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    • 专利摘要:
    The present invention relates to an antibody, or antibody fragment, specifically binding the peptide having the sequence according to SEQ ID No. 29 or a peptide having at least 60% homology with the peptide having the sequence according to SEQ ID No. 29, may be used for therapeutic purposes, in particular may be useful in the treatment of cancers expressing cytokeratin 8 (CK8).
    公开号:FR3024731A1
    申请号:FR1457704
    申请日:2014-08-08
    公开日:2016-02-12
    发明作者:Marie Alexandra Albaret；Jean-Jacques Diaz；Hichem Claude Mertani；Jean-Christophe Saurin；Claudine Vermot-Desroches；Boris Vuillermoz
    申请人:CT LEON BERARD；INTERNAT DRUG DEV BIOTECH；Centre National de la Recherche Scientifique CNRS；Universite Claude Bernard Lyon 1 UCBL；Institut National de la Sante et de la Recherche Medicale INSERM；Centre Leon Berard；Hospices Civils de Lyon HCL；
    IPC主号:
    专利说明:

    [0001] FIELD OF THE INVENTION The present invention relates to the field of antibodies that can be used for therapeutic purposes. In particular, the present invention relates to an anti-cytokeratin 8 antibody. These antibodies appear to be useful in the treatment of cancers expressing cytokeratin 8 (CK8), in particular colorectal cancers. BACKGROUND OF THE INVENTION Cytokeratin 8 (CK8) is a protein that forms the intermediate filaments of the cytoskeleton of epithelial cells. Different studies have shown that the CK8 protein is present on the membrane of different cancer cells. Thus, Godfroid et al. (Journal of Cell Science, 99: 595-607, 1991) have described the presence of CK8 proteins on the surface of cultured breast carcinoma cells. Hembrough et al. (Journal of Cell Science, 108: 1071-1082, 1995) have described the presence of CK8 or a "CK8 like" protein on the surface of hepatocytes, HepG2 cells and breast carcinoma cells. CK8 protein has also been detected on the surface of carcinomas of other cancers such as cancer of the upper digestive tract (Gires et al., Biochemical and Biophysical Research Communications, 328: 1154-1162, 2005). It has also been shown that the CK8 protein is exposed on the surface of invasive or non-invasive tumor cells in colorectal cancers (WO2010 / 136536). More particularly, it has been shown that in colorectal cancers, the appearance of invasive and / or metastatic cells is accompanied by cleavage of the exposed human CK8 protein on the surface of these cells. The N-terminal portion of the CK8 protein remains exposed on the surface of colonic adenocarcinoma cells while the C-terminal part of the protein disappears partially or totally from the surface of these cells. Colorectal cancer is a particularly invasive cancer. Although significant improvements have been achieved in the treatment of patients with metastatic colorectal cancer (CRC), this type of cancer remains a major public health problem in the whole world (Weitz et al., 2005; 365: 153-2). 65). CRC is the third most diagnosed cancer in both sexes with 940,000 estimated cases / year and the second leading cause of cancer death with approximately 500,000 cases / year (Weitz et al Lancet 2005; 365: 153-65). In France, 37,000 new patients are diagnosed with CRC each year, about half of whom will die of metastatic disease. However, approximately 50% of the 36,000 new patients already have metastases at the time of diagnosis or will develop metastases (Weitz et al Lancet 2005, 365: 153-65, Cunningham et al Lancet 2010, Chevreul Eur J Health Econ 2010). The introduction of more effective drugs (Irinotecan, Oxaliplatin) has significantly improved the treatment of patients with CRC. The last decade has also seen the emergence of molecule-based therapies, the monoclonal antibodies, directed against specific overexpressed targets (EGF or VEGF receptor) in tumor cells compared to healthy cells. These molecules significantly improve treatments.
    [0002] Various monoclonal antibodies to human CK8 protein have been described. In particular, Erlandsson et al. (J. of Molecular Recognition, 16: 157-163, 2003, Johansson et al Cancer Res 1999, 59, 48-51) have described the monoclonal antibody TS1 directed against the human CK8 protein and its anti-idiotype aTS1 as well as their use in immunotherapy for the treatment of carcinomas. Doljak et al. (Cancer Letters, 267: 75-84, 2008) have described a monoclonal antibody binding to the motif JKIAl ~ EVEIATY conserved among various cytokeratins. This monoclonal antibody binds in particular to the CK2, CK8, CK10 and CK18 proteins in MCF-7 breast cancer cells. This antibody inhibits in vitro plasminogen activation on MCF-7 cells. The monoclonal antibody M20 marketed by Sigma® has been described by Van Muijen, G. et al. (Invest., 57: 359, 1987, Waseem et al., Biochemistry 2004, 43, 1283-1295). This antibody, which specifically binds the CK8 protein, has been shown to inhibit the growth and invasiveness of tumor cancer cells that have been tested in in vitro and in vivo assays (WO2010 / 136536). In particular, in International Patent Application WO2010 / 136536, it has been shown that this antibody specifically binds to the uncleaved portion of the human CK8 protein present on the surface of colonic adenocarcinoma tumor cells.
    [0003] Although progress has been made in the identification of antigens on the surface of tumor cells and in the understanding of the mechanisms associated with the development of cancers, these advances have enabled the development of monoclonal antibodies that can significantly improve treatments. their efficiency and specificity can still be improved. Thus, needs remain for the development of therapeutic means for treating tumors whose cells express the CK8 protein on their surface, in particular for treating colorectal cancers. BRIEF DESCRIPTION OF THE INVENTION The present invention relates to an antibody, or antibody fragment, specifically binding the peptide having the sequence according to SEQ ID No. 29 or a peptide having at least 60% homology with the peptide having the Sequence according to SEQ ID No. 29. The present invention also relates to an antibody, or antibody fragment for its use in the treatment of tumors whose cells express the CK8 protein, in particular the sequence peptide according to SEQ ID No. 29, on their surface. The present invention also relates to an antigenic peptide of human CK8 having the sequence according to SEQ ID No. 29. Finally, the present invention relates to a pharmaceutical composition comprising an antibody according to the present invention and a suitable pharmaceutical carrier, in particular for its use in the treatment in the treatment of tumors whose cells express the CK8 protein, in particular the sequence peptide according to SEQ ID No. 29, on their surface. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A and 1B illustrate western blot detection of CK8 isoforms using anti-CK8 antibodies, including mouse monoclonal antibody D-A10. FIG. 2 illustrates the competition or not via peptides 1 (SEQ ID No. 1) and 2 (SEQ ID No. 2) on the cell labeling of anti-CK8 mAbs analyzed by flow cytometry. Figure 3 illustrates the effect of peptides 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2) on the detection of isoforms of the protein CK8 by Western blotting using the antibody. M20 and murine monoclonal antibody D-Al 0.
    [0004] Figure 4 illustrates the effect of anti-CK8 antibodies (50 μg / mL) including murine monoclonal antibody D-Al 0 on Isreco-1 cell invasion induced by 10% FBS. Figure 5 illustrates the effect of anti-CK8 antibodies on Isreco-1 cell proliferation. Figure 6 illustrates the effect of anti-CK8 antibodies including murine monoclonal antibody D-A105 on tumor growth (Isreco-1) in mice. Figure 7 illustrates the dose-dependent effect of murine monoclonal antibody concentration D-Al 0 on tumor growth (Isreco-1) in mice. Figure 8 illustrates the effect of anti-CK8 antibodies including murine monoclonal antibody D-Al 0 on tumor growth (HCT116) in mice.
    [0005] Figure 9 illustrates the dose-dependent effect of murine monoclonal antibody concentration D-Al 0 on tumor growth (HCT116) in mice. Figure 10 illustrates the positioning of peptides 1 (SEQ ID NO: 1), 2 (SEQ ID NO: 2), 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4) in the sequence of cytokeratin 8 human (ref UniProtKB P05787).
    [0006] Figure 11 illustrates the reactivity of the M20 antibody and the murine monoclonal antibody D-Al 0 for the different peptides 1 (SEQ ID NO: 1), 2 (SEQ ID NO: 2), 3 (SEQ ID NO: 1). ° 3) and 4 (SEQ ID No. 4) by ELISA test. Figure 12 illustrates the reactivity of mouse monoclonal antibody D-A10 for the different peptides 5-16 and 18-28 (SEQ ID NO: 5 to SEQ ID NO: 16) and (SEQ ID NO: 18 at SEQ ID No. 28) by ELISA. Figure 13A illustrates the effect of peptides 5 and 10 to 16 (SEQ ID NO: 5 and SEQ ID NO: 10 to SEQ ID NO: 16) on recognition of peptide 1 (SEQ ID NO: 1) by antibody. monoclonal mouse D-Al 0 in ELISA and FIG. 13B the effect of peptides 18 to 24 (SEQ ID No. 18 to SEQ ID No. 24) on the recognition of peptide 17 (SEQ ID No. 17) by the antibody murine monoclonal D-A10 25 in ELISA. Figure 14 illustrates the amino acid and nucleic acid sequences of the light chain variable region (VL) of murine Ac Mo D-Al 0 (variable murine light chain of anti-CK8 antibody D-A10 with description of FR1, CDR1, FR2, CDR2, FR3 and CDR3 according to IMGT®).
