit.
  • the cDNA:mRNA hybrid thus obtained was used for the amplification by PCR of the genes coding for the heavy and light chains of the Mab 7C10.
  • the PCR were carried out by using a combination of oligonucleotides specific for the heavy and light (Kappa) chains of mouse immunoglobulins.
  • the primers corresponding to the 5' ends hybridize in the region corresponding to the signal peptides (Table 3 for heavy chains, Table 4 for light chains). These primers were compiled from a large number of mouse antibody sequences found in the databanks (Jones S.T. et al, Bio/Technology 9:88-89, 1991).
  • the primers conesponding to the 3' ends hybridize in the constant regions of the heavy chains (CHI domain of the subclass IgGl, not far from the V-C junction, MHC-1 primer Table 5) and light chains (Kappa domain not far from the V-C junction, MKC primer Table 5).
  • MHV-1 5' ATGAAATGCAGCTGGGTCATSTTCTT 3' (SEQ ID No. 13)
  • MKN-1 5' ATGAAGTTGCCTGTTAGGCTGTTGGTGCT 3' (SEQ ID No. 25)
  • MKN-2 5' ATGGAGWCAGACACACTCCTGYTATGGGT 3' (SEQ ID No. 26)
  • MKV-3 5 ' ATGAGTGTGCTCACTCAGGTCCT 3 ' (SEQ ID No. 27)
  • MKV-4 5' ATGAGGRCCCCTGCTCAGWTTYTTGG 3 ' (SEQ ID No. 28)
  • MKV-5 5' ATGGATTTWCAGGTGCAGATTWTCAGCTT 3' (SEQ ID No. 29)
  • MKV-5A 5' ATGGATTTWCARGTGCAGATTWTCAGCTT 3' (SEQ ID No. 30)
  • MKV-6 5' ATGAGGTKCYYTGYTSAGYTYCTGRG 3' (SEQ ID No. 31)
  • MKV-7 5 ⁇ TGGGCWTCAAGATGGAGTCACA 3' (SEQ ID No
  • MKV-8 5' ATGTGGGGAYCTKTTTYCMMTTTTTCAAT 3 ' (SEQ ID No. 33)
  • MKV-9 5' ATGGTRTCCWCASCTCAGTTCCTT 3 ' (SEQ ID No. 34)
  • MKV-10 5' ATGTATATATGTTTGTTGTCTATTTC 3 ' (SEQ ID No. 35)
  • MKV-11 5' ATGGAAGCCCCAGCTCAGCTTCTCTT 3' (SEQ ID No. 36)
  • MKV-12A 5' ATGRAGTYWCAGACCCAGGTCTTYRT 3' (SEQ ID No. 37)
  • MKV-12B 5' ATGGAGACACATTCTCAGGTCTTTGT 3' (SEQ ID No. 38)
  • MKV-13 5' ATGGATTCACAGGCCCAGGTTCTTAT 3' (SEQ ID No. 39)
  • CHI domain of mouse gamma- 1 (IgGl subclass): A K T T P P S V Y P L (SEQ ID No.46)
  • PCR products corresponding to the variable regions of the heavy (VH) and light (VL) chains were cloned by using the "pGEM®-T Easy Vector Systems" (Promega).
  • PCR products were obtained with the MKC primer in combination with the MKV1 and MKV2 primers.
  • PCR products were obtained with the MHC-1 primer in combination with the MHV8 and MHV12 primers.
  • a thorough sequencing of the PCR products cloned in the pGem-T easy vectors revealed two different sequences for the light chain and one unique sequence for the heavy chain.
  • a) Variable region isolated from the oligo MKV1 The DNA sequence obtained is characteristic of a variable region of functional Ig. This novel sequence is therefore presumed to be that coding for 7C10 VL.
  • the DNA (SEQ ID Nos. 48 and 50) and amino acid (SEQ ID No. 49) sequences of the cDNA coding for 7C10 VL are represented in figure 14.
  • b) Variable region isolated from the oligo MKV2 The gene coding for this light chain comes from an aberrant mRNA transcript which is present in all the standard fusion partners derived from the original MOPC-21 tumor of which the mouse myeloma Sp2/Oagl4, which was used in order to produce the 7C10 hybridoma, is part.
  • This sequence contains an aberrant recombination between the V and J genes (deletion of four nucleotide bases involving a change in the reading frame) and a mutation of the invariable cysteine in position 23 to tyrosine. These changes suggest that this light chain would be nonfunctional although nevertheless transcribed to messenger RNA.
  • the DNA sequence of this pseudo light chain is not shown.
  • c) Variable region isolated from the oligos MHV8 and MHV12 The DNA sequences obtained with these two oligos are identical, apart from the sequence encoded by the oligo itself. This sequence is a novel sequence coding for a functional heavy chain presumed to be that of the monoclonal antibody 7C10.
  • the DNA (SEQ ID Nos.
  • Example 9 Construction of chimeric mouse-man genes
  • the chimeric antibody 7C10 was constructed so as to have the mouse 7C10 regions VL and VH connected to the human constant regions kappa and gamma- 1, respectively. Oligos were used in order to modify the 5' and 3' ends of the sequences flanking the DNA coding for 7C10 VL and VH in order to allow their cloning in vectors for expression in mammalian cells.
  • These vectors use the strong promoter HCMV in order effectively to transcribe the heavy and light chains of the chimeric antibody 7C10.
  • these vectors likewise contain the replication origin of SV40 allowing an effective replication of the DNA and, as a consequence, as a transitory expression of the proteins in cos cells.
  • Example 10 Expression and evaluation of the recognition activity of the IGFl receptor of the chimeric antibody 7C10
  • the two plasmids containing the DNA coding for the chimeric 7C10 antibody were cotransfected in cos-7 cells (ATCC number CRL- 1651) in order to study the transitory expression of the recombinant antibody. After incubation for 72 hours, the culture medium was removed, centrifuged in order to eliminate the cell debris and analyzed by the ELISA technique for the production of human IgGl (see Example 16) and the recognition of the receptor for IGFl (see Example 17).
  • the ELISA tests for measurement of concentrations of human IgGl /Kappa showed that the expression of the chimeric antibody 7C10 in the cos-7 cells was between 300 and 500 ng/mm, which is comparable to the values obtained with the majority of antibodies.
  • the ELISA tests for recognition of the receptor for IGFl show that the chimeric antibody recognizes it specifically and with a good relative avidity (see figures 3 A, 3B and 3C). This provides the functional proof that the good VH and VL of the 7C10 antibody have been identified.
  • this chimeric form of 7C10 appears as being an indispensable tool in the evaluation of the affinity of the humanized forms.
  • Example 11 Molecular modeling of the variable regions of the mouse antibody 7C10
  • a molecular model of the VL and VH regions of the mouse antibody 7C10 was constructed. The model is based on the crystallographic structure of the heavy chain 1 AY1 and of the light chain 2PCP.
  • Example 12 Process of humanization by CDR grafting of the variable region of the light chain of the antibody 7C10 (7C10 VL) a) Comparison of the amino acid sequence of 7C10 VL with all the known mouse VL sequences As a preliminary step to humanization by CDR grafting, the amino acid sequence of 7C10 VL was first compared with all the mouse VL sequences present in the databank of Kabat (Internet address: ftp://ftp.ebi.ac.uk/pub/ database/kabat/fasta_format/, last update of data dates from 1999). 7C10 VL has thus been identified as belonging to the subgroup II of the Kappa light chains as defined by Kabat et al.
