EP2167544A2 - Monoclonal antibody directed against the human ldl receptor - Google Patents

Monoclonal antibody directed against the human ldl receptor

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Publication number
EP2167544A2
EP2167544A2 EP07872460A EP07872460A EP2167544A2 EP 2167544 A2 EP2167544 A2 EP 2167544A2 EP 07872460 A EP07872460 A EP 07872460A EP 07872460 A EP07872460 A EP 07872460A EP 2167544 A2 EP2167544 A2 EP 2167544A2
Authority
EP
European Patent Office
Prior art keywords
antibody
seq
cells
antibody according
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07872460A
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German (de)
French (fr)
Inventor
Christophe De Romeuf
Christian Behrens
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LFB Biotechnologies SAS
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LFB Biotechnologies SAS
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Publication of EP2167544A2 publication Critical patent/EP2167544A2/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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C07K2317/72—Increased effector function due to an Fc-modification
    • 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
    • C07K2317/75—Agonist effect on antigen

Definitions

  • Malignant melanoma is a malignant tumor of the pigmentary system (melanocytes) occurring either primitively in healthy skin or by degeneration of a pre-existing nevus.
  • Melanoma is the most common skin cancer in the world. It doubles every 10 years. The majority of cases occur on healthy skin, with less than a quarter appearing on pre-existing nevi.
  • TIL Tumor Infiltrating Lymphocyte
  • Adoptive immunotherapy with TIL consists in injecting the patient with a large quantity (several billion) of cytotoxic T cells specific for melanoma antigens, which are isolated from a tumor or metastasis and expanded in vitro in a GMP laboratory (Good Manufacturing Practices). So we are in an autologous system.
  • the first clinical studies carried out in the USA with metastatic melanoma TILs achieved a response rate of around 35%, but often with rapid relapses, leading to discuss the abandonment of this passive immunotherapy approach.
  • Active immunotherapy by vaccination aims either to inject lysates of irradiated melanic cells subcutaneously (multi antigen approach), or to inject specific peptides obtained after identification of certain melanoma antigens in a restricted HLA context.
  • the objective is thus to induce a specific anti-melanoma immune response.
  • the first generation is that of multi-antigen vaccines. They are constituted by the crushed irradiated tumor cells. They thus have the advantage of being composed of several tumor antigens, which increases the chances of being able, a priori, to correspond to one of the cytotoxic T populations presented by the patient.
  • the melanoma tumor antigens are: melanocyte differentiation antigens: tyrosinase, gp 100, Melan-A / MART-I, gp 75; or tumor-specific antigens (embryonic antigens): "Melanoma Associated Antigen" MAA: Mage-1, Mage-2, Mage-3, Bage, Gage-1, Gage-2, Muc-1, Rage-1, NA- 17.
  • these antigens are generally present in small quantities, which limits their activating action.
  • These tumor tumors, injected into the patient subcutaneously or intradermally come either from the patient himself (autologous system) or from an allogeneic tumor line.
  • the stimulating action of the tumbler on the T lymphocyte cells can be intensified by adding to the latter an immune adjuvant.
  • BCG, Detox, QS-21, and MF-59 have been proposed.
  • the second generation is that of specific antigen vaccines, which rely on the principle of injecting a single tumor antigen to the patient. They induce an important cytotoxic T lymphocyte activation.
  • HLA-Al for Mage
  • HLA-A2 for NA-17, Melan-A
  • the third generation of vaccine is the use of dendritic cells for the purpose of vaccination in melanoma, and is based on the fact that these cells are excellent antigen-presenting cells. They are capable of internalizing antigens and presenting them to T cells in a class II HLA context for CD4 + and HLA class I lymphocytes for cytotoxic lymphocytes. This activation of the T lymphocyte also involves the co-stimulatory molecules CD40-CD40L and B7-CD28.
  • the principle of the development of the treatment is based on in vitro culture of CD34 + cells or monocytes isolated from the patient's blood (cytapheresis) in the presence of cytokines such as GM-CSF (Granulocyte-
  • the differentiated dendritic cells obtained are then loaded in vitro with one or more peptides. These dendritic cells, said to be loaded, then become excellent cytotoxic T cells activating the peptide-specific cells when they are reinjected into the patient, intradermally, subcutaneously and intra-ganglionally.
  • the fourth generation of vaccine is that of the modified tumor cells.
  • the melanoma tumor cell bypasses the immune system by producing immunosuppressive cytokines, masking its tumor antigens, by not expressing co-activation molecules or class I or II antigens.
  • the cytotoxic T response is thereby inhibited.
  • the principle of the tumor cell vaccine is to inject subcutaneous or intradermal patients with irradiated autologous tumor cells whose phenotypic profile has been modified to make it accessible to cytotoxic T lymphocytes.
  • the tumor cells can be transfected with: either IL-7, IL-2 and IL-12 cytokines; either GM-CSF that activates macrophages; or class I, II antigens, BL7 antigen thus allowing the recognition of tumor antigens by the cytotoxic cell.
  • phase I-II The clinical studies (phases I-II) carried out with these different vaccines of first and second generation, at the metastatic stage, relate to a limited number of patients with currently an average response rate of the order of 20%. These responses are mainly obtained on cutaneous sites, lymph nodes pulmonary and hepatic Interestingly, and specific to vaccination, prolonged response times (greater than 2 years) were noted. An important point is the delay of the clinical response. Indeed, unlike chemotherapy, the clinical regression most often appears after a stabilization phase that can last for 3 to 4 months or more.
  • Tolerance is generally good.
  • the side effects noted being essentially erythema at the injection site, autoimmune reactions of the vitiligo type.
  • variable region of each of the light chains is encoded by the murine nucleic acid sequence SEQ ID NO: 5
  • the variable region of each of the heavy chains is encoded by the murine nucleic acid sequence SEQ ID NO: 7, or by nucleic acid sequences having sufficient homology with the sequences SEQ ID NO: 5 and SEQ ID NO: 7 so that the nature and affinity of the binding to said antibody to its antigen are not modified, and whose constant regions of its light chains and heavy chains are constant regions from a non-murine species.
  • the antibodies consist of heavy chains and light chains, linked together by disulfide bridges.
  • Each chain consists, in the N-terminal position, of a variable region (or domain) (encoded by the VJ rearranged genes for the light chains and VDJ for the heavy chains) specific for the antigen against which the antibody is directed. and in the C-terminal position of a constant region consisting of a single CL domain for the light chains or several domains for the heavy chains.
  • the two heavy chains (H, Heavy) and the two light chains (L, Light) are identical to each other.
  • the light chain is composed of 2 domains, a variable domain V and a constant domain C, folded independently of each other in space. They are called VL and CL.
  • the heavy chain also comprises a V domain denoted VH and 3 or 4 C domains denoted from CH1 to CH4. Each domain comprises about 110 amino acids and is structurally comparable.
  • the 2 heavy chains are linked by disulfide bridges and each heavy chain is linked to a light chain by a disulfide bridge as well.
  • variable parts The region that determines the specificity of the antibody for the antigen is carried by the variable parts, whereas the constant parts can interact with the Fc receptors of the effector cells or molecules as complement to mediate different functional properties.
  • monoclonal antibody or “monoclonal antibody composition” refer to a preparation of antibody molecules having identical and unique specificity.
  • antibodies monoclonal antibody a complete monoclonal antibody, a fragment or a derivative of such an antibody.
  • the homology sufficient corresponds to
  • a first nucleic acid having at least 70% homology with a second reference nucleic acid will have at least 90%, preferably at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.3% 98.6%, 99%, 99.6% identity nucleotides with said second nucleic acid. reference.
  • a first polypeptide having at least 70% identity with a second reference polypeptide will have at least 90%, preferably at least 70%, 80%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 97.5%, 98%, 98.3% 98.6%, 99%, 99.6% amino acid identity with said second reference polypeptide.
  • the "percentage of homology" between two nucleic acid sequences or between two polypeptide sequences, within the meaning of the present invention, is determined by comparing the two optimally aligned sequences, through a comparison window.
  • the part of the nucleotide or amino acid sequence in the comparison window may thus comprise additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or deletions) in order to obtain an optimal alignment between the two sequences.
  • the percentage of homology is calculated by determining the number of positions at which a base same nucleic acid, or an identical amino acid residue, is observed for the two compared sequences, and then by dividing the number of positions at which there is identity between the two nucleic bases, or between the two amino acid residues, by the total number of positions in the comparison window, then multiplying the result by one hundred to obtain the percent identity nucleotides of the two sequences together, or the percent amino acid identity of the two sequences together.
  • the term "the nature and affinity of the binding of said antibody to its antigen are not modified" is on the one hand that the antibodies having a homology with the antibody whose variable region of each light chains is encoded by the sequence SEQ ID NO: 5, and whose variable region of each of the heavy chains is encoded by the sequence SEQ ID NO: 7, bind to the same antigen, and secondly that their binding affinity is at least equal to 90% of the affinity binding of the antibody whose variable region of each of the light chains is encoded by the sequence SEQ ID NO: 5, and whose variable region of each of the heavy chains is encoded by the sequence SEQ ID NO: 7.
  • nucleic acid sequences having sufficient homology with the sequences SEQ ID NO: 5 and SEQ ID NO: 7 are understood to mean that the nature and affinity of the binding of the antibody to its antigen is not modified" any nucleic acid sequence encoding a polypeptide, peptide or protein, comprising at least one immunoglobulin domain or fragment, as well as any antibody derivative having sufficient homology with the sequences SEQ ID NO: 5 and SEQ ID NO: 7 so that the nature and affinity of the binding of the antibody to its antigen are not changed.
  • the term "immunoglobulin domain” means any of the domains VL, CL, VH, CH1, CH2, CH3, CH4.
  • the antibody according to the invention may advantageously contain one or more of these domains, all the combinations between the aforementioned domains are part of the invention.
  • the term "immunoglobulin fragment” is understood to mean one of the fragments chosen from the Fab, Fab ', F (ab') 2, Fc fragment, a scFv or a CDR (Complemantarity Determining Region).
  • the enzymatic digestion of immunoglobulins by papain generates 2 identical fragments, called “Fragment Antigen Binding", and an Fc fragment (crystallizable fragment).
  • the Fc fragment is the support of the effector functions of immunoglobulins.
  • an F (ab ') 2 fragment is generated, where the two Fab fragments remain linked by two disulfide bridges, and the Fc fragment is split into several peptides.
  • the F (ab ') 2 fragment is formed of two Fab' fragments, linked by interchain disulfide bridges to form an F (ab ') 2.
  • variable regions of the heavy and light chains it is found that the sequence variability is not distributed equally. In fact, the variable regions consist on the one hand of very little variable regions called "framework" or
  • the antibody according to the invention can advantageously contain one or more of these fragments, all the combinations between the aforementioned fragments are part of the invention.
  • the antibody according to the invention contains at least one immunoglobulin domain and at least one immunoglobulin fragment, for example an Fc fragment and one or more variable or hypervariable regions.
  • antibody derivative is understood to mean any antibody, this antibody possibly comprising one or more mutations, substitutions, deletions and / or additions of one or more amino acid residues.
  • Such an addition, substitution or deletion may be located at any position in the molecule.
  • any combination of addition, substitution or deletion may be considered, provided that the resulting antibody still has at least the advantageous properties of the antibody of the invention.
  • the antibody according to the invention whose variable regions of the light and heavy chains, or at least one domain or fragment of these regions, belong to a species different from the constant regions of the light and heavy chains, is called antibody. "Chimerical”.
  • the murine nucleic acid sequences SEQ ID NO: 5 and SEQ ID NO: 7 encode the variable domain of each of the light chains and the variable domain of each of the heavy chains respectively of the antibody produced by the C7 murine hybridoma, available from ATCC (American Type Culture Collection) under number CRL-1691.
  • This hybridoma produces a mouse monoclonal antibody of IgG2b isotype directed against LDL-R.
  • the human LDL receptor is a transmembrane protein of 839 amino acids that comprises three regions: the extracellular region (1-768), the transmembrane region (768-790) and the cytoplasmic region (790-839). ). The extracellular region is divided into two subregions: the LDL binding region (1-322) and the subregion outside the LDL binding zone (323-768).
  • the murine sequences of the antibody of the invention have been chosen to encode the variable regions of the antibody according to the invention, or at least one domain or fragment of these regions, because of the specificity of the murine C7 antibody. for the LDL-R antigen.
  • the C7 antibody was generated by immunization with the extracellular domain of bovine LDL-R. It binds, apart from bovine LDL-R, to human LDL-R but does not cross-react with LDL-R in rats, mice, Chinese hamster, rabbits and dogs (Beisiegel et al. 1981 J. Biol Chem 256, 11923-11931). This property is advantageous because Monoclonal antibody production lines are very often lines from these species.
  • the recombinant antibody may advantageously be produced in a cell line originating from the lines YB2 / 0, NSO, Sp2 / 0, CHO, this list not being limiting.
  • the antibody of the invention also includes antibodies having CDR regions whose peptide sequence is chosen from the sequences SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28.
  • sequences are the sequences of the CDR regions originating from the murine C7 antibody, the sequence SEQ ID NO: 6 representing the sequence of the CDR1 of the light chain of the antibody according to the Kabat numbering, the sequence SEQ ID NO: 8 representing the CDR2 sequence of the light chain of the antibody according to the Kabat numbering, the sequence SEQ ID NO: 9 representing the CDR3 sequence of the light chain of the antibody according to the Kabat numbering, the sequence SEQ ID NO: Representing the CDR1 sequence of the light chain of the antibody according to the IMGT numbering, the sequence SEQ ID NO: 16 representing the CDR2 sequence of the light chain of the antibody according to the IMGT numbering, the sequence SEQ ID NO: 17 showing the CDR3 sequence of the light chain of the antibody according to the IMGT numbering, the sequence SEQ ID NO: 22 representing the CDR1 sequence of the heavy chain of the antibody according to the e Kabat, the sequence SEQ ID NO: 24 representing the CDR2 sequence of the heavy chain of the antibody according to the Kab
  • the invention also includes antibodies whose variable domain of each of the light chains and the variable domain of each of the heavy chains have at least 70% homology, or advantageously at least 80%, or 90% or 99% , with the sequences SEQ ID NO: 5 and SEQ ID NO: 7 respectively, the sequence modifications not modifying the specificity of the antibody. Preferably, these sequence modifications do not decrease its affinity for its target.
  • the antibody according to the invention also has constant regions of its light and heavy chains belonging to a non-murine species.
  • non-murine mammals all families and species of non-murine mammals are likely to be used, and in particular humans, monkeys, murids (except the mouse), suids, cattle, equines, felids , canids, for example, and birds.
  • the antibodies according to the invention can be constructed using standard techniques of recombinant DNA, which are well known to those skilled in the art, and more particularly by using the "chimeric" antibody construction techniques described, for example, in Morrison et al. ., Proc. Natl. Acad. Sci. USA, 81, pp. 6851-55 (1984), where recombinant DNA technology is used to replace the constant region of a heavy chain and / or the constant region of a light chain of an antibody from a non-human mammal. with the regions of a human immunoglobulin.
  • An expression vector is a nucleic acid molecule in which the murine nucleic acid sequence encoding the variable domain of each of the heavy or light chains of the antibody and the nucleic acid sequence, preferably human, encoding for the constant region of each of the heavy or light chains of the antibody were inserted, in order to introduce and maintain them in a host cell. It allows the expression of these foreign nucleic acid fragments in the host cell because it has essential sequences (promoter, polyadenylation sequence, selection gene) to this expression.
  • the vector may be, for example, a plasmid, an adenovirus, a retrovirus or a bacteriophage
  • the host cell may be any mammalian cell, for example SP2 / 0, YB2 / 0, IR983F, Namalwa human myeloma, PER. C6, CHO lines, especially CHO-KI, CHO-Lec10, CHO-Lee1, CHO-Lecl3, CHO Pro-5, CHO dhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293- HEK, BHK, K6H6, NSO, SP2 / O-Ag14 and P3X63Ag8.653.
  • variable regions For the construction of expression vectors of the chimeric antibodies according to the invention, appropriate synthetic signal sequences and restriction sites can be fused to the variable regions during the amplification reactions. PCR. The variable regions are then combined with the constant regions of an antibody, preferably a human IgG1.
  • the genes thus constructed are cloned to place them under the control of a promoter, for example the RSV (Rous Sarcoma Virus), CMV (cytomegalovirus), MLP (Major Late Promoter) promoter, this list not being limiting, and in particular upstream of a polyadenylation site, using two separate vectors (one for each chain).
  • the vectors are also provided with selection genes known to those skilled in the art, such as, for example, the dhfr (dihydrofolate reductase) gene or the neomycin resistance gene.
  • the chimeric antibodies according to the invention may be produced by cotransfection in a host cell of the light chain expression vector and the heavy chain expression vector using a method well known to those skilled in the art (for example calcium phosphate co-precipitation, electroporation, microinjection, etc.).
  • the cells can be put in a selective medium, for example in RPMI medium (Invitrogen, ref 21875-034) containing 5% dialysis serum (Invitrogen, ref 10603-017), 500 ⁇ g / ml G418 (Invitrogen, P / N 10131-027) and 25 nM methotrexate (Sigma, M8407).
  • the supernatants of the resistant transfection wells are screened for the presence of chimeric immunoglobulin (Ig) by ELISA assay specific for human Ig or other species if the constant part is from another species.
  • the transfectants producing the most antibodies are amplified and their supernatants redosed by ELISA in order to estimate their productivity and to select the 3 best producers for limiting dilution cloning (40 cells / plate).
  • variable region of each of the light chains of the antibody according to the invention has the peptide sequence SEQ ID NO: 10 and the variable region of each of the heavy chains of the antibody according to the invention has the peptide sequence SEQ ID NO: 11.
  • the peptide sequence SEQ ID NO: 10 is the peptide sequence deduced from the nucleotide sequence SEQ ID NO: 5 and the peptide sequence SEQ ID NO: 11 is the sequence deduced from the nucleotide sequence SEQ ID NO: 7.
  • the constant regions of each of the light chains and of each of the heavy chains of the antibody according to the invention are human constant regions.
  • This preferred embodiment of the invention makes it possible to reduce the immunogenicity of the antibody in humans and thereby improve its effectiveness during its therapeutic or prophylactic administration in humans.
  • the constant region of each of the light chains of the antibody according to the invention is of type K.
  • Any allotype is suitable for carrying out the invention, for example Km (I), Km (I, 2), Km (I, 2, 3) or Km (3).
  • the constant region of each of the light chains of the antibody according to the invention is of type ⁇ .
  • the constant region of each of the heavy chains of the antibody is of type ⁇ .
  • the constant region of each of the chains The heavy of the antibody can be of type ⁇ 1, of type ⁇ 2, of type ⁇ 3, these three types of constant regions having the particularity of fixing the human complement, or of type ⁇ 4.
  • Antibodies possessing a constant region of each of the heavy Y-type chains belong to the IgG class.
  • Immunoglobulin type G (IgG); are heterodimers consisting of two heavy chains and two light chains, linked together by disulfide bridges.
  • Each chain consists, in the N-terminal position, of a region or variable domain (encoded by the rearranged genes VJ for the light chain and VDJ for the heavy chain) specific for the antigen against which the antibody is directed, and in the C-terminal position, a constant region consisting of a single CL domain for the light chain or 3 domains (CH1, CH2 and CH3) for the heavy chain.
  • variable domains V H and V L and of the constant domains CH 1 and CL of the heavy and light chains forms the Fab parts, which are connected to the Fc region by a very flexible hinge region allowing each Fab to be fixed to its antigenic target while the Fc region, mediator of the effector properties of the antibody remains accessible to effector molecules such as Fc ⁇ R receptors, neonatal Fc receptor (FcRn) and CIq.
  • Fc region consisting of the 2 globular domains C ⁇ 2 and C ⁇ 3 , is glycosylated at the C ⁇ 2 domain with the presence, on each of the 2 chains, of a biantennary ⁇ 7-glycan linked to Asn 297.
  • the constant region of each of the heavy chains of the antibody is of ⁇ 1 type, since such an antibody shows an ability to generate ADCC (Antibody-Dependent Cellular Cytotoxicity) activity in the largest number of individuals (human ).
  • ADCC Antibody-Dependent Cellular Cytotoxicity
  • any allotype is suitable for carrying out the invention, for example GIm (3), GIm (1, 2, 17), GIm (I, 17) or GIm (1,3).
  • the constant region of each of the heavy chains of the antibody is of ⁇ 1 type, and it is encoded by the human nucleic acid sequence SEQ ID NO: 23, the constant region of each of its light chains being encoded by the human nucleic acid sequence SEQ ID NO: 21, or a sequence homologous to the sequence SEQ ID NO: 23 or SEQ ID NO: 21, or a fragment of said sequences.
  • an antibody has a murine variable region and a human constant region, with heavy chains of ⁇ 1 type. This antibody therefore belongs to the subclass of IgG1.
  • This antibody has two light chains whose variable domain is encoded by the murine nucleic acid sequence SEQ ID NO: 5 and the human constant region is encoded by the nucleic acid sequence SEQ ID NO: 21, and two heavy chains of which the variable domain is encoded by the murine nucleic acid sequence SEQ ID NO: 7 and the constant region is encoded by the human nucleic acid sequence SEQ ID NO: 23.
  • each of the light chains of the antibody according to the invention is encoded by the murine-human chimeric nucleic acid sequence SEQ ID NO: 13, and each of the heavy chains is encoded by the chimeric nucleic acid sequence.
  • murine-human SEQ ID NO: 19 The murine-human chimeric nucleic acid sequence SEQ ID NO: 13 encoding each of the light chains of the antibody is obtained by fusion of the murine nucleic acid sequence SEQ ID NO: 5 coding for the variable domain of each of the light chains of the antibody and the human nucleic acid sequence SEQ ID NO: 21 coding for the constant region of each of the light chains of the antibody.
  • the murine-human chimeric nucleic acid sequence SEQ ID NO: 19 coding for each of the heavy chains of the antibody is obtained by melting the murine nucleic acid sequence SEQ ID NO: 7 coding for the variable domain of each of the heavy chains of the antibody and the human nucleic acid sequence SEQ ID NO: 23 encoding the constant region of each of the heavy chains of the antibody.
  • each of the light chains of the antibody is encoded by the murine-human chimeric nucleic acid sequence SEQ ID NO: 13 and each of the heavy chains is encoded by the sequence of murine-human chimeric nucleic acid SEQ ID NO: 19,
  • the peptide sequence of each of the light chains, deduced from the nucleic acid sequence SEQ ID NO: 13 is the sequence SEQ ID NO: 14
  • the peptide sequence of each heavy chains, deduced from the nucleic acid sequence SEQ ID NO: 19 is the sequence SEQ ID NO: 20.
  • the invention also includes antibodies each of which has light chains encoded by a murine-human chimeric nucleic acid sequence having at least 70% homology to the murine-human chimeric nucleic acid sequence SEQ ID NO: 13 and each of the heavy chains encoded by a murine-human chimeric nucleic acid sequence has at least 70% homology, or preferably at least 80%, or 90% or 99%, with the murine chimeric nucleic acid sequence.
  • -human SEQ ID NO: 19 these modifications not altering the specificity of the antibody nor its effector activities.
  • the antibody according to the invention is coupled to a toxin.
  • the toxin is, for example, diphtheria toxin or ricin, this list not being limiting.
  • the binding between the antibody according to the invention and the toxin is sufficiently strong to prevent the systemic release of the toxin and also sufficiently labile, so that the toxin is released into the target cells.
  • the antibody is coupled to a radioisotope.
  • the presence of the radioisotope greatly increases the cytotoxicity.
  • Two isotopes are mainly used: iodine-131 (beta and gamma emitter), whose half-life is relatively long (8 days) and which exerts a tumoricidal effect on about 1 mm around the tumor cell that has fixed the antibody according to the invention.
  • Iodine 131 has the advantage of making it possible to perform imaging, but requires compliance with radiation protection measures.
  • tumoricidal effects over a distance of 5 mm.
  • the antibody of the invention is produced in a rat hybridoma cell line.
  • the line producing the antibody according to the invention is an important characteristic since it confers on the antibody some of its particular properties. Indeed, the means of expression of the antibodies is at the origin of the post-translational modifications, in particular modifications of the glycosylation, which may vary from one cell line to another, and thus confer different functional properties on antibodies having identical primary sequences.
  • the antibody is produced in the YB2 / 0 rat cell line (YB2 / 3HL.P2.GH.16Ag.2O cell, deposited at the American Type Culture Collection as ATCC number CRL-1662). .
  • a preferred antibody according to the invention is the EMAB604 antibody produced by hybridoma R604 (deposited on November 14, 2006 under the number 1-3692 at the CNCM - Pasteur Institute, 25 rue du Professeur Roux, 75724 Paris Cedex 15, France) .
  • the variable region of each of the light chains of the monoclonal antibody produced by the hybridoma R604 is encoded by the nucleic acid sequence SEQ ID NO: 5, and the variable region of each of the heavy chains of the monoclonal antibody produced by hybridoma R604 is encoded by the nucleic acid sequence SEQ ID NO: 7.
  • a particular object of the invention relates to a monoclonal antibody binding to LDL-R and allowing the recruitment of effector cells.
  • this antibody is EMAB604, or any chimeric, humanized or human antibody having functional characteristics identical to the EMAB604 antibody.
  • this antibody is produced by the R604 cell line.
  • Another subject of the invention relates to the expression vector of the light chain of an antibody according to the invention, of sequence SEQ ID NO: 12.
  • This vector is the vector allowing the expression of an antibody according to the invention.
  • invention in which the light chain is encoded by the SED ID NO: 13 nucleic acid sequence, the deduced peptide sequence of which is the sequence SEQ ID NO: 14.
  • This vector is a nucleic acid molecule in which the sequence of murine nucleic acid SEQ ID NO: 5 coding for the variable domain of each of the light chains of the antibody and the nucleic acid sequence SEQ ID NO: 21 encoding the constant region of each of the light chains of the antibody were inserted, to introduce and maintain them in a host cell.