    [0007] Figure 15 illustrates the amino acid and nucleic acid sequences of the heavy chain (VH) variable region of murine Ac Mo D-Al 0 (variable murine heavy chain of anti-CK8 D antibody). -A10 with description of FR1, CDR1, FR2, CDR2, FR3 and CDR3 according to IMGT®). DEFINITIONS The term "antibody" as used in the description of the present invention refers to monoclonal (Ac Mo) or polyclonal antibodies. The antibodies may be chimeric, humanized or conjugated antibodies. The term "monoclonal antibody" as used in the description of the present invention refers to an antibody derived from a substantially homogeneous antibody population.
    [0008] More particularly, the individual antibodies of a population are identical with the exception of a few naturally occurring mutations that may occur in minute proportions. In other words, a monoclonal antibody is a homogeneous antibody resulting from the proliferation of a single cell clone (e.g. a hybridoma, a eukaryotic host cell transfected with a DNA molecule encoding the homogeneous antibody, a prokaryotic host cell transfected with a DNA molecule encoding the homogeneous antibody, etc.) and which is generally characterized by heavy chains of one and the same class and subclass, and light chains of a single type . The monoclonal antibodies are highly specific and are directed against a single antigen. In addition, unlike polyclonal antibody preparations which conventionally comprise different antibodies directed against different determinants, or epitopes, each monoclonal antibody is directed against a single epitope of the antigen. The term "chimeric antibody (s)" as used in the description of the present invention refers to antibodies comprising the constant parts of the heavy and light chains of an antibody of a given species, for example a human antibody. on which are grafted the variable parts of the heavy and light chains of an antibody of a heterologous species, for example a murine antibody. The term "humanized antibody (s)" as used in the description of the present invention refers to human antibodies in which the hypervariable regions (CDRs) are replaced by the hypervariable regions (CDRs) of an antibody of heterogeneous origin. , for example of murine origin, which constitute the binding site of the antigen. Since some amino acids located in regions adjacent to CDR regions (FR regions) play a role in the structure of the antigen binding site, human antibodies can be further modified (CDR) to include amino acids. of the FR region of the antibody of heterogeneous origin. The term "conjugated antibodies" as used in the description of the present invention refers to artificial mixed molecules in which components are associated with an antibody capable of selectively recognizing an antigen. These components may be cytotoxic drugs, anticancer agents, toxins, fragments and / or radioelements. The term "antibody fragment (s)" as used in the description of the present invention refers to portions of antibodies (as opposed to whole antibodies) which are functional, i.e. portions of antibodies capable of binding an antigen. Examples of antibody fragments include Fv fragments (consisting of heavy and light chain variable regions of an antibody), ScFv (divalent single variable variable fragment), Fab (consisting of whole and partial light chain) of the heavy chain), F (ab ') 2 (consisting of two Fab fragments connected by the hinge region) or any fragment whose half-life duration would have been increased by chemical modification. The term "cancer" as used in the description of the present invention refers to all malignant neoplastic formations, regardless of the histological nature. There are two major categories of malignancies: carcinomas of epithelial origin and sarcomas of conjunctive origin. Malignant tumors are composed of atypical cells, invasive or with a capacity for dissemination, generally characterized by an autonomous capacity of growth, an imprecise delimitation, a capacity of invasion of the neighboring tissues and vessels and a tendency to disseminate by the production of metastases. The term "polynucleotide" as used in the description of the present invention denotes a single-stranded nucleotide chain or its complementary which may be of the DNA or RNA type, or a double-stranded nucleotide chain which may be of the cDNA (complementary) or genomic type. . The term "epitope" as used in the description of the present invention refers to a molecule that can be recognized by a paratope (variable portion of an antibody or membrane receptor of T-cells: TCR) to determine whether it belongs to the domain of the self or the domain of no-self.
    [0009] The term "homology" as used in this specification refers to the similarity between peptide sequences. The peptide sequences may have a deletion, addition or substitution of at least one amino acid with respect to the reference polypeptide. The percentage of homology is based on the percentage identity of the sequences. LIST OF SEQUENCES SEQ ID No. 1 designates the following peptide: AEQRGELAIKDANAKLSELEAALQRAKQD-C SEQ ID No. 2 designates the following peptide: AEQRGELAIKDANAKLSELE-C SEQ ID No. 3 designates the following peptide: AALQRAKQD-C SEQ ID N 4 denotes the following peptide: SELEAALQRAKQD-C SEQ ID NO: 5 designates the following peptide: AEQRGELAIKDANAKLSELEAALQRAKQD SEQ ID No. 6 designates the following peptide: AALQRAKQD SEQ ID No. 7 designates the following peptide: EAALQRAKQD SEQ ID No. 8 designates the following peptide: LEAALQRAKQD SEQ ID No. 9 designates the following peptide: ELEAALQRAKQD SEQ ID No. 10 designates the following peptide: SELEAALQRAKQD SEQ ID No. 11 designates the following peptide: LSELEAALQRAKQD 20 SEQ ID N 12 designates the following peptide: KLSELEAALQRAKQD SEQ ID No. 13 designates the following peptide: AKLSELEAALQRAKQD SEQ ID No. 14 designates the following peptide: NAKLSELEAALQRAKQD SEQ ID No. 15 designates the following peptide: ANAKLSELEAALQRAKQD SEQ ID No. 16 means the following peptide: DANAKLSELEAALQRAKQD SEQ ID NO: 17 designates the following peptide: C-AIKDANAKLSELEAALQRAKQD SEQ ID No. 18 designates the following peptide: AIKDANAKLSELEAALQRAKQ SEQ ID No. 19 designates the following peptide: AIKDANAKLSELEAALQRAK SEQ ID NO: 20 refers to the following peptide: AIKDANAKLSELEAALQRA SEQ ID NO: 21 designates the following peptide: AIKDANAKLSELEAALQR SEQ ID NO: 22 designates the following peptide: AIKDANAKLSELEAALQ SEQ ID No. 23 designates the following peptide: AIKDANAKLSELEAAL 3024731 8 SEQ ID NO: 24 designates the following peptide: AIKDANAKLSELEAA SEQ ID No. 25 designates the following peptide: AIKDANAKLSELEA SEQ ID No. 26 designates the following peptide: AIKDANAKLSELE SEQ ID No. 27 designates the following peptide: AIKDANAKLSEL SEQ ID NO: 28 denotes the following peptide: AIKDANAKLSE SEQ ID NO: 29 designates the following peptide: LSELEAAL SEQ ID No. 30 designates the following nucleotide sequence: AAC ATT GTTATGACC CAGGCC GCAC CC TC TGTAC CT GTCAC TCC TGGAGAGTCAGT ATCCATCTCCTGCAGGTCTAGTAAGAGTCTTCTGTATAGTAATGGCAACACTTATT 10 T GTATTGGTTC CT GCAGAGGC CAGGCCAGTCTCC TCAGC GCC TGATATATTATAT G TCCAACCTTGCC TCAGGAGTCC CAGAC AGGTTCAGTGGCAGAGGGTCAGGAACT G ATTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTG TATGCAAAGTCTAGAATATCCTTTCACG SEQ ID NO: 31 denotes the following peptide: 15 NIVIVIT SAQF SVPVTPGE SVSIS CRS SKSLLYSNGNTYLYWFLQRPGQSPQRLIYYMSN LAS GVPDRF S GRGS GTDF TLRISRVEAEDVGVYYCMQ SLEYPF TF GGGTKLEIK SEQ ID No. 32 denotes the following nucleotide sequence: GAAGTGCAGCTGTTGGAGACTGGAGGAGGCTTGGTGCAACCGGGGGGGTCACGG GGACTCTCTTGTGAAGGCTCAGGGTTTACTTTTAGTGGCTTCTGGATGAGCTGGGT 20 TCGACAGACACC TGGGAAGAC CC TGGAGTGGATTGGAGACATTAATTC TGATGGC AGTGCAAT AAAATACGCACC AT CCATAAAGGAT C GATTCACT AT CTTCAGAGACA AT GACAAGAGCACC CTGTAC CTGCAGAT GAGC AATGTGCGATCT GAGGACACAGC CACGTATTTCTGTATCGCCCATTACTCCGGTGGGGGGTTTGCTTACTGGGGTCAAG GAACCTCGGTCACCGTCTCCTCA 25 SEQ ID NO: 33 denotes the following peptide: EVQLLETGGGLVQPGGS GLR S CEGS GF TF S GFWM ## STR2 ## SEQ ID NO: 34 designates the following peptide: KSLLYSNGNTY SEQ ID NO: 35 designates the following peptide: YMS SEQ ID No. 36 designates the peptide: ## STR2 ## SEQ ID No. 37 designates the following peptide: GFTFSGFW SEQ ID NO: 38 designates the following peptide: INSDGSAI SEQ ID No. 39 designates the following peptide: IAHYSGGGFAY DETAILED DESCRIPTION OF THE INVENTION Antibody and Antibody fragments The inventors have developed antibodies, or antibody fragments, which specifically bind the peptide having the amino acid sequence from position 353 to position 360 of the human CK8 protein (ref Uni-ProtKB P05787) or the peptide having at least 60%, or at least 70% or even at least 85%, homology with said sequence. The amino acid sequence from position 353 to position 360 of the human CK8 protein is shown in SEQ ID NO: 29. By "specifically", it is understood that the antibodies, or fragments of antibodies, bind only the human CK8 peptide having said sequence or exhibiting at least 60%, or at least 70% or even at least 85%, of homology with said sequence. It is understood that the antibodies, or antibody fragments, may bind any protein / peptide fragment comprising said sequence. The present invention thus relates to antibodies, or antibody fragments, specifically binding the peptide according to the sequence SEQ ID No. 29 or a peptide having at least 60%, or at least 70% or even at least 85%, of homology with the peptide having the sequence according to SEQ ID No. 29. These antibodies or antibody fragments are referred to in the description of the present invention as "antibodies or antibody fragments of the present invention". The antibodies, or antibody fragments, of the present invention (anti-CK8 antibody, or anti-CK8 antibody fragments) have been shown to effectively inhibit the invasive capabilities of tumor cells in vitro and effectively inhibit tumor growth. in vivo. The antibodies of the present invention are preferably monoclonal antibodies. These monoclonal antibodies can be antibodies of murine, chimeric or humanized origin. They can be obtained according to standard methods well known to those skilled in the art.