  • the first column indicates the position of the amino acid residue according to Kabat et al. (1991); the second column (#) indicates the position of the amino acid residue in the regular sequence; the third column (FR or CDR) was made in order easily to identify the segments of the skeleton (FRl, FR2, FR3 and FR4) and the CDR segments (CDR1, CDR2 and CDR3) ("CDR" for "Complementarity-Determining Region") with the three CDRs separating the four FRs; the fourth column (Mouse light chain 7C10) represents the amino acid sequence (SEQ ID No.
  • the fifth column (Human germinal line DPK15/A19) represents the amino acid sequence (SEQ ID No. 59) of the kappa II human V light chain of the germinal line;
  • the sixth column represents the amino acid sequence (SEQ ID No. 58) of the V L region of the human antibody GM607;
  • the seventh and eighth columns (remodeled human 7C10 1 and 2) represent the amino acid sequences of the humanized 1 and 2 antibody 7C10 VL (respectively SEQ ID Nos. 61 and 65).
  • “*" indicates the parts of the canonical structure of the CDR loop such as defined by Chothia et al. (Nature, 342, 877-883, 1989).
  • Example 13 Process of humanization by CDR grafting of the variable region of the heavy chain of the antibody 7C10 (7C10 VH) a) Comparison of the amino acid sequence of 7C10 VH with all of the known mouse VH sequences As a preliminary stage in humanization by CDR grafting, the amino acid sequence of 7C10 VH was first compared with all the mouse VH sequences present in the Kabat databank (Internet address: ftp://ftp.ebi.ac.uk/ pub/database/kabat/fasta_format/, last update of data dates from 1999). 7C10 VH has thus been identified as belonging to the subgroup 1(A) of the heavy chains as defined by Kabat et al (1991).
  • VH regions of mouse monoclonal antibodies having a sequence identity ranging up to 90.5% were identified (AN03'CL (SEQ ID No. 70), see figure 22).
  • AN03'CL SEQ ID No. 70
  • Residue 17 (Kabat's numbering) Thr for the consensus sequence of subgroup 1(A) and Ser in 7C10 VH, is located on the surface of the molecule with respect to the interface with the constant region. This residue does not seem to be important.
  • Residue 27 (Kabat's numbering), Asp for the consensus sequence of subgroup 1(A) and Tyr in 7C10 VH, is a canonical residue for the CDR 1. Tyr in this position is not rare and is probably critical for maintaining CDR 1 in its good conformation.
  • Residue 84 (Kabat's numbering), Thr for the consensus sequence of the subgroup 1(A) and Asn in 7C10 VH. Asn was found 93 times in mouse VH and 3 times in human VH. According to the molecular model, it is a surface residue remote from the paratope. The numbering of the amino acids is that of Kabat et al. (1991).
  • AN03'CL represents the sequence of the heavy chain of a mouse antibody (access number in the Kabat databank is P001289).
  • Mouse 7C10 VH had the greatest sequence homology with the human VH regions of the subgroup II as defined by Kabat et al. (1991).
  • VH regions of monoclonal antibodies of human origin were identified having a sequence identity ranging up to 67.3% (human VH FURl'CL (SEQ ID No. 73, see figure 23) over the whole of the 98 amino acids encoded by the variable gene (that is to say apart from CDR3 and region J).
  • the sequence encoded by the germinal line 4.22 VH IV was chosen as a human sequence receptive of the CDRs (according to the definition of Kabat) of mouse 7C10 VH rather than VH FURl'CL because in comparing the sequences of 4.22 VH IV and VH FURl'CL with that of the consensus sequence of the human subgroup II (human Kabat sg II (SEQ ID No. 72), see figure 23 and table 7), no atypical residue in the framework regions (Rch) could be identified for 4.22 VH IV although the presence of two atypical residues (Gin and Arg in positions 81 and 82A according to the nomenclature of Kabat, respectively) were identified in the sequence encoded by VH FURl'CL.
  • All the residues of the Rchs are therefore of human origin including the residues 30, 48, 67 and 71 (Kabat's nomenclature) which have been conserved (see Table 7, figure 24 for the amino acid sequence (SEQ ID No. 83) and figure 27 for the DNA sequence (SEQ ID Nos. 84 and 86) and the amino acid sequence' comprising the peptide signal (SEQ ID No. 85)).
  • This humanized form 3 is therefore totally humanized (apart, of course, from the CDRs themselves as defined by Kabat) since all the residues of the Rchs are those encoded by the VH gene of the germinal line 4.22 VH IV.
  • the first column indicates the position of the amino acid residue according to Kabat et al. (1991); the second column (FR or CDR) was made in order easily to identify the segments of the skeleton (FRl, FR2, FR3 and FR4) and the CDR segments (CDR1, CDR2 and CDR3) with the three CDRs separating the four FRs; the third column (Mouse heavy chain 7C10) represents the amino acid sequence (SEQ ID No. 69) of the V H region of the mouse antibody 7C10; the fourth column (Germinal line 4.22 VH IV) represents the amino acid sequence of the gene 4.22 VH IV (Sanz et al., 1989) (SEQ ID No.
  • the fifth column (human FURl'CL VH, kabat accession number N020619) represents the amino acid sequence (SEQ ID No. 73) [lacuna] IgMK antilamin B of human origin (Mariette et al., 1993);
  • the sixth, seventh and eighth columns (remodeled human 7C10 1, 2 and 3) represent the amino acid sequences of the VH region of remodeled human 7C10 respectively for the versions 1 (SEQ ID No. 75), 2 (SEQ ID No. 79) and 3 (SEQ ID No. 83).
  • "*" indicates the parts of the canonical structure of the CDR loop such as defined by Chothia et al. (1989).
  • Example 14 Construction of the genes coding for the humanized versions 1 of 7C10 VL and VH by assembly of ohgonucleotides a) Principle The genes (leader peptide + variable regions VDJ for VH or VJ for VK) coding for the humanized variable regions were synthesized by solid-phase assembly on magnetic beads coated with streptavidin. The genes coding for humanized 7C10 VH (445 base pairs) and humanized 7C10 VL (433 base pairs) are constructed by fusing two fragments of DNA owing to the presence of a Kpnl restriction site present in the two sequences and situated almost halfway along the gene (at 200 and 245 nucleotides with respect to the 5' end of the gene for VL and VH, respectively).
  • the two fragments which are fused together are themselves assembled by an assembly technique which consists in using phosphorylated oligonucleotides (approximately 30-35 mer) hybridized two by two (one oligo sense and the other antisense, with a homology of approximately 50%) in such a way that they overlap during elongation.
  • a first oligonucleotide biotinylated in the 5' position is attached to the magnetic beads and then the pairs of phosphorylated oligonucleotides are added one by one.
  • the phosphodiester linkage between the juxtaposed phosphorylated oligonucleotides is produced by the enzyme T4 DNA ligase.
  • the genes thus synthesized de novo can be cloned directly (by digestion with restriction enzymes compatible with the expression vector chosen) or amplified by PCR in order to obtain more material as a prelude to directional cloning by enzymatic digestion.
  • the sequence of the gene thus constructed by de novo assembly is then verified by automatic sequencing of the DNA.
  • Oligonucleotides phosphorylated in the 5' position or biotinylated in the 5' position whose concentration was adjusted to 100 ⁇ M were ordered from MWG Biotech (see the sequences of the oligonucleotides used in Table 7 for the construction of humanized 7C10 VL, and Table 9 for the construction of humanized 7C10 VH).
