ANTIBODY AGAINST GALECTIN 9 AND INHIBITOR OF THE SUPPRESSIVE ACTIVITY OF T REGULATORY LYMPHOCYTES

专利摘要:
The present invention relates to an anti-galectin-9 antibody and an inhibitor of regulatory T-cell suppressive activity, as well as to the use of this antibody for the treatment of diseases associated with the suppressive activity of regulatory T-cells, including treatment of cancer.
公开号:FR3021970A1
申请号:FR1455177
申请日:2014-06-06
公开日:2015-12-11
发明作者:Nadira Delhem；Pierre Busson；Olivier Morales；Clement Barjon；Dhafer Mrizak
申请人:CELLVAX；Centre National de la Recherche Scientifique CNRS；Universite Lille 1 Sciences et Technologies；Institut Gustave Roussy (IGR)；Universite Lille 2 Droit et Sante；Universite Paris Sud Paris 11；
IPC主号:

专利说明:

[0001] The present invention relates to an anti-galectin-9 antibody and an inhibitor of regulatory T-cell suppressive activity, and to the use of this antibody for the treatment of diseases associated with the suppressive activity of regulatory T-cells.
[0002] Human T lymphocytes are characterized by the expression of a membrane marker called CD3 and a specific receptor, the TCR (T cell receptor), which is directly involved in the specific recognition of an antigen. This antigenic recognition by the naive T lymphocyte induces the activation of the primary immune response resulting in a modification of the phenotype and the activity of the T lymphocytes.
[0003] Different types of lymphocyte populations T will develop, for example effector cells or effector T cells, which will provide specialized functions to defend the body. Thus, CD4 + T lymphocytes, also called helper T cells, secrete major cytokines that help in particular B cells in their humoral function (production of specific antibodies) and CD8 + T lymphocytes in their cytotoxic activity. Another population of CD4 + T cells consists of natural regulatory T cells, hereafter referred to more briefly as "regulatory T cells". They overexpress constitutively the CD25 molecule (the regulatory T lymphocytes can thus also be called "CD4 + CD25 +") and the Foxp3 transcription factor. This low percentage of CD4 + CD25 + T cells has the particularity of negatively regulating the actors of the immune response who would have recognized various autoantigens by their TCRs. Regulatory T lymphocytes thus play a major role in the physiology of the immune system, in particular to protect the body against the emergence of autoimmune diseases. However, it has been pointed out that, in pathological situations, regulatory T lymphocytes can induce inappropriate immunosuppression, which then promotes tumor growth or the persistence of infectious pathogens (viruses, bacteria, parasites, etc.). Numerous studies have thus shown that regulatory T cells decrease anti-tumor or anti-viral immune responses, in particular by inappropriately inhibiting the activity of effector T cells, thus favoring the persistence of viruses and the tumor progression in a large scale. majority of cancers. The mechanisms by which regulatory T cells exert their suppressive effects on effector T cells are still poorly understood. However, various studies have highlighted different mechanisms by which regulatory T cells could suppress the immune response. Among the possible explanations, for example, studies have shown that Foxp3 + regulatory T lymphocytes can lyse effector T lymphocytes via the production of granzymes / perforins (1) or by labeling effector lymphocytes to IL-2 or by inhibiting the proliferation of effector T cells, in particular by expressing surface molecules such as galectin 1 which interacts with receptors expressed on effector T lymphocytes and induces cell cycle arrest of effector T lymphocytes (2). The pathophysiological role of Treg in cancers has thus encouraged the emergence of a new anti-tumor therapeutic strategy. It consists in neutralizing the inhibitory factors of the immune response, and in particular the regulatory T lymphocytes, or in other words to break the tolerance towards the tumor antigens. Indeed, in order to reverse the balance between regulatory T cells and effector T cells in the control of anti-tumor immunity, many teams have sought to develop therapeutic strategies to inhibit CD4 + CD25 + regulatory T cells. , in particular by targeting, with monoclonal antibodies, surface molecules expressed by these regulatory T cells, and especially those involved in the suppressive activity of these regulatory T cells.