    [0010] Monoclonal antibodies, particularly of murine origin, can be prepared according to the techniques described in the Antibodies manual (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988). or prepared or from hybridomas. Such techniques are well known to those skilled in the art. In particular, the monoclonal antibodies of the present invention can be obtained for example from cells of an animal immunized with a fragment of the human CK8 protein comprising the peptide having SEQ ID NO: 29. The fragments of the human CK8 protein may be produced according to the usual procedures by expression in a recombinant host organism or by peptide synthesis, for example. The monoclonal antibodies according to the invention may for example be purified on an affinity column on which has previously been immobilized a fragment of human CK8 comprising the peptide of SEQ ID No. 29. Other purification techniques well known to those skilled in the art may be used simultaneously or successively. Chimeric antibodies can be prepared using genetic recombination techniques. For example, the chimeric antibody can be made by cloning a recombinant DNA having a promoter and a sequence coding for the variable region of a non-human, particularly murine, monoclonal antibody, and a sequence coding for the human antibody constant region. . A chimeric antibody of the invention encoded by such a recombinant gene will for example be a mouse-human chimera, the specificity of this antibody being determined by the variable region derived from murine DNA and its isotype determined by the constant region derived from the murine DNA. Human DNA. Methods for preparing chimeric antibodies are widely described in the literature. The humanized antibodies can be prepared by techniques known to those skilled in the art.
    [0011] The antibody fragments of the present invention can be obtained from the antibodies as previously described by enzymatic digestion, for example by means of pepsin or papain, and / or by cleavage of the disulfide bridges by chemical reduction. Alternatively, the antibody fragments of the present invention can be obtained by genetic recombination or peptide synthesis techniques. These methods are well known to those skilled in the art.
    [0012] The antibodies, or antibody fragments, of the present invention are preferably antibodies, or antibody fragments selected from murine, chimeric, and humanized antibodies, preferably having an optimized sequence. The expression "optimized sequence" indicates in particular that the codons coding for the constituent amino acids of the protein of interest (for example, the antibody variable domains) can be optimized for better recognition by the translation machinery in a dedicated cell type. In this regard, the amino acid sequence of the protein encoded by the optimized sequence is identical to the non-optimized sequence, but the nucleotide sequence is different.
    [0013] The antibodies, or antibody fragments, of the present invention are preferably antibodies, or antibody fragments, humanized. The antibodies, or antibody fragments, of the present invention may comprise at least one of the following sequences: SEQ ID NO: 31 or SEQ ID NO: 33 and their derivatives Antibodies, or antibody fragments, of the present invention The invention may comprise variable regions encoded by polynucleotides having the variable murine light chain nucleotide sequence (SEQ ID No. 30) and / or the variable murine heavy chain nucleotide sequence (SEQ ID No. 32). SEQ ID No. 30 designates the nucleotide sequence of the variable murine light chain of an antibody according to the present invention (anti-CK8 antibody D-A10 with description of FR1, CDR1, FR2, CDR2, FR3 and CDR3 according to the invention. IMGT® shown in Figure 14). SEQ ID NO: 31 designates the amino acid sequence of the variable murine light chain of an antibody according to the present invention (anti-CK8 antibody D-A10 with description of FR1, CDR1, FR2, CDR2, FR3 and CDR3 d after IMGT® shown in FIG. 14).
    [0014] SEQ ID NO: 32 designates the nucleotide sequence of the variable murine heavy chain of an antibody according to the present invention (anti-CK8 antibody D-A10 with description of FR1, CDR1, FR2, CDR2, FR3 and CDR3. after IMGT® shown in Figure 15).
    [0015] SEQ ID NO: 33 designates the amino acid sequence of the variable murine heavy chain of an antibody according to the present invention (anti-CK8 antibody D-A10 with description of FR1, CDR1, FR2, CDR2, FR3 and CDR3. according to IMGT® shown in Figure 15).
    [0016] The sequences of the CDR regions are defined in accordance with IMGT®, Kabat® or the common numbering system which retains the common sequences between IMGT Kabat © (see examples section). The CDR regions of murine monoclonal anti-CK8 antibody D-Al 0 (mD-Al 0) according to the present invention are shown in Table 1. SEQ ID NO: IMGT SEQ sequence SEQ ID No: Kabat SEQ ID No: Common Numbering System VL mD-A10 CDR1 34 KSLLYSNG NTY 34K RSSKSLLYSNGN TYLY 34C KSLLYSNGNTY CDR2 35 YMS 35K YMSNLAS 35C YMS CDR3 36 MQSLEYPFT 36K MQSLEYPFT 36C MQSLEYPFT VH MD-A10 CDR1 37 GF TF GFW 37K GFWMS 37C GFW CDR2 38 INSDGSAI 38K DINSDGSAIKYAP SIKD 38C INSDGSAI CDR3 39 IAHYSGGG FAY 39K HYSGGGFAY 39C HYSGGGFAY Table 1: Numbering of CDR sequences.
    [0017] By definition, these CDR regions include variant CDR regions, by deletion, substitution or addition of one or more amino acids, which variant maintains the specificity of the original CDR. The common numbering system provides a CDR definition having short amino acid sequences or the definition of minimal CDR. This polypeptide comprises one or two binding domains comprising a pair of VII and TL chains in which the VL chain contains a CDR1 region of sequence SEQ ID No. 34, CDR2 of sequence SEQ ID No. 35, CDR3 of sequence SEQ ID N 36; and the chain VII comprising a CDR1 region of sequence SEQ ID No. 37, CDR2 of sequence SEQ ID No. 38 and a CDR3 region of sequence SEQ ID No. 39. This polypeptide specifically binds to the epitope of CK8 (SEQ ID NO: 29). In this example, this polypeptide comprises these two binding domains.