  • the oligonucleotides were hybridized in pairs (an equimolar mixture, 500 pmol, of a sense oligo and of an antisense oligo in the buffer T4 DNA ligase is heated to 95°C for 5 minutes and then allowed to cool on the bench to ambient temperature) according to a scheme described in Table 10.
  • the first biotinylated oligonucleotide is attached to magnetic beads coated with streptavidin (Dynabeads M-280 streptavidin, Dynal product No. 112-05).
  • 500 pmol of the biotinylated oligonucleotide in a 15 mM NaCl solution are added to 50 l of the decanted beads (use of a magnet holder) previously washed twice with 100 ⁇ l of TE IX buffer (Tris-EDTA 100X buffer: 1 M Tris-HCI, pH 8, 0.1 M EDTA, Sigma T-9285). After incubation at 37°C for 15 min, the beads are washed twice with the wash buffer (10 mM Tris-HCI pH 7.6, 10 mM EDTA and 50 mM NaCl) and the pairs of hybridized oligo-nucleotides are then added one by one.
  • TE IX buffer Tris-EDTA 100X buffer: 1 M Tris-HCI, pH 8, 0.1 M EDTA, Sigma T-9285
  • the mixture On each readdition of a pair of oligonucleotides, the mixture is heated to 95°C for 5 min and then allowed to cool on the bench to ambient temperature. Once ambient temperature is reached, 2 ⁇ l of 10 U/ ⁇ l T4 DNA ligase (Biolabs) are added and the mixture is incubated for 20 min at 37°C. The beads are then washed (wash buffer) and the following pairs of oligonucleotides are then added in succession. The last unpaired oligo (antisense) is assembled in the following fashion.
  • biotin 5 ' -GTCAGAACGCGTGCCGCC SEQ ID No. 87
  • biotin 5 ' -GTCAGAACGCGTGCCGCC SEQ ID No. 117
  • de novo assembly de novo assembly of the MlUI-Kpnl fragment of the Kpnl-BamHI fragment of 7C10 VL humanized 1 of 7C10 VL humanized 1 Biotinylated oligo leader Biotinylated oligo 7C10 L M1UI 7C10 VL Kpnl
  • de novo assembly de novo assembly of the MlUI-Kpnl fragment of the Kpnl-BamHI fragment of 7C10 VL humanized 1 of 7C10 VL humanized 1
  • Example 15 Construction of the genes coding for the humanized versions 2 of 7C10 VL and 7C10 VH and 3 of 7C10 VH by directed mutagenesis
  • the humanized version 2 of 7C10 VH was obtained by directed mutagenesis of - Ill -
  • the humanized version 3 of 7C10 VH was obtained by site-directed mutation of the residues 30 and 71 (according to Kabat's nomenclature) of version 2 likewise using the system QuikChangeTM. This construction is carried out in two stages. At first, the residue 30 on version 2 was mutated with the aid of the primers 7C10HhumanizedQCT30 sense and antisense (see Table 11). Subsequently, this version mutated at the residue 30 was itself mutated at the residue 71 by using the pair of primers 7C10HhumanizedlV67QCR71 sense and antisense (see Table 11).
  • the humanized version 2 of 7C10 VL was obtained by site-directed mutation of the residue 2 (according to Kabat's nomenclature) of version 1 by using the system QuikChangeTM.
  • the residue 2 on version 1 was mutated by using the pair of primers 7C10LhumanizedlQCV2 sense and antisense (see Table 11).
  • Example 16 Transfection of the cos7 cells by electroporation
  • the mammalian expression vectors containing the chimeric or humanized versions of the heavy and light chains of the antibody 7C10 were tested in cos7 cells for the transitory expression of the recombinant antibodies 7C10.
  • the DNA was introduced into the cos cells by electroporation with the aid of a BioRad instrument (Gene Pulsar). 10
  • the DNA (10 ⁇ g of each vector) is added to aliquots of 0.8 ml of cos cells at a concentration of 1 x 10 7 cells per ml in PBS buffer (without Ca++ and Mg++). A pulsation of 1900 volts and a capacity of 25 ⁇ F was delivered.
  • transfected cos cells are then added to 8 ml of DMEM medium containing 5% of calf serum and incubated at 37°C for 72 hours.
  • the supernatant is then collected, centrifuged in order to eliminate the cell debris and tested by ELISA for the measurement of its concentration of recombinant antibody 7C10 of IgGl/human Kappa type.
  • Example 17 ELISA method for measuring the concentrations of recombinant antibody IgGl/human Kappa present in the supernatant of the cos transfectants
  • the supematants produced by transitory expression in cos7 cells were tested for the presence of 7C10 antibody of IgGl/human Kappa type.
  • 96-well ELISA plates (Maxisorb, Nunc) were coated with a goat anti-human IgG polyclonal antibody (specific for the gamma Fc fragment, Jackson Immuno-Research Laboratories hie, #109-005-098).
  • the supematants of cos cells were diluted in series and added to the coated wells.
  • Example 18 ELISA method for determining the recognition activity of 7C10 recombinant antibodies of human IgGl/Kappa type on the receptor for IGFl (IGF-IR)
  • IGF-IR human IgGl/Kappa type on the receptor for IGFl
  • the cos7 culture supematants were tested for their capacity to recognize IGF-IR by an ELISA method.
  • 96-well ELISA plates (Dynex Immulon 2HB) were coated with 100 ⁇ l per well of a solution of PBS containing 0.31 ng/ ⁇ l of IGF-IR (Human Insulin- Like Growth Factor I soluble Receptor, R & D Systems, #391-GR) by incubation for one night at 4°C.
  • the plates were saturated by the addition of a solution of PBS containing 0.5%> gelatin solution and incubation at 37°C for 1 hour. After three washes with PBS, the samples of cos supematants to be tested, previously diluted in series in PBS containing 0.1 % gelatin and 0.05%) Tween 20, were added to the plates.
  • Example 19 Determination of the recognition activity of IGF1-R by different versions of the humanized 7C10 antibody by "CDR grafting"
  • Figure 28 shows the results of an ELISA test of recognition of the IGF-IR (see Example 18) from supematants of the cos7 cells whose concentration of IgGl/human Kappa had been previously determined by ELISA (see Example 17).
  • the titration curves of the four recombinant antibodies tested overlap perfectly indicating that their relative affinities for IGF-IR are very similar. It is therefore concluded from this that the humanized form 1 of 7C10, composed of the humanized light chain 1 (1 single mouse residue present in the framework regions) in combination with the humanized heavy chain 1 (4 mouse residues present in the framework regions), specifically recognizes the
  • IGF-IR and has an affinity very similar to that of the chimeric antibody (mouse variable regions).
  • residue 2 accordinging to Kabat's nomenclature
  • Figure 29 shows the results of the ELISA test for recognition of the IGF-IR (see Example 18) from supematants of cos7 cells whose concentration of IgGl/human Kappa had been previously determined by ELISA (see Example 17).
  • the two humanized versions 1 and 2 of the light chain had been combined successively with humanized 7C10 VH 1.
  • the titration curves of the two combinations are superimposed indicating that the mutation of residue 2 of the light chain, which has been changed from one valine in the humanized version 1 to an isoleucine in the humanized form 2, apparently has no influence on the relative affinity of recognition of the IGFl receptor.