  • any mammalian cell may be used as a host cell, that is to say as a cell expressing the antibody according to the invention, for example YB2 / 0, CHO, CHO dhfr- (for example CHO DX BlI, CHO DG44), CHO Lecl3, SP2 / 0, NSO, 293, BHK or COS.
  • Another subject of the invention relates to the expression vector of the heavy chain of an antibody according to the invention, of sequence SEQ ID NO: 18.
  • This vector is the vector allowing the expression of an antibody according to the invention. invention in which the heavy chain is encoded by the SED ID NO: 19 nucleic acid sequence, the deduced peptide sequence of which is the sequence SEQ ID NO: 20.
  • This vector is a nucleic acid molecule in which the sequence of murine nucleic acid SEQ ID NO: 7 coding for the variable domain of each of the heavy chains of the antibody and the human nucleic acid sequence SEQ ID NO: 23 coding for the constant region of each of the heavy chains of the antibody have have been inserted, to introduce and maintain them in a host cell.
  • the vector may be for example a plasmid, an adenovirus, a retrovirus or a bacteriophage
  • the host cell may be any mammalian cell, for example YB2 / 0, CHO, CHO dhfr- (CHO DX BlI, CHO DG44), CHO Lecl3, SP2 / 0, NSO, 293, BHK or COS.
  • an antibody produced by coexpression of these vectors in the YB2 / 0 cell is illustrated by the anti-LDL-R antibody EMAB604, produced by the clone R604 (deposited under the CNCM I-3692 registration number at the CNCM). .
  • This antibody has a high cytotoxic activity, found in an ADCC test.
  • the EMAB604 antibody induces the secretion of IL-2 by the Jurkat-CD16 cell line, this test being used to demonstrate the ability of the antibodies to activate the CDl ⁇ receptor.
  • the EMAB604 antibody which can be produced by culturing the clone R604 in a culture medium and under conditions allowing the expression of the previously described vectors, is therefore a most interesting tool likely to advance the therapy and the diagnosis of melanomas.
  • Another subject of the invention relates to a stable cell line producing an antibody according to the invention as described above.
  • the stable cell line according to the invention which is characterized in that the cell line in which the antibody is expressed, is selected from the group consisting of: SP2 / 0, YB2 / 0 (YB2 / 3HL cell .P2.GIl .16Ag.20, deposited at the American Type Culture Collection as ATCC number CRL-1662), SP2 / 0-AG14 (ATCC CRL-1581), IR983F, Namalwa human myeloma, PERC6, CHO lines.
  • the stable cell line of the invention incorporated the two expression vectors previously described.
  • Another subject of the invention relates to the hybridoma R604 deposited under the registration number CNCM 1-3692 to the National Collection of Cultures of Microorganisms (CNCM, Pasteur Institute, 25 rue du Dondel Roux, 75724 Paris Cedex 15) .
  • the antibody according to the invention allows the recruitment of effector immune cells.
  • Such an antibody by its good specificity and good affinity, is a tool that can be used to mediate reactions of ADCC (Antibody Dependent Cellular-mediated Cytotoxicity).
  • ADCC Antibody Dependent Cellular-mediated Cytotoxicity
  • the antibody according to the invention has a good affinity for LDL-R and also allows the recruitment of effector immune cells.
  • effector immune cell means a cell which causes the destruction of the cells on which the antibody according to the invention is bound (“target cells").
  • the anti-LDL-R EMAB604 antibody has the ability to interact strongly with the Fcgamma RIIIa or CD16 receptor expressed by NK cells. This binding is at least three times higher than that of the anti-CD20 Rituxan® antibody and comparable to that of the anti-CD20 antibody produced by the LFB's EMABLing platform. This strong attachment to the receiver CD16 makes it possible to envisage optimized cytotoxic capacities for the anti-LDL-R EMAB604 antibody.
  • ADCC Antibody-Dependent CeIl-mediated Cytotoxicity
  • HT144 melanoma
  • GUY 17.2 cells dependent on the interaction of its Fc part with the low affinity CD16 receptor expressed on NK cells (Natural Killer).
  • ADCC Antibody-Dependent CeIl-mediated Cytotoxicity
  • CD16 is also expressed by macrophages and in a monocyte subpopulation, which makes it possible to envisage the action of anti-LDL-R EMAB604 antibody by induction of cellular cytotoxicity via the monocytic line.
  • this strong binding of the anti-LDL-R EMAB604 antibody to the CD1 ⁇ receptor confers on it the presence in the presence of HT144 cells, the capacity to induce the secretion of interleukin-2 (IL-2) by the Jurkat line transfected with CDl ⁇ (Jurkat-CDl ⁇ ). Indeed, the commitment of the Fc part of the antibody fixed on its target (HT144) induces an activation signal which results in the secretion of IL-2 by Jurkat-CD16.
  • IL-2 interleukin-2
  • the C7 antibody was obtained by immunizing mice with partially purified bovine LDL-R. It has been shown to be a good competitor of LDL, and therefore has an affinity for LDL-R comparable to that of the natural LDL-R ligand.
  • ADCC Antibody Dependent Cellular Cytotoxicity
  • the target cells according to the invention are tumor cells, such as sarcomas, myelomas, melanomas, lymphomas, leukemias, this list not being limiting.
  • tumor cells such as sarcomas, myelomas, melanomas, lymphomas, leukemias, this list not being limiting.
  • studies have shown a correlation between the increase in the level of expression of LDL-R by cells and certain cancers. It turns out that patients with certain cancers have hypocholesterolemia. This hypocholesterolemia is the consequence of overuse of cholesterol by cancer cells. For their survival, the latter induce an increase in LDL receptor expression level (LDL-R) in the tumor organs (Henricksson et al, 1989).
  • LDL-R LDL receptor expression level
  • the anti-LDL-R EMAB604 antibody was shown to bind to HT144 and GUY 17.2 melanoma lines.
  • the invention relates to a monoclonal antibody directed against the human LDL (Low Density Lipoprotein) receptor, capable of inducing specific lysis of melanomas.
  • LDL Low Density Lipoprotein
  • Another subject of the invention is the use of an antibody according to the invention, to activate in vitro, ex vivo or in vivo, the Fc ⁇ RIII receptors of effector immune cells or to cause the secretion of cytokines or chemokines by effector cells.
  • the antibodies of the invention can be used for their ability to activate the Fc ⁇ RIIIA receptor by their Fc region. This is of considerable interest because this receptor is expressed on the surface of cells called "effector cells”: the binding of the Fc region of the antibody to its receptor carried by the effector cell causes the activation of Fc ⁇ RIIIA and the destruction of target cells.
  • the effector cells are, for example, NK (Natural Kilier) cells, macrophages, neutrophils, CD8 lymphocytes, T ⁇ lymphocytes, NKT cells, eosinophils, basophils or mast cells.
  • Another particular object of the invention is an antibody as described above for its use as a medicament.
  • the antibody used binds to the human LDL receptor, and allows the recruitment of effector cells.
  • Another subject of the invention is the use of a monoclonal antibody directed against the human LDL receptor for the manufacture of a medicament for the treatment of cancer, such as cancer of the prostate, breast, liver, pancreas, ovaries, colon, lung, stomach and leukaemias.
  • the antibody according to the invention targets LDL-R specifically.
  • the antibody according to the invention by binding to this receptor, will generate a lysis reaction of the target cancer cells, in particular by ADCC against the target cancer cells and allow the lysis of the latter.
  • the lysed cells will be near-specific cancer cells, healthy cells not over-expressing or little LDL-R and thus being preserved.
  • the cancers treated with the antibody according to the invention are the cancers for which the LDL receptor is overexpressed on the surface of the cancer cells, and this with respect to the corresponding healthy cells.
  • the treated cancer is melanoma.
  • the invention also relates to the use of a monoclonal antibody directed against the human LDL receptor, or an antibody as defined above, for the manufacture of a medicament for the treatment of melanoma.
  • the cancerous target cells may be lysed by the effector cells recruited during the ADCC reaction, healthy cells expressing little or no LDL-R or, if appropriate, being previously treated to induce LDL-internalization. R so as not to be recognized by the antibody and thus be preserved.
  • the malignant melanoma treated may be extensive superficial melanoma, which has the appearance of a polychromatic patch whose contours and surface are irregular, and which correspond to the horizontal development phase of malignant melanoma, and nodular melanomas characterized clinically by a prominent melanoma tumor and histologically by a well circumscribed proliferation which invades the dermis immediately.
  • the antibody of the invention is capable of inducing specific lysis of melanomas from HT144 (HTB-63) (HLA A1, Aw24, B13, B15, Cw3, DRw4, DRw7) and GUY-17.2, dependent lines. of CD16.
  • Another subject of the invention relates to the use of an antibody of the invention in combination with one or more other antibodies directed against one or more other antigens expressed on the melanoma cells.
  • antigens can be expressed on lymphoid cells and are selected from HLA-DR, CD20, CD22, CD23, CD25, CD30, CD33 and CD40.
  • Another object of the invention relates to the use of an antibody of the invention in combination with one or more other drugs commonly used for the treatment of cancers such as cytotoxic drugs and cytostatigues (chemotherapy).
  • cytotoxic drugs and cytostatigues (chemotherapy).
  • chemotherapy chemotherapy.
  • tyrosine kinase receptor inhibitors including EGF (endothelial growth factor) receptors, insulin receptors, PDGF (Platelet-derived growth factor) receptors, FGF receptors (fibroblast growth factor) and VEGF (vascular endothelial growth factor) receptors, this list not being limiting.
  • the use of the antibodies of the invention is carried out in combination, in vitro, ex vivo or in vivo, with cells expressing Fc ⁇ R, such as NK (Watural Killer) cells, NKT (Natural Killer T) cells, Ty ⁇ lymphocytes, macrophages, monocytes, any cell genetically modified to express CD16, or dendritic cells.
  • NK Wild Killer
  • NKT Natural Killer T
  • Another subject of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one antibody according to the invention as described above and a pharmaceutically acceptable excipient and / or carrier.
  • This pharmaceutical composition is intended to target cancer cells, including those overexpressing LDL-R. As these cancer cells express on their surface a quantity of LDL receptors greater than the quantity of receptors expressed by the healthy cells, the drug thus prepared will be preferentially bound by the cancer cells.
  • the excipient may be any solution, such as saline, physiological, isotonic, buffered, etc., as well as any suspension, gel, powder, etc., compatible with a pharmaceutical use and known to those skilled in the art.
  • the compositions according to the invention may also contain one or more agents or vehicles chosen from dispersants, solubilizers, stabilizers, surfactants, preservatives, and the like.
  • the composition of the invention further comprises at least one antibody directed against another antigen present on the target cells of the antibodies.
  • the composition of the invention further comprises an anti-HLA-DR antibody.
  • an anti-HLA-DR antibody express the HLA-DR antigen on their surface.
  • a composition comprising a mixture of anti-LDL-R and anti-HLA DR antibodies in proportions which may represent 5, 25, 50, 75 or 95% of either antibody relative to the total weight of the The composition is particularly advantageous for the treatment of patients with melanoma.
  • compositions can be administered in different ways and under different forms.
  • the administration can be carried out by any conventional route for this type of therapeutic approach, in particular by the systemic route, in particular by intravenous, intradermal, intratumoral, subcutaneous, intraperitoneal, intramuscular, intraarterial injection, etc. .
  • intratumoral injection or injection into an area close to the tumor or irrigating the tumor may be mentioned.
  • Administration may also be oral, mucosal or topical.
  • the doses may vary according to the number of administrations, the association with other active ingredients, the stage of evolution of the pathology, etc.
  • Another subject of the invention is the use of the antibody according to the invention in immunohistochemical analyzes of cancerous, healthy or cirrhosis tissues, or in Western Blot analyzes, in ELISA or in vivo quantification test, ex. vivo or in vi tro.
  • FIG. 2 Inhibition of the ADCC activity induced by 10 ⁇ g / ml of the anti-LDL-R (EMAB604) antibodies and HLA-DR-CHO on the GUY-17.2 melanoma line by the murine anti-CD1 ⁇ antibody (250 ⁇ g / ml).
  • EMB604 anti-LDL-R
  • FIG. 4 Inhibition of the ADCC Activity Induced by 10 ⁇ g / ml of the Anti-LDL-R (EMAB604) and Anti-HLA-DR-CHO Antibodies on the HT-144 Melanoma Line by the Murine Anti-CD1 ⁇ Antibodies (250 ⁇ g / ml).
  • EMB604 Anti-LDL-R
  • FIG. 4 Inhibition of the ADCC Activity Induced by 10 ⁇ g / ml of the Anti-LDL-R (EMAB604) and Anti-HLA-DR-CHO Antibodies on the HT-144 Melanoma Line by the Murine Anti-CD1 ⁇ Antibodies (250 ⁇ g / ml).
  • FIG. 5 Secretion of IL-2 by the Jurkat-CDl ⁇ cell induced by the anti-LDL-R antibodies (EMAB604) and anti-HLA-DR CHO in the presence of the melanoma line GUY 17.2.
  • FIG. 6 Secretion of IL-2 by the Jurkat-CDl ⁇ cell induced by the anti-LDL-R ( ⁇ MAB604) and anti-HLA-DR CHO antibodies in the presence of the HT-144 melanoma line.
  • Figure 7 Percent fixation of anti-LDL-R (EMAB604), anti-CD20 produced in YB2 / 0 and Rituxan® on the CD16 receptor expressed by NK cells. The results are expressed for a concentration of 10 and 50 ⁇ g / ml of antibody.
  • SEQ ID NO: 5 murine nucleic acid sequence coding for the variable region of each of the light chains of the antibody
  • SEQ ID NO: 6 CDR1 sequence of the light chain of the antibody according to the Kabat numbering
  • SEQ ID NO: 7 murine nucleic acid sequence coding for the variable region of each of the heavy chains of the antibody
  • SEQ ID NO: 8 CDR2 sequence of the light chain of the antibody according to the Kabat numbering
  • SEQ ID NO: 9 CDR3 sequence of the light chain of the antibody according to Kabat numbering
  • SEQ ID NO: 10 peptide sequence of the variable region of each of the light chains of the antibody
  • SEQ ID NO: 11 Peptide sequence of the variable region of each of the heavy chains of
  • SEQ ID NO: 12 nucleic acid sequence corresponding to the expression vector of the light chain of the antibody
  • SEQ ID NO: 13 chimeric murine-human nucleic acid sequence coding for each of the light chains of the antibody
  • SEQ ID NO: 14 peptide sequence of each of the light chains of the antibody deduced from the acid sequence nucleic acid SEQ ID NO: 13,
  • SEQ ID NO: 15 CDR1 sequence of the light chain of the antibody according to the IMGT numbering
  • SEQ ID NO: 16 CDR2 sequence of the light chain of the antibody according to the IMGT numbering
  • SEQ ID NO: 17 CDR3 sequence of the light chain of the antibody according to the IMGT numbering
  • SEQ ID NO: 18 nucleic acid sequence corresponding to the expression vector of the heavy chain of the antibody according to the invention
  • SED ID NO: 19 nucleic acid sequence encoding the heavy chain of the antibody according to
  • SEQ ID NO: 20 Peptide sequence of the heavy chain of the antibody deduced from the SEQ ID sequence
  • SEQ ID NO: 19 nucleic acid sequence coding for the constant region of each of the light chains of the antibody
  • SEQ ID NO: 22 CDR1 sequence of the heavy chain of the antibody according to the Kabat numbering
  • SEQ ID NO: 23 human nucleic acid sequence coding for the constant region of each of the heavy chains of the ⁇ 1 type antibody
  • SEQ ID NO: 24 CDR2 sequence of the antibody heavy chain according to the numbering from Kabat
  • SEQ ID NO: 25 CDR3 sequence of the heavy chain of the antibody according to the Kabat numbering
  • SEQ ID NO: 26 CDR1 sequence of the heavy chain of the antibody according to the IMGT numbering
  • SEQ ID NO: 27 CDR2 sequence of the heavy chain of the antibody according to the IMGT numbering
  • SEQ ID NO: 28 CDR3 of the heavy chain of the antibody according to the IMGT numbering.
  • SEQ ID NO: 31 Peptide sequence of the constant region of each of the light chains of the antibody, deduced from SEQ ID NO: 21, and
  • SEQ ID NO: 34 Peptide sequence of the constant region of each of the heavy chains of the antibody deduced from SEQ ID NO: 23.
  • RNA of the C7 murine hybridoma producing an IgG2b, ⁇ -type immunoglobulin was extracted (Nucleospin RNA Macherey-Nagel kit 740609.250).
  • VK light
  • VH heavy
  • 5'RACE Rapid Amplification of cDNA Ends
  • a first reverse transcription step was first performed using a primer located in the 5 'region of murine CK OR ⁇ 2b constant regions.
  • a poly-dC sequence was then added at 3 'to the synthesized cDNAs before performing the amplification of the VK and VH regions using a 5' primer recognizing the poly-DC sequence and a 3 'primer. located in the murine CK OR ⁇ 2b constant regions 5 'of the reverse transcription primer.
  • the primers used for these two steps are as follows:
  • VH and VK PCR products thus obtained were cloned into the vector pCR4Blunt-T0P0 (Zero blunt TOPO PCR cloning kit, Invitrogen, K2875-20) and then sequenced.
  • the nucleotide sequence of the VK region of the C7 murine antibody is indicated under the sequence SEQ ID NO: 5 and the deduced peptide sequence is the sequence SEQ ID NO: 10.
  • the VK gene belongs to the V ⁇ l subgroup [Almagro JC et al Immunogenetics (1998), 47: 355-363].
  • the CDR1, CDR2 and CDR3 sequences of the VK region of the C7 murine antibody defined according to the Kabat numbering [Kabat et al. "Sequences of Proteins of Iiranunological Interest", NIH Publication, 91-3242 (1991)], are indicated under the following sequences: SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.
  • This definition different from that of Kabat based on the analysis of sequence variability alone, takes into account and combines the characterization of hypervariable loops [Chothia C. and Lesk AMJ Mol. Biol. 196: 901-17 (1987)] and structural analysis of antibodies by crystallography.
  • the nucleotide sequence of the VH region of C7 is the sequence SEQ ID NO: 7 and the peptide sequence which is deduced therefrom is the sequence SEQ ID NO: 11.
  • the VH gene belongs to the VH1 subgroup
  • CDR3 of the VH region of the murine C7 antibody defined according to Kabat numbering [Kabat et al., "Sequences of Proteins of Immunological Interest", NIH Publication, 91-3242 (1991)], are indicated under the following sequences SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 25, respectively.
  • SEQ ID NO: 26 SEQ ID NO: 27 and SEQ ID NO: 28, respectively.
  • This definition different from that of Kabat based on the analysis of sequence variability alone, takes into account and combines the characterization of hypervariable loops [Chothia C. and Lesk AMJ Mol. Biol. 196: 901-17 (1987)] and structural analysis of antibodies by crystallography.
  • VK sequence cloned into the sequencing vector pCR4Blunt-T0P0 was amplified using the following cloning primers:
  • the underlined sequence corresponds to the Spe I restriction site, the bold sequence corresponds to a Kozak consensus sequence, the initiator ATG is in italics.
  • VK antisense primer SEQ ID NO: 30
  • This primer joins the murine VK (in italic) and human constant region (CK) sequences (in bold).
  • the underlined sequence corresponds to the Dra III restriction site.
  • the VK PCR product thus obtained contains the sequence encoding the natural signal peptide of the murine C7 antibody.
  • This VK PCR was then cloned between the Spe I and Dra III sites of the 5 'light chain chimeric vector of the human CK constant region, whose nucleic sequence is the sequence SEQ ID NO: 21 and the deduced peptide sequence is the sequence SEQ ID NO: 31.
  • the human CK sequence of this chimeric vector was previously modified by silent mutagenesis to create a Dra III restriction site to allow cloning of murine VK sequences.
  • This chimerization vector contains an RSV promoter and a bGH (Growth Hormone) polyadenylation sequence as well as the dhfr (dihydrofolate reductase) selection gene
  • sequence of the light chain of the chimeric antibody EMAB604 encoded by this vector is presented in SEQ ID NO: 13 for the nucleotide sequence and corresponds to the deduced peptide sequence SEQ ID NO: 14.
  • VH sequence cloned into the pCR4Blunt-TOPO vector was first amplified using the following cloning primers:
  • VH antisense primer SEQ ID NO: 33
  • This primer joins the murine VH sequences (in italics) and the human Gl constant region (in bold).
  • the underlined sequence corresponds to the Apa I restriction site.
  • the amplified VH fragment contains the sequence encoding the natural signal peptide of the murine antibody C1.
  • This VH PCR amplification product was then cloned between the Spe I and Apa I sites of the chimeric vector containing the 5 'heavy chain.
  • the human ⁇ 1 constant region whose nucleic sequence is the sequence SEQ ID NO: 23 and the deduced peptide sequence is the sequence SEQ ID NO: 34.
  • This chimerization vector contains an RSV promoter and a bGH (Growth Hormone) bGH polyadenylation sequence as well as the neo selection gene.
  • sequence of the heavy chain of the chimeric antibody EMAB604 encoded by this vector is presented in SEQ ID NO: 19 for the nucleotide sequence and in sequence SEQ ID NO: 20 for the deduced peptide sequence.
  • Example 2 Creation of a Cell Line Derived from the YB2 / 0 Line Producing the Chimeric Antibody EMAB604
  • the rat YB2 / 0 (ATCC # CRL-1662) line was cultured in EMS medium (Invitrogen, Cat # 041-95181M) containing 5% fetal calf serum (JRH Biosciences, Cat # 12103-78P).
  • Electrobuffer medium (CeIl Projects, ref EB-110) with 25 ⁇ g of Avi II linearized K463-26-C7 light chain vector, and 26 , 7 ⁇ g of heavy chain vector H-463-27-C7 linearized with Not I.
  • the applied electroporation conditions were 230 volts and 960 microfarads for a 0.5 ml and 0.4 cm wide cuvette.
  • the contents of the electroporation cuvette were then distributed over 5 P96 plates with a density of 5000 cells / well.
  • RPMI Invitrogen, ref
  • the production of the chimeric antibody EMAB604 was carried out by expansion of the culture in EMS medium containing 5% of serum depleted in bovine Ig. (Invitrogen, ref 16250-078) and 500 ⁇ g / ml of G418 (Invitrogen, ref.10131-027), obtained by dilution with 2x10E5 cells / ml in 25 cm 2 , 75 cm 2 and 175 cm 2 flasks then roll-type bottle. After reaching maximum volume (0.9 L), the culture was continued until the cell viability was less than 50%. After production, the chimeric EMAB604 antibody was purified by protein A affinity chromatography and monitored by polyacrylamide gel electrophoresis.
  • the target cells (lines HT-144 or GUY 17.2) are incubated with different concentrations of antibodies (0, 0.1, 1, 10 ⁇ g / ml) and the effector cells (NK cells) purified by a negative depletion kit (NK CeIl Isolation Kit, Myltenyi, Paris, France) from peripheral blood of healthy donors. After 4 hours of incubation, the cytotoxic activity induced by the antibodies is measured by colorimetry by assaying in the supernatants the activity of the lactate dehydrogenase (LDH) enzyme released by the lysed cells. The results are expressed as a percentage of specific lysis as a function of the antibody concentration. The values of Emax (percentage of maximal lysis), as well as the EC50 values (amount of antibody inducing 50% of the maximum lysis), are calculated using the software PRISM (Graphpad Software).
  • EXAMPLE 4 ADCC Activity of the EMAB604 and Anti-HLA-DR Antibodies Produced by the CHO Line on the GUY 17.2 Melanoma Line (FIG.
  • the EMAB604 antibody induces a specific lysis of the GUY-17.2 line greater than that induced by an anti-HLA-DR antibody produced in CHO.
  • the maximum lysis values (Emax) are 19 and 15% for the EMAB604 and anti-HLA-DR CHO antibodies.
  • the corresponding EC50s (amount of antibody required to reach 50% of maximal lysis) are 0.45 and 5.71 ⁇ g / ml respectively, showing that the activity of the EMAB604 antibody is approximately 10 times stronger than that of the anti-HLA-DR antibody produced in CHO.
  • the ADCC activity is dependent on CD16 since it is inhibited for all the antibodies in the presence of the murine antibody 3G8 (anti-CD1 ⁇ ) (FIG.
  • EXAMPLE 5 ADCC Activity of the EMAB604 and Anti-HLA-DR Antibodies Produced by the CHO Line on the HT-144 Melanoma Line (FIG.
  • the EMAB604 antibody of the invention induces specific lysis of the HT-144 melanoma line that is greater than that induced by an anti-HLA-DR antibody produced in CHO.
  • the maximum lysis values (Emax) are 18 and 17% for the EMAB604 and anti-HLA-DR CHO antibodies.
  • the corresponding EC50s are 0.45 and 1.45 ⁇ g / ml respectively, showing that the activity of the antibody EMAB604 is approximately 8 times stronger than that of the anti-HLA-DR antibody produced in CHO.
  • the ADCC activity is dependent on CD16 since it is inhibited for all antibodies in the presence of murine antibody 3G8 (anti-CD1 ⁇ ) (FIG.
  • This test uses a Jurkat line transfected with the human CD16 receptor (FcgammaRIIIa) as an effector cell (Jurkat-CD16).
  • the technique is based on measuring the secretion of interleukin-2 (IL-2) by the Jurkat-CD16 line induced by the commitment of CD16 with the tested antibodies fixed on the target cells (melanoma lines HT-144 or GUY 17.2).
  • ng / ml of the antibodies tested are added to the target cells (1.5 ⁇ 10 5 cells / ml) in the presence of Jurkat-CD16 cells (5 ⁇ 10 6 cells / ml) and 10 ng / ml of the acetate of phorbol myristate (PMA) for 18 hours at 37 ° C. in 5% CO 2 -
  • the culture plates are then centrifuged and the IL-2 released in the culture supernatant quantified by ELISA (Quantikine IL-2, R & D, Abingdon, UK). The final result is expressed in absorbance unit (OD).