    [0018] The antibody or antibody fragment according to the present invention may be an F (ab ') 2, Fab, Fv, ScFv fragment or a monoclonal antibody or any antibody fragment. The antibodies, or antibody fragments, of the present invention have been shown to effectively inhibit the invasive capabilities of tumor cells in vitro and effectively inhibit tumor growth in vivo. In particular, the inventors have shown according to the present invention that murine monoclonal antibody D-A10 (referred to either as "DA-10" or "mD-Al 0"), effectively inhibits the invasive capacities of Isreco-1 cells in vitro ( Figure 4) and effectively inhibits tumor growth of Isreco 1 and HCT116 cells in mice (Figures 6 to 9). In particular, the inventors have shown that the D-Al 0 antibody in vitro inhibits the invasive capabilities of Isreco-1 cells, while the D-D6 antibody has no effect. This antagonistic activity is also observed with the M20 antibody (FIG. 4). In vivo, only the D-Al 0 and M 20 antibodies also control the development of tumors. If the D-A10 and M20 antibodies both recognize the human CK8 protein and have antagonistic properties, it has been shown that the D-Al 0 and M 2 0 antibodies do not recognize the same epitope (FIG. 2, FIG. 3, FIG. 11). ). Indeed, the antibody M20 3024731 specifically recognizes the peptide having the sequence corresponding to the amino acids of position 338 to 366 of human CK8 (SEQ ID No. 1), but does not detect the peptides having either the sequence corresponding to the amino acids from position 338 to 357 of the human CK8 protein (SEQ ID NO: 2), the sequence corresponding to the amino acids of position 358 to 5,366 of the human CK8 protein (SEQ ID NO: 3), or the sequence corresponding to the amino acids of position 354 to 366 of the human CK8 protein (SEQ ID NO: 4). While the murine monoclonal antibody D-A10 recognizes both the peptide having the amino acid sequence of position 338 to 366 of human CK8 (SEQ ID NO: 1) and the peptide having the sequence corresponding to amino acids from position 354 to 366 of human CK8 (SEQ ID NO: 4), and does not detect peptides having either the amino acid sequence of position 338 to 357 of human CK8 (SEQ ID NO: 2) or the sequence corresponding to the amino acids of position 358 to 366 of human CK8 (SEQ ID NO: 3), (fig.
    [0019] In addition, the detection in either flow cytometry (FIG. 2) or by western blot (FIG. 3) of the isoforms of the human CK8 protein by the M20 antibody is not inhibited by the peptide having the sequence corresponding to the amino acids. from position 338 to 366 of human CK8 (SEQ ID NO: 1), nor by the peptide having the sequence corresponding to amino acids of position 338 to 357 of human CK8 (SEQ ID NO: 2) whereas detection isoforms of human CK8 by the murine monoclonal antibody (Mab) D-A10 is strongly inhibited by the peptide having the sequence according to SEQ ID No. 1 but not by the peptide having the sequence according to SEQ ID No. 2 and that MAb D-D6 is inhibited both by the two peptides having the sequence according to SEQ ID No. 1 and SEQ ID No. 2.
    [0020] The positioning of peptides 1 (SEQ ID NO: 1), 2 (SEQ ID NO: 2), 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4) on the sequence of human cytokeratin 8 (ref. Uni-ProtKB P05787) is shown in Figure 10. Figure 12 depicts the reactivity of murine monoclonal antibody D-A10 for N- and C-terminal anti-CK8 peptides (Peptide 5-16 and 18-28, SEQ ID NO: 5 to SEQ ID NO: 16 and SEQ ID NO: 18 to SEQ ID NO: 28). The D-Al 0 antibody binds to peptides 5, 11 to 3024731 and peptides 18, 19 to 23. The leucine at position 353 of peptide 11 is essential for recognition by the antibody. Indeed, the antibody D-Al 0 does not bind to the peptide 10. The leucine at position 360 of the peptide 23 is essential for recognition by the antibody. Indeed, the antibody D-Al 0 does not bind to the peptide 24. The essential presence of the leucines at position 353 and 360 of the epitope sequence is confirmed by the non-competition of the peptides (SEQ ID N 10) and 24 (SEQ ID NO: 24) for recognition by murine monoclonal antibody D-Al 0 in ELISA (FIGS. 13A and 13B) when this competition is carried out with peptide 1 and peptide 17 respectively. The epitope recognized by the murine monoclonal antibody D-A10 anti-cytokeratin 8 is therefore the 8 amino acid sequence "LSELEAAL" corresponding to the amino acids of position 353 at position 360 (SEQ ID No. 29). The set of amino acid sequences as well as the recognition or not by the murine monoclonal antibody D-Al 0 of the peptides 1, 5 to 28 (SEQ ID No. 1, SEQ ID No. 5 to SEQ ID No. 28) are shown in Table 5.
    [0021] In some embodiments, the antibody of the present invention is murine monoclonal antibody D-A10. The present invention also relates to an antibody, or antibody fragment, specifically binding to the peptide having the amino acid sequence of position 353 at position 360 of the human CK8 protein, i.e. specifically binding to the peptide having the sequence as shown in SEQ ID No. 29 for use in the treatment of tumors whose cells express the CK8 protein on their surface, in particular whose cells express the sequence peptide according to SEQ ID No. 29 on their surface.
    [0022] In particular, the antibody, or antibody fragments, of the present invention can be used to treat colorectal, ovarian, breast, lung, testicular, pancreatic, nervous, and lymph node cancers. kidney, and / or head-neck cancers. Colorectal, ovarian, breast, lung, testicular, pancreatic, nervous system, lymph node, kidney, and / or head-neck cancers are characterized by the presence of tumor cells expressing CK8 protein. on their surface.
    [0023] In particular, the antibodies, or fragments of antibodies, of the present invention can be used to treat colorectal, ovarian, breast, lung, testicular, pancreatic, nervous system, lymph node cancers. kidney, and / or invasive and / or metastatic head-neck cancers.
    [0024] Preferably, the antibodies, or antibody fragments, used in therapy are humanized antibodies. Peptides The invention also relates to an antigenic peptide of human CK8 having the sequence according to SEQ ID No. 29. Such a peptide can be used in vaccination. Polynucleotides The invention also relates to polynucleotides encoding the human CK8 derived peptide having the amino acid sequence of position 353 at position 360 of the human CK8 protein (SEQ ID NO: 29). Preferably, the polynucleotides of the invention are of the DNA type, in particular of double-stranded DNA. The term "polynucleotide" also refers to modified polynucleotides.
    [0025] The polynucleotides of the present invention are isolated or purified from their natural environment. Preferably, the polynucleotides of the present invention may be prepared by standard molecular biology techniques as described by Sambrook et al. (Molecular Cloning: A Labratory Manual, 1989) or by chemical synthesis.
    [0026] The invention also relates to polynucleotides encoding the variable regions of the D-A10 antibody having the nucleotide sequence of the variable murine light chain (SEQ ID No. 30) and the nucleotide sequence of the variable murine heavy chain ( SEQ ID No. 32). Thus, the present invention also relates to polynucleotides which can include one of the following sequences: SEQ ID No. 30 or SEQ ID No. 32.
    [0027] The amino acid and nucleic acid sequences of the heavy chain and light chain variable regions of the D-A10 antibody are shown in Table 2. VL nucleic acid sequence VH nucleic acid sequence D-A10 SEQ ID NO: 30 SEQ ID NO: 32 Amino acid sequence VL Amino acid sequence VH D-A10 SEQ ID NO: 31 SEQ ID NO: 33 Table 2: Numbering of VL or VH sequences to amino acids and nucleic acids. Pharmaceutical Compositions The invention also relates to a pharmaceutical composition comprising an antibody, or antibody fragments, of the present invention.
    [0028] The pharmaceutical compositions may comprise an antibody or antibody fragment and a suitable pharmaceutical carrier. Examples of suitable pharmaceutical vehicles include solvents, gelatin, starch, lactose, magnesium stearate, talc, gum arabic or the like. The compositions may further comprise dispersing agents, wetting agents, suspending agents, solubilizing agents, stabilizers, preservatives, taste correctors and / or sweeteners. The compositions may be formulated for administration to mammals, in particular to humans. They may be intended to be administered orally, sublingually, subcutaneously, intramuscularly, intravenously, transdermally, locally or rectally. The compositions are then presented in all appropriate forms according to the desired modes of administration. They can therefore be in the form of a solution or an oral or injectable liquid suspension, or in solid or semi-solid form, powders, 3024731 18 tablets, capsules, granules, dragees, capsules. , sprays, cachets, pills, tablets, or pasta. The dosage varies according to the treatment and the condition in question. The invention also relates to vaccine compositions comprising a peptide according to the present invention and a suitable adjuvant. In vaccination, adjuvants are conventionally defined as substances capable of potentiating or modulating the immune response against one or more co-administered antigens. The vaccines are usually inoculated by injection, but they can be ingested orally or by nasal spray.
    [0029] The invention relates to pharmaceutical or vaccine compositions for the prevention and / or treatment of tumors whose cells express the CK8 protein on their surface, in particular colorectal cancers, ovaries, breast, lung, testicles. , pancreas, nervous system, lymph nodes, kidney, and / or head-neck cancers.