  • the humanized form 2 of the light chain of 7C10 thus forms one version where no mouse residue (apart from CDRs) has been conserved.
  • This version, totally humanized represents the preferred version of 7C10 VL.
  • the totally humanized version of the 7C10 light chain (humanized version 2, see above) was tested in combination with the three humanized versions of the heavy chain of 7C10.
  • Figure 30 shows the results of the ELISA test for recognition of the IGF-IR from supematants of cos7 cells whose concentration of IgGl/human Kappa had been previously determined by ELISA (see Example 17).
  • the titration curves are very similar and virtually overlap with the reference curve corresponding to the chimeric antibody, indicating that the three humanized versions 1, 2 and 3 of 7C10 VH give an identical relative affinity for IGF-IR when they are combined with humanized 7C10 VL 2.
  • This last form constitutes the ultimate humanized version since no mouse residue is present at the same time in the heavy and light chains.
  • Example 20 Expression of EGFR and of IGF-IR on the surface of A549 cells
  • the synergy of action obtained by the coadministration of two MABs directed respectively against IGF-IR and EGFR was studied in nude mice carrying a non-small cell lung tumor established by subcutaneous injection (s.c.) of A549 cells (lung carcinoma cell line).
  • A549 cells lung carcinoma cell line.
  • labeling for FACS reading of these cells was carried out with, respectively, the murine 7C10 anti- IGF-IR MAB (figure 37B) and the murine 225 anti-EGFR MAB (figure 37D).
  • the cells were saturated for 30 min at 4°C with a solution of PBS 10% FCS (fetal calf serum), washed and then incubated for 30 min at 4°C with the MAB of interest. After 3 new washes, the secondary anti-species antibody coupled to FITC (fluorescein isothiocyanate) is added. After incubation for 30 min, reading on the FACS (Fluorescence Activated Cells Sorter) is carried out at 520 nm (excitation 488 nm).
  • FACS Fluorescence Activated Cells Sorter
  • Example 21 Synergy of action of an anti-IGF-IR MAB and of an anti-EGFR MAB coadministered in vivo, in the nude mouse in the context of an antitumor treatment
  • nude mice are grafted s.c. with 5.10 6 A549 cells. Five days after the cell graft, the tumors are measured and a homogeneous batch of mice in terms of tumor volume is formed. Starting from this batch, groups of 6 mice are generated at random. These mice will be treated intraperitoneally (i.p.), twice per week with each of the MAB 7C10 and 225 individually at the dose of 250 ⁇ g/mouse or with the two MAB in coadministration.
  • the MAB 9G4 is administered as an experiment isotype control.
  • the results presented in figure 38 show that each of the antibodies 7C10 and 225 administered alone is capable of inducing a significant decrease in the tumor growth in vivo. It can be noted that the two MAB tested have a comparable activity on the growth of the tumor A549.
  • Example 22 Study of the antitumor activity of the murine antibodies 7C10 and 225 coadministered in mice orthotopically implanted with A549 cells
  • the use of orthotopic models for the evaluation of the antitumor activity presents a particular interest with respect to the process of metastatic dissemination of a tumor.
  • 10 6 A549 cells non-small cell lung cancer
  • 10 6 A549 cells were implanted in the intrapleural cavity of nude mice. It is to be noted that the consequence of this type of tumor implantation is a metastatic dissemination similar to that observed in man and leads to the death of the animals.
  • Figure 39 shows that the administration of the antibodies 225 and 7C10 alone allows a comparable and a significant gain in survival to be observed. In a surprising fashion, the coadministration of these two antibodies increases in a considerable fashion the survival of the animals suggesting that this treatment could have an impact on the metastatic dissemination of the tumor cells.
  • Example 23 7C10 and 7H2HM inhibit the phosphorylation of the tyrosine of the ⁇ chain of IGF-IR and of IRS-I MCF7 cells are cultured for 24 hours at 5.10 4 cells/cm 2 (75 cm 2 plates, COSTAR) in 20 ml of RPMI without phenol red, mixed with 5 mM of glutamine, penicillin/ streptomycin (respectively 100 U/100 ⁇ g/ml) and 10% of fetal calf serum.
  • the cells were incubated for 12 hours in medium (RPMI) without phenol red, devoid of fetal calf serum and mixed with 5 mM of glutamine, penicillin/streptomycin, bovine serum albumin at 0.5 ⁇ g/ml (Sigma A-8022) and transferrin at 5 ⁇ g/ml (Sigma T8158).
  • medium RPMI
  • the cells were first incubated at 37°C for 2 minutes with blocking antibodies (10 ⁇ g/ml) and then IGFl (Sigma 13769, 50 ng/ml) was added for two additional minutes. The reaction was stopped by aspiration of the incubation medium and the plates were laid on ice.
  • the cells were solubilized by addition of 0.5 ml of lysis buffer (50 mM tris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate), mixed with protease inhibitors (1 tablet per 50 ml, Boehringer Ref.: 1697 498), and phosphatase inhibitors (Calbiochem Ref.: 524625 (1/100)).
  • the cells were scraped off and the suspension was recovered and placed on a shaker at 4°C for 1.5 hours.
  • the solutions were centrifuged at 12,000 rpm for ten minutes (4°C) and the protein concentrations of the supematants were quantified by BCA.
  • the agarose beads were washed twice with 1 ml of lysis buffer, twice with a wash buffer 1 (50 mM tris-HCl pH 7.5; 500 mM NaCl; 0.1% Nonidet P40; 0.05% sodium deoxycholate (Boehringer 1 332 597), mixed with protease inhibitors and phosphatase inhibitors) and once with a wash buffer 2 (50 mM tris-HCl; 0.1% Nonidet P40; 0.05% sodium deoxycholate (Boehringer Ref.: 1 332 597), mixed with protease inhibitors and phosphatase inhibitors 1/100).
  • wash buffer 1 50 mM tris-HCl pH 7.5; 500 mM NaCl; 0.1% Nonidet P40; 0.05% sodium deoxycholate (Boehringer 1 332 597), mixed with protease inhibitors and phosphatase inhibitors 1/100.
  • the immunoprecipitates were resuspended in a Laemmli buffer, heated to 100°C for 5 minutes.
  • the supematants were analyzed by electrophoresis on polyacrylamide SDS gel (8% Novex EC6015).
  • the proteins were transferred to a nitrocellulose membrane followed by either an immunoblot with anti-phosphotyrosine antibodies conjugated to HRP (upstate Biotechnology 4G10) or beta anti-chain of IGF-IR or anti-IRS-1 (Santa Cruz Ref.: sc 8038) followed by an anti-rabbit antibody conjugated to HRP.
  • HRP upstate Biotechnology 4G10
  • beta anti-chain of IGF-IR or anti-IRS-1 Santa Cruz Ref.: sc 8038
  • the imprints were revealed by chemiluminescence (Amersham RPN 2209) followed by autoradiography on Kodak X-mat AR films.
  • Figure 40A represents MCF7 cells nonstimulated (0) or stimulated either with IGFl (50 ng/ml) alone (0+IGF-I) or combined with monoclonal or humanized anti-IGF- IR antibodies (10 ⁇ g/ml) 7C10, 1H7, 7H2HM.
  • the antibodies 9G4 or hlgGl are murine or human immunoglobulins of isotype IgGl used as an experiment negative control.