  • EXAMPLE 7 Ability of the EMAB604 and anti-HLA-DR antibodies produced by the CHO line on the GUY 17.2 melanoma line to induce the secretion of IL-2 by the Jurkat-CD16 line (FIG.
  • the EMAB604 antibody induces a secretion of IL-2 in the presence of the GUY 17.2 superior line to the same antibody produced in CHO.
  • the maximum lysis values (Emax) are 1.35 and 0.28 OD units for the EMAB604 and anti-HLA-DR CHO antibodies.
  • the R297 anti-Rhesus D antibody produced in YB2 / 0 serves as a negative control against GUY 17.2 cells.
  • EXAMPLE 8 Ability of the EMAB604 and Anti-HLA-DR Antibodies Produced by the HT-144 Melanoma Line to Induce the Secretion of IL-2 by the Jurkat-CD16 Line (FIG.
  • the EMAB604 antibody induces an IL-2 secretion in the presence of the higher HT-144 line to the same antibody produced in CHO.
  • the maximum lysis values (Emax) are 1.2 and 0.49 OD units for the EMAB604 and anti-HLA-DR CHO antibodies.
  • the R297 anti-Rhesus D antibody produced in YB2 / 0 serves as a negative control against HT-144 cells.
  • the binding to the CD1 ⁇ receptor is evaluated by a competition test of murine anti-CD1 ⁇ (3G8) antibody.
  • NK cells The effector cells (NK cells) are purified by the negative depletion kit (NK CeIl Isolation Kit, Myltenyi, Paris, France) from peripheral blood of healthy donors. Then, the NK cells are incubated with variable concentrations (0, 10 and 50 ⁇ g / ml) of antibodies to be evaluated and the anti-CD1 ⁇ (3G8) antibody coupled to a fluorochrome (3G8-PE) at a fixed concentration.
  • NK CeIl Isolation Kit Myltenyi, Paris, France
  • the EMAB604 antibody binds strongly to the CD1 ⁇ of the NK cells in a manner comparable to that of an anti-CD20 antibody produced by the EMABling platform (and described in the patent application WO 2006064121).
  • This fixation (62%) is approximately 3 times greater than that of Rituxan® (18.5%), an antibody produced in the CHO line, at a concentration of 50 ⁇ g / ml ( Figure 7).

Abstract

The invention relates to a monoclonal antibody directed against the human LDL (Low Density Lipoprotein) receptor, in which the variable region of each of the light chains is coded by the murine nucleic acid sequence SEQ ID NO : 5, the variable region of each of the heavy chains is coded by the murine nucleic acid sequence SEQ ID NO : 7, or by nucleic acid sequences having a sufficient homology with the sequences SEQ ID NO : 5 and SEQ ID NO : 7 so that the nature and the affinity of the bond between the antibody and its antigene are not modified, while the constant regions of the light chains and the heavy chains thereof are constant regions from a non-murine species.

Description

Anticorps monoclonal dirigé contre le récepteur humain des LDLMonoclonal antibody directed against the human LDL receptor
Le mélanome malin est une tumeur maligne du système pigmentaire (mélanocytes ) survenant soit primitivement en peau saine, soit par dégénérescence d'un naevus préexistant.Malignant melanoma is a malignant tumor of the pigmentary system (melanocytes) occurring either primitively in healthy skin or by degeneration of a pre-existing nevus.
Le mélanome est le cancer cutané dont la fréquence augmente le plus dans le monde. Elle double tous les 10 ans. La majorité des cas survient sur une peau saine, moins d'un quart apparaissant sur naevus préexistant .Melanoma is the most common skin cancer in the world. It doubles every 10 years. The majority of cases occur on healthy skin, with less than a quarter appearing on pre-existing nevi.
Chaque année, aux Etats-Unis, plus de 47 000 personnes sont nouvellement diagnostiquées avec un mélanome et 7 700 d'entre elles mourront de ce cancer agressif de la peau.Every year in the United States, more than 47,000 people are newly diagnosed with melanoma and 7,700 of them will die from this aggressive skin cancer.
En France, 4 000 à 5 000 cas sont découverts tous les ans, et 1 000 personnes en meurent'.In France, 4 000 to 5 000 cases are discovered each year, and 1,000 people die.
La prévention et la détection précoce du mélanome sont donc essentielles.Prevention and early detection of melanoma are therefore essential.
En effet, si des recherches sont effectuées pour traiter le mélanome, le traitement standard du mélanome repose encore sur la chirurgie. Il peut s'agir d'une exérèse initiale qui doit comporter des marges variables en fonction de 1 ' épaisseur de la tumeur, ou d'une reprise chirurgicale pour adapter les marges d'exérèse à l'épaisseur de la tumeur s'il y a besoin. Si le bilan met en évidence une ou plusieurs métastases, il faudra les traiter soit par ablation chirurgicale soit par chimiothérapie.Indeed, if research is done to treat melanoma, standard treatment of melanoma is still based on surgery. It may be an initial excision which must have variable margins depending on the thickness of the tumor, or a surgical revision to adapt the margins of excision to the thickness of the tumor if there requires. If the assessment reveals one or more metastases, it will be necessary to treat them either by surgical removal or by chemotherapy.
Les espoirs actuels dans le traitement du mélanome se tournent vers les nouvelles approches par thérapie cellulaire.Current hopes in the treatment of melanoma are turning to new approaches by cell therapy.
Le rationnel de leur utilisation repose sur deux constatations : d'une part, des régressions spontanées de mélanomes primitifs ou de métastases cutanées ont été rapportées dans la littérature, laissant supposer que le système immunitaire joue un rôle important dans le développement de cette tumeur ; d'autre part, et ce depuis quelques années, des antigènes spécifiques des cellules tumorales de mélanome ont été isolés .The rational of their use is based on two observations: on the one hand, spontaneous regressions primary melanomas or cutaneous metastases have been reported in the literature, suggesting that the immune system plays an important role in the development of this tumor; on the other hand, and for some years now, antigens specific for melanoma tumor cells have been isolated.
La thérapie cellulaire appliquée au mélanome fait appel à 2 types de traitement : l'immunothérapie adoptive, processus consistant à administrer à un patient des médicaments dotés d'une « activité antitumorale » afin de stimuler son système immunitaire et d'aider son corps à combattre plus efficacement le cancer, par «TIL» ou (Tumeur Infiltrante Lymphocytaire - Tumor Infiltrating Lymphocyte), et l'immunothérapie active représentée par la vaccination.Cell therapy for melanoma involves two types of treatment: adoptive immunotherapy, the process of administering to a patient drugs with "antitumor activity" to stimulate the immune system and help the body fight. more effectively, by "TIL" or (Tumor Infiltrating Lymphocyte), and active immunotherapy represented by vaccination.
L'immunothérapie adoptive par TIL consiste à injecter au malade une grande quantité (plusieurs milliards) de cellules T cytotoxiques , spécifiques des antigènes de mélanome, qui sont isolées à partir d'une tumeur ou métastase et expandues in vitro dans un laboratoire GMP (Good Manufacturing Practices) . On est donc dans un système autologue. Les premières études cliniques réalisées aux USA avec les TIL au stade métastatique du mélanome ont obtenu un taux de réponse de l'ordre de 35 %, mais souvent avec des rechutes rapides , amenant à discuter l'abandon de cette approche par immunothérapie passive.Adoptive immunotherapy with TIL consists in injecting the patient with a large quantity (several billion) of cytotoxic T cells specific for melanoma antigens, which are isolated from a tumor or metastasis and expanded in vitro in a GMP laboratory (Good Manufacturing Practices). So we are in an autologous system. The first clinical studies carried out in the USA with metastatic melanoma TILs achieved a response rate of around 35%, but often with rapid relapses, leading to discuss the abandonment of this passive immunotherapy approach.
Toutefois, la notion d'efficacité sur une masse tumorale «résiduelle», évoquée avec les traitements par cytokines, notamment interféron alpha, a amené à se poser la question de son application aux TIL.However, the notion of efficacy on a "residual" tumor mass, evoked with cytokine treatments, notably interferon alpha, has raised the question of its application to TIL.
Parallèlement à cette approche par TIL, est développée l'approche par injection de clones dirigés contre des antigènes de mélanome, essentiellement Melan-A et tyrosinase. Des résultats préliminaires au stade métastatique ont été obtenus, mais il a surtout été démontré que les clones injectés chez un patient pouvaient d'une part être retrouvés au site métastatique, et d'autre part qu'ils étaient amplifiés chez les patients après injection.Parallel to this approach by TIL, is developed the approach by injection of directed clones against melanoma antigens, primarily Melan-A and tyrosinase. Preliminary results at the metastatic stage were obtained, but it was mainly demonstrated that the clones injected into a patient could be found on the one hand at the metastatic site, and on the other hand that they were amplified in the patients after injection.
L'immunothérapie active par vaccination vise soit à injecter des lysats de cellules mélaniques irradiées en sous-cutané (approche multi antigènes) , soit à injecter des peptides spécifiques obtenus après identification de certains antigènes du mélanome dans un contexte HLA restreint. L'objectif est ainsi d'induire une réponse immunitaire spécifique anti- mélanome.Active immunotherapy by vaccination aims either to inject lysates of irradiated melanic cells subcutaneously (multi antigen approach), or to inject specific peptides obtained after identification of certain melanoma antigens in a restricted HLA context. The objective is thus to induce a specific anti-melanoma immune response.
Aujourd'hui, on peut schématiquement distinguer 4 générations de vaccins dans le mélanome. La première génération est celle des vaccins multi-antigènes . Ils sont constitués par le broyât de cellules tumorales irradiées. Ils ont ainsi l'avantage d'être constitués de plusieurs antigènes de tumeurs ce qui augmente les chances de pouvoir a priori correspondre à l'une des populations T cytotoxique présentées par le malade.Today, we can schematically distinguish four generations of vaccines in melanoma. The first generation is that of multi-antigen vaccines. They are constituted by the crushed irradiated tumor cells. They thus have the advantage of being composed of several tumor antigens, which increases the chances of being able, a priori, to correspond to one of the cytotoxic T populations presented by the patient.
Les antigènes de tumeur du mélanome sont : soit des antigènes de différenciation mélanocytaire : tyrosinase, gp 100, Melan-A /MART-I, gp 75 ; soit des antigènes spécifiques de tumeur (antigènes embryonnaires) : «Melanoma Associated Antigen» MAA : Mage-1, Mage-2, Mage-3, Bage, Gage-1, Gage-2, Muc-1, Rage-1, NA-17. En revanche, ces antigènes sont généralement présents en petites quantités, ce qui en limite l'action activatrice. Ces broyats tumoraux, injectés au malade en sous-cutané ou intradermique, proviennent soit du malade lui-même (système autologue) , soit d'une lignée tumorale allogénique. L'action stimulatrice du broyât tumoral sur les cellules lymphocytaires T peut être intensifiée en ajoutant à ce dernier un adjuvant immunitaire. Ainsi, en tant qu'adjuvants, ont été proposés le BCG, Detox, le QS-21, et le MF-59.The melanoma tumor antigens are: melanocyte differentiation antigens: tyrosinase, gp 100, Melan-A / MART-I, gp 75; or tumor-specific antigens (embryonic antigens): "Melanoma Associated Antigen" MAA: Mage-1, Mage-2, Mage-3, Bage, Gage-1, Gage-2, Muc-1, Rage-1, NA- 17. On the other hand, these antigens are generally present in small quantities, which limits their activating action. These tumor tumors, injected into the patient subcutaneously or intradermally, come either from the patient himself (autologous system) or from an allogeneic tumor line. The stimulating action of the tumbler on the T lymphocyte cells can be intensified by adding to the latter an immune adjuvant. Thus, as adjuvants, BCG, Detox, QS-21, and MF-59 have been proposed.
La deuxième génération est celle des vaccins antigènes spécifiques, qui reposent sur le principe de l'injection d'un seul antigène de tumeur au malade. Ils induisent de ce fait une importante activation lymphocytaire T cytotoxique. En revanche, leur utilisation est soumise à deux conditions : d'une part, une restriction HLA (HLA-Al pour Mage, HLA-A2 pour NA-17, Melan-A) ; d'autre part, l'expression par la tumeur ou la métastase, de l'antigène correspondant au peptide que 1 ' on veut inj ecter .The second generation is that of specific antigen vaccines, which rely on the principle of injecting a single tumor antigen to the patient. They induce an important cytotoxic T lymphocyte activation. On the other hand, their use is subject to two conditions: on the one hand, an HLA restriction (HLA-Al for Mage, HLA-A2 for NA-17, Melan-A); on the other hand, the expression by the tumor or the metastasis of the antigen corresponding to the peptide that one wants to inject.
Il faut donc que la lésion soit accessible à une biopsie qui permet d'identifier par PCR la présence ou non de l'antigène. Ceci bien sûr limite les possibilités de faire bénéficier un malade d'un vaccin. Jusqu'à ce jour, les protocoles cliniques ont essentiellement été réalisés avec les peptides Mage-3 , Mage-1, Melan-A/Mart-1, NA-17, tyrosinase et NY-SOl.It is therefore necessary that the lesion be accessible to a biopsy which makes it possible to identify by PCR the presence or absence of the antigen. This of course limits the possibilities of providing a patient with a vaccine. To date, clinical protocols have essentially been performed with the Mage-3, Mage-1, Melan-A / Mart-1, NA-17, tyrosinase and NY-SO1 peptides.
La troisième génération de vaccin est l'utilisation de cellules dendritiques dans un but de vaccination dans le mélanome, et repose sur le fait que ces cellules sont d'excellentes cellules présentatrices d'antigènes. Elles sont capables d' internaliser des antigènes et de les présenter aux lymphocytes T dans un contexte HLA de classe II pour les lymphocytes CD4+ et HLA de classe I pour les lymphocytes cytotoxiqu.es . Cette activation du lymphocyte T fait par ailleurs intervenir des molécules co-stimulatrices CD40-CD40L et B7-CD28. Le principe de la mise au point du traitement repose sur la culture in vitro de cellules CD34+ ou de monocytes isolés à partir du sang du malade (cytaphérèse) en présence de cytokines comme le GM-CSF (Granulocyte-The third generation of vaccine is the use of dendritic cells for the purpose of vaccination in melanoma, and is based on the fact that these cells are excellent antigen-presenting cells. They are capable of internalizing antigens and presenting them to T cells in a class II HLA context for CD4 + and HLA class I lymphocytes for cytotoxic lymphocytes. This activation of the T lymphocyte also involves the co-stimulatory molecules CD40-CD40L and B7-CD28. The principle of the development of the treatment is based on in vitro culture of CD34 + cells or monocytes isolated from the patient's blood (cytapheresis) in the presence of cytokines such as GM-CSF (Granulocyte-
Macrophage Colony-Stimulating Factor et l'IL-13Macrophage Colony-Stimulating Factor and IL-13
(Interleukine-13) . Les cellules dendritiques différenciées obtenues sont ensuite chargées in vitro avec un ou plusieurs peptideε . Ces cellules dendritiques, dites chargées, deviennent alors d'excellentes cellules activatrices des lymphocytes T cytotoxiques spécifiques du peptide lorsqu'elles sont réinjectées au malade, en intradermique, sous-cutané et intra-ganglionnaire.(Interleukin-13). The differentiated dendritic cells obtained are then loaded in vitro with one or more peptides. These dendritic cells, said to be loaded, then become excellent cytotoxic T cells activating the peptide-specific cells when they are reinjected into the patient, intradermally, subcutaneously and intra-ganglionally.
La quatrième génération de vaccin est celle des cellules tumorales modifiées .The fourth generation of vaccine is that of the modified tumor cells.
La cellule tumorale mélanique contourne le système immunitaire en produisant des cytokines immunosuppressives , en masquant ses antigènes de tumeur, en n'exprimant pas les molécules de co- activation ou les antigènes de classe I ou II. La réponse cytotoxique T se trouve de ce fait inhibée. Le principe du vaccin par cellule tumorale consiste à injecter au malade en sous-cutané ou intradermique des cellules tumorales autologues irradiées dont le profil phénotypique a été modifié pour la rendre accessible aux lymphocytes T cytotoxiques. C'est ainsi que les cellules tumorales peuvent être transfectées par : soit des cytokines IL-7, IL-2 et IL-12 ; soit le GM- CSF qui active les macrophages ; soit des antigènes de classe I, II, l'antigène BL7 permettant ainsi la reconnaissance des antigènes de tumeur par la cellule cytotoxique.The melanoma tumor cell bypasses the immune system by producing immunosuppressive cytokines, masking its tumor antigens, by not expressing co-activation molecules or class I or II antigens. The cytotoxic T response is thereby inhibited. The principle of the tumor cell vaccine is to inject subcutaneous or intradermal patients with irradiated autologous tumor cells whose phenotypic profile has been modified to make it accessible to cytotoxic T lymphocytes. Thus, the tumor cells can be transfected with: either IL-7, IL-2 and IL-12 cytokines; either GM-CSF that activates macrophages; or class I, II antigens, BL7 antigen thus allowing the recognition of tumor antigens by the cytotoxic cell.
Les études cliniques (phases I-II) réalisées avec ces différents vaccins de première et deuxième génération, au stade métastatique, portent sur un nombre limité de patients avec actuellement un taux de réponse moyen de l'ordre de 20 %. Ces réponses sont surtout obtenues sur des sites cutanés, ganglionnaires pulmonaires et hépatiques . Fait intéressant et spécifique à la vaccination, des durées de réponse prolongée (supérieures à 2 ans) ont été notées. Un point important est le délai de la réponse clinique. En effet, contrairement aux chimiothérapies, la régression clinique n'apparaît le plus souvent qu'après une phase de stabilisation pouvant durer 3 à 4 mois, voire plus.The clinical studies (phases I-II) carried out with these different vaccines of first and second generation, at the metastatic stage, relate to a limited number of patients with currently an average response rate of the order of 20%. These responses are mainly obtained on cutaneous sites, lymph nodes pulmonary and hepatic Interestingly, and specific to vaccination, prolonged response times (greater than 2 years) were noted. An important point is the delay of the clinical response. Indeed, unlike chemotherapy, the clinical regression most often appears after a stabilization phase that can last for 3 to 4 months or more.
La tolérance est en général bonne. Les effets secondaires notés étant essentiellement un érythème au site d'injection, des réactions auto-immunes de type vitiligo.Tolerance is generally good. The side effects noted being essentially erythema at the injection site, autoimmune reactions of the vitiligo type.
Toutefois, le faible taux de réponse moyen des thérapies actuelles implique un besoin important pour de nouveaux outils permettant d'élargir la gamme de traitement des mélanomes malins .However, the low average response rate of current therapies implies a significant need for new tools to expand the range of treatment for malignant melanoma.
C'est dans le but de répondre à ce problème technique que la Demanderesse a mis au point un anticorps monoclonal, fragment d'anticorps monoclonal ou dérivé d'anticorps monoclonal, dirigé contre le récepteur humain des LDL (Low Density Lipoprotein) , dont la région variable de chacune des chaînes légères est codée par la séquence d'acide nucléique murine SEQ ID NO : 5, la région variable de chacune des chaînes lourdes est codée par la séquence d'acide nucléique murine SEQ ID NO : 7, ou par des séquences d'acide nucléique présentant une homologie suffisante avec les séquences SEQ ID NO : 5 et SEQ ID NO : 7 pour que la nature et l'affinité de la liaison audit anticorps à son antigène ne soient pas modifiées, et dont les régions constantes de ses chaînes légères et de ses chaînes lourdes sont des régions constantes provenant d'une espèce non-murine. Les anticorps sont constitués de chaînes lourdes et de chaînes légères, liées entre elles par des ponts disulfures. Chaque chaîne est constituée, en position N-terminale, d'une région (ou domaine) variable (codée par les gènes réarrangés V-J pour les chaînes légères et V-D-J pour les chaînes lourdes) spécifique de l'antigène contre lequel l'anticorps est dirigé, et en position C-terminale, d'une région constante, constituée d'un seul domaine CL pour les chaînes légères ou de plusieurs domaines pour les chaînes lourdes .In order to answer this technical problem, the Applicant has developed a monoclonal antibody, a fragment of monoclonal antibody or monoclonal antibody derivative, directed against the human LDL (Low Density Lipoprotein) receptor. variable region of each of the light chains is encoded by the murine nucleic acid sequence SEQ ID NO: 5, the variable region of each of the heavy chains is encoded by the murine nucleic acid sequence SEQ ID NO: 7, or by nucleic acid sequences having sufficient homology with the sequences SEQ ID NO: 5 and SEQ ID NO: 7 so that the nature and affinity of the binding to said antibody to its antigen are not modified, and whose constant regions of its light chains and heavy chains are constant regions from a non-murine species. The antibodies consist of heavy chains and light chains, linked together by disulfide bridges. Each chain consists, in the N-terminal position, of a variable region (or domain) (encoded by the VJ rearranged genes for the light chains and VDJ for the heavy chains) specific for the antigen against which the antibody is directed. and in the C-terminal position of a constant region consisting of a single CL domain for the light chains or several domains for the heavy chains.
Les deux chaînes lourdes (H, Heavy) et les deux chaînes légères (L, Light) sont identiques entre elles. La chaîne légère est composée de 2 domaines, un domaine variable V et un domaine constant C, repliés indépendamment l'un de l'autre dans l'espace. On les appelle VL et CL. La chaîne lourde comporte également un domaine V noté VH et 3 ou 4 domaines C noté de CHl à CH4. Chaque domaine comprend environ 110 acides aminés et est structuré de manière comparable. Les 2 chaînes lourdes sont liées par des ponts disulfures et chaque chaîne lourde est liée à une chaîne légère par un pont disulfure également.The two heavy chains (H, Heavy) and the two light chains (L, Light) are identical to each other. The light chain is composed of 2 domains, a variable domain V and a constant domain C, folded independently of each other in space. They are called VL and CL. The heavy chain also comprises a V domain denoted VH and 3 or 4 C domains denoted from CH1 to CH4. Each domain comprises about 110 amino acids and is structurally comparable. The 2 heavy chains are linked by disulfide bridges and each heavy chain is linked to a light chain by a disulfide bridge as well.
La région qui détermine la spécificité de l'anticorps pour l'antigène est portée par les parties variables, alors que les parties constantes peuvent interagir avec les récepteurs Fc des cellules effectrices ou des molécules comme le complément pour médier différentes propriétés fonctionnelles. Aux fins de l'invention, les expressions « anticorps monoclonal » ou « composition d'anticorps monoclonal » se réfèrent à une préparation de molécules d'anticorps possédant une spécificité identique et unique. En outre, dans l'ensemble de la description, des revendications et des figures de la présente demande, on entend par « anticorps monoclonal », un anticorps monoclonal complet, un fragment ou un dérivé d'un tel anticorps.The region that determines the specificity of the antibody for the antigen is carried by the variable parts, whereas the constant parts can interact with the Fc receptors of the effector cells or molecules as complement to mediate different functional properties. For purposes of the invention, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to a preparation of antibody molecules having identical and unique specificity. In addition, throughout the description, claims and figures of the present application, the term "antibodies monoclonal antibody, a complete monoclonal antibody, a fragment or a derivative of such an antibody.
De manière particulièrement avantageuse, dans le cadre de l'invention, l 'homologie suffisante (pour que la nature et l'affinité de la liaison dudit anticorps à son antigène ne soient pas modifiées) correspond àParticularly advantageously, in the context of the invention, the homology sufficient (so that the nature and affinity of the binding of said antibody to its antigen are not modified) corresponds to
70%-100% d'homologie.70% -100% homology.
Selon l'invention, un premier acide nucléique ayant au moins 70 % d'homologie avec un second acide nucléique de référence, possédera au moins 90 %, de préférence au moins 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97,5%, 98%, 98,3% 98,6%, 99%, 99,6% d'identité en nucléotides avec ledit second acide nucléique de référence.According to the invention, a first nucleic acid having at least 70% homology with a second reference nucleic acid will have at least 90%, preferably at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.3% 98.6%, 99%, 99.6% identity nucleotides with said second nucleic acid. reference.
Selon l'invention, un premier polypeptide ayant au moins 70 % d'identité avec un second polypeptide de référence, possédera au moins 90 %, de préférence au moins 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97,5%, 98%, 98,3% 98,6%, 99%, 99,6% d'identité en acides aminés avec ledit second polypeptide de référence.According to the invention, a first polypeptide having at least 70% identity with a second reference polypeptide will have at least 90%, preferably at least 70%, 80%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 97.5%, 98%, 98.3% 98.6%, 99%, 99.6% amino acid identity with said second reference polypeptide.
Le « pourcentage d'homologie » entre deux séquences d'acide nucléique ou entre deux séquences de polypeptide, au sens de la présente invention, est déterminé en comparant les deux séquences alignées de manière optimale, à travers une fenêtre de comparaison.The "percentage of homology" between two nucleic acid sequences or between two polypeptide sequences, within the meaning of the present invention, is determined by comparing the two optimally aligned sequences, through a comparison window.
La partie de la séquence nucléotidique ou d'acides aminés dans la fenêtre de comparaison peut ainsi comprendre des additions ou des délétions (par exemple des « gaps ») par rapport à la séquence de référence (qui ne comprend pas ces additions ou ces délétions) de manière à obtenir un alignement optimal entre les deux séquences .The part of the nucleotide or amino acid sequence in the comparison window may thus comprise additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or deletions) in order to obtain an optimal alignment between the two sequences.
Le pourcentage d'homologie est calculé en déterminant le nombre de positions auxquelles une base nucléique identique, ou un résidu d'acide aminé identique, est observé pour les deux séquences comparées, puis en divisant le nombre de positions auxquelles il y a identité entre les deux bases nucléiques, ou entre les deux résidus d'acides aminés, par le nombre total de positions dans la fenêtre de comparaison, puis en multipliant le résultat par cent afin d'obtenir le pourcentage d'identité en nucléotides des deux séquences entre elles, ou le pourcentage d'identité en acides aminés des deux séquences entre elles.The percentage of homology is calculated by determining the number of positions at which a base same nucleic acid, or an identical amino acid residue, is observed for the two compared sequences, and then by dividing the number of positions at which there is identity between the two nucleic bases, or between the two amino acid residues, by the total number of positions in the comparison window, then multiplying the result by one hundred to obtain the percent identity nucleotides of the two sequences together, or the percent amino acid identity of the two sequences together.