    [0030] The invention also relates to methods of therapeutic treatment of tumors whose cells express the CK8 protein on their surface, in particular colorectal cancers, ovaries, breast, lung, testes, pancreas, nervous system, lymph nodes, kidney, and / or head-neck cancers, comprising administering to an individual an effective amount of an antibody, or antibody fragment, according to the present invention. The invention finally relates to the use of antibodies, or antibody fragment, according to the present invention for the manufacture of medicaments for the treatment of tumors whose cells express CK8 protein on their surface, in particular colorectal cancers, ovaries, breast, lung, testes, pancreas, nervous system, lymph nodes, kidney, and / or head-neck cancers. EXAMPLES The M20 antibody referred to below is the antibody commercially available as C5301 (Sigma) and the E8 antibody is the commercially available antibody as ab28050 (abcam).
    [0031] Preparation of Anti-CK8 Antibodies Anti-CK8 antibodies are generated using hybridoma methods (Zola et al., Aust, Exp Biol Med Sci 1981, 59: 303-6). 5, 7, 10, 15, 20 amino acid compounds recognized by the M20 antibody were synthesized, these peptide fragments comprise at least one epitope corresponding to positions 338 to 367 of human CK8. M20-like peptides, that is to say peptides containing substantially all (peptide 1) or part (peptide 2) the sequence recognized by the antibody M20.Their sequences are as follows: Peptide 1: AEQRGELAIKDANAKLSELEAALQRAKQD -C (SEQ ID NO: 1) Peptide 2: AEQRGELAIKDANAKLSELE-C (SEQ ID NO: 2) The immunization was carried out on 3 OF1 mice with a mixture of the 2 peptides coupled to KLH. was detected positive by ELISA assays for CK8 peptides and protein CK8 from cell fraction enriched in CK8 were selected. The splenocytes were fused with the mouse myeloma line X63-Ag8.653. After cloning of the hybridoma, two murine Ac MB were obtained D-Al 0 and D-D6. Clone D-A10 or clone D-D6 was injected into the peritoneum of a Nude mouse. The murine ascites fluid was purified by protein A affinity chromatography column (protein A affinity for Fc part of murine IgG). The IgGs were eluted at acidic pH and then transferred to PBS. After concentration, the PBS solution containing IgG was filtered and the assay was performed at 280 nm. Cell culture The established human colon (CC) cancer cells Isreco-1, Isreco-2, Isreco-3, HCT116 + 1 +, HCT116 - / - and HT29 were cultured in Dulbecco's modified Eagle's medium (Sigma , St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (SFB) (Sigma, St Quentin Fallavier, France), 4 mM L-glutamine (Sigma, St Quentin Fallavier, France) and 50 U / m1.50 μg / ml penicillin - streptomycin (Sigma, St Quentin Fallavier, France).
    [0032] The established human breast adenocarcinoma cells MCF-7 (available from ECACC) are grown in Minimum Essential Eagle Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% fetal bovine serum (SFB). ) inactivated by heat (Sigma, St Quentin Fallavier, France), 2 mM L-glutamine (Sigma, St Quentin Fallavier, France), 100 U / mL, 100 μg / mL penicillin-streptomycin (Sigma, St Quentin) Fallavier, France) and 1% Non Essential Amino Acids (Sigma, St Quentin Fallavier, France). Nalm-6 established human B-precursor cell leukemia cells (available from DSMZ), established Raji's human Burkitt's lymphoma cells (available from ECACC), established non-Hodgkin's human lymphoma cells RL 10 ( available from DSMZ), U937 established human histiocytic lymphoma cells (available from ECACC), established human acute lymphoblastic leukemia Cem cells (available from DSMZ), established human acute lymphoblastic leukemia cells Molt4 (available from from DSMZ), established Fravel human plasma cells (available from ECACC) grown in RPMI-1640 (Sigma, St. Quentin Fallavier, France) are grown in RPMI-1640 (Sigma, St Quentin Fallavier). , France) supplemented with 10% heat-inactivated fetal bovine serum (SFB) (Sigma, St Quentin Fallavier, France), 2 mM L-glutamine (Sigma, St Quentin Fallavier, France), 100 U / m L, 100 μg / ml penicillin - streptomycin (Sigma, St Quentin Fallavier, France).
    [0033] CK8 cell expression by flow cytometry This example describes methods for investigating CK8 cell surface expression of cells and for determining epitopes following antibody or peptide competition analyzed by CK8. flow cytometry. 2.105 cells for 96 wells are incubated with a dilution of an unconjugated anti-CK8 monoclonal antibody at 10 μg / ml and then diluted to 1/10. Unbound antibodies are removed by washing with PBS (Invitrogen, Villebon sur Yvette, France) supplemented with 1% bovine albumin (Sigma, St Quentin Fallavier, France). Then, the cells are centrifuged (5 min at 400 g) and the bound antibody is detected with a polyclonal goat anti mouse Ig (Fab ') 2 conjugated to fluorescein isothiocyanate (FITC) (MP Biomedical, Illkirch, France) at 4 ° C for 30 minutes. After washing, the cells are centrifuged (5 min at 400 g) and resuspended in 300 μl of PBS. The bound detection antibody is quantified on a FACSCAN device (BD Biosciences, Rungis, France), (FL1 channel, 2000 events per acquisition). During the experiment, the controls of the respective isotypes are included to exclude non-specific binding events. The expression of CK8 is determined following the analysis of labeling with anti-CK8 antibodies (1E8, M20, D-D6 or D-A10) in a manner similar to the cell labeling observed with irrelevant antibodies of the same isotype. The results of the experiments (percentage and intensity of labeling) are illustrated in the table below with a concentration of mAb or at 5 μg / ml. Various cancer cell lines such as colon or breast cancer express the CK8 antigens. No expression is observed on lymphoma or leukemia cells. Cell line Cell line type 1E8 M20 D D6 D-A10 Name 0 / IFM% IFM% IFM% IFM ISRECO-1 1-1Lrrn colon carcinoma 45f; 2 144 - i. No. 421, No. 4, ISRECO-2, 1, 11, 1, 1, 2, 4, 4, 4, 4, 4, 473,.... i. :: 1 402 4; , 341, 47f .3 -., .., 4. HCT116 + / + - .- co: on c.:::irc.Jr ,, 1; 4- 127, 1), 334 'HCT116-1-Fluman (1), (3). ) 3851 - '- -2 411.' HT29 Huinanc ...; 1; .... ar.I.:n.Dcart.;:!), - jrr ..; 49: 284,,, 361, MCF-7 B .tdenocarcinoma 77-412 1 4 17 4/64 51 + 1-23 388, - ', 50 +/- 23: 197 Nalyl; -1 precursor leu k -, 0 + / 0 J 0 + 10 - 0 +/- 0 Raji 1nn Bu!;, :: ..;, P.Inpholla 0 + / 0 - RL, ', H. , r;.) homan F3rf, 0 + / 0 - u +: 0 + / 0 - 0 +/- 0 0 +/- 0 ..rnphoma 0 +/- 0 - 0 +/- 0 ^ ____ 0c.as, r. ..: Molt4 'innn .- (: irte 4) hcbase: r- ieukaemia (.. -) Fravel Flu m an ria 0: I I ..: .4 04 u 0; PBMNC Hnpnoc, i.4., ..., i, 0+ -0 .. 0:. ; 04 Competition Competition Antibodies and peptides analyzed by flow cytometry. Briefly, 2.105 Isreco-1 cells for 96 wells are incubated in the presence of an anti-CK8 antibody with or without CK8-related peptides (peptide 1 or peptide 2 as described above) tested at different concentrations and incubated at 4 ° C. C for 30 minutes. Only the data with 1.25 μg / ml of an anti-CK8 antibody are shown. Unbound antibodies are removed by washing with PBS (Invitrogen, Villebon sur Yvette, France) supplemented with 1% bovine albumin (Sigma, St Quentin Fallavier, France). The cells are then centrifuged (5 min at 400 g) and the bound antibody is detected and detected with a polyclonal goat anti mouse Ig (Fab ') 2 conjugated to fluorescein isothiocyanate (FITC) (MP 10 Biomedical, Illkirch, France) at 4 ° C for 30 minutes. After washing, the cells are centrifuged (5 min at 400 g) and resuspended in 300 μl of PBS. The bound detection antibody is quantified on a FACSCAN device (BD Biosciences, Rungis, France), (FL1 channel, 2000 events per acquisition). During the experiment, the controls of the respective isotypes are included to exclude non-specific binding events.
    [0034] The results of the experiments are presented in FIG. 2. The Isreco-1 cell staining (2.105 cells / ml) with the antibody 1E8, M20, D-D6 or D-A10 is carried out in the presence or absence of peptide 1 or peptide. 2. The percent inhibition of labeling intensity of the tested Antibody at 12.5 μg / mL in the presence of different peptide concentrations is reported.