  • the beta chains of the IGF-IR were immunoprecipitated and blotted with phosphorylated anti-tyrosine antibodies.
  • FIG. 40B represents MCF7 cells nonstimulated (0) or stimulated either with IGFl (50 ng/ml) alone (0+IGF-I) or combined with monoclonal or humanized anti-IGF- IR antibodies (10 ⁇ g/ml) 7C10, 1H7, 7H2HM.
  • the antibodies 9G4 or hlgGl are murine or human immunoglobulins of isotype IgGl used as an experiment negative control.
  • the IRS-1 was immunoprecipitated and blotted with phosphorylated anti-tyrosine antibodies.
  • the results obtained show that the monoclonal antibodies 7C 10, 7H2HM and 1 H7 inhibit the phosphorylation of the tyrosine of the IRS-1.
  • Example 24 7C10 and 7H2HM induces the internalization of the IGF-IR MCF7 and A549 cells were suspended to 1.10 7 cells/ml in PBS with 10% of fetal calf serum (FACS buffer). 1.10 6 cells were incubated for 30 minutes at 37°C with the monoclonal antibodies at 10 ⁇ g/ml (7C10, 7G3, 9G4) or at 20 ⁇ g/ml for 7H2HM. After washing, the cells were labeled at 4°C for 30 minutes with a biotinylated anti- IGF-IR (monoclonal antibody 12B1) and finally incubated at 4°C for 30 minutes with a conjugate of streptavidin-488 alexa Fluor ® .
  • FACS buffer fetal calf serum
  • Figure 41 shows the A549 cells without coloration (1 st peak), the A549 cells incubated with 7C10 or 7H2HM (2 nd peak) and the A549 cells incubated with an irrelevant mouse or rat IgGl (3 r peak). A decrease by two of the surface expression of the IGF-IR by the cells is seen when the cells have been previously incubated with 7C10 or 7H2HM.
  • Example 25 7C10 and 7H2HM induce the degradation of the IGF-IR MCF-7 cells were cultured for 24 hours at 10.10 cells/cm (75 cm , Costar) in 15 ml of complete medium. Next, the cultures were washed three times with PBS and incubated for 12 hours with medium devoid of serum. Next, the cells were incubated with cycloheximide at 25 ⁇ g/ml alone or with 10 ⁇ g/ml of monoclonal antibody 7C10, 9G4, 7G3 or of IGFl (50 ng/ml).
  • the cells before incubation with the monoclonal antibodies, the cells were treated for 1 hour at 37°C with MG-132 (10 ⁇ M, Calbiochem 474791) in order to inhibit the proteasome activities. After incubation, the cells were washed and solubilized by addition of a lysis buffer. 20 ⁇ g of proteins were analyzed by electrophoresis on polyacrylamide gel at 8% of SDS and transferred to a nitrocellulose membrane followed by a beta anti-chain immunoblot of the IGF-IR such as described further above. The analysis by Westem-blot (figure 42A) of the integrity of the IGF-IR shows that 7C10 and 7H2HM induce the degradation of the receptor while the natural ligand does not cause any degradation of the latter.
  • Example 26 Evaluation of 7C10 and h7C10 abihty to bind to IGF-IR and hybrid- R.
  • Example 26.1 Evaluation of 7C10 and h7C10 ability to immunoprecipitate IGF-IR and hvbrid-R purified from transfected cells respectivelv with IGF-IR and IR-A or IGF-IR and IR-B (thereafter referred as R+/IR-A or R+/IR-B
  • the goal of this study is to evaluate the ability of 7C10 and h7C10 to immunoprecipitate IGF-IR, IR or Hybrid-R.
  • 7C10 and h7C10 are compared to 17-69 (which recognizes both IGF-IR well and Hybrid-R).
  • Method The used cells for this study are listed thereafter: - R+: R- fibroblasts stably transfected with the IGF-IR (IGF-IR) cDNA
  • R+/IR-B R- fibroblasts stably co-transfected with the IGFl and the insulin receptor isoform B cDNA and, therefore, expressing hybrid receptors A (Hybrid-RsB)
  • Hybrid-RsB hybrid receptors A
  • Example 26-2 Displacement analysis of IGFl on IGF-IR by 7C10. h7C10 and 1H7 IGF-IR from R+ cell lysates were immunocaptured in Maxisorb plates coated with 17-69 antibody. 19 I-IGF1 (Fig. 44) was then allowed to bind to immunocaptured receptors in the absence or the presence of increasing concentrations of unlabeled ligand (IGFl or IGF2) or antibodies (7C10, h7C10, 1H7, 9G4). Results are plotted as percent of maximal specific binding.
  • Example 26-3 Displacement analysis of IGFl on Hybrid-RsA by 7C10. h7C10 and 1H7 Hybrid-RsA from R+/IR-A cell lysates were immunocaptured in Maxisorb plates coated with anti IR antibody 83-7.
  • 125I-IGF1 (Fig. 45) was then allowed to bind to immunocaptured receptors in the absence or the presence of increasing concentrations of unlabeled ligand (IGFl or IGF2) or antibodies (7C10, h7C10, 1H7, 9G4). Results are plotted as percent of maximal specific binding. Both 7C10 and h7C10 displace labeled IGFl with a very similar efficiency with IC 50 values less than 100 nM, and in this example of about 2.0 nM for each. By comparison, 1H7 was much less effective with an IC 50 value up to 50 nM, preferably up to 100 nM (Fig. 45).
  • Example 26-4 Displacement analysis of IGFl on Hybrid-RsB by 7C10.
  • h7C10 and 1H7 Hybrid-RsB from R-/IR-B cell lysates were immunocaptured in Maxisorb plates coated with 83-7 antibody.
  • 125I-IGF1 (Fig. 46) was then allowed to bind to immunocaptured receptors in the absence or the presence of increasing concentrations of IGFl and IGF2 or antibodies (7C10, h7C10, 1H7, 9G4). Results are plotted as percent of maximal binding.
  • Both 7C10 and h7C10 displace labeled IGFl with a very similar efficiency with IC50 values less tha 100 nM, and in this example of about 1.5 and 2.5 respectively.
  • 1H7 was much less effective with an IC50 value up to 100 nM (Fig. 46).
  • EXEMPLE 27 Intemalization and degradation studies of the IGF-IR Intemalization and degradation studies were analyzed by FACS and western- blot amalysis. Intemalization studies were performed by FACS analysis using a murine biotinylated anti-IGF-IR monoclonal antibody (Mab) thereafter described as 12B1 MAb and binding to an epitope different from the one recognized by 7C10 and h7C10 antibodies. The 7G3 MAb, a non neutralizing anti-IGF-IR was introduced as negative control. Both antibodies were generated in our laboratory. Confluent MCF-7 cells were trypsinized and lxl 0 6 cells from each cellular suspension was plated in 96-well plates in FACS buffer.
  • Mob murine biotinylated anti-IGF-IR monoclonal antibody
  • Plates were incubated, either with or without 25 ⁇ g/ml of cycloheximide (Calbiochem), 30 min at 37°C with either IGFl (50 ng/ml) or with 10 ⁇ g/ml of 7C10, 7G3, h7C10, mlgGl, hlgGl. Cells incubated with FACS buffer alone were used to determine the basal level of expression of the IGF-IR. Then cells were washed twice and 12 ⁇ g/ml of biotinylated- 12B1 MAb were added to the plate.