L'alignement optimal des séquences pour la comparaison peut être réalisé de manière informatique à l'aide d'algorithmes connus. De manière tout à fait préférée, le pourcentage d'identité de séquence est déterminé à l'aide du logiciel CLUSTAL W (version 1.82), les paramètres étant fixés comme suit : (1) CPU MODE = ClustalW mp ;The optimal alignment of the sequences for the comparison can be performed in a computer manner using known algorithms. Most preferably, the percentage of sequence identity is determined using the CLUSTAL W software (version 1.82), the parameters being set as follows: (1) CPU MODE = ClustalW mp;
(2) ALIGNMENT = « full » ; (3) OUTPUT FORMAT = « aln w/numbers » ; (4) OUTPUT ORDER = « aligned » ; (5) COLOR ALIGNMENT = « no » ; (6) KTUP (word size) = « default » ; (7) WINDOW LENGTH = « default » ; (8) SCORE TYPE = « percent » ; (9) TOPDIAG = « default » ; (10) PAIRGAP = « default » ; (11) PHYLOGENETIC TREE/TREE TYPE = « none » ; (12) MATRIX(2) ALIGNMENT = "full"; (3) OUTPUT FORMAT = "aln w / numbers"; (4) OUTPUT ORDER = "aligned"; (5) COLOR ALIGNMENT = "no"; (6) KTUP (word size) = "default"; (7) WINDOW LENGTH = "default"; (8) SCORE TYPE = "percent"; (9) TOPDIAG = "default"; (10) PAIRGAP = "default"; (11) PHYLOGENETIC TREE / TREE TYPE = "none"; (12) MATRIX
« default » ; (13) GAP OPEN = « default » ; (14) END GAPS = « default » ; (15) GAP EXTENSION « default » ; (16) GAP DISTANCES = « default » ; (17) TREE TYPE = « cladogram » et (18) TREE GRAP DISTANCES = « hide »."Default"; (13) GAP OPEN = "default"; (14) END GAPS = "default"; (15) GAP EXTENSION "default"; (16) GAP DISTANCES = "default"; (17) TREE TYPE = "cladogram" and (18) TREE GRAP DISTANCES = "hide".
Selon l'invention, on entend par « la nature et l'affinité de la liaison dudit anticorps à son antigène ne soient pas modifiées » le fait d'une part que les anticorps présentant une homologie avec l'anticorps dont la région variable de chacune des chaînes légères est codée par la séquence SEQ ID NO : 5, et dont la région variable de chacune des chaînes lourdes est codée par la séquence SEQ ID NO : 7, se lient au même antigène, et d'autre part que leur affinité de liaison est au moins égale à 90% de l'affinité de liaison de l'anticorps dont la région variable de chacune des chaînes légères est codée par la séquence SEQ ID NO : 5, et dont la région variable de chacune des chaînes lourdes est codée par la séquence SEQ ID NO : 7.According to the invention, the term "the nature and affinity of the binding of said antibody to its antigen are not modified" is on the one hand that the antibodies having a homology with the antibody whose variable region of each light chains is encoded by the sequence SEQ ID NO: 5, and whose variable region of each of the heavy chains is encoded by the sequence SEQ ID NO: 7, bind to the same antigen, and secondly that their binding affinity is at least equal to 90% of the affinity binding of the antibody whose variable region of each of the light chains is encoded by the sequence SEQ ID NO: 5, and whose variable region of each of the heavy chains is encoded by the sequence SEQ ID NO: 7.
On entend par « séquences d'acide nucléique présentant une homologie suffisante avec les séquences SEQ ID NO : 5 et SEQ ID NO : 7 pour que la nature et l'affinité de la liaison de l'anticorps à son antigène ne soit pas modifiée » toute séquence d'acide nucléique codant pour un polypeptide, peptide ou protéine, comprenant au moins un domaine ou fragment d'immunoglobuline, ainsi que tout dérivé d'anticorps présentant une homologie suffisante avec les séquences SEQ ID NO : 5 et SEQ ID NO : 7 pour que la nature et l'affinité de la liaison de l'anticorps à son antigène ne soient pas modifiées."Nucleic acid sequences having sufficient homology with the sequences SEQ ID NO: 5 and SEQ ID NO: 7 are understood to mean that the nature and affinity of the binding of the antibody to its antigen is not modified" any nucleic acid sequence encoding a polypeptide, peptide or protein, comprising at least one immunoglobulin domain or fragment, as well as any antibody derivative having sufficient homology with the sequences SEQ ID NO: 5 and SEQ ID NO: 7 so that the nature and affinity of the binding of the antibody to its antigen are not changed.
A ce titre, on entend par « domaine d'immunoglobuline » l'un quelconque des domaines VL, CL, VH, CHl, CH2 , CH3 , CH4. L'anticorps selon 1 ' invention pouvant avantageusement contenir un ou plusieurs de ces domaines, toutes les combinaisons entre les domaines précédemment cités font partie de l ' invention. On entend par « fragment d'immunoglobuline » un des fragments choisis parmi le fragment Fab, Fab' , F(ab')2, Fc, un scFv ou un CDR (Complemantarity Determining Région) .As such, the term "immunoglobulin domain" means any of the domains VL, CL, VH, CH1, CH2, CH3, CH4. The antibody according to the invention may advantageously contain one or more of these domains, all the combinations between the aforementioned domains are part of the invention. The term "immunoglobulin fragment" is understood to mean one of the fragments chosen from the Fab, Fab ', F (ab') 2, Fc fragment, a scFv or a CDR (Complemantarity Determining Region).
La digestion enzymatique des immunoglobulines par la papaïne génère 2 fragments identiques, qu'on appelle « fragment Fab » (Fragment Antigen Binding) , et un fragment Fc (fragment cristallisable) . Le fragment Fc est le support des fonctions effectrices des immunoglobulines .The enzymatic digestion of immunoglobulins by papain generates 2 identical fragments, called "Fragment Antigen Binding", and an Fc fragment (crystallizable fragment). The Fc fragment is the support of the effector functions of immunoglobulins.
Par digestion à la pepsine, un fragment F(ab')2 est généré, où les deux fragments Fab restent liés par deux ponts disulfure, et le fragment Fc est scindé en plusieurs peptides . Le fragment F(ab')2 est formé de deux fragments Fab' , liés par des ponts disulfure intercaténaires pour former un F(ab')2.By digestion with pepsin, an F (ab ') 2 fragment is generated, where the two Fab fragments remain linked by two disulfide bridges, and the Fc fragment is split into several peptides. The F (ab ') 2 fragment is formed of two Fab' fragments, linked by interchain disulfide bridges to form an F (ab ') 2.
Quant aux régions variables des chaînes lourdes et légères, on constate que la variabilité de séquence n'est pas distribuée de manière égale. En effet, les régions variables sont constituées d'une part de régions très peu variables nommées « charpente » ouAs for the variable regions of the heavy and light chains, it is found that the sequence variability is not distributed equally. In fact, the variable regions consist on the one hand of very little variable regions called "framework" or
« framework » (FR) au nombre de 4 (FR 1 à FR4) et d'autre part de régions dans lesquelles la variabilité est extrême : il s'agit des régionsFramework (FR) 4 (FR 1 to FR4) and secondly regions in which the variability is extreme: these are the regions
« hypervariables », ou CDR, au nombre de 3 (CDRl à"Hypervariable", or CDR, 3 (CDR1 to
CDR3 ) .CDR3).
Ainsi, l'anticorps selon l'invention pouvant avantageusement contenir un ou plusieurs de ces fragments, toutes les combinaisons entre les fragments précédemment cités font partie de l'invention.Thus, the antibody according to the invention can advantageously contain one or more of these fragments, all the combinations between the aforementioned fragments are part of the invention.
Dans un aspect particulier de l'invention, l'anticorps selon l'invention contient au moins un domaine d' immunoglobuline et au moins un fragment d' immunoglobuline, par exemple un fragment Fc et une ou plusieurs régions variables ou hypervariables .In a particular aspect of the invention, the antibody according to the invention contains at least one immunoglobulin domain and at least one immunoglobulin fragment, for example an Fc fragment and one or more variable or hypervariable regions.
Enfin, on entend par « dérivé d'anticorps » tout anticorps, cet anticorps pouvant comprendre une ou plusieurs mutations, substitutions, délétions et/ou additions d'un ou plusieurs résidus d'acides aminés.Finally, "antibody derivative" is understood to mean any antibody, this antibody possibly comprising one or more mutations, substitutions, deletions and / or additions of one or more amino acid residues.
Un tel ajout, substitution ou délétion peut être localisé à n'importe quelle position dans la molécule.Such an addition, substitution or deletion may be located at any position in the molecule.
Dans le cas où plusieurs acides aminés ont été ajoutés, substitués ou délétés, toute combinaison d'ajout, de substitution ou de délétion peut être considérée, à condition que l'anticorps résultant présente toujours au moins les propriétés avantageuses de l'anticorps de l'invention.In the case where several amino acids have been added, substituted or deleted, any combination of addition, substitution or deletion may be considered, provided that the resulting antibody still has at least the advantageous properties of the antibody of the invention.
L'anticorps selon l'invention, dont les régions variables des chaînes légères et lourdes, ou au moins un domaine ou fragment de ces régions, appartiennent à une espèce différente des régions constantes des chaînes légères et des chaînes lourdes, est qualifié d'anticorps « chimérique ».The antibody according to the invention, whose variable regions of the light and heavy chains, or at least one domain or fragment of these regions, belong to a species different from the constant regions of the light and heavy chains, is called antibody. "Chimerical".
Les séquences d'acide nucléique murineε SEQ ID NO : 5 et SEQ ID NO : 7 codent pour le domaine variable de chacune des chaînes légères et le domaine variable de chacune des chaînes lourdes respectivement de l'anticorps produit par l'hybridome murin C7 , disponible à l'ATCC (American Type Culture Collection) sous le numéro CRL-1691. Cet hybridome produit un anticorps monoclonal murin d' isotype IgG2b dirigé contre le LDL-R.The murine nucleic acid sequences SEQ ID NO: 5 and SEQ ID NO: 7 encode the variable domain of each of the light chains and the variable domain of each of the heavy chains respectively of the antibody produced by the C7 murine hybridoma, available from ATCC (American Type Culture Collection) under number CRL-1691. This hybridoma produces a mouse monoclonal antibody of IgG2b isotype directed against LDL-R.
Le récepteur humain des LDL (LDL-R) est une protéine transmembranaire de 839 acides aminés qui comprend trois régions : la région extra-cellulaire (1-768), la région transmembranaire (768-790) et la région cytoplasmique (790-839). La région extracellulaire est divisée en deux sous-régions : celle de liaison des LDL (1-322) et la sous-région en dehors de la zone de liaison des LDL (323-768) .The human LDL receptor (LDL-R) is a transmembrane protein of 839 amino acids that comprises three regions: the extracellular region (1-768), the transmembrane region (768-790) and the cytoplasmic region (790-839). ). The extracellular region is divided into two subregions: the LDL binding region (1-322) and the subregion outside the LDL binding zone (323-768).
Les séquences murines de l'anticorps de l'invention ont été choisies pour coder les régions variables de l'anticorps selon l'invention, ou au moins un domaine ou fragment de ces régions , en raison de la spécificité de l'anticorps murin C7 pour l'antigène LDL-R. L'anticorps C7 a été généré par immunisation avec le domaine extracellulaire du LDL-R bovin. Il se fixe, en dehors du LDL-R bovin, au LDL-R humain mais il ne cross-réagit pas avec les LDL-R du rat, de la souris, du hamster chinois, du lapin et du chien (Beisiegel et al. 1981 J. Biol. Chem. 256, 11923-11931) . Cette propriété est avantageuse car les lignées de production d'anticorps monoclonaux sont très souvent des lignées provenant de ces espèces . Ainsi, l'anticorps recombinant peut être avantageusement produit dans une lignée cellulaire provenant des lignées YB2/0, NSO, Sp2/0, CHO, cette liste n'étant pas limitative.The murine sequences of the antibody of the invention have been chosen to encode the variable regions of the antibody according to the invention, or at least one domain or fragment of these regions, because of the specificity of the murine C7 antibody. for the LDL-R antigen. The C7 antibody was generated by immunization with the extracellular domain of bovine LDL-R. It binds, apart from bovine LDL-R, to human LDL-R but does not cross-react with LDL-R in rats, mice, Chinese hamster, rabbits and dogs (Beisiegel et al. 1981 J. Biol Chem 256, 11923-11931). This property is advantageous because Monoclonal antibody production lines are very often lines from these species. Thus, the recombinant antibody may advantageously be produced in a cell line originating from the lines YB2 / 0, NSO, Sp2 / 0, CHO, this list not being limiting.
L'anticorps de l'invention s'entend aussi des anticorps possédant des régions CDR dont la séquence peptidique est choisie parmi les séquences SEQ ID NO : 6, SEQ ID NO : 8, SEQ ID NO : 9, SEQ ID NO : 15, SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 22, SEQ ID NO : 24, SEQ ID NO : 25, SEQ ID NO : 26, SEQ ID NO : 27, SEQ ID NO : 28.The antibody of the invention also includes antibodies having CDR regions whose peptide sequence is chosen from the sequences SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28.
Ces séquences sont les séquences des régions CDR provenant de l'anticorps murin C7 , la séquence SEQ ID NO : 6 représentant la séquence du CDRl de la chaîne légère de l'anticorps selon la numérotation de Kabat, la séquence SEQ ID NO : 8 représentant la séquence du CDR2 de la chaîne légère de l'anticorps selon la numérotation de Kabat, la séquence SEQ ID NO : 9 représentant la séquence du CDR3 de la chaîne légère de l'anticorps selon la numérotation de Kabat, la séquence SEQ ID NO : 15 représentant la séquence du CDRl de la chaîne légère de l'anticorps selon la numérotation IMGT, la séquence SEQ ID NO : 16 représentant la séquence du CDR2 de la chaîne légère de l'anticorps selon la numérotation IMGT, la séquence SEQ ID NO : 17 représentant la séquence du CDR3 de la chaîne légère de l'anticorps selon la numérotation IMGT, la séquence SEQ ID NO : 22 représentant la séquence du CDRl de la chaîne lourde de l'anticorps selon la numérotation de Kabat, la séquence SEQ ID NO : 24 représentant la séquence du CDR2 de la chaîne lourde de l'anticorps selon la numérotation de Kabat, la séquence SEQ ID NO : 25 représentant la séquence du CDR3 de la chaîne lourde de l'anticorps selon la numérotation de Kabat, la séquence SEQ ID NO : 26 représentant la séquence du CDRl de la chaîne lourde de l'anticorps selon la numérotation IMGT, la séquence SEQ ID NO : 27 représentant la séquence du CDR2 de la chaîne lourde de l'anticorps selon la numérotation IMGT, la séquence SEQ ID NO : 28 représentant la séquence du CDR3 de la chaîne lourde de l'anticorps selon la numérotation IMGT.These sequences are the sequences of the CDR regions originating from the murine C7 antibody, the sequence SEQ ID NO: 6 representing the sequence of the CDR1 of the light chain of the antibody according to the Kabat numbering, the sequence SEQ ID NO: 8 representing the CDR2 sequence of the light chain of the antibody according to the Kabat numbering, the sequence SEQ ID NO: 9 representing the CDR3 sequence of the light chain of the antibody according to the Kabat numbering, the sequence SEQ ID NO: Representing the CDR1 sequence of the light chain of the antibody according to the IMGT numbering, the sequence SEQ ID NO: 16 representing the CDR2 sequence of the light chain of the antibody according to the IMGT numbering, the sequence SEQ ID NO: 17 showing the CDR3 sequence of the light chain of the antibody according to the IMGT numbering, the sequence SEQ ID NO: 22 representing the CDR1 sequence of the heavy chain of the antibody according to the e Kabat, the sequence SEQ ID NO: 24 representing the CDR2 sequence of the heavy chain of the antibody according to the Kabat numbering, the sequence SEQ ID NO: 25 representing the CDR3 sequence of the heavy chain of the antibody according to the numbering of Kabat, the sequence SEQ ID NO: 26 representing the CDR1 sequence of the heavy chain of the antibody according to the IMGT numbering, the sequence SEQ ID NO: 27 representing the CDR2 sequence of the heavy chain of the antibody according to the IMGT numbering, the sequence SEQ ID NO: 28 representing the CDR3 sequence of the heavy chain of the antibody according to the IMGT numbering.
L'invention s'entend aussi des anticorps dont le domaine variable de chacune des chaînes légères et le domaine variable de chacune des chaînes lourdes possèdent au moins 70% d'homologie, ou avantageusement au moins 80%, ou encore 90% ou 99% , avec les séquences SEQ ID NO : 5 et SEQ ID NO : 7 respectivement, les modifications de séquence ne modifiant pas la spécificité de l'anticorps. De préférence, ces modifications de séquences ne diminuent pas son affinité pour sa cible.The invention also includes antibodies whose variable domain of each of the light chains and the variable domain of each of the heavy chains have at least 70% homology, or advantageously at least 80%, or 90% or 99% , with the sequences SEQ ID NO: 5 and SEQ ID NO: 7 respectively, the sequence modifications not modifying the specificity of the antibody. Preferably, these sequence modifications do not decrease its affinity for its target.
L'anticorps selon l'invention possède en outre des régions constantes de ses chaînes légères et lourdes appartenant à une espèce non-murine. A cet égard, toutes les familles et espèces de mammifères non-murins sont susceptibles d'être utilisées, et en particulier l'homme, le singe, les muridés (sauf la souris), les suidés, les bovidés, les équidés, les félidés, les canidés, par exemple, ainsi que les oiseaux.The antibody according to the invention also has constant regions of its light and heavy chains belonging to a non-murine species. In this respect, all families and species of non-murine mammals are likely to be used, and in particular humans, monkeys, murids (except the mouse), suids, cattle, equines, felids , canids, for example, and birds.
Les anticorps selon l'invention peuvent être construits en utilisant les techniques standard de l 'ADN recombinant , bien connues de l'homme du métier, et plus particulièrement en utilisant les techniques de construction des anticorps « chimériques » décrites par exemple dans Morrison et al., Proc. Natl . Acad. Sci. U. S.A., 81, pp. 6851-55 (1984), où la technologie de 1 'ADN recombinant est utilisée pour remplacer la région constante d'une chaîne lourde et/ou la région constante d'une chaîne légère d'un anticorps provenant d'un mammifère non-humain avec les régions correspondantes d'une immunoglobuline humaine. De tels anticorps et leur mode de préparation ont également été décrits dans la demande de brevet EP 173 494, dans le document Neuberger, M. S. et al., Nature 312 (5995) : 604-8 (1985), ainsi que dans le document EP 125 023 par exemple. Des méthodes pour générer des anticorps chimériques sont largement disponibles pour l'homme du métier. Par exemple, les chaînes lourdes et légères de l'anticorps peuvent être exprimées séparément en utilisant un vecteur pour chaque chaîne, ou bien être intégrées dans un seul vecteur.The antibodies according to the invention can be constructed using standard techniques of recombinant DNA, which are well known to those skilled in the art, and more particularly by using the "chimeric" antibody construction techniques described, for example, in Morrison et al. ., Proc. Natl. Acad. Sci. USA, 81, pp. 6851-55 (1984), where recombinant DNA technology is used to replace the constant region of a heavy chain and / or the constant region of a light chain of an antibody from a non-human mammal. with the regions of a human immunoglobulin. Such antibodies and their method of preparation have also been described in patent application EP 173,494, in Neuberger, MS et al., Nature 312 (5995): 604-8 (1985), as well as in EP 125 023 for example. Methods for generating chimeric antibodies are widely available to those skilled in the art. For example, the heavy and light chains of the antibody may be expressed separately using a vector for each chain, or may be integrated into a single vector.
Un vecteur d'expression est une molécule d'acide nucléique dans laquelle la séquence d'acide nucléique murine codant pour le domaine variable de chacune des chaînes lourdes ou légères de l'anticorps et la séquence d'acide nucléique, de préférence humaine, codant pour la région constante de chacune des chaînes lourdes ou légères de l'anticorps ont été insérées, afin de les introduire et de les maintenir dans une cellule hôte. Il permet l'expression de ces fragments d'acide nucléique étrangers dans la cellule hôte car il possède des séquences indispensables (promoteur, séquence de polyadénylation, gène de sélection) à cette expression. Le vecteur peut être par exemple un plasmide, un adénovirus, un rétrovirus ou un bactériophage, et la cellule hôte peut être toute cellule de mammifère, par exemple SP2/0, YB2/0, IR983F, le myélome humain Namalwa, PER. C6, les lignées CHO, notamment CHO-K-I, CHO-LeclO, CHO-Lee1, CHO- Lecl3, CHO Pro-5, CHO dhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, BHK, K6H6, NSO, SP2/0-Ag 14 et P3X63Ag8.653.An expression vector is a nucleic acid molecule in which the murine nucleic acid sequence encoding the variable domain of each of the heavy or light chains of the antibody and the nucleic acid sequence, preferably human, encoding for the constant region of each of the heavy or light chains of the antibody were inserted, in order to introduce and maintain them in a host cell. It allows the expression of these foreign nucleic acid fragments in the host cell because it has essential sequences (promoter, polyadenylation sequence, selection gene) to this expression. The vector may be, for example, a plasmid, an adenovirus, a retrovirus or a bacteriophage, and the host cell may be any mammalian cell, for example SP2 / 0, YB2 / 0, IR983F, Namalwa human myeloma, PER. C6, CHO lines, especially CHO-KI, CHO-Lec10, CHO-Lee1, CHO-Lecl3, CHO Pro-5, CHO dhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293- HEK, BHK, K6H6, NSO, SP2 / O-Ag14 and P3X63Ag8.653.
Pour la construction de vecteurs d'expression des anticorps chimériques selon l'invention, des séquences signal synthétiques et des sites de restriction appropriés peuvent être fusionnés aux régions variables au cours des réactions d'amplification par PCR. Les régions variables sont ensuite combinées avec les régions constantes d'un anticorps, de manière préférentielle une IgGl humaine. Les gènes ainsi construits sont clones pour les placer sous le contrôle d'un promoteur, par exemple le promoteur RSV (Rous Sarcoma Virus), CMV (cytomegalovirus) , MLP (Major Late Promoter) , cette liste n'étant pas limitative, et en amont d'un site de polyadénylation, en utilisant deux vecteurs séparés (un pour chaque chaîne) . Les vecteurs sont également munis de gènes de sélection connus de l'homme du métier, tels que par exemple le gène dhfr (dihydrofolate reductase) ou le gène de résistance à la néomycine.For the construction of expression vectors of the chimeric antibodies according to the invention, appropriate synthetic signal sequences and restriction sites can be fused to the variable regions during the amplification reactions. PCR. The variable regions are then combined with the constant regions of an antibody, preferably a human IgG1. The genes thus constructed are cloned to place them under the control of a promoter, for example the RSV (Rous Sarcoma Virus), CMV (cytomegalovirus), MLP (Major Late Promoter) promoter, this list not being limiting, and in particular upstream of a polyadenylation site, using two separate vectors (one for each chain). The vectors are also provided with selection genes known to those skilled in the art, such as, for example, the dhfr (dihydrofolate reductase) gene or the neomycin resistance gene.
Les anticorps chimériques selon l'invention peuvent être produits par co-transfection dans une cellule hôte du vecteur d'expression de la chaîne légère et du vecteur d'expression de la chaîne lourde en utilisant une méthode bien connue de l'homme du métier (par exemple la co-précipitation au phosphate de calcium, l ' électroporation, la micro-injection, etc.) . A l'issue de la transfection, les cellules peuvent être mises en milieu sélectif, par exemple en milieu RPMI (Invitrogen, ref 21875-034) contenant 5% de sérum dialyse (Invitrogen, réf. 10603-017), 500 μg/ml de G418 (Invitrogen, réf. 10131-027) et 25 nM de methotrexate (Sigma, réf. M8407) . Les surnageants des puits de transfection résistants sont criblés pour la présence d' immunoglobuline (Ig) chimérique par dosage ELISA spécifique des séquences Ig humaines ou d'une autre espèce si la partie constante provient d'une autre espèce. Les transfectants produisant le plus d'anticorps sont amplifiés et leur surnageant redosé par ELISA afin d'estimer leur productivité et de sélectionner les 3 meilleurs producteurs pour le clonage par dilution limite (40 cellules / plaque) .The chimeric antibodies according to the invention may be produced by cotransfection in a host cell of the light chain expression vector and the heavy chain expression vector using a method well known to those skilled in the art ( for example calcium phosphate co-precipitation, electroporation, microinjection, etc.). At the end of the transfection, the cells can be put in a selective medium, for example in RPMI medium (Invitrogen, ref 21875-034) containing 5% dialysis serum (Invitrogen, ref 10603-017), 500 μg / ml G418 (Invitrogen, P / N 10131-027) and 25 nM methotrexate (Sigma, M8407). The supernatants of the resistant transfection wells are screened for the presence of chimeric immunoglobulin (Ig) by ELISA assay specific for human Ig or other species if the constant part is from another species. The transfectants producing the most antibodies are amplified and their supernatants redosed by ELISA in order to estimate their productivity and to select the 3 best producers for limiting dilution cloning (40 cells / plate).