    [0035] Detection of isoforms of the CK8 protein by western-blot after competition with CK8 peptides or non-CK8 antibodies In Western blot experiments (WB), five micrograms of proteins from a colorectal cell lysate Isreco-1 cancer cells are separated by SDS-PAGE electrophoresis and transferred to nitrocellulose membrane. The membrane is saturated with a solution of TBS-T (20 mM Tris-HCl pH 7, 130 mM NaCl, 0.1Tween 20) containing 5% milk, and then incubated with various anti-CK8 antibodies diluted in a solution of TBS. -T containing 2.5% milk. The M20 antibody (50 μg / ml) or the murine monoclonal antibodies such as 1 E8 (1/500), D-A10 (1 μg / ml) or D-D6 (1 μg / ml) are incubated for 1 hour with the membrane.
    [0036] Primary antibodies are revealed with secondary anti-mouse antibodies coupled to peroxidase (HRP). The results are shown in Figure 1.
    [0037] The isoforms of the CK8 protein are differently detected according to the anti-CK8 antibodies tested. The entire form of the CK8 protein (CK8-E) is revealed by all the antibodies. The C-terminal truncated form of the CK8 protein (CK8-C) revealed by the M20 and D-A10 antibodies, while the CK8-directed 1E8 antibody to the C-terminal end does not recognize it. The different isoforms, as well as their estimated molecular mass and their corresponding amino acid sequence are shown in Table 3. In WB competition experiments, M20 antibodies (11.tg / ml) or murine monoclonal antibodies such as D -Al 0 (11.tg / ml) or D-D6 (11.tg / ml) are previously incubated for 2 hours at room temperature with peptide 1 or peptide 2 with a molecular ratio of 1/100 (1 molecule of antibody per 100 peptide molecules) in TBS-T solution. The antibody-peptide mixture is then contacted with the membranes in a TBS-T solution with 2.5% milk for 1 h for HRP-coupled secondary anti-mouse antibody revelation. The results are shown in Figure 3. E: CK8 Integral Form I: CK8 Indeterminate Isoform C: C Cterminal CK8 Isoform The isoforms of CK8 are shown in Table 3 below. Protein isoform of estimated CK8 MM Corresponding sequence (kDa) (AA) E '(long form extension N 57 1-511 tem) E - orrne enter) 54 1-483 I (indeterminate form) 51,49,48, 47, 46 2 C (C-terminal cleaved form) 43 Ismod 1-393 Table 3: CK8 protein isoforms 3024731 Effect of murine monoclonal antibody D-A10 on the invasive capacities of Isreco-1 in cellulo cancerous colorectal cells : Analysis of cancer cell invasion by real-time measurement of cell impedance (xCELLi2ence-ACEA BiosciencesTM system) 5 The real-time measurement system, xCELLigence (ACEA BiosciencesTM), is based on the measurement of the cell impedance generated by attaching cells to microelectrodes. The system used to test the invasion of cancer cells is the RTCA DP system. This system uses 16-well CIM plates. Wells consist of a lower chamber and an upper chamber. The upper chamber is punctured by pores of 81 microns in diameter placed above the microelectrodes. When measuring the invasive capacity of Isreco-1 cells, the lower chamber is filled with culture medium with 10% fetal calf serum (FCS). Isreco-1 cells (20,000 cells) in the presence of medium without FBS are placed in the upper chamber previously covered with a layer of matrigel (matrigel ™, BD biosciences) with or without anti-CK8 antibody (M20, D- A10 or D-D6 at 50 μg / ml.) During invasion, the cells, attracted by the SVF gradient established between the lower chamber and the upper chamber, digest the matrigel before migrating through the pores and contacting the microelectrodes The measurement of the impedance generated after the invasion is carried out every 15 minutes for 70 hours and recorded by a computer connected to the RTCA DP apparatus.The results of the measurements of the invasive capacity of the cells are presented in FIG. Figure 4. The percentage inhibition of the invasive capacity of Isreco-1 cells is reported with a standard deviation for 3 independent experiments Analysis of the viability of the cells following the determination of the ATP level.
    [0038] Cell viability is determined from the quantification of ATP, an indicator of metabolically active cells. Detection is based on the use of the CellTiterGloe kit (Promega, Charbonnières-les Bains, France). Within minutes, after loss of membrane integrity, cells lose the ability to synthesize ATP and endogenous ATPases destroy all remaining ATP, resulting in a sharp fall in ATP levels. Isreco-1 cells (5 × 10 4 cells / ml) are incubated alone for 72 hours or in the presence of an anti-CK8 antibody or a control isotype antibody (B-Z1 IG1g, B-E4 3024731 IgG2a). The CellTiter-Glo® reagent is added directly to the cells in culture at a ratio of 50 μl of reagent per 200 μl of culture medium. The assay plates are incubated at room temperature for 10 minutes and the bioluminescent signal is recorded using a Mithras LB940 standard multiwell fluorometer, (Berthold, Thoiry, France). The results of the experiments to determine the activity of the anti-CK8 antibodies are presented in FIG. 5. The Isreco-1 cells (2.104 cells / ml) are cultured for 72 hours in the presence or absence of anti-CK8 Ab (M20). , 1E8, D-D6 or D-A10) or in the presence of irrelevant murine Ac of the same isotype (B-Z1, B-E4). Cell viability is determined by quantitation of ATP. The percentage inhibition of cell proliferation of Isreco-1 cells is reported, with a standard deviation for 2 independent experiments. Only the 1E8 antibody is able to neutralize Isreco-1 cell proliferation. Determination of the CDR and FR regions of murine monoclonal antibody D-A10. The cDNA corresponding to the variable region of the hybridoma is obtained using two approaches. The first approach is to use in the PCR a set of degenerate primers associated with N-terminal amino acids generated since the N-terminal sequencing and the second approach is to use in the PCR a set of degenerate primers generated by the base IMGT® primer data and the specific primers previously described (Essono et al., J Immunol Methods, 2003; 203: 279: 25-66, Wang et al., Mol Immunol, 1991; 28: 1387-97). . The sequence of the N-terminal variable region is determined by Edman degradation. Extraction of the total RNA is performed using the Tri Reagent kit according to the protocol described by the Sigma supplier. The amplified VL and VH fragments are cloned into the TOPO-TA cloning vector (Invitrogen) for sequence analysis by the dideoxytermination method (Sanger et al., Nature 1977, 265: 687-95). Then, the antibody constructs are amplified by PCR and cloned into the expression vector. The CDR and FR regions of the antibodies are determined according to the different numbering approaches such as the IMGT numbering system (ImMunoGeneTics Information System® http://imgt.cines.fr), Kabat or the common numbering system. However, the CDR regions determined by IMGT for a given antibody are not necessarily identical to the CDRs defined by the other numbering systems. The CDRs of the variable domain 3024731 and the frame regions were identified by the inventor, using the IMGT numbering systems. The positions are numbered according to IMGT® and Kabat® index (amino acid sequences of region V and identical sequence segments in antibodies of different specificities). The relative contributions of VH and VL genes, minigenes and complementarity determining regions binding to antibody combining sites are analyzed (Kabat et al., NIH Publ 1991, 91-3242, Vol.1, 647). -669). Effect of murine monoclonal antibody D-A10 on the tumor growth of Isreco-1 colorectal cancer cells implanted subcutaneously in mice. The Isreco-1 cells (10 × 10 6) and their complete culture medium (10% fetal calf serum and 1% penicillin-streptomycin) were injected subcutaneously into 12 SCID CB17 mice. After 15 days, the tumor formed reached a volume between 100 and 150 mm 3. At this time, the mice were separated into 4 groups of 3 mice. Each group received or not specific treatment by intraperitoneal injection opposite the tumor. Once a week for 4 weeks (days 15, 22, 29 and 36 black arrows on the graph), the mice received an injection of 30 mg / kg of the reference antibody M20, MAb D-A10, D -D6 or have not received an injection (control group). For each mouse, the volume of the tumor was measured twice a week throughout the experiment. The average volume of each group of 20 mice is represented on the graph with a standard deviation corresponding to 3 mice per group. The results are shown in Figure 6. Dose-dependent effect of murine monoclonal antibody D-A10 concentration on tumor growth of Iscreco-1 colorectal cancer cells implanted subcutaneously in mice. Tumor fragments formed from Isreco-1 cancerous colorectal cells were subcutaneously implanted into 18 SCID CB17 mice. After 15 days, the tumor reached a volume between 100 and 150 mm3. At this time, the mice were separated into 6 groups of 3 mice. Each group received or not specific treatment by intraperitoneal injection opposite the tumor. Once a week for 4 weeks (days 15, 22, 29 and 36-black arrows on the graph), the mice received an injection of 1 mg / kg, 3 mg / kg, 10 mg / kg, 30 mg / kg 28 or 60 mg / kg of the murine monoclonal antibody (MAb) D-Al 0 or did not receive an injection (control group). For each mouse, the volume of the tumor was measured twice a week throughout the experiment. The average volume of each group of mice is represented on the graph with a standard deviation corresponding to 3 mice per group.