  • cycloheximide Calbiochem
  • 7.5 X 10 6 cells were plated in 75 cm 2 flasks in 15 ml of complete medium (red phenol-free RPMI and Ham-F12K respectively for MCF-7 and A549 both supplemented with 10% FCS and 1% L-Glutamine). Twenty four hours after plating, cells were washed 3 times with PBS and incubated for 24 additional hours at 37°C. Then medium was removed and cells incubated either lh, 4h or 16h at 37°C with 15 ml of serum-free medium with or without antibodies to be tested or with IGF-I.
  • Figures 47 A and 47B represent the study of antibody induced degradation of the IGF-IR.
  • experiments were done without cyclohexemide as the above experiment shows that no difference was observed in presence or in absence of this compound.
  • 7C10 and h7C10 cause a comparable intemalization of the IGF-IR in both A549 (A) and MCF-7 (B) cells.
  • MCF-7 cells the maximal intemalization was observed after four hours incubation with 7C10 and h7C10, whereas, for A549 the maximal intemalization is observed as earlier as 1 hour. No degradation was observed when cells were incubated either with IGF-I, 7G3 or murine (mlgGl) or human (hlgGl) isotype control.
  • EXEMPLE 28 Study of the degradation pathway of IGF- IR 7.5 X 10 6 MCF-7 cells were plated in 75 cm 2 flasks in 15 ml of complete medium (red phenol-free RPMI supplemented with 10% FCS and 1% L-Glutamine). Twenty four hours after plating, cells were washed 3 times with PBS and incubated for 24 additional hours at 37°C in 15 ml serum-free medium . Then medium was removed and cells incubated for two hours in 7.5 ml of serum-free medium either containing 30 ⁇ M MG115 or DMSO. Then, 7.5 ml of serum-free medium with or without h7C10, hlgGl or IGFl were added for 4 additionnal hours.
  • Figure 48 shows the obtained results.
  • IGFl human isotype control
  • MG115 human isotype control
  • h7C10 also down regulate, in addition to the proteasome pathway as previously described, via other known and described pathways for anti-IGF-IR antibodies, i.e. liso some-dependent and/or endocytic pathways.
  • Such a property, if validated, is of particular interest as it would demonstrate the capacity of the h7C10 to interact with different signalization/degradation pathways, and thus its therapeutic efficacy. Supplementary studies are in progress in order to validate this hypothesis.
  • Example 29 Anti-tumoral activity of the murine antibody 7C10 co-administrated with an anti- VEGF antibody on mice orthopically implanted with A549 cells
  • A549 NSCLC One million of A549 NSCLC were implanted through the chest wall into the left pleural cavity space of 6 weeks old Swiss nude mice following the protocol described by Klaus-Berthier et al. (Kraus-Berthier, L., Jan, M., Guilbaud, N., Naze, M., Pierre, A., and Atassi, G. Histology and sensitivity to anticancer drugs of two human non-small cell lung carcinomas implanted in the pleural cavity of nude mice. Clin. Cancer Res. 6(l):297-304, 20O0).
  • mice Seven days after the cell injection, mice were treated i.p. with a loading dose of 250 ⁇ g of antibodies, and them, twice a week with 125 ⁇ g of antibodies.
  • antibodies were mixed prior to the injection.
  • the anti- VEGF antibody used was an IgG2b, clone 26503.11 commercialized by SIGMA. It was described as a neutralizing antibody (Ferrara N. et al., Biochem. Res. Com. 161:851, 1999; Ferrara et al., Endocrinol. Review, 13:18, 1992; Leung D.W. et al., Science 246:1306, 1989).
  • Figure 48 shows that a combined therapy increase dramatically the time survival compared to untreated mice or to mice treated with single therapy.
  • the T/C% are calculated according the following formula, [MEDIAN OF
  • the obtained T/C% are about 134% and 144% for the 7C10 and anti-VEF antibody respectively.
  • the T/C% is 188%.
  • the co-administration of 7C10 + anti- VEGF antibodies increase the mice survival.
  • Example 30 Production of deoxyvinblastine 4'-R deoxyvinblastine (structure see below Scheme 1) is obtained by ionic reduction of anhydrovinblastine according to a process known to those skilled in the art (Lafitte C et al., Tetrahedron Letters, 1998, Volume 39, pp. 8281-8282).
  • 4'-S deoxyvinblastine, or 4'-S deoxyleurosidine is obtained by catalytic hydrogenation of anhydrovinblastine according to the technique also known to those skilled in the art (De-Bruyn A. et al., Bulletin of the Belgian Chemical Society, 1983, Volume 92, number 5, pp 485-494).
  • Example 31 Deacetylation of Vinca dimeric alkaloids
  • Deoxyvinblastine or deoxyleurosidine is dissolved and stirred for 4 hours at 50°C in 30 ml of methanol containing 1.2 equivalents of sodium methoxide. This solution is then poured into ice-cold water in order to precipitate the compound formed. After filtration, washing with water and drying under vacuum at 40°C, 4- deacetyldeoxyvinblastine or 4-deacetyldeoxyleurosidine is obtained, with a purity of greater than 95%.
  • Example 32 Direct coupling of 4 '-deoxyvinblastine (4' R) or 4 '-deoxyleurosidine (4' S) by reaction of a 4-carboxyhydrazide function on the pre-oxidized anti-IGF- IR antibodies
  • the 4 '-deoxyvinblastine or the 4'-deoxyleurosidine is treated with anhydrous hydrazine in solution in methanol and at ambient temperature.
  • the reaction is monitored by Analytical High Performance Liquid Chromatography (HPLC) and, when 95% of the starting alkaloid has reacted, the reaction medium is treated with water in order for the 4'-deoxyvinblastine-3-deacetyl-4-carbohydrazide or the 4'-deoxyleurosidine-3- deacetyl-4-carbohydrazide to be separated by filtration. After silica gel chromatography and then crystallization, the 4'- deoxyvinblastine-3-deacetyl-4-carbohydrazide or the 4'-deoxyleurosidine-3-deacetyl-4- carbohydrazide is greater than 96% pure.
  • HPLC Analytical High Performance Liquid Chromatography
  • the anti-IGF-IR antibody is oxidized under cold conditions in a sodium acetate buffer by treatment with sodium meta-periodate. After exclusion chromatography, the oxidized anti-IGF-IR antibody, in solution in an acetate buffer, is treated under cold conditions with the 4'-deoxyvinblastine-3-deacetyl-4-carbohydrazide or the 4'- deoxyleurosidine-3-deacetyl-4-carbohydrazide.
  • the immunoconjugate thus obtained is separated from the unconjugated residual Vinca alkaloid and purified by exclusion chromatography with a phosphate buffer at pH 7.4, and then intensive dialysis. The absence of free Vinca alkaloid is verified by analytical HPLC.
  • the immunoconjugate is characterized on an SDS PAGE-type electrophoresis gel (Coomassie blue and/or silver nitrate), by exclusion chromatography (SEC, UV at 280 nm) and by MALDI-TOF mass spectrometry.
  • SEC exclusion chromatography
  • MALDI-TOF mass spectrometry MALDI-TOF mass spectrometry.
  • the mapping of the coupling sites is carried out by means of analysis by liquid chromatography coupled to mass spectrometry (LC MS), subsequent to enzyme digestion (trypsin and PNGase F) (Laguzza et al, J. MED. CHEM., 1989, 32:548).