Un mode de réalisation particulier sera illustré plus loin. Préférentiellement , la région variable de chacune des chaînes légères de l ' anticorps selon l ' invention possède la séquence peptidique SEQ ID NO : 10 et la région variable de chacune des chaînes lourdes de l'anticorps selon l'invention possède la séquence peptidique SEQ ID NO : 11. La séquence peptidique SEQ ID NO : 10 est la séquence peptidique déduite de la séquence nucléotidique SEQ ID NO : 5 et la séquence peptidique SEQ ID NO : 11 est la séquence déduite de la séquence nucléotidique SEQ ID NO : 7.A particular embodiment will be illustrated below. Preferably, the variable region of each of the light chains of the antibody according to the invention has the peptide sequence SEQ ID NO: 10 and the variable region of each of the heavy chains of the antibody according to the invention has the peptide sequence SEQ ID NO: 11. The peptide sequence SEQ ID NO: 10 is the peptide sequence deduced from the nucleotide sequence SEQ ID NO: 5 and the peptide sequence SEQ ID NO: 11 is the sequence deduced from the nucleotide sequence SEQ ID NO: 7.
De manière préférée, les régions constantes de chacune des chaînes légères et de chacune des chaînes lourdes de l'anticorps selon l'invention sont des régions constantes humaines . Ce mode de réalisation préféré de l ' invention permet de diminuer l'immunogénicité de l'anticorps chez l'homme et par là même d'améliorer son efficacité lors de son administration thérapeutique ou prophylactique chez l ' homme .Preferably, the constant regions of each of the light chains and of each of the heavy chains of the antibody according to the invention are human constant regions. This preferred embodiment of the invention makes it possible to reduce the immunogenicity of the antibody in humans and thereby improve its effectiveness during its therapeutic or prophylactic administration in humans.
Dans un mode de réalisation préféré de l'invention, la région constante de chacune des chaînes légères de l'anticorps selon l'invention est de type K. Tout allotype convient à la réalisation de l'invention, par exemple Km(I), Km(I, 2), Km(I, 2, 3) ou Km (3 ) .In a preferred embodiment of the invention, the constant region of each of the light chains of the antibody according to the invention is of type K. Any allotype is suitable for carrying out the invention, for example Km (I), Km (I, 2), Km (I, 2, 3) or Km (3).
Dans un autre mode de réalisation de l'invention, la région constante de chacune des chaînes légères de l'anticorps selon l'invention est de type λ.In another embodiment of the invention, the constant region of each of the light chains of the antibody according to the invention is of type λ.
Dans un aspect particulier de l'invention, et notamment lorsque les régions constantes de chacune des chaînes légères et de chacune des chaînes lourdes de l'anticorps selon l'invention sont des régions humaines, la région constante de chacune des chaînes lourdes de l'anticorps est de type γ. Selon cette variante, la région constante de chacune des chaînes lourdes de l'anticorps peut être de type γl, de type γ2, de type γ3, ces trois types de régions constantes présentant la particularité de fixer le complément humain, ou encore de type γ4. Les anticorps possédant une région constante de chacune des chaînes lourdes de type Y appartiennent à la classe des IgG. Les immunoglobulines de type G (IgG) ; sont des hétérodimères constitués de deux chaînes lourdes et de deux chaînes légères, liées entre elles par des ponts disulfures. Chaque chaîne est constituée, en position N-terminale, d'une région ou domaine variable (codée par les gènes réarrangés V-J pour la chaîne légère et V-D-J pour la chaîne lourde) spécifique de l'antigène contre lequel l'anticorps est dirigé, et en position C-terminale, d'une région constante, constituée d'un seul domaine CL pour la chaîne légère ou de 3 domaines (CHl, CH2 et CH3 ) pour la chaîne lourde. L'association des domaines variables VH et VL et des domaines constantes CHi et CL des chaînes lourdes et légères forme les parties Fab, qui sont connectées à la région Fc par une région charnière très flexible permettant à chaque Fab de se fixer à sa cible antigénique tandis que la région Fc, médiatrice des propriétés effectrices de l'anticorps reste accessible aux molécules effectrices telles que les récepteurs FcγR, le récepteur Fc néonatal (FcRn) et le CIq. La région Fc, constituée des 2 domaines globulaires Cγ2 et Cγ3, est glycosylée au niveau du domaine Cγ2 avec la présence, sur chacune des 2 chaînes, d'un Λ7-glycanne biantenné, lié à l'Asn 297.In a particular aspect of the invention, and especially when the constant regions of each of the light chains and of each of the heavy chains of the antibody according to the invention are human regions, the constant region of each of the heavy chains of the antibody is of type γ. According to this variant, the constant region of each of the chains The heavy of the antibody can be of type γ1, of type γ2, of type γ3, these three types of constant regions having the particularity of fixing the human complement, or of type γ4. Antibodies possessing a constant region of each of the heavy Y-type chains belong to the IgG class. Immunoglobulin type G (IgG); are heterodimers consisting of two heavy chains and two light chains, linked together by disulfide bridges. Each chain consists, in the N-terminal position, of a region or variable domain (encoded by the rearranged genes VJ for the light chain and VDJ for the heavy chain) specific for the antigen against which the antibody is directed, and in the C-terminal position, a constant region consisting of a single CL domain for the light chain or 3 domains (CH1, CH2 and CH3) for the heavy chain. The association of the variable domains V H and V L and of the constant domains CH 1 and CL of the heavy and light chains forms the Fab parts, which are connected to the Fc region by a very flexible hinge region allowing each Fab to be fixed to its antigenic target while the Fc region, mediator of the effector properties of the antibody remains accessible to effector molecules such as FcγR receptors, neonatal Fc receptor (FcRn) and CIq. The Fc region, consisting of the 2 globular domains Cγ 2 and Cγ 3 , is glycosylated at the Cγ2 domain with the presence, on each of the 2 chains, of a biantennary Λ7-glycan linked to Asn 297.
De manière préférée, la région constante de chacune des chaînes lourdes de l'anticorps est de type γl, car un tel anticorps montre une capacité à engendrer une activité ADCC (Antibody-Dependent Cellular Cytotoxicity) chez le plus grand nombre d'individus (humains). A cet égard, tout allotype convient à la réalisation de l'invention, par exemple GIm(3), GIm (1, 2, 17), GIm(I, 17) ou GIm(1,3).Preferably, the constant region of each of the heavy chains of the antibody is of γ1 type, since such an antibody shows an ability to generate ADCC (Antibody-Dependent Cellular Cytotoxicity) activity in the largest number of individuals (human ). In this respect, any allotype is suitable for carrying out the invention, for example GIm (3), GIm (1, 2, 17), GIm (I, 17) or GIm (1,3).
Dans un aspect particulier de l'invention, la région constante de chacune des chaînes lourdes de l'anticorps est de type γl, et elle est codée par la séquence d'acide nucléique humaine SEQ ID NO : 23, la région constante de chacune de ses chaînes légères étant codée par la séquence d'acide nucléique humaine SEQ ID NO : 21, ou une séquence homologue à la séquence SEQ ID NO : 23 ou SEQ ID NO : 21, ou un fragment desdites séquences. Ainsi, un tel anticorps possède une région variable murine et une région constante humaine, avec des chaînes lourdes de type γl . Cet anticorps appartient donc à la sous-classe des IgGl . Cet anticorps possède deux chaînes légères dont le domaine variable est codé par la séquence d'acide nucléique murine SEQ ID NO : 5 et la région constante humaine est codée par la séquence d'acide nucléique SEQ ID NO : 21, et deux chaînes lourdes dont le domaine variable est codé par la séquence d'acide nucléique murine SEQ ID NO : 7 et la région constante est codée par la séquence d'acide nucléique humaine SEQ ID NO : 23.In a particular aspect of the invention, the constant region of each of the heavy chains of the antibody is of γ1 type, and it is encoded by the human nucleic acid sequence SEQ ID NO: 23, the constant region of each of its light chains being encoded by the human nucleic acid sequence SEQ ID NO: 21, or a sequence homologous to the sequence SEQ ID NO: 23 or SEQ ID NO: 21, or a fragment of said sequences. Thus, such an antibody has a murine variable region and a human constant region, with heavy chains of γ1 type. This antibody therefore belongs to the subclass of IgG1. This antibody has two light chains whose variable domain is encoded by the murine nucleic acid sequence SEQ ID NO: 5 and the human constant region is encoded by the nucleic acid sequence SEQ ID NO: 21, and two heavy chains of which the variable domain is encoded by the murine nucleic acid sequence SEQ ID NO: 7 and the constant region is encoded by the human nucleic acid sequence SEQ ID NO: 23.
De manière préférentielle, chacune des chaînes légères de l'anticorps selon l'invention est codée par la séquence d'acide nucléique chimère murine-humaine SEQ ID NO : 13 , et chacune des chaînes lourdes est codée par la séquence d'acide nucléique chimère murine-humaine SEQ ID NO : 19. La séquence d'acide nucléique chimère murine-humaine SEQ ID NO : 13 codant pour chacune des chaînes légères de l'anticorps est obtenue par fusion de la séquence d'acide nucléique murine SEQ ID NO : 5 codant pour le domaine variable de chacune des chaînes légères de l'anticorps et de la séquence d'acide nucléique humaine SEQ ID NO : 21 codant pour la région constante de chacune des chaînes légères de l'anticorps. La séquence d'acide nucléique chimère murine-humaine SEQ ID NO : 19 codant pour chacune des chaînes lourdes de l'anticorps est obtenue par fusion de la séquence d'acide nucléique murine SEQ ID NO : 7 codant pour le domaine variable de chacune des chaînes lourdes de l'anticorps et de la séquence d'acide nucléique humaine SEQ ID NO : 23 codant pour la région constante de chacune des chaînes lourdes de l'anticorps.Preferably, each of the light chains of the antibody according to the invention is encoded by the murine-human chimeric nucleic acid sequence SEQ ID NO: 13, and each of the heavy chains is encoded by the chimeric nucleic acid sequence. murine-human SEQ ID NO: 19. The murine-human chimeric nucleic acid sequence SEQ ID NO: 13 encoding each of the light chains of the antibody is obtained by fusion of the murine nucleic acid sequence SEQ ID NO: 5 coding for the variable domain of each of the light chains of the antibody and the human nucleic acid sequence SEQ ID NO: 21 coding for the constant region of each of the light chains of the antibody. The murine-human chimeric nucleic acid sequence SEQ ID NO: 19 coding for each of the heavy chains of the antibody is obtained by melting the murine nucleic acid sequence SEQ ID NO: 7 coding for the variable domain of each of the heavy chains of the antibody and the human nucleic acid sequence SEQ ID NO: 23 encoding the constant region of each of the heavy chains of the antibody.
Dans un tel aspect particulier de l'invention, lorsque chacune des chaînes légères de l'anticorps est codée par la séquence d'acide nucléique chimère murine-humaine SEQ ID NO : 13 , et que chacune des chaînes lourdes est codée par la séquence d'acide nucléique chimère murine-humaine SEQ ID NO : 19, la séquence peptidique de chacune des chaînes légères, déduite de la séquence d'acide nucléique SEQ ID NO : 13, est la séquence SEQ ID NO : 14 et la séquence peptidique de chacune des chaînes lourdes, déduite de la séquence d'acide nucléique SEQ ID NO : 19, est la séquence SEQ ID NO : 20.In such a particular aspect of the invention, when each of the light chains of the antibody is encoded by the murine-human chimeric nucleic acid sequence SEQ ID NO: 13, and each of the heavy chains is encoded by the sequence of murine-human chimeric nucleic acid SEQ ID NO: 19, the peptide sequence of each of the light chains, deduced from the nucleic acid sequence SEQ ID NO: 13, is the sequence SEQ ID NO: 14 and the peptide sequence of each heavy chains, deduced from the nucleic acid sequence SEQ ID NO: 19, is the sequence SEQ ID NO: 20.
L'invention s'entend aussi des anticorps dont chacune des chaînes légères codées par une séquence d'acide nucléique chimère murine-humaine possède au moins 70% d'homologie avec la séquence d'acide nucléique chimère murine-humaine SEQ ID NO : 13 et chacune des chaînes lourdes codées par une séquence d'acide nucléique chimère murine-humaine possède au moins 70% d'homologie, ou avantageusement au moins 80%, ou encore 90% ou 99%, avec la séquence d'acide nucléique chimère murine-humaine SEQ ID NO : 19, ces modifications n'altérant ni la spécificité de l'anticorps ni ses activités effectrices.The invention also includes antibodies each of which has light chains encoded by a murine-human chimeric nucleic acid sequence having at least 70% homology to the murine-human chimeric nucleic acid sequence SEQ ID NO: 13 and each of the heavy chains encoded by a murine-human chimeric nucleic acid sequence has at least 70% homology, or preferably at least 80%, or 90% or 99%, with the murine chimeric nucleic acid sequence. -human SEQ ID NO: 19, these modifications not altering the specificity of the antibody nor its effector activities.
De manière avantageuse, l'anticorps selon l'invention est couplé à une toxine. La toxine est, par exemple, la toxine diphtérique ou la ricine, cette liste n'étant pas limitative. La liaison entre l'anticorps selon l'invention et la toxine est suffisamment forte pour éviter la libération systémique de la toxine et aussi suffisamment labile, afin que la toxine soit libérée dans les cellules cibles .Advantageously, the antibody according to the invention is coupled to a toxin. The toxin is, for example, diphtheria toxin or ricin, this list not being limiting. The binding between the antibody according to the invention and the toxin is sufficiently strong to prevent the systemic release of the toxin and also sufficiently labile, so that the toxin is released into the target cells.
Dans un autre aspect de l'invention, l'anticorps est couplé à un radio-isotope. La présence du radio- isotope accroît considérablement la cytotoxicité. Deux isotopes sont essentiellement utilisés : l'iode 131 (émetteur bêta et gamma) , dont la demi-vie est relativement longue (8 jours) et qui exerce un effet tumoricide sur environ 1 mm autour de la cellule tumorale ayant fixé l'anticorps selon l'invention.In another aspect of the invention, the antibody is coupled to a radioisotope. The presence of the radioisotope greatly increases the cytotoxicity. Two isotopes are mainly used: iodine-131 (beta and gamma emitter), whose half-life is relatively long (8 days) and which exerts a tumoricidal effect on about 1 mm around the tumor cell that has fixed the antibody according to the invention.
L'iode 131 présente l'avantage de rendre possible la réalisation d'une imagerie, mais nécessite le respect des mesures de radio-protection. L'yttrium 90Iodine 131 has the advantage of making it possible to perform imaging, but requires compliance with radiation protection measures. Yttrium 90
(émetteur bêta), dont la demi-vie est plus brève (2,5 jours) , exerce des effets tumoricides sur une distance de 5 mm.(beta emitter), whose half-life is shorter (2.5 days), exerts tumoricidal effects over a distance of 5 mm.
De manière avantageuse, l'anticorps de l'invention est produit dans une lignée cellulaire d'hybridome de rat. La lignée productrice de l'anticorps selon l'invention est une caractéristique importante puisqu'elle confère à l'anticorps certaines de ses propriétés particulières. En effet, le moyen d'expression des anticorps est à l'origine des modifications post-traductionnelles, notamment des modifications de la glycosylation, qui peuvent varier d'une lignée cellulaire à l'autre, et ainsi conférer des propriétés fonctionnelles différentes à des anticorps ayant pourtant des séquences primaires identiques . Dans un certain mode de réalisation, l'anticorps est produit dans la lignée de rat YB2/0 (cellule YB2/3HL.P2.GH.16Ag.2O, déposée à l 'American Type Culture Collection sous le numéro ATCC CRL-1662) .Advantageously, the antibody of the invention is produced in a rat hybridoma cell line. The line producing the antibody according to the invention is an important characteristic since it confers on the antibody some of its particular properties. Indeed, the means of expression of the antibodies is at the origin of the post-translational modifications, in particular modifications of the glycosylation, which may vary from one cell line to another, and thus confer different functional properties on antibodies having identical primary sequences. In one embodiment, the antibody is produced in the YB2 / 0 rat cell line (YB2 / 3HL.P2.GH.16Ag.2O cell, deposited at the American Type Culture Collection as ATCC number CRL-1662). .
Un anticorps préféré selon l'invention est l'anticorps EMAB604 produit par l'hybridome R604 (déposé le 14 Novembre 2006 sous le numéro 1-3692 à la CNCM - Institut Pasteur, 25 rue du Professeur Roux, 75724 Paris Cedex 15, France) . La région variable de chacune des chaînes légères de l'anticorps monoclonal produit par l'hybridome R604 est codée par la séquence d'acide nucléique SEQ ID NO : 5, et la région variable de chacune des chaînes lourdes de 1 ' anticorps monoclonal produit par l'hybridome R604 est codée par la séquence d'acide nucléique SEQ ID NO : 7.A preferred antibody according to the invention is the EMAB604 antibody produced by hybridoma R604 (deposited on November 14, 2006 under the number 1-3692 at the CNCM - Pasteur Institute, 25 rue du Professeur Roux, 75724 Paris Cedex 15, France) . The variable region of each of the light chains of the monoclonal antibody produced by the hybridoma R604 is encoded by the nucleic acid sequence SEQ ID NO: 5, and the variable region of each of the heavy chains of the monoclonal antibody produced by hybridoma R604 is encoded by the nucleic acid sequence SEQ ID NO: 7.
Un objet particulier de l'invention concerne un anticorps monoclonal se liant au LDL-R et permettant le recrutement de cellules effectrices . De préférence, cet anticorps est le EMAB604, ou tout anticorps chimérique, humanisé ou humain possédant des caractéristiques fonctionnelles identiques à l'anticorps EMAB604. Avantageusement, cet anticorps est produit par la lignée cellulaire R604.A particular object of the invention relates to a monoclonal antibody binding to LDL-R and allowing the recruitment of effector cells. Preferably, this antibody is EMAB604, or any chimeric, humanized or human antibody having functional characteristics identical to the EMAB604 antibody. Advantageously, this antibody is produced by the R604 cell line.
Un autre objet de l'invention se rapporte au vecteur d'expression de la chaîne légère d'un anticorps selon l'invention, de séquence SEQ ID NO : 12. Ce vecteur est le vecteur permettant l'expression d'un anticorps selon l'invention dont la chaîne légère est codée par la séquence d'acide nucléique SED ID NO : 13, dont la séquence peptidique déduite est la séquence SEQ ID NO : 14. Ce vecteur est une molécule d'acide nucléique dans laquelle la séquence d'acide nucléique murine SEQ ID NO : 5 codant pour le domaine variable de chacune des chaînes légères de 1 ' anticorps et la séquence d'acide nucléique SEQ ID NO : 21 codant pour la région constante de chacune des chaînes légères de l'anticorps ont été insérées, afin de les introduire et de les maintenir dans une cellule hôte. II permet l'expression de ces fragments d'acide nucléique étrangers dans la cellule hôte car il possède des séquences indispensables (promoteur, séquence de polyadénylation, gène de sélection) à cette expression. De tels vecteurs sont bien connus de l'homme du métier, et peuvent être un adénovirus, un rétrovirus , un plasmide ou un bactériophage, cette liste n'étant pas limitative. De plus, toute cellule de mammifère peut être utilisée comme cellule hôte, c'est-à-dire comme cellule exprimant l'anticorps selon l'invention, par exemple YB2/0, CHO, CHO dhfr- (par exemple CHO DX BlI, CHO DG44) , CHO Lecl3 , SP2/0, NSO, 293, BHK ou COS.Another subject of the invention relates to the expression vector of the light chain of an antibody according to the invention, of sequence SEQ ID NO: 12. This vector is the vector allowing the expression of an antibody according to the invention. invention in which the light chain is encoded by the SED ID NO: 13 nucleic acid sequence, the deduced peptide sequence of which is the sequence SEQ ID NO: 14. This vector is a nucleic acid molecule in which the sequence of murine nucleic acid SEQ ID NO: 5 coding for the variable domain of each of the light chains of the antibody and the nucleic acid sequence SEQ ID NO: 21 encoding the constant region of each of the light chains of the antibody were inserted, to introduce and maintain them in a host cell. It allows the expression of these foreign nucleic acid fragments in the host cell because it has essential sequences (promoter, polyadenylation sequence, selection gene) to this expression. Such vectors are well known to those skilled in the art, and may be an adenovirus, a retrovirus, a plasmid or a bacteriophage, this list not being limiting. In addition, any mammalian cell may be used as a host cell, that is to say as a cell expressing the antibody according to the invention, for example YB2 / 0, CHO, CHO dhfr- (for example CHO DX BlI, CHO DG44), CHO Lecl3, SP2 / 0, NSO, 293, BHK or COS.
Un autre objet de l'invention se rapporte au vecteur d'expression de la chaîne lourde d'un anticorps selon l'invention, de séquence SEQ ID NO : 18. Ce vecteur est le vecteur permettant l'expression d'un anticorps selon l'invention dont la chaîne lourde est codée par la séquence d'acide nucléique SED ID NO : 19, dont la séquence peptidique déduite est la séquence SEQ ID NO : 20. Ce vecteur est une molécule d'acide nucléique dans laquelle la séquence d'acide nucléique murine SEQ ID NO : 7 codant pour le domaine variable de chacune des chaînes lourdes de l'anticorps et la séquence d'acide nucléique humaine SEQ ID NO : 23 codant pour la région constante de chacune des chaînes lourdes de l'anticorps ont été insérées, afin de les introduire et de les maintenir dans une cellule hôte. Il permet l'expression de ces fragments d'acide nucléique étrangers dans la cellule hôte car il possède des séquences indispensables (promoteur, séquence de polyadénylation, gène de sélection) à cette expression. Tout comme indiqué précédemment, le vecteur peut être par exemple un plasmide, un adénovirus , un rétrovirus ou un bactériophage, et la cellule hôte peut être toute cellule de mammifère, par exemple YB2/0, CHO, CHO dhfr- (CHO DX BlI, CHO DG44) , CHO Lecl3, SP2/0, NSO, 293, BHK ou COS.Another subject of the invention relates to the expression vector of the heavy chain of an antibody according to the invention, of sequence SEQ ID NO: 18. This vector is the vector allowing the expression of an antibody according to the invention. invention in which the heavy chain is encoded by the SED ID NO: 19 nucleic acid sequence, the deduced peptide sequence of which is the sequence SEQ ID NO: 20. This vector is a nucleic acid molecule in which the sequence of murine nucleic acid SEQ ID NO: 7 coding for the variable domain of each of the heavy chains of the antibody and the human nucleic acid sequence SEQ ID NO: 23 coding for the constant region of each of the heavy chains of the antibody have have been inserted, to introduce and maintain them in a host cell. It allows the expression of these foreign nucleic acid fragments in the host cell because it has essential sequences (promoter, polyadenylation sequence, selection gene) to this expression. As indicated above, the vector may be for example a plasmid, an adenovirus, a retrovirus or a bacteriophage, and the host cell may be any mammalian cell, for example YB2 / 0, CHO, CHO dhfr- (CHO DX BlI, CHO DG44), CHO Lecl3, SP2 / 0, NSO, 293, BHK or COS.
Un anticorps produit par co-expression de ces vecteurs dans la cellule YB2/0 est illustré par l'anticorps anti-LDL-R EMAB604, produit par le clone R604 (déposé sous le numéro d'enregistrement CNCM I- 3692 à la CNCM) . Cet anticorps possède une forte activité cytotoxique, relevée dans un test ADCC. De plus, l'anticorps EMAB604 induit la sécrétion d'IL-2 par la lignée cellulaire Jurkat-CD16, ce test étant utilisé pour démontrer la capacité d'activation du récepteur CDlβ par les anticorps. L'anticorps EMAB604, pouvant être produit par culture du clone R604 dans un milieu de culture et à des conditions permettant l'expression des vecteurs précédemment décrits, est donc un outil des plus intéressants susceptibles de faire progresser la thérapie et le diagnostic des mélanomes .An antibody produced by coexpression of these vectors in the YB2 / 0 cell is illustrated by the anti-LDL-R antibody EMAB604, produced by the clone R604 (deposited under the CNCM I-3692 registration number at the CNCM). . This antibody has a high cytotoxic activity, found in an ADCC test. In addition, the EMAB604 antibody induces the secretion of IL-2 by the Jurkat-CD16 cell line, this test being used to demonstrate the ability of the antibodies to activate the CDlβ receptor. The EMAB604 antibody, which can be produced by culturing the clone R604 in a culture medium and under conditions allowing the expression of the previously described vectors, is therefore a most interesting tool likely to advance the therapy and the diagnosis of melanomas.
Un autre objet de l'invention se rapporte à une lignée cellulaire stable produisant un anticorps selon l'invention tel que décrit précédemment.Another subject of the invention relates to a stable cell line producing an antibody according to the invention as described above.
De manière avantageuse, la lignée cellulaire stable selon l'invention, qui est caractérisée en ce que la lignée cellulaire dans laquelle l'anticorps est exprimé, est choisie parmi le groupe consistant en : SP2/0, YB2/0 (cellule YB2/3HL.P2.GIl .16Ag.20, déposée à 1 'American Type Culture Collection sous le numéro ATCC CRL-1662), SP2/0-AG14 (ATCC CRL-1581), IR983F, le myélome humain Namalwa, PERC6, les lignées CHO, notamment CHO-K-I, CHO-LeclO, CHO-Lecl, CH0-Lecl3 , CHO Pro-5, CHO dhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, BHK, K6H6 , NSO, SP2/0-Ag 14 et P3X63Ag8.553.Advantageously, the stable cell line according to the invention, which is characterized in that the cell line in which the antibody is expressed, is selected from the group consisting of: SP2 / 0, YB2 / 0 (YB2 / 3HL cell .P2.GIl .16Ag.20, deposited at the American Type Culture Collection as ATCC number CRL-1662), SP2 / 0-AG14 (ATCC CRL-1581), IR983F, Namalwa human myeloma, PERC6, CHO lines. including CHO-KI, CHO-LeclO, CHO-Lecl, CHO-Lecl3, CHO Pro-5, CHO dhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, BHK, K6H6, NSO, SP2 / O-Ag 14 and P3X63Ag8.553.
La lignée cellulaire stable de l'invention a intégré les deux vecteurs d'expression précédemment décrits .The stable cell line of the invention incorporated the two expression vectors previously described.
Un autre objet de l'invention se rapporte à l'hybridome R604 déposé sous le numéro d'enregistrement CNCM 1-3692 à la Collection Nationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) .Another subject of the invention relates to the hybridoma R604 deposited under the registration number CNCM 1-3692 to the National Collection of Cultures of Microorganisms (CNCM, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) .