    [0039] The results are shown in Figure 7. Effect of murine monoclonal antibody D-A10 on tumor growth of subcutaneous HCT116 cancerous colorectal cancer cells in mice. 10x106 HCT116 cells and their complete culture medium (10% fetal calf serum and 1% penicillin-streptomycin) were injected subcutaneously into 12 SCID CB17 mice. After 6 days, the tumor formed reached a volume between 100 and 150 mm 3. At this time, the mice were separated into 4 groups of 3 mice. Each group received or not specific treatment by intraperitoneal injection opposite the tumor. Once a week for 3 weeks (days 6, 13 and 20-black arrows on the graph), the mice were injected with 30 mg / kg of the standard antibody M20, MAb D-A10, MAb D -D6 or have not received an injection (control group). For each mouse, the volume of the tumor was measured twice a week throughout the experiment. The average volume of each group of mice is represented on the graph with a standard deviation corresponding to 3 mice per group. The results are shown in Figure 8.
    [0040] Dose-dependent effect of murine monoclonal antibody D-A10 concentration on tumor growth of subcutaneous HCT116 cancerous colorectal cancer cells in mice. 10x106 HCT116 cells and their complete culture medium (10% fetal calf serum and 1% penicillin-streptomycin) were injected subcutaneously into 18 SCID CB17 mice. After 10 days, the tumor formed reached a volume between 100 and 150 mm 3. At this time, the mice were separated into 6 groups of 3 mice. Each group received or not specific treatment by intraperitoneal injection opposite the tumor. Once a week for 3 weeks (days 10, 17, and 24 black arrows on the graph), the mice received an injection of 1 mg / kg, 3 mg / kg, 10 mg / kg, 30 mg / kg or 60 mg / kg of the murine monoclonal antibody (MAb) D-A10 or did not receive an injection (control group). For each mouse, the volume of the tumor was measured twice a week throughout the experiment. The average volume of each group of mice is represented on the graph with a standard deviation corresponding to 3 mice per group. The results are shown in Figure 9.
    [0041] Determination of the Specificity of Anti-CK8 Ab by ELISA General Principle The CK8 peptides, coupled or not with bovine serum albumin (BSA) at the SH group of C-terminal cysteine, are immobilized at the bottom of the wells. a 96 well plate overnight. The wells are then saturated with a solution of phosphate buffered saline (PBS) containing 2.5% milk and incubated with dilutions of the anti-CK8 antibodies with a solution of PBS-Tween containing 0.5% BSA for 2 hours. AntiCK8 antibodies are then revealed by peroxidase-coupled murine anti-IgG secondary antibodies (HRP). The absorbance is read at 450 nm.
    [0042] The specific conditions for the different experiments performed are detailed below. Reactivity of the murine monoclonal antibody D-Al 0 or the M20 antibody for the different peptides 1 (SE () ID No. 1), 2 (SE () ID No. 2), 3 (SE () ID N 3) and 4 (SEQ ID NO: 4) of human CK8.
    [0043] Peptides 1, 2, 3 and 4 of human CK8 were coupled to BSA at the SH group of C-terminal cysteine and immobilized at the bottom of 96-well plate wells. The complete protein sequence of human cytokeratin 8 (Uni-ProtKB ref P05787), 483 amino acids (aa), is shown in FIG. 10. The positions of the sequences of peptides 1, 2, 3 and 4 are indicated by gray arrows. and blacks on the sequence of human CK8: Peptide 1 (SEQ ID NO: 1): AEQRGELAIKDANAKLSELEAALQRAKQD-C (aa 338- aa 366) Peptide 2 (SEQ ID NO: 2): AEQRGELAIKDANAKLSELE-C (aa 338- aa 357 Peptide 3 (SEQ ID NO: 3): AALQRAKQD-C (aa 358- aa 366) Peptide 4 (SEQ ID NO: 4): SELEAALQRAKQD-C (aa 354- aa 366) Various concentrations (10, 1 and 0) 1 μg / ml) of the M20 antibody or of the murine monoclonal antibody D-A10 were incubated for 2 hours at room temperature with either peptide 1, peptide 2, peptide 3 or peptide. 4 and used for ELISA detection. The optical density (OD) values read at 450 nm are presented on the graph. The results are shown in Figure 11 and presented in Table 4 below. The recognition or not by the M20 antibody or the murine monoclonal antibody D-Al 0 of the peptides 1 to 4 is respectively indicated by a + or - sign. Peptide (M17 ') Sequence CK8 () Reactivity of antibody Peptide 1 Peptide 2 Peptide 3 Peptide 4 M20 D-A10 333-AEQRGELAIKDANAKLSELEAALQRAKQD-C-356 338-AEQRGELAIKDANAKLSELE-C-357 358-AALQRAKQD-C-356 354-SELEAALQRAKQD Table 4: Coding and amino acid sequence of peptides 1, 2, 3 and 4 of human CK8, as well as their recognition or not by the M20 antibody or the murine monoclonal antibody D-Al 0 (sign + or - respectively). Reactivity of murine monoclonal antibody D-Al 0 for the different peptides 5 to 16 and 18 to 28 (SEQ ID NO: 5 to SEQ ID NO: 16 and SEQ ID NO: 18 to SEQ ID NO: 28) of CK8 human. Peptides 5 to 16 and 18 to 28 of human CK8 were immobilized on the bottom of 96-well plate wells. Different concentrations (10, 1, 0.1, 0.01, 0.001 and 0.0001 lag / ml) of the murine monoclonal antibody D-Al 0 were incubated for 2 h at room temperature with the different peptides and used for one hour. ELISA detection. The optical density (OD) values read at 450 nm are presented on the graph. The results are shown in Figure 12. The coding, sequence and recognition of murine monoclonal antibody D-A10 are shown in Table 5. Effect of peptides 5, 10 to 16 and 18 to 24 (SEQ ID NO: 5 and SEQ ID NO: 10 to SEQ ID NO: 16 and SEQ ID NO: 18 to SEQ ID NO: 24) on the recognition of peptides CK8 1 and 17 (SEQ ID NO: 1 and SEQ ID NO: 17) by the mouse monoclonal antibody D-A10.
    [0044] When the murine monoclonal antibody D-A10 is put in competition with different CK8 peptides, the antibody-peptide mixture is incubated for 30 minutes before being placed in the presence of the peptide to be detected and immobilized at the bottom of the wells. . The murine monoclonal antibody D-Al 0 is either in the presence of peptides 5 and 10 to 16 (SEQ ID No. 5 and SEQ ID No. 10 to SEQ ID No. 16) for the recognition of peptide 1 (SEQ ID N 1) or is placed in the presence of peptides 18 to 24 (SEQ ID No. 18 to SEQ ID No. 24) for the recognition of peptide 17.
    [0045] Peptide 1 of human CK8 was coupled to BSA at the SH group of C-terminal cysteine and immobilized at the bottom of 96-well plate wells. The murine monoclonal antibody D-Al 0 at a concentration of 0.03 lag / ml was incubated for 30 minutes in the presence of peptides 5 and 10 to 16 at different concentrations (10; 1; 0.1; 0.01; 0.001 and 0.0001 lag / mL). The peptide-antibody mixture or the antibody alone was then placed in contact for 2 hours at room temperature with peptide 1 and used for ELISA detection. The optical density (OD) values read at 450 nm are presented on the graph. The results are shown in Figure 13A. The coding, the sequence and their recognition by murine monoclonal antibody D-Al 0 are presented in Table 5.
    [0046] Peptide 17 of human CK8 was coupled to BSA at the SH group of N-terminal cysteine and immobilized at the bottom of 96-well plate wells. The murine monoclonal antibody D-Al 0 at the concentration of 0.01 lag / ml was incubated for 30 minutes in the presence of peptides 18 to 24 at different concentrations (10, 1, 0.1, 0.01, 0.001 and 0.0001 lag / mL). The peptide-antibody mixture or the antibody alone was then placed in contact for 2 hours at room temperature with peptide 17 and used for ELISA detection. The optical density (OD) values read at 450 nm are presented on the graph. The results are shown in Figure 13B and Table 5 below. The recognition or not by murine monoclonal antibody D-A10 of peptides 1 and 5 to 28 is indicated by a + or - sign, respectively.