  • Example 33 Coupling of the 4 '-deoxyvinblastine (4' R) or the 4 '-deoxyleurosidine (4' S) to the anti-IGF-IR antibodies by virtue of succinic anhydride
  • the 3 -deacetyl-4' -deoxyvinblastine or the 3 -deacetyl-4' -deoxyleurosidine is treated with succinic anhydride in pyridine for 24 hours at 20°C.
  • the reaction is monitored by analytical HPLC and, when 95% of the starting alkaloid has reacted, the reaction medium is treated with water in order to precipitate the 3 -deacetyl-4 '- deoxyvinblastine hemisuccinate or the 3 -deacetyl-4 '-deoxyleurosidine hemisuccinate. After filtration and drying, the compound is purified by reverse-phase preparative HPLC using C18 grafted silica and an eluent made up of acetonitrile, methanol and ammonium acetate buffer.
  • the 3 -deacetyl-4 '-deoxyvinblastine hemisuccinate or the 3 -deacetyl-4 '- deoxyleurosidine hemisuccinate is treated with hydroxybenzotriazole and dicyclohexylcarbodiimide in dimethylformamide at ambient temperature for 24 hours and in the presence of a catalytic amount of dimethylaminopyridine. After filtration, the solution is mixed with the anti-IGF-IR monoclonal antibody at pH 8.6 for 4 hours. The immunoconjugate is separated from the unconjugated Vinca alkaloid by exclusion chromatography with a phosphate buffer at pH 7.4. Intensive dialysis makes it possible to eliminate the unconjugated Vinca alkaloid.
  • the immunoconjugate is characterized by SDS PAGE gel electrophoresis, by exclusion chromatography and by MALDI TOF mass spectrometry.
  • the mapping of the coupling sites is carried out by means of liquid chromatography analysis coupled to mass spectrometry (LC MS), subsequent to enzyme (trypsin) digestion, compared to a reference tryptic map obtained for the non-derived monoclonal antibody (Schneck et al., Clin. Pharmacol. Ther., 1990, 47:36; Rowland et al., Cancer. Immunol. Imrnunother., 1985, 19:1).
  • Example 34 Coupling of the 4 '-deoxyvinblastine (4' R) or the 4 '-deoxyleurosidine (4' S) on a nitrogen-containing residue of the anti-IGF-IR antibodies by virtue of a disulphide bridge included in the linkage
  • the 3 -deacetyl-4 '-deoxyvinblastine or the 3-deacetyl-4'-deoxyleurosidine is treated, in methylene chloride, at ambient temperature for 24 hours, in the presence of a catalytic amount of dimethylaminopyridine, with a large excess of 3- methyldisulphanylpropanoic acid and a large excess of dicyclohexylcarbodiimide.
  • reaction medium is treated conventionally and the 3 -deacetyl-4 '-deoxyvinblastine 3- methyldisulphanylpropanoate or the 3-deacetyl-4'-deoxyleurosidine 3- methyldisulphanylpropanoate is then purified by reverse-phase preparative HPLC using C18 grafted silica and an eluent made up of acetonitrile, methanol and ammonium acetate buffer.
  • the 3 -deacetyl-4 '-deoxyvinblastine 3-methyldisulphanylpropanoate or the 3- deacetyl-4' -deoxyleurosidine 3-methyldisulphanylpropanoate is treated with dithiothreitol in a mixture of water and methanol so as to obtain 3 -deacetyl-4 '- deoxyvinblastine 3-sulphanylpropanoate or 3 -deacetyl-4 '-deoxyleurosidine 3- sulphanylpropanoate, which is purified by reverse-phase preparative HPLC using C18 grafted silica and an eluent made up of acetonitrile, methanol and ammonium acetate buffer.
  • the anti-IGF-IR antibody is derivatized with N-succinimidyl 4-(2- pyridyldithio)propanoate (the trade name of which is SPDP) in a 50 mM potassium phosphate buffer, pH 6.5, containing 50 mM NaCl and 2 mM EDTA, for 90 minutes.
  • SPDP N-succinimidyl 4-(2- pyridyldithio)propanoate
  • SPDP sodium pyridyldithiopropanoate
  • the immunoconjugate is isolated by exclusion chromatography and is characterized on an SDS PAGE electrophoresis gel, by exclusion chromatography and by MALDI TOF mass spectrometry (Ojima et al., J. Med. Chem., 2002, 45:5320).
  • Example 35 Coupling of the 4 '-deoxyvinblastine (4' R) or the 4 '-deoxyleurosidine (4' S) to the anti-IGF-IR antibodies by virtue of a terminal hydrazide function carried by a linkage connected to the Vinca alkaloid
  • the 3 -deacetyl-4 '-deoxyvinblastine or the 3-deacetyl-4'-deoxyleurosidine is treated, in methylene chloride at ambient temperature for 24 hours, in the presence of a catalytic amount of dimethylaminopyridine, with an excess of methyl monoester of 1,6- hexanedicarboxylic acid and an excess of dicyclohexylcarbodiimide.
  • reaction medium is treated conventionally and the 3 -deacetyl-4 '-deoxyvinblastine 3- methoxycarbonyl pentanoate or the 3-deacetyl-4'-deoxyleurosidine 3-methoxycarbonyl pentanoate is then purified by reverse-phase preparative HPLC using C18 grafted silica and an eluent made up of acetonitrile, methanol and ammonium acetate buffer.
  • the 3-deacetyl-4'-deoxyvinblastine 3-methoxycarbonyl pentanoate or the 3- deacetyl-4'-deoxyleurosidine 3-methoxycarbonyl pentanoate is treated by default with anhydrous hydrazine in solution in methanol at ambient temperature.
  • the reaction is monitored by analytical HPLC and, when 70% of the starting alkaloid has reacted, the reaction medium is evaporated and the 3 -deacetyl-4 '-deoxyvinblastine 3- hydrazinocarbonyl pentanoate or the 3 -deacetyl-4 '-deoxyleurosidine 3- hydrazinocarbonyl pentanoate is purified by reverse-phase preparative HPLC using C18 grafted silica and an eluent made up of acetonitrile, methanol and ammonium acetate buffer.
  • Example 36 Activity, compared in vivo, of the 7C10 and h7C10 antibodies on the A549 and MCF-7 models
  • the latter was compared with 7C10 in the MCF-7 oestrogen-dependent breast tumor model and in the A549 non-small-cell lung tumor model.
  • 5.10 6 A549 cells were implanted subcutaneously in nude mice. Five days after this implantation, the tumors were measured and groups of 6 mice were formed.
  • Figures 50 and 51 show, as expected, that significant inhibition of tumor growth is observed with the 7C10 murine antibody.
  • the activity observed is of exactly the same intensity as that observed with its murine counterpart, whatever the model used. This datum indicates that the humanization has not modified the properties of the antibody generated.
  • Example 37 Demonstration of the compared activities of vinblastine, of vincristine, of 4' S deoxyvinblastine and of 4' R deoxyleurosidine
  • the greater activity of the (4' R) deoxyvinblastine and of the (4' S) deoxyleurosidine was demonstrated in vivo against intravenously-grafted P388 murine leukaemia and compared with the activity of vinblastine and of vincristine tested under the same conditions.
  • the protocol for this test is described by Kruczynski A. et al., Cancer Chemotherapy and Pharmacology, 1998, volume 41, pages 437 to 447. To do this, a total of 1O 6 P388 murine leukaemia cells were implanted i.v.
  • the in vivo activity of compounds is expressed by the increase in survival time.