De manière avantageuse, l'anticorps selon l'invention permet le recrutement de cellules immunitaires effectrices .Advantageously, the antibody according to the invention allows the recruitment of effector immune cells.
Un tel anticorps, de par sa bonne spécificité et sa bonne affinité, est un outil pouvant servir à médier des réactions d'ADCC (Antibody Dépendent Cellular-mediated Cytotoxicity) . En effet, l'anticorps selon l'invention présente une bonne affinité pour le LDL-R et permet d'autre part le recrutement de cellules immunitaires effectrices. Aux fins de l'invention, on entend par « cellule immunitaire effectrice » une cellule qui provoque la destruction des cellules sur lesquelles l'anticorps selon l'invention est lié (« cellules cibles ») .Such an antibody, by its good specificity and good affinity, is a tool that can be used to mediate reactions of ADCC (Antibody Dependent Cellular-mediated Cytotoxicity). Indeed, the antibody according to the invention has a good affinity for LDL-R and also allows the recruitment of effector immune cells. For the purposes of the invention, the term "effector immune cell" means a cell which causes the destruction of the cells on which the antibody according to the invention is bound ("target cells").
L'anticorps anti-LDL-R EMAB604 a la capacité d' interagir fortement avec le récepteur Fcgamma RIIIa ou CD16 exprimé par les cellules NK. Cette fixation est au moins trois fois supérieure à celle de l'anticorps anti-CD20 Rituxan® et comparable à celle l'anticorps anti-CD20 produit par la plateforme EMABLing du LFB. Cette forte fixation au récepteur CD16 permet d'envisager des capacités cytotoxiques optimisées pour l'anticorps anti-LDL-R EMAB604.The anti-LDL-R EMAB604 antibody has the ability to interact strongly with the Fcgamma RIIIa or CD16 receptor expressed by NK cells. This binding is at least three times higher than that of the anti-CD20 Rituxan® antibody and comparable to that of the anti-CD20 antibody produced by the LFB's EMABLing platform. This strong attachment to the receiver CD16 makes it possible to envisage optimized cytotoxic capacities for the anti-LDL-R EMAB604 antibody.
Ainsi il a été montré que cet anticorps induisait une lyse cellulaire par ADCC (Antibody-Dependent CeIl- mediated Cytotoxicity) des cellules HT144 (mélanome) et GUY 17.2 dépendante de l'interaction de sa partie Fc avec le récepteur de faible affinité CD16 exprimé sur les cellules NK (Natural Killer) . En effet, cette ADCC est inhibée en présence d'un anticorps anti-CDlβThus it has been shown that this antibody induces cell lysis by ADCC (Antibody-Dependent CeIl-mediated Cytotoxicity) of HT144 (melanoma) and GUY 17.2 cells dependent on the interaction of its Fc part with the low affinity CD16 receptor expressed on NK cells (Natural Killer). Indeed, this ADCC is inhibited in the presence of an anti-CDlβ antibody
(clone 3G8) . Il est à noter que le CD16 est également exprimé par les macrophages et dans une sous population de monocytes ce qui permet d'envisager l'action de l'anticorps anti-LDL-R EMAB604 par induction d'une cytotoxicité cellulaire via des cellules de la lignée monocytaire.(clone 3G8). It should be noted that CD16 is also expressed by macrophages and in a monocyte subpopulation, which makes it possible to envisage the action of anti-LDL-R EMAB604 antibody by induction of cellular cytotoxicity via the monocytic line.
De plus, cette forte fixation de l'anticorps anti-LDL-R EMAB604 au récepteur CDlβ lui confère en présence de cellules HT144, la capacité d'induire la sécrétion d' interleukine-2 (IL-2) par la lignée Jurkat transfectée avec le CDlβ (Jurkat-CDlβ) . En effet, l'engagement de la partie Fc de l'anticorps fixé sur sa cible (HT144) induit un signal d'activation qui se traduit par la sécrétion d'IL-2 par Jurkat-CD16.In addition, this strong binding of the anti-LDL-R EMAB604 antibody to the CD1β receptor confers on it the presence in the presence of HT144 cells, the capacity to induce the secretion of interleukin-2 (IL-2) by the Jurkat line transfected with CDlβ (Jurkat-CDlβ). Indeed, the commitment of the Fc part of the antibody fixed on its target (HT144) induces an activation signal which results in the secretion of IL-2 by Jurkat-CD16.
L'anticorps C7 a été obtenu par immunisation de souris avec le LDL-R bovin partiellement purifié. Il a été montré qu'il est un bon compétiteur des LDL, et présente donc une affinité pour le LDL-R comparable à celle du ligand naturel des LDL-R.The C7 antibody was obtained by immunizing mice with partially purified bovine LDL-R. It has been shown to be a good competitor of LDL, and therefore has an affinity for LDL-R comparable to that of the natural LDL-R ligand.
Ces anticorps se lient à la cellule-cible par leur fragment variable et se lient aux cellules effectrices par leur fragment constant. Cette relation dépendante des anticorps entre les cellules cibles et les cellules effectrices provoque la lyse des cellules cibles par un mécanisme de type ADCC (Antibody Dépendent Cellular Cytotoxicity) .These antibodies bind to the target cell by their variable fragment and bind to the effector cells by their constant fragment. This antibody-dependent relationship between the target cells and the effector cells causes lysis of the cells targets by an ADCC (Antibody Dependent Cellular Cytotoxicity) mechanism.
De manière avantageuse, les cellules cibles selon l'invention sont des cellules tumorales, telles que les sarcomes, les myélomes, les mélanomes, les lymphomes , les leucémies, cette liste n'étant pas limitative. En effet, des études ont montré une corrélation entre l ' augmentation du niveau d'expression du LDL-R par les cellules et certains cancers. Il s'avère que des patients atteints de certains cancers présentent une hypocholestérolémie. Cette hypocholestérolémie est la conséquence d'une sur-utilisation du cholestérol par les cellules cancéreuses. Pour leur survie, ces dernières induisent une augmentation du niveau d'expression du récepteur des LDL (LDL-R) au sein des organes tumoraux (Henricksson et al, 1989) . On peut citer notamment le cancer de la prostate, du sein, du foie, du pancréas, des ovaires, du colon, du poumon, de l'estomac et les leucémies .Advantageously, the target cells according to the invention are tumor cells, such as sarcomas, myelomas, melanomas, lymphomas, leukemias, this list not being limiting. Indeed, studies have shown a correlation between the increase in the level of expression of LDL-R by cells and certain cancers. It turns out that patients with certain cancers have hypocholesterolemia. This hypocholesterolemia is the consequence of overuse of cholesterol by cancer cells. For their survival, the latter induce an increase in LDL receptor expression level (LDL-R) in the tumor organs (Henricksson et al, 1989). These include cancer of the prostate, breast, liver, pancreas, ovaries, colon, lung, stomach and leukaemias.
Par conséquent, les cellules cancéreuses surexprimant le LDL-R seront donc des cibles préférées de l'anticorps selon l'invention. Ainsi il a été montré que l'anticorps anti-LDL-R EMAB604 se fixait sur les lignées de mélanomes HT144 et GUY 17.2.Therefore, cancer cells overexpressing LDL-R will therefore be preferred targets of the antibody of the invention. Thus, the anti-LDL-R EMAB604 antibody was shown to bind to HT144 and GUY 17.2 melanoma lines.
Ainsi, l'invention se rapporte à un anticorps monoclonal dirigé contre le récepteur humain des LDL (Low Density Lipoprotein) , capable d'induire une lyse spécifique des mélanomes .Thus, the invention relates to a monoclonal antibody directed against the human LDL (Low Density Lipoprotein) receptor, capable of inducing specific lysis of melanomas.
Un autre objet de l'invention est l'utilisation d'un anticorps selon l'invention, pour activer in vitro, ex vivo ou in vivo, les récepteurs FcγRIII de cellules immunitaires effectrices ou provoquer la sécrétion de cytokines ou chimiokines par des cellules effectrices. En effet, les anticorps de l'invention peuvent être utilisés pour leur capacité à activer par leur région Fc le récepteur FcγRIIIA. Ceci représente un intérêt considérable, car ce récepteur est exprimé à la surface de cellules appelées « cellules effectrices » : la liaison de la région Fc de l'anticorps à son récepteur porté par la cellule effectrice provoque l'activation du FcγRIIIA et la destruction des cellules cibles. Les cellules effectrices sont par exemple des cellules NK (Natural Kilier) , des macrophages, des neutrophiles , les lymphocytes CD8 , les lymphocytes Tγδ, les cellules NKT, les éosinophiles , les basophiles ou les mastocytes .Another subject of the invention is the use of an antibody according to the invention, to activate in vitro, ex vivo or in vivo, the FcγRIII receptors of effector immune cells or to cause the secretion of cytokines or chemokines by effector cells. . Indeed, the antibodies of the invention can be used for their ability to activate the FcγRIIIA receptor by their Fc region. This is of considerable interest because this receptor is expressed on the surface of cells called "effector cells": the binding of the Fc region of the antibody to its receptor carried by the effector cell causes the activation of FcγRIIIA and the destruction of target cells. The effector cells are, for example, NK (Natural Kilier) cells, macrophages, neutrophils, CD8 lymphocytes, Tγδ lymphocytes, NKT cells, eosinophils, basophils or mast cells.
Un autre objet particulier de l'invention est un anticorps tel que décrit précédemment pour son utilisation comme médicament.Another particular object of the invention is an antibody as described above for its use as a medicament.
Dans un aspect particulier de l'invention, l'anticorps utilisé se lie au récepteur humain des LDL, et permet le recrutement de cellules effectrices.In a particular aspect of the invention, the antibody used binds to the human LDL receptor, and allows the recruitment of effector cells.
Un autre objet de l'invention est l'utilisation d'un anticorps monoclonal dirigé contre le récepteur humain des LDL pour la fabrication d'un médicament destiné au traitement du cancer, tel que le cancer de la prostate, du sein, du foie, du pancréas, des ovaires, du colon, du poumon, de l'estomac et les leucémies. En effet, l'anticorps selon l'invention cible le LDL-R de manière spécifique. A cet égard, l'anticorps selon l'invention, en se liant à ce récepteur, va engendrer une réaction de lyse des cellules cibles cancéreuses, notamment par ADCC contre les cellules cibles cancéreuses et permettre la lyse de ces dernières. Ainsi, les cellules lysées seront de manière quasi-spécifiques les cellules cancéreuses, les cellules saines ne sur-exprimant pas ou peu le LDL-R et étant ainsi préservées. De manière avantageuse, les cancers traités au moyen de l'anticorps selon l'invention sont les cancers pour lesquels le récepteur des LDL est surexprimé à la surface des cellules cancéreuses, et ce par rapport aux cellules saines correspondantes .Another subject of the invention is the use of a monoclonal antibody directed against the human LDL receptor for the manufacture of a medicament for the treatment of cancer, such as cancer of the prostate, breast, liver, pancreas, ovaries, colon, lung, stomach and leukaemias. Indeed, the antibody according to the invention targets LDL-R specifically. In this respect, the antibody according to the invention, by binding to this receptor, will generate a lysis reaction of the target cancer cells, in particular by ADCC against the target cancer cells and allow the lysis of the latter. Thus, the lysed cells will be near-specific cancer cells, healthy cells not over-expressing or little LDL-R and thus being preserved. Advantageously, the cancers treated with the antibody according to the invention are the cancers for which the LDL receptor is overexpressed on the surface of the cancer cells, and this with respect to the corresponding healthy cells.
De manière particulièrement avantageuse, le cancer traité est le mélanome.Particularly advantageously, the treated cancer is melanoma.
Ainsi, l'invention se rapporte également à l'utilisation d'un anticorps monoclonal dirigé contre le récepteur humain des LDL, ou d'un anticorps tel que défini ci-avant, pour la fabrication d'un médicament destiné au traitement du mélanome. Les cellules cibles, cancéreuses, pourront être lysées par les cellules effectrices recrutées lors de la réaction d'ADCC, les cellules saines exprimant peu ou pas de LDL-R ou étant, le cas échéant, préalablement traitées pour induire l' internalisation du LDL-R de façon à ne pas être reconnues par l'anticorps et être ainsi préservées .Thus, the invention also relates to the use of a monoclonal antibody directed against the human LDL receptor, or an antibody as defined above, for the manufacture of a medicament for the treatment of melanoma. The cancerous target cells may be lysed by the effector cells recruited during the ADCC reaction, healthy cells expressing little or no LDL-R or, if appropriate, being previously treated to induce LDL-internalization. R so as not to be recognized by the antibody and thus be preserved.
Les mélanomes malins traités peuvent être les mélanome superficiels extensifs, qui ont l'aspect d'une tache polychrome dont les contours et la surface sont irréguliers, et qui correspondent à la phase de développement horizontal du mélanome malin, et les mélanomes nodulaires caractérisés cliniquement par une tumeur mélanique saillante et histologiquement par une prolifération bien circonscrite qui envahit d'emblée le derme.The malignant melanoma treated may be extensive superficial melanoma, which has the appearance of a polychromatic patch whose contours and surface are irregular, and which correspond to the horizontal development phase of malignant melanoma, and nodular melanomas characterized clinically by a prominent melanoma tumor and histologically by a well circumscribed proliferation which invades the dermis immediately.
Avantageusement, l'anticorps de l'invention est capable d'induire une lyse spécifique de mélanomes issus des lignées HT144 (HTB-63) (HLA Al, Aw24, B13 , B15, Cw3, DRw4, DRw7) et GUY-17.2, dépendante du CD16.Advantageously, the antibody of the invention is capable of inducing specific lysis of melanomas from HT144 (HTB-63) (HLA A1, Aw24, B13, B15, Cw3, DRw4, DRw7) and GUY-17.2, dependent lines. of CD16.
Un autre objet de l'invention se rapporte à l'utilisation d'un anticorps de l'invention en combinaison avec un ou plusieurs autre anticorps dirigés contre un ou plusieurs autres antigènes exprimés sur les cellules de mélanome. De tels antigènes peuvent être exprimés sur les cellules lymphoïdes et sont choisis parmi le HLA-DR, le CD20, le CD22, CD23, CD25, CD30, CD33 et le CD40.Another subject of the invention relates to the use of an antibody of the invention in combination with one or more other antibodies directed against one or more other antigens expressed on the melanoma cells. Such antigens can be expressed on lymphoid cells and are selected from HLA-DR, CD20, CD22, CD23, CD25, CD30, CD33 and CD40.
Un autre objet de l'invention se rapporte à l'utilisation d'un anticorps de l'invention en combinaison avec un ou plusieurs autres médicaments couramment utilisés pour le traitement des cancers tels que les médicaments cytotoxiques et cytostatigues (chimiothérapie) . Ces médicaments sont bien connus de l'homme du métier, parmi lesquels on peut citer, pour les agents cytotoxiques, les agents alkylants et moutardes azotées, les dérivés du platine, les agents intercalant, les antimétabolites, les inhibiteurs de Topoisomérases, les poisons du fuseau, et, pour les agents cytostatiques, les inhibiteurs de récepteurs à la tyrosine kinase, notamment les récepteurs à EGF (endothelial growth factor) , les récepteurs à insuline, les récepteurs à PDGF (Platelet-derived growth factor) , les récepteurs à FGF (fibroblast growth factor) et les récepteurs à VEGF (vascular endothelial growth factor) , cette liste n'étant pas limitative.Another object of the invention relates to the use of an antibody of the invention in combination with one or more other drugs commonly used for the treatment of cancers such as cytotoxic drugs and cytostatigues (chemotherapy). These medicaments are well known to those skilled in the art, among which, for cytotoxic agents, mention may be made of alkylating agents and nitrogenous mustards, platinum derivatives, intercalating agents, antimetabolites, topoisomerase inhibitors, poisons of the present invention. spindle, and, for cytostatic agents, tyrosine kinase receptor inhibitors, including EGF (endothelial growth factor) receptors, insulin receptors, PDGF (Platelet-derived growth factor) receptors, FGF receptors (fibroblast growth factor) and VEGF (vascular endothelial growth factor) receptors, this list not being limiting.
Dans un aspect particulier de l'invention, l'utilisation des anticorps de l'invention s'effectue en association, in vitro, ex vivo ou in vivo, avec des cellules exprimant des FcγR, telles que les cellules NK (Watural Killer) , les cellules NKT (Natural Killer T), les lymphocytes Tyδ, les macrophages, les monocytes, toute cellule génétiquement modifiée pour exprimer le CD16, ou les cellules dendritiques .In a particular aspect of the invention, the use of the antibodies of the invention is carried out in combination, in vitro, ex vivo or in vivo, with cells expressing FcγR, such as NK (Watural Killer) cells, NKT (Natural Killer T) cells, Tyδ lymphocytes, macrophages, monocytes, any cell genetically modified to express CD16, or dendritic cells.
Un autre objet de l'invention concerne une composition pharmaceutique comprenant au moins un anticorps selon l'invention tel que décrit ci-dessus et un excipient et/ou un véhicule pharmaceutiquement acceptables. Cette composition pharmaceutique a pour vocation de cibler les cellules cancéreuses, notamment celles surexprimant le LDL-R. Ces cellules cancéreuses exprimant à leur surface une quantité de récepteurs au LDL supérieure à la quantité de récepteurs exprimés par les cellules saines, le médicament ainsi préparé sera préférentiellement lié par les cellules cancéreuses .Another subject of the invention relates to a pharmaceutical composition comprising at least one antibody according to the invention as described above and a pharmaceutically acceptable excipient and / or carrier. This pharmaceutical composition is intended to target cancer cells, including those overexpressing LDL-R. As these cancer cells express on their surface a quantity of LDL receptors greater than the quantity of receptors expressed by the healthy cells, the drug thus prepared will be preferentially bound by the cancer cells.
L'excipient peut être toute solution, telle qu'une solution saline, physiologique, isotonique, tamponnée, etc., ainsi que toute suspension, gel, poudre, etc., compatible avec un usage pharmaceutique et connu de l'homme du métier. Les compositions selon l'invention peuvent en outre contenir un ou plusieurs agents ou véhicules choisis parmi les dispersants, solubilisants, stabilisants, surfactants, conservateurs , etc . Avantageusement, la composition de l'invention comprend en outre au moins un anticorps dirigé contre un autre antigène présent sur les cellules cibles des anticorps .The excipient may be any solution, such as saline, physiological, isotonic, buffered, etc., as well as any suspension, gel, powder, etc., compatible with a pharmaceutical use and known to those skilled in the art. The compositions according to the invention may also contain one or more agents or vehicles chosen from dispersants, solubilizers, stabilizers, surfactants, preservatives, and the like. Advantageously, the composition of the invention further comprises at least one antibody directed against another antigen present on the target cells of the antibodies.
Avantageusement, la composition de l'invention comprend en outre un anticorps anti-HLA-DR. En effet, les lignées de mélanomes HT144 et GUY 17.2 expriment l'antigène HLA-DR à leur surface. Une composition comprenant un mélange d'anticorps anti-LDL-R et anti- HLA DR dans des proportions pouvant représenter 5, 25, 50, 75 ou 95% de l'un ou de l'autre anticorps par rapport au poids total de la composition est particulièrement avantageuse pour le traitement de patients atteints de mélanomes .Advantageously, the composition of the invention further comprises an anti-HLA-DR antibody. Indeed, HT144 and GUY 17.2 melanoma lines express the HLA-DR antigen on their surface. A composition comprising a mixture of anti-LDL-R and anti-HLA DR antibodies in proportions which may represent 5, 25, 50, 75 or 95% of either antibody relative to the total weight of the The composition is particularly advantageous for the treatment of patients with melanoma.
Par ailleurs, les compositions peuvent être administrées de différentes manières et sous différentes formes. L'administration peut être réalisée par toute voie classique pour ce type d'approche thérapeutique, notamment par voie systémique, en particulier par injection intraveineuse, intradermique, intra-tumorale, sous- cutanée, intra-péritonéale, intramusculaire, intra- artérielle, etc. On peut citer par exemple l'injection intra-tumorale ou l'injection dans une zone proche de la tumeur ou irriguant la tumeur. L'administration peut également être orale, mucosale ou topique.Moreover, the compositions can be administered in different ways and under different forms. The administration can be carried out by any conventional route for this type of therapeutic approach, in particular by the systemic route, in particular by intravenous, intradermal, intratumoral, subcutaneous, intraperitoneal, intramuscular, intraarterial injection, etc. . For example, intratumoral injection or injection into an area close to the tumor or irrigating the tumor may be mentioned. Administration may also be oral, mucosal or topical.
Les doses peuvent varier en fonction du nombre d'administrations, de l'association à d'autres principes actifs, du stade d'évolution de la pathologie, etc.The doses may vary according to the number of administrations, the association with other active ingredients, the stage of evolution of the pathology, etc.
Un autre objet de l'invention est l'utilisation de l'anticorps selon l'invention dans les analyses immunohistochimiques de tissus cancéreux, sains ou cirrhoses, ou dans les analyses en Western Blot, en ELISA ou en test de quantification in vivo, ex vivo ou in vi tro .Another subject of the invention is the use of the antibody according to the invention in immunohistochemical analyzes of cancerous, healthy or cirrhosis tissues, or in Western Blot analyzes, in ELISA or in vivo quantification test, ex. vivo or in vi tro.
D'autres aspects et avantages de l'invention seront décrits dans les exemples qui suivent, qui doivent être considérés comme illustratifs et ne limitent pas la portée de l'invention.Other aspects and advantages of the invention will be described in the examples which follow, which should be considered as illustrative and do not limit the scope of the invention.
Figuresfigures
Figure 1 : Activité ADCC des anticorps anti-LDL-R (EMAB604) et anti-HLA-DR CHO sur la lignée de mélanome GUY 17.2. Les résultats sont exprimés en pourcentage de lyse (moyenne +/-SEM, n=2) en fonction de la concentration d'anticorps.Figure 1: ADCC activity of anti-LDL-R (EMAB604) and anti-HLA-DR CHO antibodies on the GUY 17.2 melanoma line. The results are expressed as percentage lysis (mean +/- SEM, n = 2) as a function of the antibody concentration.
Figure 2 : Inhibition de l'activité ADCC induite par 10 μg/ml des anticorps anti-LDL-R (EMAB604) et anti- HLA-DR-CHO sur la lignée de mélanome GUY-17.2 par l'anticorps anti-CDlβ murin 3G8 (250 μg/ml) .FIG. 2: Inhibition of the ADCC activity induced by 10 μg / ml of the anti-LDL-R (EMAB604) antibodies and HLA-DR-CHO on the GUY-17.2 melanoma line by the murine anti-CD1β antibody (250 μg / ml).
Figure 3 : Activité ADCC des anticorps anti-LDL-R (EMAB604) et anti-HLA-DR CHO sur la lignée de mélanome HT-144. Les résultats sont exprimés en pourcentage de lyse (moyenne +/-SEM, n=2) en fonction de la concentration d' anticorps .Figure 3: ADCC activity of anti-LDL-R (EMAB604) and anti-HLA-DR CHO antibodies on the HT-144 melanoma line. The results are expressed as a percentage of lysis (mean +/- SEM, n = 2) as a function of the concentration of antibodies.
Figure 4 : Inhibition de l'activité ADCC induite par 10 μg/ml des anticorps anti-LDL-R (EMAB604) et anti- HLA-DR-CHO sur la lignée de mélanome HT-144 par l'anticorps anti-CDlβ murin 3G8 (250 μg/ml).FIG. 4: Inhibition of the ADCC Activity Induced by 10 μg / ml of the Anti-LDL-R (EMAB604) and Anti-HLA-DR-CHO Antibodies on the HT-144 Melanoma Line by the Murine Anti-CD1β Antibodies (250 μg / ml).
Figure 5 : Sécrétion d'IL-2 par la cellule Jurkat-CDlβ induite par les anticorps anti-LDL-R (EMAB604) et anti-HLA-DR CHO en présence de la lignée de mélanome GUY 17.2.FIG. 5: Secretion of IL-2 by the Jurkat-CDlβ cell induced by the anti-LDL-R antibodies (EMAB604) and anti-HLA-DR CHO in the presence of the melanoma line GUY 17.2.
Figure 6 : Sécrétion d'IL-2 par la cellule Jurkat-CDlβ induite par les anticorps anti-LDL-R (ΞMAB604) et anti-HLA-DR CHO en présence de la lignée de mélanome HT-144.FIG. 6: Secretion of IL-2 by the Jurkat-CDlβ cell induced by the anti-LDL-R (ΞMAB604) and anti-HLA-DR CHO antibodies in the presence of the HT-144 melanoma line.
Figure 7 : Pourcentage de fixation des anticorps anti- LDL-R (EMAB604) , anti-CD20 produit dans YB2/0 et Rituxan® sur le récepteur CDl6 exprimé par les cellules NK. Les résultats sont exprimés pour une concentration de 10 et 50 μg/ml d'anticorps.Figure 7: Percent fixation of anti-LDL-R (EMAB604), anti-CD20 produced in YB2 / 0 and Rituxan® on the CD16 receptor expressed by NK cells. The results are expressed for a concentration of 10 and 50 μg / ml of antibody.