    [0047] Peptide (N °) Sequence CK8 (aa) Reactivity of antibody D-A10 Peptide 1333-AEQRGELAIKDANAKLSELEAALQRAKO, DC-366 Peptide 5 338-AEQRGELAIKDANAKLSELEAALQRAKQD-366 Peptide 6 358-AALQRAKQD-366 Peptide 7 357-E) n ,, I, QRAKCID-356 Peptide 8,555-LEAALORAKC, D-365 Peptide 9,355-ELEAALORAKOD-366 Peptide 10 354-SELEAALORAKOD-366 Peptide 11 353-LSELEMLQRAKQD-366 Peptide 12 352-KLSELEAALQRAKQD-366 Peptide 13 351-AKLSE LEAALQRAKQD -366 Peptide 14 350-NAKLSELEAALQRAKQD-365 Peptide 15 349-ANAKLSELEAALQRAKQD-366 Peptide 16 348 -DANAKLSELEAALQRAKQD-3E E Peptide 17 345-C-AIKDANAKLSELEAALQRAKQD-365 Peptide 18 345-AIKDANAKLSELEAALQRAK0, -355 Peptide 19 345-AIKDANIAKLSELEAALO, RAK-364 Peptide 20 345-AKDANAKLSELEAALQRA-363 Peptide 21 345-AIKDANAKLSELEAALQR-362 Peptide 22 345-AIKDANAKLSELEAALQ-361 Peptide 23 345-4KDANAIKLSELEAAL-360 Peptide 24 345-AIKDANAKLSELEAA-35 Peptide 25 345-AIKDANAKLSELEA-358 Peptide 26 345-AIKDANAKLSELE-357 Peptide 27 345-AIKDANAK _SEL-356 Peptide 28 345-4 KDANAKLSE-355 Ta Figure 5: Coding and amino acid sequence of peptides 1 and 5 to 28 of human CK8, their recognition or not by murine monoclonal antibody D-Al 0 (+ sign or respectively). 5
    权利要求:
    Claims (13)
    [0001]
    REVENDICATIONS1. An antibody, or antibody fragment, specifically binding the peptide having the sequence according to SEQ ID No. 29 or a peptide having at least 60% homology with the peptide having the sequence according to SEQ ID No. 29.
    [0002]
    2. Antibody or antibody fragment according to claim 1, characterized in that it is a monoclonal antibody. 10
    [0003]
    3. Antibody, or antibody fragment, according to claim 1 or 2, characterized in that it is a humanized antibody.
    [0004]
    4. Antibody, or antibody fragment, according to claim 1, characterized in that it comprises at least one of the following sequences: SEQ ID No. 31 or SEQ ID No. 33.
    [0005]
    5. Antibody, or antibody fragment, according to claim 1, characterized in that it comprises variable regions encoded by polynucleotides having the nucleotide sequence of the variable murine light chain (SEQ ID No. 30) and / or the sequence nucleotide of the variable murine heavy chain (SEQ ID No.32).
    [0006]
    6. Antibody, or antibody fragment, according to one of claims 1 to 5, for its use in the treatment of tumors whose cells express the CK8 protein, in particular the peptide of sequence according to SEQ ID No.29, to their surface. 25
    [0007]
    7. Antibody, or fragment of antibody, according to one of claims 1 to 6 for its use in the treatment of colorectal cancers, ovaries, breast, testes, lung, kidney, nervous system, ganglia lymphatic, pancreas, head and neck.
    [0008]
    8. The antibody or antibody fragment of claim 7 for use in the treatment of invasive cancers. 15 20 3024731 34
    [0009]
    9. Antibody, or antibody fragment, according to one of claims 1 to 7, for its use in the treatment of colorectal cancers, preferably invasive colorectal cancers.
    [0010]
    10. Antigenic peptide of human CK8 having the sequence according to SEQ ID No. 29.
    [0011]
    11. Pharmaceutical composition characterized in that it comprises an antibody according to one of claims 1 to 5 and a suitable pharmaceutical vehicle.
    [0012]
    12. Pharmaceutical composition according to claim 11 for use in the treatment of tumors whose cells express the CK8 protein, in particular the sequence peptide according to SEQ ID No. 29, on their surface.
    [0013]
    13. Pharmaceutical composition according to claims 11 or 12 for use in the treatment of colorectal cancers.
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    同族专利:
    公开号 | 公开日
    CA2959671A1|2016-02-11|
    WO2016020553A1|2016-02-11|
    IL250507D0|2017-03-30|
    US10533046B2|2020-01-14|
    EP3189078A1|2017-07-12|
    IL250507A|2021-10-31|
    EP3189078B1|2020-11-04|
    KR20170082495A|2017-07-14|
    FR3024731B1|2019-06-14|
    EA201790341A1|2017-06-30|
    EA036368B1|2020-10-30|
    CN107001452B|2021-01-08|
    JP2017534661A|2017-11-24|
    CN107001452A|2017-08-01|
    US20180237508A1|2018-08-23|
    JP6689847B2|2020-05-27|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2010136536A1|2009-05-27|2010-12-02|Universite Claude Bernard Lyon 1|Anti-ck8 antibodies to be used for treating colorectal cancers and identifying metastatic and/or invasive phenotypes|
    US8168181B2|2006-02-13|2012-05-01|Alethia Biotherapeutics, Inc.|Methods of impairing osteoclast differentiation using antibodies that bind siglec-15|
    EP2602265A1|2011-12-07|2013-06-12|Centre National de la Recherche Scientifique |Antibodies anti-sPLA2-X and uses thereof|
    FR3024731B1|2014-08-08|2019-06-14|International - Drug - Development - Biotech|ANTI-CK8 ANTIBODIES FOR USE IN THE TREATMENT OF CANCERS|FR3024731B1|2014-08-08|2019-06-14|International - Drug - Development - Biotech|ANTI-CK8 ANTIBODIES FOR USE IN THE TREATMENT OF CANCERS|
    EP3444272A1|2017-08-17|2019-02-20|International-Drug-Development-Biotech|Treatment of ck8 positive cancers in relation with k-ras gene status|
    CN111018984B|2019-11-26|2021-09-21|山东立菲生物产业有限公司|anti-CK 8 monoclonal antibody and application thereof|
    CN113234156A|2021-05-25|2021-08-10|宁波赛珀生物技术有限公司|Anti-myoglobin antibody and preparation method and application thereof|
    法律状态:
    2015-08-26| PLFP| Fee payment|Year of fee payment: 2 |
    2016-02-12| PLSC| Publication of the preliminary search report|Effective date: 20160212 |
    2016-08-12| PLFP| Fee payment|Year of fee payment: 3 |
    2017-05-30| PLFP| Fee payment|Year of fee payment: 4 |
    2018-10-18| PLFP| Fee payment|Year of fee payment: 5 |
    2019-07-22| PLFP| Fee payment|Year of fee payment: 6 |
    2020-07-16| PLFP| Fee payment|Year of fee payment: 7 |
    2021-07-09| PLFP| Fee payment|Year of fee payment: 8 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1457704|2014-08-08|
    FR1457704A|FR3024731B1|2014-08-08|2014-08-08|ANTI-CK8 ANTIBODIES FOR USE IN THE TREATMENT OF CANCERS|FR1457704A| FR3024731B1|2014-08-08|2014-08-08|ANTI-CK8 ANTIBODIES FOR USE IN THE TREATMENT OF CANCERS|
    CN201580048278.9A| CN107001452B|2014-08-08|2015-08-10|anti-CK 8 antibodies for the treatment of cancer|
    EP15748040.1A| EP3189078B1|2014-08-08|2015-08-10|Anti-ck8 antibodies for use in the treatment of cancers|
    KR1020177006409A| KR20170082495A|2014-08-08|2015-08-10|Anti-Ck8 antibodies for use in the treatment of cancers|
    PCT/EP2015/068400| WO2016020553A1|2014-08-08|2015-08-10|Anti-ck8 antibodies for use in the treatment of cancers|
    CA2959671A| CA2959671A1|2014-08-08|2015-08-10|Anti-ck8 antibodies for use in the treatment of cancers|
    US15/502,469| US10533046B2|2014-08-08|2015-08-10|Anti-CK8 antibodies for use in the treatment of colorectal cancers|
    EA201790341A| EA036368B1|2014-08-08|2015-08-10|Anti-ck8 antibodies for use in the treatment of cancers|
    JP2017526766A| JP6689847B2|2014-08-08|2015-08-10|Anti-CK8 antibody for use in the treatment of cancer|
    IL250507A| IL250507A|2014-08-08|2017-02-08|Anti-ck8 antibodies for use in the treatment of cancers|
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