  • the survival time is expressed by the T/C at a dose expressed in mg per kg (mg/kg).
  • the T/C corresponds to the ratio, multiplied by 100, of the median of the survival time of the treated animals to the median of the survival time of the control animals.
  • a T/C of 120 corresponds to a minimum level for concluding that activity is present.
  • a T/C of between 120 and 175 makes it possible to conclude that there is significant activity and a T/C above 175 makes it possible to conclude that there is a high level of anti-leukaemia activity.
  • a T/C below 75 expresses toxicity of the test compound at the dose administered.
  • Table 13 below gives the results obtained with a minimum of 7 and a maximum of 15 treated mice for each group of animals treated with a Vinca alkaloid or for the control group.
  • Table 13 gives the results of T/C values obtained for each Vinca alkaloid tested.
  • Figures 52 and 53 show the greater anti-leukaemia activity of the 4'R and 4'S deoxyvinblastines compared to vinblastine and vincristine.
  • Example 38 Demonstration of the in vivo antitumor activity of 4' R- and 4' S- deoxyvinblastine conjugated with IGR-IR antibodies on human tumors of various origins
  • 4' R and (4' S) deoxyvinblastine
  • RDV deoxyvinblastine
  • the tumors can be measured and the animals are distributed randomly into 6 groups of 6 mice and treated according to the following protocol: - h7C10: twice a week at a rate of 250 ⁇ g/dose throughout the entire duration of the experiment; - RDV and SDV: 4 intraperitoneal injections 7 days apart at the dose of 0.35 mg/kg, which corresponds to the dose of each of the compounds present in the conjugates;
  • the groups of animals given the chemotherapy compounds coupled to the antibody receive respectively 0.35 mg/kg of each of the chemotherapy agents and 250 ⁇ g/dose of antibodies. These conjugates are administered according to the same modes as the groups given the chemotherapy compounds alone;
  • the animals of the control batch are given injections of PBS, administered according to the same frequency.
  • the weight of the mice and the tumor volume are evaluated twice a week.
  • the tumor volumes are calculated according to the formula: % (length . width . height).
  • the results are shown in Figure 53.
  • the animals given only RDV or SDV evolve in the same manner as the control group, which seems coherent with respect to the optimum doses usually injected for these two compounds, which are respectively 20 mg/kg and 2.5 mg/kg.
  • a very significant inhibition of the tumor growth is observed. This inhibition is significantly greater than that observed with the antibody alone, administered at the same concentration.
  • EXEMPLE 39 In vivo down-regulation of IGF-IR To confirm if previous in vitro observations (Examples 24 and 25) of down- regulation of IGF-IR levels by 7C10 and h7C10 was one of the mechanisms responsible for inhibition of tumor growth in vivo, the effect of h7C10 on IGF-IR levels in mice with MCF-7 xenograft tumors was tested. Nine mice bearing a xenograft tumor were studied. Tumors were resected from 3 mice before treatment (described as TO in Figure 59). Then 3 mice received i.p. injections of 1 mg of A2CHM and 3 other mice received i.p. injections of a hlgGl used as isotype control.
  • mice Six hours after treatment, mice were sacrificed and tumors harvested. Tumor samples were frozen in liquid nitrogen and homogeneized in a lysis buffer. A total of 50 ⁇ g of tumor extracts immunoblotted for total IGF-IRb levels. In this experiment, he cytokeratin 19 (CK19) was immunoblotted with an antibody recognizing specifically the CK19 from MCF-7 cells as control of the amount of tumor protein loaded in each lane.
  • Figure 59 shows that the amount of protein loaded is comparable in all lanes excepted in lane 9 which could not be considered as interpretable. All MCF-7 xenograft tumors had high levels of IGF-IR in non treated mice (lanes 1-3).
  • Example 40 CeU lines with IGF-IR overexpressed and/or abnormally overactivated: The following cell lines are known as overexpressing and/or displaying overactivated IGF-IR : - Prostate (PC3 , DO 145), - breast (MCF-7, T47D, BT20, ZR-75-1 , MDA-MB-231 , - lung (A549, A427, SK-LU-1) - colon (HT29, Colo205, CaCo-2), - thyroid (BC-PAP, FRO, ARO), - ovarian (SK-OV-3), - pancreas (BxPC3, MiaPaCa-2, LN36), - renal, adrenal cancer, sarcomas (SK-ES-1), medulloblastoma (Daoy, TE- 671, D283 Med), - retinoblastoma, multiple myeloma (MM- IS, MM-1R), melanoma (SK-MEL-
  • 9G4 was used as irrelevant isotype control.
  • Anti-mouse MAb coupled to FITC was used for revelation of the bound 7C10.
  • beads from DAKO QIFIKIT bearing quantified amounts of CD5 receptors were used for standard curve detennination. After FACS analysis, beads yield a MFI. A standard curve relating MFI and receptor number was determined. MFI measured after cell staining with 7C10 was reported on the standard curve and receptor number on each tumor cell line was determined. Quantification of the number of IGF-IR on tumor cell line surface shows that all the cell lines analyzed express between 1200 to 96000 receptors/cell.
  • Example 41 Cell lines expressing hybrid-R: The following cell lines are known as overexpressing and/or displaying overactivated hybrid-R and/or IGF-IR (Siddle et al.Horm. Res. 41: 56-65, 1994 ; Lynn Seely et al. Endocrinology. 136: 1635-1641, 1995 and Pandini et al. Clinical Cancer Res. 5 : 1935-1944.
  • Example 42 To determine the variation of IGF-IR between normal and tumor tissues, paraffin embedded section of either breast or lung cancer have been stained with a biotinylated-conjugated anti-IGF-IR monoclonal antibody. The result shown in figure 58 demonstrate that samples from patients with either lung or breast cancer display significantly higher levels of IGF-IR.

Abstract

La présente invention se rapporte à de nouveaux anticorps pouvant se lier spécifiquement (i) au récepteur hybride anti-insuline/IGF-I humain (R hybride) ou (ii) à la fois audit R hybride et au récepteur du facteur de croissance insulinoïde I humain (IGF-IR), et/ou étant apte à inhiber spécifiquement l'activité tyrosine kinase dudit R hybride ou à la fois du R hybride et de IGF-IR, et notamment à des anticorps monoclonaux d'origine murine, chimérique et humanisée, ainsi qu'aux séquences d'acides aminés et d'acides nucléiques codant pour ces anticorps. En outre, l'invention se rapporte à l'application de ces anticorps comme médicament dans le traitement et/ou la prévention des cancers à surexpression, ou à activation anormale, de R hybride ou à la fois de R hybride et de IGF-IR, ou de toute pathologie liée à cette surexpression ou à cette activation anormale, dudit récepteur, ainsi que dans des procédés et des trousses de diagnostic de maladies liées à la surexpression de R hybride ou à la fois de R hybride et de IGF-IR. Enfin, l'invention se rapporte à des produits et/ou compositions comportant des anticorps de ce type en association avec des anticorps anti-EGFR et/ou des composés et/ou des agents anti-cancéreux ou des agents conjugués avec des toxines, et leur application au traitement et/ou à la prévention de certains cancers.
PCT/IB2004/004360 2003-12-16 2004-12-16 Nouveau recepteur hybride anti-insuline/igf-i ou recepteur hybride anti-insuline/igf-i et anticorps igf-ir et applications WO2005058967A2 (fr)

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