Figure 8 : Séquences :Figure 8: Sequences:
SEQ ID NO : 5 : séquence d'acide nucléique murine codant pour la région variable de chacune des chaînes légères de l'anticorps, SEQ ID NO : 6 : séquence du CDRl de la chaîne légère de l'anticorps selon la numérotation de Kabat, SEQ ID NO : 7 : séquence d'acide nucléique murine codant pour la région variable de chacune des chaînes lourdes de l ' anticorps ,SEQ ID NO: 5: murine nucleic acid sequence coding for the variable region of each of the light chains of the antibody, SEQ ID NO: 6: CDR1 sequence of the light chain of the antibody according to the Kabat numbering, SEQ ID NO: 7: murine nucleic acid sequence coding for the variable region of each of the heavy chains of the antibody,
SEQ ID NO : 8 : séquence du CDR2 de la chaîne légère de l'anticorps selon la numérotation de Kabat,SEQ ID NO: 8: CDR2 sequence of the light chain of the antibody according to the Kabat numbering,
SEQ ID NO : 9 : séquence du CDR3 de la chaîne légère de l'anticorps selon la numérotation de Kabat, SEQ ID NO : 10 : séquence peptidique de la région variable de chacune des chaînes légères de l'anticorps,SEQ ID NO: 9: CDR3 sequence of the light chain of the antibody according to Kabat numbering, SEQ ID NO: 10: peptide sequence of the variable region of each of the light chains of the antibody,
SEQ ID NO : 11 : séquence peptidique de la région variable de chacune des chaînes lourdes deSEQ ID NO: 11: Peptide sequence of the variable region of each of the heavy chains of
1 ' anticorps,The antibody,
SEQ ID NO : 12 : séquence d'acide nucléique correspondant au vecteur d'expression de la chaîne légère de l'anticorps,SEQ ID NO: 12: nucleic acid sequence corresponding to the expression vector of the light chain of the antibody,
SEQ ID NO : 13 : séquence d'acide nucléique chimère murine-humaine codant pour chacune des chaînes légères de l'anticorps, SEQ ID NO : 14 : séquence peptidique de chacune des chaînes légères de l'anticorps déduite de la séquence d'acide nucléique SEQ ID NO : 13,SEQ ID NO: 13: chimeric murine-human nucleic acid sequence coding for each of the light chains of the antibody, SEQ ID NO: 14: peptide sequence of each of the light chains of the antibody deduced from the acid sequence nucleic acid SEQ ID NO: 13,
SEQ ID NO : 15 : séquence du CDRl de la chaîne légère de l'anticorps selon la numérotation IMGT, SEQ ID NO : 16 : séquence du CDR2 de la chaîne légère de l'anticorps selon la numérotation IMGT,SEQ ID NO: 15: CDR1 sequence of the light chain of the antibody according to the IMGT numbering, SEQ ID NO: 16: CDR2 sequence of the light chain of the antibody according to the IMGT numbering,
SEQ ID NO : 17 : séquence du CDR3 de la chaîne légère de l'anticorps selon la numérotation IMGT,SEQ ID NO: 17: CDR3 sequence of the light chain of the antibody according to the IMGT numbering,
SEQ ID NO : 18 : séquence d'acide nucléique correspondant au vecteur d'expression de la chaîne lourde de l'anticorps selon l'invention,SEQ ID NO: 18: nucleic acid sequence corresponding to the expression vector of the heavy chain of the antibody according to the invention,
SED ID NO : 19 : séquence d'acide nucléique codant pour la chaîne lourde de l'anticorps selonSED ID NO: 19: nucleic acid sequence encoding the heavy chain of the antibody according to
1 ' invention, SEQ ID NO : 20 : séquence peptidique de la chaîne lourde de l'anticorps déduite de la séquence SEQ ID1, SEQ ID NO: 20: Peptide sequence of the heavy chain of the antibody deduced from the SEQ ID sequence
NO : 19 , SEQ ID NO : 21 : séquence d'acide nucléique codant pour la région constante de chacune des chaîne légères de l'anticorps,NO: 19, SEQ ID NO: 21: nucleic acid sequence coding for the constant region of each of the light chains of the antibody,
SEQ ID NO : 22 : séquence du CDRl de la chaîne lourde de l'anticorps selon la numérotation de Kabat,SEQ ID NO: 22: CDR1 sequence of the heavy chain of the antibody according to the Kabat numbering,
SEQ ID NO : 23 : séquence d'acide nucléique humaine codant pour la région constante de chacune des chaînes lourdes de l'anticorps de type γl, SEQ ID NO : 24 : séquence du CDR2 de la chaîne lourde de l'anticorps selon la numérotation de Kabat,SEQ ID NO: 23: human nucleic acid sequence coding for the constant region of each of the heavy chains of the γ1 type antibody, SEQ ID NO: 24: CDR2 sequence of the antibody heavy chain according to the numbering from Kabat,
SEQ ID NO : 25 : séquence du CDR3 de la chaîne lourde de l'anticorps selon la numérotation de Kabat, SEQ ID NO : 26 : séquence du CDRl de la chaîne lourde de l'anticorps selon la numérotation IMGT, SEQ ID NO : 27 : séquence du CDR2 de la chaîne lourde de l'anticorps selon la numérotation IMGT, SEQ ID NO : 28 : du CDR3 de la chaîne lourde de l'anticorps selon la numérotation IMGT. SEQ ID NO : 31 : séquence peptidique de la région constante de chacune des chaîne légères de l'anticorps, déduite de SEQ ID NO : 21, et SEQ ID NO : 34 : séquence peptidique de la région constante de chacune des chaîne lourdes de l'anticorps déduite de SEQ ID NO : 23.SEQ ID NO: 25: CDR3 sequence of the heavy chain of the antibody according to the Kabat numbering, SEQ ID NO: 26: CDR1 sequence of the heavy chain of the antibody according to the IMGT numbering, SEQ ID NO: 27 CDR2 sequence of the heavy chain of the antibody according to the IMGT numbering, SEQ ID NO: 28: CDR3 of the heavy chain of the antibody according to the IMGT numbering. SEQ ID NO: 31: Peptide sequence of the constant region of each of the light chains of the antibody, deduced from SEQ ID NO: 21, and SEQ ID NO: 34: Peptide sequence of the constant region of each of the heavy chains of the antibody deduced from SEQ ID NO: 23.
ExemplesExamples
Exemple 1 : Construction des vecteurs d' expression de l'anticorps chimérique anti-LDL-R C7Example 1 Construction of Expression Vectors for the Chimeric Anti-LDL-R C7 Antibody
A. Détermination de la séquence leader des régions variables de l ' anticorps murin ClA. Determination of the leader sequence of the variable regions of the murine antibody C1
L'ARN total de l'hybridome murin C7 produisant une immunoglobuline de type IgG2b,κ a été extrait (kit Nucleospin RNA Macherey-Nagel réf. 740609.250). Après transcription inverse, les domaines variables des chaînes légères (VK) et lourdes (VH) de l'anticorps C7 ont été amplifiés par la technique de 5'RACE (Rapid Amplification of cDNA Ends) (kit 5'RACE, Invitrogen réf. 18374.041) .The total RNA of the C7 murine hybridoma producing an IgG2b, κ-type immunoglobulin was extracted (Nucleospin RNA Macherey-Nagel kit 740609.250). After reverse transcription, the variable domains of the light (VK) and heavy (VH) chains of the C7 antibody were amplified by the 5 'RACE (Rapid Amplification of cDNA Ends) technique (5'RACE kit, Invitrogen 18374.041).
Brièvement, une première étape de transcription inverse a été tout d'abord réalisée en utilisant une amorce localisée dans la région 5' des régions constantes CK OU γ2b murines . Une séquence poly-dC a été ensuite ajoutée en 3' des ADNc synthétisés avant de réaliser l'amplification des régions VK et VH à l'aide d'une amorce 5' reconnaissant la séquence poly- dC et d'une amorce 3', localisée dans les régions constantes CK OU γ2b murines en 5' de l'amorce de transcription inverse. Les amorces utilisées pour ces deux étapes sont les suivantes :Briefly, a first reverse transcription step was first performed using a primer located in the 5 'region of murine CK OR γ2b constant regions. A poly-dC sequence was then added at 3 'to the synthesized cDNAs before performing the amplification of the VK and VH regions using a 5' primer recognizing the poly-DC sequence and a 3 'primer. located in the murine CK OR γ2b constant regions 5 'of the reverse transcription primer. The primers used for these two steps are as follows:
1. amorces de transcription inverse a. Amorce antisens spécifique Kappa murin1. reverse transcription primers a. Murine Kappa specific antisense primer
5'- ACT GCC ATC AAT CTT CCA CTT GAC -3' (SEQ ID NO : 1) b. Amorce antisens spécifique γ2b murin5'- ACT GCC ATC AAT CTT CCA CTT GAC -3 '(SEQ ID NO: 1) b. Specific antisense primer γ2b murine
5'- GTGTAGAGTCCAGACTGCAGGAG -3' (SEQ ID NO : 2)5'-GTGTAGAGTCCAGACTGCAGGAG -3 '(SEQ ID NO: 2)
2. amorces de PCR 5 ' RACE a. Amorce antisens spécifique Kappa murin 5'- TTGTTCAAGAAGCACACGACTGAGGCAC -3' (SEQ ID NO : 3) b. Amorce antisens spécifique γ2b murin2. 5 'RACE PCR primers a. Murine Kappa specific antisense primer 5'-TTGTTCAAGAAGCACACGACTGAGGCAC -3 '(SEQ ID NO: 3) b. Specific antisense primer γ2b murine
5'- CACTGACTCAGGGAAGTAGCCCTTG -3' (SEQ ID NO : 4)5'-CACTGACTCAGGGAAGTAGCCCTTG -3 '(SEQ ID NO: 4)
Les produits de PCR VH et VK ainsi obtenus ont été clones dans le vecteur pCR4Blunt-T0P0 (Zéro blunt TOPO PCR cloning kit, Invitrogen, réf. K2875-20) puis séquences .The VH and VK PCR products thus obtained were cloned into the vector pCR4Blunt-T0P0 (Zero blunt TOPO PCR cloning kit, Invitrogen, K2875-20) and then sequenced.
La séquence nucléotidique de la région VK de l'anticorps murin C7 est indiquée sous la séquence SEQ ID NO : 5 et la séquence peptidique déduite est la séquence SEQ ID NO : 10. Le gène VK appartient au sous-groupe Vκl [Almagro JC et al Immunogenetics (1998), 47 : 355-363]. Les séquences des CDRl, CDR2 et CDR3 de la région VK de l'anticorps murin C7, définies selon la numérotation de Kabat [Kabat et al . , "Séquences of Proteins of Iiranunological Interest", NIH Publication, 91-3242 (1991) ] , sont indiquées sous les séquences suivantes : SEQ ID NO : 6, SEQ ID NO : 8 et SEQ ID NO : 9, respectivement. Les séquences des CDRl- IMGT, CDR2-IMGT et CDR3-IMGT de la région VK de l'anticorps murin Ql, définies selon l'analyse IMGT (international ImMunoGeneTics database) [Lefranc, M.- P. et al., Dev. Comp. Immunol . , 27, 55-77 (2003)] sont indiquées sous les séquences suivantes : SEQ ID NO : 15, SEQ ID NO : 16 et SEQ ID NO : 17, respectivement. Cette définition, différente de celle de Kabat fondée sur la seule analyse de variabilité des séquences, prend en compte et combine la caractérisation des boucles hypervariables [Chothia C. and Lesk A.M. J. Mol. Biol. 196 : 901-17 (1987)] et l'analyse structurale des anticorps par cristallographie.The nucleotide sequence of the VK region of the C7 murine antibody is indicated under the sequence SEQ ID NO: 5 and the deduced peptide sequence is the sequence SEQ ID NO: 10. The VK gene belongs to the Vκl subgroup [Almagro JC et al Immunogenetics (1998), 47: 355-363]. The CDR1, CDR2 and CDR3 sequences of the VK region of the C7 murine antibody, defined according to the Kabat numbering [Kabat et al. "Sequences of Proteins of Iiranunological Interest", NIH Publication, 91-3242 (1991)], are indicated under the following sequences: SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The CDR1-IMGT, CDR2-IMGT and CDR3-IMGT sequences of the VK region of the murine antibody Q1, defined according to IMGT analysis (International ImMunoGeneTics database) [Lefranc, M.P. et al., Dev. Comp. Immunol. , 27, 55-77 (2003)] are indicated under the following sequences: SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively. This definition, different from that of Kabat based on the analysis of sequence variability alone, takes into account and combines the characterization of hypervariable loops [Chothia C. and Lesk AMJ Mol. Biol. 196: 901-17 (1987)] and structural analysis of antibodies by crystallography.
La séquence nucléotidique de la région VH de C7 est la séquence SEQ ID NO : 7 et la séquence peptidique qui en est déduite est la séquence SEQ ID NO : 11. Le gène VH appartient au sous-groupe VHlThe nucleotide sequence of the VH region of C7 is the sequence SEQ ID NO: 7 and the peptide sequence which is deduced therefrom is the sequence SEQ ID NO: 11. The VH gene belongs to the VH1 subgroup
[Honjo T. and Matsuda F. in « Immunoglobulin gènes ».[Honjo T. and Matsuda F. in "Immunoglobulin Genes".
Honjo T. and Alt F.W. eds, Académie Press, LondonHonjo T. and Alt F.W. eds, Academy Press, London
(1996), ppl45-171] . Les séquences des CDRl, CDR2 et(1996), ppl45-171]. The sequences of CDR1, CDR2 and
CDR3 de la région VH de l'anticorps murin C7 , définies selon la numérotation de Kabat [Kabat et al., "Séquences of Proteins of Immunological Interest", NIH Publication, 91-3242 (1991)], sont indiquées sous les séquences suivantes : SEQ ID NO : 22, SEQ ID NO : 24 et SEQ ID NO : 25, respectivement. Les séquences des CDRl-IMGT, CDR2-IMGT et CDR3-IMGT de la région VH de l'anticorps murin Ql, définies selon l'analyse IMGT (international ImMunoGeneTics database) [Lefranc, M.- P. et al., Dev. Comp. Immunol . , 27, 55-77 (2003)] sont indiquées sous les séquences suivantes : SEQ ID NO : 26, SEQ ID NO : 27 et SEQ ID NO : 28, respectivement. Cette définition, différente de celle de Kabat fondée sur la seule analyse de variabilité des séquences, prend en compte et combine la caractérisation des boucles hypervariables [Chothia C. and Lesk A.M. J. Mol. Biol. 196 : 901-17 (1987)] et l'analyse structurale des anticorps par cristallographie.CDR3 of the VH region of the murine C7 antibody, defined according to Kabat numbering [Kabat et al., "Sequences of Proteins of Immunological Interest", NIH Publication, 91-3242 (1991)], are indicated under the following sequences SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 25, respectively. The CDR1-IMGT, CDR2-IMGT and CDR3-IMGT sequences of the VH region of the murine antibody Q1, defined according to IMGT analysis (International ImMunoGeneTics database) [Lefranc, M.- P. et al., Dev. Comp. Immunol. , 27, 55-77 (2003)] are indicated in the following sequences: SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively. This definition, different from that of Kabat based on the analysis of sequence variability alone, takes into account and combines the characterization of hypervariable loops [Chothia C. and Lesk AMJ Mol. Biol. 196: 901-17 (1987)] and structural analysis of antibodies by crystallography.
B. Construction des vecteurs d'expression chaîne lourde et chaîne légère de l'anticorps chimérique EMAB604B. Construction of the Heavy Chain and Light Chain Expression Vectors of the EMAB604 Chimeric Antibody
1. Vecteur chaîne légère Kappa1. Kappa light chain vector
La séquence VK clonée dans le vecteur de séquençage pCR4Blunt-T0P0 a été amplifiée à l'aide des amorces de clonage suivantes :The VK sequence cloned into the sequencing vector pCR4Blunt-T0P0 was amplified using the following cloning primers:
a) amorce sens VK (SEQ ID NO : 29)a) sense primer VK (SEQ ID NO: 29)
5'- ATCGAACTAGTGCCGCCACCΛΓGAAGTTGCCTGTTAGGCT -3'5'- ATCGAACTAGTGCCGCCACCΛΓGAAGTTGCCTGTTAGGCT -3 '
La séquence soulignée correspond au site de restriction Spe I, la séquence en gras correspond à une séquence consensus de Kozak, l 'ATG initiateur est en italiques.The underlined sequence corresponds to the Spe I restriction site, the bold sequence corresponds to a Kozak consensus sequence, the initiator ATG is in italics.
b) amorce antisens VK (SEQ ID NO : 30)b) VK antisense primer (SEQ ID NO: 30)
5 '-GATGAAGACACTTGGTGCAGCCACAGTTCGΓΓΓGAΓTΓCCAGCΓΓGGTGCCΓ -3 '5 '-GATGAAGACACTTGGTGCAGCCACAGTTCGΓΓΓGAΓTΓCCAGCΓΓGGTGCCΓ -3'
Cette amorce réalise la jonction des séquences VK murine (en italique) et région constante (CK) humaine (en gras) . La séquence soulignée correspond au site de restriction Dra III. Le produit de PCR VK ainsi obtenu contient la séquence codant le peptide signal naturel de l'anticorps murin C7. Cette PCR VK a été ensuite clonée entre les sites Spe I et Dra III du vecteur de chimérisation chaîne légère en 5' de la région constante CK humaine, dont la séquence nucléique est la séquence SEQ ID NO : 21 et la séquence peptidique déduite est la séquence SEQ ID NO : 31. La séquence CK humaine de ce vecteur de chimérisation a été préalablement modifiée par mutagénèse silencieuse afin de créer un site de restriction Dra III pour permettre le clonage de séquences VK murines. Ce vecteur de chimérisation contient un promoteur RSV et une séquence de polyadénylation bGH (bovine Growth Hormone) ainsi que le gène de sélection dhfr (dihydrofolate reductase) .This primer joins the murine VK (in italic) and human constant region (CK) sequences (in bold). The underlined sequence corresponds to the Dra III restriction site. The VK PCR product thus obtained contains the sequence encoding the natural signal peptide of the murine C7 antibody. This VK PCR was then cloned between the Spe I and Dra III sites of the 5 'light chain chimeric vector of the human CK constant region, whose nucleic sequence is the sequence SEQ ID NO: 21 and the deduced peptide sequence is the sequence SEQ ID NO: 31. The human CK sequence of this chimeric vector was previously modified by silent mutagenesis to create a Dra III restriction site to allow cloning of murine VK sequences. This chimerization vector contains an RSV promoter and a bGH (Growth Hormone) polyadenylation sequence as well as the dhfr (dihydrofolate reductase) selection gene.
La séquence de la chaîne légère de l ' anticorps chimérique EMAB604 codée par ce vecteur est présenté en SEQ ID NO : 13 pour la séquence nucléotidique et correspond à la séquence peptidique déduite SEQ ID NO : 14.The sequence of the light chain of the chimeric antibody EMAB604 encoded by this vector is presented in SEQ ID NO: 13 for the nucleotide sequence and corresponds to the deduced peptide sequence SEQ ID NO: 14.
2. Vecteur chaîne lourde2. Vector heavy chain
Une démarche similaire a été appliquée pour la chimérisation de la chaîne lourde de l'anticorps EMAB604.A similar approach has been applied for the chimericization of the heavy chain of the EMAB604 antibody.
La séquence VH clonée dans le vecteur pCR4Blunt- TOPO a été tout d'abord amplifiée à l'aide des amorces de clonage suivantes :The VH sequence cloned into the pCR4Blunt-TOPO vector was first amplified using the following cloning primers:
a) amorce sens VH (SEQ ID NO : 32)a) sense primer VH (SEQ ID NO: 32)
5'- ATCGAGCTAGCGCCGCCACC.ΛTGGAATGGCCTTGTATCTT -3' La séquence soulignée correspond au site de restriction Nhe I, la séquence en gras correspond à une séquence consensus de Kozak, l'ATG initiateur est en italiques.5'- ATCGAGCTAGCGCCGCCACC.ΛTGGAATGGCCTTGTATCTT -3 ' The underlined sequence corresponds to the Nhe I restriction site, the bold sequence corresponds to a Kozak consensus sequence, the initiator ATG is in italics.
b) amorce antisens VH (SEQ ID NO : 33)b) VH antisense primer (SEQ ID NO: 33)
5 ' - ACCGATGGGCCCTTGGTGGAGGCTGAGGAGACTGTGAGAGT -3 '5 '- ACCGATGGGCCCTTGGTGGAGGCTGAGGAGACTGTGAGAGT -3'
Cette amorce réalise la jonction des séquences VH murine (en italique) et région constante Gl humaine (en gras ) .This primer joins the murine VH sequences (in italics) and the human Gl constant region (in bold).
La séquence soulignée correspond au site de restriction Apa I.The underlined sequence corresponds to the Apa I restriction site.
Le fragment VH amplifié contient la séquence codant le peptide signal naturel de l'anticorps murin Cl. Ce produit d'amplification PCR VH a été ensuite clone entre les sites Spe I et Apa I du vecteur de chimérisation contenant la chaîne lourde en 5 ' de la région constante γl humaine dont la séquence nucléique est la séquence SEQ ID NO : 23 et la séquence peptidique déduite est la séquence SEQ ID NO : 34. Ce vecteur de chimérisation contient un promoteur RSV et une séquence de polyadénylation bGH (bovine Growth Hormone) ainsi que le gène de sélection neo .The amplified VH fragment contains the sequence encoding the natural signal peptide of the murine antibody C1. This VH PCR amplification product was then cloned between the Spe I and Apa I sites of the chimeric vector containing the 5 'heavy chain. the human γ1 constant region whose nucleic sequence is the sequence SEQ ID NO: 23 and the deduced peptide sequence is the sequence SEQ ID NO: 34. This chimerization vector contains an RSV promoter and a bGH (Growth Hormone) bGH polyadenylation sequence as well as the neo selection gene.
La séquence de la chaîne lourde de l'anticorps chimérique EMAB604 codée par ce vecteur est présentée en SEQ ID NO : 19 pour la séquence nucléotidique et en séquence SEQ ID NO : 20 pour la séquence peptidique déduite .The sequence of the heavy chain of the chimeric antibody EMAB604 encoded by this vector is presented in SEQ ID NO: 19 for the nucleotide sequence and in sequence SEQ ID NO: 20 for the deduced peptide sequence.
Exemple 2 : Création d' une lignée cellulaire dérivée de la lignée YB2 / 0 productrice de l ' anticorps chimérique EMAB604 La lignée de rat YB2/0 (ATCC # CRL-1662) a été cultivée en milieu EMS (Invitrogen, réf. 041-95181M) contenant 5% de sérum de veau fœtal (JRH Biosciences, réf. 12103-78P) . Pour la transfection, 5 millions de cellules ont été électroporées (électroporateur Biorad, modèle 1652077) en milieu Electrobuffer (CeIl Projects, réf. EB-110) avec 25 μg de vecteur chaîne légère K463-26-C7 linéarisés par Avi II, et 26,7 μg de vecteur chaîne lourde H-463-27-C7 linéarisés par Not I. Les conditions d' électroporation appliquées étaient de 230 volts et 960 microfarads pour une cuvette de 0,5 ml et de largeur 0,4cm. Le contenu de la cuvette d' électroporation a été ensuite réparti sur 5 plaques P96 avec une densité de 5000 cellules/puits. La mise en milieu sélectif RPMI (Invitrogen, réfExample 2 Creation of a Cell Line Derived from the YB2 / 0 Line Producing the Chimeric Antibody EMAB604 The rat YB2 / 0 (ATCC # CRL-1662) line was cultured in EMS medium (Invitrogen, Cat # 041-95181M) containing 5% fetal calf serum (JRH Biosciences, Cat # 12103-78P). For transfection, 5 million cells were electroporated (Biorad electroporator, model 1652077) in Electrobuffer medium (CeIl Projects, ref EB-110) with 25 μg of Avi II linearized K463-26-C7 light chain vector, and 26 , 7 μg of heavy chain vector H-463-27-C7 linearized with Not I. The applied electroporation conditions were 230 volts and 960 microfarads for a 0.5 ml and 0.4 cm wide cuvette. The contents of the electroporation cuvette were then distributed over 5 P96 plates with a density of 5000 cells / well. Putting in selective medium RPMI (Invitrogen, ref
21875-034) contenant 5% de sérum dialyse (Invitrogen, réf. 10603-017) des cellules, 500 μg/ml de G41821875-034) containing 5% dialyzed serum (Invitrogen, ref 10603-017) cells, 500 μg / ml G418
(Invitrogen, réf. 10131-027) et 25 nM de méthotrexate(Invitrogen, ref 10131-027) and 25 nM methotrexate
(Sigma, réf. M8407), a été réalisée 3 jours après la transfection.(Sigma, M8407), was performed 3 days after transfection.
Les surnageants des puits de transfection résistants ont été criblés pour la présence d' immunoglobuline (Ig) chimérique par dosage ELISA spécifique des séquences Ig humaines. Les 16 transfectants produisant le plus d'anticorps ont été amplifiés en plaques P24 et ont été évalués pour leur capacité de production (productivité et production maximale) et pour le taux de fucose des IgG produites . Le cloïde R604 CHlO (productivité : 8,lpcd, production maximale 38,5μg/ml, taux de fucose des IgG de 25,5-26,8%), dénommé ci-après « R604», a été sélectionné pour la production de l'anticorps chimérique EMAB604. La production de l'anticorps chimérique EMAB604 a été réalisée par expansion de la culture en milieu EMS contenant 5% de sérum dépiété en Ig bovines (Invitrogen, réf. 16250-078) et 500 μg/ml de G418 (Invitrogen, réf. 10131-027), obtenue par dilution à 2xl0E5 cellules/ml en flacons de 25 cm2, 75 cm2 et 175 cm2 puis en flacon de type roller. Après avoir atteint le volume maximal (0,9 1), la culture a été poursuivie jusqu'à ce que la viabilité cellulaire soit inférieure à 50%. Après production, l'anticorps chimérique EMAB604 a été purifié par chromatographie d'affinité sur protéine A et contrôlé par électrophorèse en gel de polyacrylamide.Supernatants from the resistant transfection wells were screened for the presence of chimeric immunoglobulin (Ig) by ELISA assay specific for human Ig sequences. The 16 transfectants producing the most antibodies were amplified in P24 plates and were evaluated for their production capacity (productivity and maximum production) and for the fucose level of the IgG produced. The clone R604 CH10 (productivity: 8, lpcd, maximum production 38.5 μg / ml, fucose IgG level of 25.5-26.8%), hereinafter referred to as "R604", was selected for the production of the chimeric antibody EMAB604. The production of the chimeric antibody EMAB604 was carried out by expansion of the culture in EMS medium containing 5% of serum depleted in bovine Ig. (Invitrogen, ref 16250-078) and 500 μg / ml of G418 (Invitrogen, ref.10131-027), obtained by dilution with 2x10E5 cells / ml in 25 cm 2 , 75 cm 2 and 175 cm 2 flasks then roll-type bottle. After reaching maximum volume (0.9 L), the culture was continued until the cell viability was less than 50%. After production, the chimeric EMAB604 antibody was purified by protein A affinity chromatography and monitored by polyacrylamide gel electrophoresis.
Exemple 3 : Protocole de mise en œuvre du test d'ADCCExample 3: Implementation Protocol of the ADCC Test
Les cellules cibles (lignées HT-144 ou GUY 17.2) sont incubées avec différentes concentrations d'anticorps (0, 0.1, 1, 10 μg/ml) et les cellules effectrices (cellules NK) purifiées par un kit de déplétion négative (NK CeIl Isolation Kit, Myltenyi, Paris, France) à partir de sang périphérique de donneurs sains. Après 4 heures d'incubation, l'activité cytotoxique induite par les anticorps est mesurée par colorimétrie en dosant dans les surnageants, l'activité de l'enzyme lactate déshydrogénase (LDH) libérée par les cellules lysées . Les résultats sont exprimés en pourcentage de lyse spécifique en fonction de la concentration d'anticorps. Les valeurs d'Emax (pourcentage de lyse maximale), ainsi que les valeurs d'EC50 (quantité d'anticorps induisant 50% de la lyse maximale), sont calculées à l'aide du logiciel PRISM (Graphpad Software) .The target cells (lines HT-144 or GUY 17.2) are incubated with different concentrations of antibodies (0, 0.1, 1, 10 μg / ml) and the effector cells (NK cells) purified by a negative depletion kit (NK CeIl Isolation Kit, Myltenyi, Paris, France) from peripheral blood of healthy donors. After 4 hours of incubation, the cytotoxic activity induced by the antibodies is measured by colorimetry by assaying in the supernatants the activity of the lactate dehydrogenase (LDH) enzyme released by the lysed cells. The results are expressed as a percentage of specific lysis as a function of the antibody concentration. The values of Emax (percentage of maximal lysis), as well as the EC50 values (amount of antibody inducing 50% of the maximum lysis), are calculated using the software PRISM (Graphpad Software).
Exemple 4 : Activité ADCC des anticorps EMAB604 et anti-HLA-DR produits par la lignée CHO sur la lignée de mélanome GUY 17.2 (Fig. 1) : L'anticorps EMAB604 induit une lyse spécifique de la lignée GUY-17.2 supérieure à celle induite par un anticorps anti-HLA-DR produit dans CHO.EXAMPLE 4 ADCC Activity of the EMAB604 and Anti-HLA-DR Antibodies Produced by the CHO Line on the GUY 17.2 Melanoma Line (FIG. The EMAB604 antibody induces a specific lysis of the GUY-17.2 line greater than that induced by an anti-HLA-DR antibody produced in CHO.
Les valeurs de lyse maximales (Emax) sont de 19 et 15% pour les anticorps EMAB604 et anti-HLA-DR CHO. Les EC50 correspondantes (quantité d'anticorps requise pour atteindre 50% de la lyse maximale) sont de 0,45 et 5,71 μg/ml respectivement, montrant que l'activité de l'anticorps EMAB604 est environ 10 fois plus forte que celle de l'anticorps anti-HLA-DR produit dans CHO.The maximum lysis values (Emax) are 19 and 15% for the EMAB604 and anti-HLA-DR CHO antibodies. The corresponding EC50s (amount of antibody required to reach 50% of maximal lysis) are 0.45 and 5.71 μg / ml respectively, showing that the activity of the EMAB604 antibody is approximately 10 times stronger than that of the anti-HLA-DR antibody produced in CHO.
Calcul des valeurs ADCC Emax (lyse maximale) et EC50 (concentration d'anticorps requise pour obtenir 50% du Emax) obtenus après modélisation (sigmoïde) de la courbe par le logiciel PRISM.Calculation of the ADCC Emax (maximum lysis) and EC50 (antibody concentration required to obtain 50% Emax) values obtained after modeling (sigmoid) of the curve by the PRISM software.
L'activité ADCC est dépendante du CD16 puisqu'elle est inhibée pour tous les anticorps en présence de l'anticorps murin 3G8 (anti-CDlβ) (Fig.2).The ADCC activity is dependent on CD16 since it is inhibited for all the antibodies in the presence of the murine antibody 3G8 (anti-CD1β) (FIG.
Exemple 5 : Activité ADCC des anticorps EMAB604 et anti-HLA-DR produits par la lignée CHO sur la lignée de mélanome HT-144 (Fig. 3)EXAMPLE 5 ADCC Activity of the EMAB604 and Anti-HLA-DR Antibodies Produced by the CHO Line on the HT-144 Melanoma Line (FIG.
L'anticorps EMAB604 de l'invention induit une lyse spécifique de la lignée de mélanome HT-144 supérieure à celle induite par un anticorps anti-HLA- DR produit dans CHO.The EMAB604 antibody of the invention induces specific lysis of the HT-144 melanoma line that is greater than that induced by an anti-HLA-DR antibody produced in CHO.
Les valeurs de lyse maximales (Emax) sont de 18 et 17% pour les anticorps EMAB604 et anti-HLA-DR CHO. Les EC50 correspondantes sont de 0,45 et 1,45 μg/ml respectivement, montrant que l'activité de l'anticorps EMAB604 est environ 8 fois plus forte que celle de l'anticorps anti-HLA-DR produit dans CHO.The maximum lysis values (Emax) are 18 and 17% for the EMAB604 and anti-HLA-DR CHO antibodies. The corresponding EC50s are 0.45 and 1.45 μg / ml respectively, showing that the activity of the antibody EMAB604 is approximately 8 times stronger than that of the anti-HLA-DR antibody produced in CHO.
Calcul des valeurs ADCC Emax (lyse maximale) et EC50 (concentration d'anticorps requise pour obtenir 50% du Emax) obtenus après modélisation (sigmoïde) de la courbe par le logiciel PRISM.Calculation of the ADCC Emax (maximum lysis) and EC50 (antibody concentration required to obtain 50% Emax) values obtained after modeling (sigmoid) of the curve by the PRISM software.
L'activité ADCC est dépendante du CD16 puisqu'elle est inhibée pour tous les anticorps en présence de l'anticorps murin 3G8 (anti-CDlβ) (Fig.The ADCC activity is dependent on CD16 since it is inhibited for all antibodies in the presence of murine antibody 3G8 (anti-CD1β) (FIG.
4) .4).
Exemple 6 : Protocole de mise en œuvre du test de sécrétion d' IL-2 (Fig. 5 et 6)Example 6: Protocol for implementing the IL-2 secretion test (Figs 5 and 6)
Ce test utilise une lignée Jurkat transfectée avec le récepteur CD16 (FcgammaRIIIa) humain comme cellule effectrice (Jurkat-CDl6) . La technique est basée sur la mesure de la sécrétion d' interleukine 2 (IL-2) par la lignée Jurkat-CD16 induite par l'engagement du CD16 avec les anticorps testés fixés sur les cellules cibles (lignées de mélanome HT-144 ou GUY 17.2). Pour cela, 25 ng/ml des anticorps testés sont ajoutées aux cellules cibles (1.5 x 105 cellules/ml) en présence des cellules Jurkat-CD16 (5 x 106 cellules /ml) et de 10 ng/ml de l'acétate de phorbol myristate (PMA) pendant 18 heures à 370C en 5% CO2- Les plaques de culture sont ensuite centrifugées et l'IL-2 libérée dans le surnageant de culture quantifiée par ELISA (Quantikine IL-2, R&D, Abingdon, UK) . Le résultat final est exprimé en unité d' absorbance (DO).This test uses a Jurkat line transfected with the human CD16 receptor (FcgammaRIIIa) as an effector cell (Jurkat-CD16). The technique is based on measuring the secretion of interleukin-2 (IL-2) by the Jurkat-CD16 line induced by the commitment of CD16 with the tested antibodies fixed on the target cells (melanoma lines HT-144 or GUY 17.2). For this, 25 ng / ml of the antibodies tested are added to the target cells (1.5 × 10 5 cells / ml) in the presence of Jurkat-CD16 cells (5 × 10 6 cells / ml) and 10 ng / ml of the acetate of phorbol myristate (PMA) for 18 hours at 37 ° C. in 5% CO 2 - The culture plates are then centrifuged and the IL-2 released in the culture supernatant quantified by ELISA (Quantikine IL-2, R & D, Abingdon, UK). The final result is expressed in absorbance unit (OD).
HT 144 R297 EMAB604 HLA-DR CHOHT144 R297 EMAB604 HLA-DR CHO
25 ng/ml 0 1,199 0,4925 ng / ml 0 1,199 0.49
GUY 17.2 R297 EMAB604 HLA-DR CHOGUY 17.2 R297 EMAB604 HLA-DR CHO
25 ng/ml 0,007 1,353 0,281 Tableau de résultats correspondant aux figures 5 et 625 ng / ml 0.007 1.353 0.281 Table of results corresponding to FIGS. 5 and 6
Exemple 7 : Capacité des anticorps EMAB604 et anti- HLA-DR produits par la lignée CHO sur la lignée de mélanome GUY 17.2 à induire la sécrétion d'IL-2 par la lignée Jurkat-CD16 (Fig. 5) .EXAMPLE 7 Ability of the EMAB604 and anti-HLA-DR antibodies produced by the CHO line on the GUY 17.2 melanoma line to induce the secretion of IL-2 by the Jurkat-CD16 line (FIG.
L'anticorps EMAB604 induit une sécrétion d'IL-2 en présence de la lignée GUY 17.2 supérieure au même anticorps produit dans CHO. Les valeurs de lyse maximales (Emax) sont de 1.35 et 0,28 unités DO pour les anticorps EMAB604 et anti- HLA-DR CHO.The EMAB604 antibody induces a secretion of IL-2 in the presence of the GUY 17.2 superior line to the same antibody produced in CHO. The maximum lysis values (Emax) are 1.35 and 0.28 OD units for the EMAB604 and anti-HLA-DR CHO antibodies.
L'anticorps R297, anti-Rhésus D produit dans YB2/0, sert de témoin négatif vis-à-vis des cellules GUY 17.2.The R297 anti-Rhesus D antibody produced in YB2 / 0 serves as a negative control against GUY 17.2 cells.
Exemple 8 : Capacité des anticorps EMAB604 et anti- HLA-DR produits par la lignée de mélanome HT-144 à induire la sécrétion d'IL-2 par la lignée Jurkat-CD16 (Fig.6)EXAMPLE 8 Ability of the EMAB604 and Anti-HLA-DR Antibodies Produced by the HT-144 Melanoma Line to Induce the Secretion of IL-2 by the Jurkat-CD16 Line (FIG.
L'anticorps EMAB604 induit une sécrétion d'IL-2 en présence de la lignée HT-144 supérieure au même anticorps produit dans CHO.The EMAB604 antibody induces an IL-2 secretion in the presence of the higher HT-144 line to the same antibody produced in CHO.
Les valeurs de lyse maximales (Emax) sont de 1.2 et 0,49 unités DO pour les anticorps EMAB604 et anti-HLA- DR CHO. L'anticorps R297, anti-Rhésus D produit dans YB2/0, sert de témoin négatif vis-à-vis des cellules HT-144.The maximum lysis values (Emax) are 1.2 and 0.49 OD units for the EMAB604 and anti-HLA-DR CHO antibodies. The R297 anti-Rhesus D antibody produced in YB2 / 0 serves as a negative control against HT-144 cells.
Exemple 9 : Protocole du test de fixation au récepteur CDl6Example 9: Protocol of the CD16 receptor binding test
La fixation au récepteur CDlβ est évaluée par un test de compétition de l'anticorps murin anti-CDlβ (3G8).The binding to the CD1β receptor is evaluated by a competition test of murine anti-CD1β (3G8) antibody.
Les cellules effectrices (cellules NK) sont purifiées par le kit de déplétion négative (NK CeIl Isolation Kit, Myltenyi, Paris, France), à partir de sang périphérique de donneurs sains . Puis , les cellules NK sont incubées avec des concentrations variables (0, 10 et 50 μg/ml) d'anticorps à évaluer et l'anticorps anti-CDlβ (3G8) couplé à un fluorochrome (3G8-PE) à concentration fixe.The effector cells (NK cells) are purified by the negative depletion kit (NK CeIl Isolation Kit, Myltenyi, Paris, France) from peripheral blood of healthy donors. Then, the NK cells are incubated with variable concentrations (0, 10 and 50 μg / ml) of antibodies to be evaluated and the anti-CD1β (3G8) antibody coupled to a fluorochrome (3G8-PE) at a fixed concentration.
Après lavage, la fixation du 3G8-PE sur le récepteur CDlβ des cellules NK est évaluée par cytométrie en flux. Les résultats sont exprimés en pourcentage de fixation, 100% de fixation correspondant à l'inhibition totale de la fixation de l'anticorps anti-CDlβ (3G8) murin.After washing, the binding of 3G8-PE on the CD1β receptor of NK cells is evaluated by flow cytometry. The results are expressed as percentage fixation, 100% fixation corresponding to the total inhibition of the binding of the murine anti-CD16 (3G8) antibody.
L'anticorps EMAB604 se fixe fortement sur le CDlβ des cellules NK et cela d'une façon comparable à celle d'un anticorps anti-CD20 produit par la plateforme EMABling (et décrit dans la demande de brevet WO 2006064121) . Cette fixation (62%) est environ 3 fois supérieure à celle de Rituxan® (18,5%), anticorps produit dans la lignée CHO, à la concentration de 50 μg/ml (Fig. 7) . The EMAB604 antibody binds strongly to the CD1β of the NK cells in a manner comparable to that of an anti-CD20 antibody produced by the EMABling platform (and described in the patent application WO 2006064121). This fixation (62%) is approximately 3 times greater than that of Rituxan® (18.5%), an antibody produced in the CHO line, at a concentration of 50 μg / ml (Figure 7).

Claims

Revendications claims
1. Anticorps monoclonal, fragment d'anticorps monoclonal ou dérivé d'anticorps monoclonal, dirigé contre le récepteur humain des LDL (Low Density Lipoprotein) , caractérisé en ce que la région variable de chacune de ses chaînes légères est codée par la séquence d'acide nucléique murine SEQ ID NO : 5, la région variable de chacune de ses chaînes lourdes est codée par la séquence d'acide nucléique murine SEQ ID NO : 7, ou par des séquences d'acide nucléique présentant une homologie suffisante avec les séquences SEQ ID NO : 5 et SEQ ID NO : 7 pour que la nature et l'affinité de la liaison dudit anticorps à son antigène ne soient pas modifiées, et les régions constantes de ses chaînes légères et de ses chaînes lourdes sont des régions constantes provenant d'une espèce non-murine.Monoclonal antibody, monoclonal antibody fragment or monoclonal antibody derivative, directed against the human LDL receptor (Low Density Lipoprotein), characterized in that the variable region of each of its light chains is encoded by the sequence of Murine nucleic acid SEQ ID NO: 5, the variable region of each of its heavy chains is encoded by the murine nucleic acid sequence SEQ ID NO: 7, or by nucleic acid sequences having sufficient homology with the SEQ sequences And the constant regions of its light and a non-murine species.
2. Anticorps selon la revendication 1, caractérisé en ce que les régions constantes de chacune de ses chaînes légères et de chacune de ses chaînes lourdes sont des régions constantes humaines .2. Antibody according to claim 1, characterized in that the constant regions of each of its light chains and each of its heavy chains are human constant regions.
3. Anticorps selon l'une quelconque des revendications 1 ou 2, caractérisé en ce que la région constante de chacune de ses chaînes légères est de type K.3. Antibody according to any one of claims 1 or 2, characterized in that the constant region of each of its light chains is of type K.
4. Anticorps selon l'une quelconque des revendications précédentes, caractérisé en ce que la région constante de chacune de ses chaînes lourdes est de type γ. 4. Antibody according to any one of the preceding claims, characterized in that the constant region of each of its heavy chains is of type γ.
5. Anticorps selon la revendication 4, caractérisé en ce que la région constante de chacune de ses chaînes lourdes est de type γl .5. Antibody according to claim 4, characterized in that the constant region of each of its heavy chains is of type γl.
6. Anticorps selon la revendication 5, caractérisé en ce que la région constante de chacune de ses chaînes lourdes est de type γl et est codée par la séquence d'acide nucléique SEQ ID NO : 23 et en ce que la région constante de chacune de ses chaînes légères est codée par la séquence d'acide nucléique SEQ ID NO : 21.6. Antibody according to claim 5, characterized in that the constant region of each of its heavy chains is of γ1 type and is encoded by the nucleic acid sequence SEQ ID NO: 23 and in that the constant region of each of its light chains is encoded by the nucleic acid sequence SEQ ID NO: 21.
7. Anticorps selon l'une quelconque des revendications précédentes, caractérisé en ce que chacune de ses chaînes légères est codée par la séquence d'acide nucléique chimérique SEQ ID NO : 13 et en ce que chacune de ses chaînes lourdes est codée par la séquence d'acide nucléique chimérique SEQ ID NO : 19.7. Antibody according to any one of the preceding claims, characterized in that each of its light chains is encoded by the chimeric nucleic acid sequence SEQ ID NO: 13 and in that each of its heavy chains is encoded by the sequence chimeric nucleic acid SEQ ID NO: 19.
8. Anticorps selon la revendication 7 , caractérisé en ce que la séquence peptidique déduite de la séquence SEQ ID NO : 13 est la séquence SEQ ID NO : 14 et en ce que la séquence peptidique déduite de la séquence SEQ ID NO : 19 est la séquence SEQ ID NO : 20.8. Antibody according to claim 7, characterized in that the peptide sequence deduced from the sequence SEQ ID NO: 13 is the sequence SEQ ID NO: 14 and in that the peptide sequence deduced from the sequence SEQ ID NO: 19 is the sequence SEQ ID NO: 20.
9. Anticorps selon l'une quelconque des revendications précédentes, caractérisé en ce qu'il est produit par une lignée cellulaire d'hybridome de rat.An antibody according to any one of the preceding claims, characterized in that it is produced by a rat hybridoma cell line.
10. Anticorps selon la revendication 9, caractérisé en ce qu'il est produit dans l'hybridome de rat YB2/0 (cellule YB2/3HL.P2.GIl .16Ag.20 , déposée à l 'American Type Culture Collection sous le numéro ATCC CRL-1662) . 10. Antibody according to claim 9, characterized in that it is produced in the rat hybridoma YB2 / 0 (cell YB2 / 3HL.P2.GI1 .16Ag.20, deposited at the American Type Culture Collection under the number ATCC CRL-1662).
11. Anticorps selon la revendication 10, caractérisé en ce qu'il s'agit de l'anticorps EMAB604 produit par le clone R604 déposé sous le numéro d'enregistrement CNCM 1-3692 à la Collection Nationale de Cultures de Microorganismes (CNCM) .11. Antibody according to claim 10, characterized in that it is the EMAB604 antibody produced by the clone R604 deposited under the registration number CNCM 1-3692 to the National Collection of Cultures of Microorganisms (CNCM).
12. Vecteur d'expression de la chaîne légère d'un anticorps tel que défini selon l'une quelconque des revendications 1 à 11 de séquence SEQ ID NO : 12.12. Expression vector of the light chain of an antibody as defined according to any one of claims 1 to 11 of sequence SEQ ID NO: 12.
13. Vecteur d'expression de la chaîne lourde d'un anticorps tel que défini selon l'une quelconque des revendications 1 à 11 de séquence SEQ ID NO : 18.13. Expression vector of the heavy chain of an antibody as defined according to any one of claims 1 to 11 of sequence SEQ ID NO: 18.
14. Lignée cellulaire stable exprimant un anticorps selon l'une quelconque des revendications 1 à 11.A stable cell line expressing an antibody according to any one of claims 1 to 11.
15. Lignée cellulaire stable selon la revendication 14, caractérisée en ce que la lignée cellulaire dans laquelle l'anticorps est exprimé, est choisie parmi le groupe consistant en : SP2/0, YB2/0, IR983F, le myélome humain Namalwa, PERC6, les lignées CHO, notamment CHO-K- 1, CHO-LeclO, CHO-Lecl, CHO-Lecl3 , CHO Pro-5, CHO dhfr-, Wil-2, Jurkat, Vero, MoIt-4, COS-7, 293- HEK, BHK, K6H6, NSO, SP2/0-Ag 14 et P3X63Ag8.653.The stable cell line according to claim 14, characterized in that the cell line in which the antibody is expressed is selected from the group consisting of: SP2 / 0, YB2 / 0, IR983F, Namalwa human myeloma, PERC6, CHO lines, in particular CHO-K-1, CHO-LeclO, CHO-Lecl, CHO-Lecl3, CHO Pro-5, CHO dhfr-, Wil-2, Jurkat, Vero, MoIt-4, COS-7, 293- HEK, BHK, K6H6, NSO, SP2 / O-Ag14 and P3X63Ag8.653.
16. Lignée cellulaire stable selon la revendication 14 ou 15, ayant intégré les deux vecteurs d'expression selon les revendications 12 et 13. 16. The stable cell line according to claim 14 or 15, having integrated the two expression vectors according to claims 12 and 13.
17. Clone R604 déposé sous le numéro d'enregistrement CNCM 1-3692 à la Collection Nationale de Cultures de Microorganismes (CNCM) .17. Clone R604 registered under registration number CNCM 1-3692 at the National Collection of Cultures of Microorganisms (CNCM).
18. Fragment d'ADN de séquence SEQ ID NO : 19 codant pour la chaîne lourde d'un anticorps selon l'une quelconque des revendications 1 à 11.18. DNA fragment of sequence SEQ ID NO: 19 encoding the heavy chain of an antibody according to any one of claims 1 to 11.
19. Fragment d'ADN de séquence SEQ ID NO : 13 codant pour la chaîne légère d'un anticorps selon l'une des revendications 1 à 11.19. DNA fragment of sequence SEQ ID NO: 13 encoding the light chain of an antibody according to one of claims 1 to 11.
20. Utilisation d'un anticorps selon l'une quelconque des revendications 1 à 11, pour activer In vitro les récepteurs FcγRIII de cellules immunitaires effectrices ou provoquer la sécrétion de cytokines ou de chimiokines par des cellules effectrices .20. Use of an antibody according to any one of claims 1 to 11 for activating in vitro the FcγRIII receptors of effector immune cells or causing the secretion of cytokines or chemokines by effector cells.
21. Anticorps selon l'une quelconque des revendications 1 à 11, pour son utilisation comme médicament .An antibody according to any one of claims 1 to 11 for use as a medicament.
22. Utilisation d'un anticorps selon l'une quelconque des revendications 1 à 11, pour la fabrication d'un médicament destiné au traitement du cancer .22. Use of an antibody according to any one of claims 1 to 11 for the manufacture of a medicament for the treatment of cancer.
23. Utilisation d'un anticorps selon l'une quelconque des revendications 1 à 11, pour la fabrication d'un médicament destiné au traitement d'un mélanome.23. Use of an antibody according to any one of claims 1 to 11 for the manufacture of a medicament for the treatment of melanoma.
24. Utilisation d'un anticorps monoclonal dirigé contre le récepteur humain des LDL pour la fabrication d'un médicament destiné au traitement d'un mélanome. 24. Use of a monoclonal antibody directed against the human LDL receptor for the manufacture of a medicament for the treatment of melanoma.
25. Utilisation d'un anticorps selon la revendication 23 ou 24, en combinaison avec un ou plusieurs autre (s) anticorps dirigé (s) contre un ou plusieurs autre (s) antigène (s) exprimé (s) sur les cellules de mélanome.25. Use of an antibody according to claim 23 or 24, in combination with one or more other antibodies directed against one or more other antigen (s) expressed on the melanoma cells. .
26. Utilisation d'un anticorps selon la revendication 25, caractérisée en ce que ledit antigène exprimé sur les cellules de mélanome est choisi parmi le HLA-DR, CD20, CD22, CD23, CD25, CD30, CD33 et le CD40.26. Use of an antibody according to claim 25, characterized in that said antigen expressed on the melanoma cells is selected from HLA-DR, CD20, CD22, CD23, CD25, CD30, CD33 and CD40.
27. Utilisation selon l'une quelconque des revendications 22 à 25, in vitro ou ex vivo, d'un anticorps tel que défini selon l'une quelconque de ces revendications, en association avec des cellules exprimant des FcγR, telles que les cellules NK (Natural Killer) , les cellules NKT (Natural Killers T) , les lymphocytes Tγδ, les macrophages, les monocytes, toute cellule génétiquement modifiée pour exprimer le CDlβ ou les cellules dendritiques .27. Use according to any one of claims 22 to 25, in vitro or ex vivo, of an antibody as defined according to any one of these claims, in association with cells expressing FcγR, such as NK cells. (Natural Killer), NKT (Natural Killers T) cells, Tγδ lymphocytes, macrophages, monocytes, any cell genetically modified to express CD1β or dendritic cells.
28. Composition pharmaceutique comprenant au moins un anticorps selon l'une quelconque des revendications 1 à 11 et au moins un excipient et/ou au moins un véhicule pharmaceutiquement acceptables.28. A pharmaceutical composition comprising at least one antibody according to any one of claims 1 to 11 and at least one excipient and / or at least one pharmaceutically acceptable carrier.
29. Composition selon la revendication 28, caractérisée en ce qu'elle comprend en outre au moins un anticorps dirigé contre un autre antigène présent sur les cellules cibles desdits anticorps . 29. Composition according to claim 28, characterized in that it further comprises at least one antibody directed against another antigen present on the target cells of said antibodies.
30. Composition selon l'une des revendications 28 et 29, caractérisée en ce qu'elle comprend en outre un anticorps anti- HLA-DR. 30. Composition according to one of claims 28 and 29, characterized in that it further comprises an anti-HLA-DR antibody.
EP07872460A 2006-12-29 2007-12-28 Monoclonal antibody directed against the human ldl receptor Withdrawn EP2167544A2 (en)

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