[0004] For example, rodent analyzes have shown that inhibition of CD4 + CD25 + regulatory T cells by a monoclonal antibody directed against the IL-2 alpha receptor (CD25) promotes activation and expansion of T cells. effector inhibiting tumor growth (3). However, CD25 is also expressed by activated effector T cells. Thus this strategy is to be taken with caution insofar as it can also promote the elimination of effector T cells. It has also been documented that activation of GITR signaling via an anti-GITR antibody is capable of inhibiting the suppressive activity of regulatory T cells (4). In this context, the use of an anti-GITR antibody in the treatment of murine tumors made it possible to increase the anti-tumor response of the CD4 + and CD8 + T lymphocytes, and this more effectively when the tumor is already installed. However, activated effector T cells also express GITR. Therefore, there is also a risk of effector T-cell suppression when using an anti-GITR antibody. The strategy to inhibit regulatory T cells has also been contemplated by the use of an anti-CTLA4 antibody, a marker expressed by regulatory T cells. Thus, in a mouse model presenting CTLA-4 KO (KnockOut) in regulatory T cells, or when using anti-CTLA-4 antibodies, an increase in lymphocyte activity was shown T effectors and a decrease in suppression mediated by regulatory T cells, leading to inhibition of tumor growth (5). However, activated effector T cells also express the CTLA-4 marker. Therefore, there is, again, a risk of suppression of effector T cells by the use of an anti-GITR antibody. Thus, despite the promising effect of these different molecules, one of the obstacles to the specific depletion of regulatory T cells is the lack of specificity of their surface markers. Indeed, the CD25, CTLA-4 or GITR surface proteins, expressed by regulatory T cells, are also markers of activation of effector T lymphocytes. The use of these proteins as targets for the depletion of regulatory T cells thus has the undesirable effect of eliminating numerous CD4 + and CD8 + effector T cells, which are essential for tumor regression. In this context, it remains very difficult to specifically target regulatory T cells in therapeutic protocols. Therefore, there is still a need for compounds to selectively and effectively inhibit the suppressive activity of regulatory T cells. One of the aims of the invention is thus to provide an antibody which is directed against a marker specific for regulatory T cells and which allows the inhibition of the suppressive activity of regulatory T cells, without altering the function of effector T cells. The inventors have the merit of having demonstrated that a molecule, galectin 9, is expressed specifically by regulatory T cells during activation and that an antibody directed against this molecule makes it possible to inhibit suppressive activity of regulatory T cells, and this specifically, that is to say without inhibiting effector T cells.
[0005] It has thus been discovered that such an anti-galectin-9 antibody and inhibitor of regulatory T-cell suppressive activity could be used in the treatment of diseases associated with the suppressive activity of regulatory T cells. The term "disease associated with the suppressive activity of regulatory T cells" is understood to mean any disease in which the suppressive activity of regulatory T lymphocytes plays a role, in particular by promoting the development or persistence of the disease. In particular, the suppressive activity of regulatory T cells has been shown to promote the development of tumors. The invention therefore more particularly targets cancers in which the suppressive activity of T lymphocytes plays a role.
[0006] Galectin 9, which can be more concisely called "gal9", is part of the galectin family. Galectins, or type S lectins, constitute a family consisting of fifteen members in vertebrates, including ten in humans. Galectin 9 interacts preferentially with the beta-galactoside residues of glycoproteins and glycolipids.
[0007] In humans, galectin 9 exists in three isoforms, long, medium and short. Several studies have been conducted to determine the link between the development of cancers and galectins, including galectin 9. However, it is considered in most of these studies that galectin 9 has a cytotoxic activity against activated T cells ( whether CD4 + or CD8 +) and has no cytotoxic activity against unactivated T cells. For example, patent application EP1586325 is based on the assumption that, in vitro, galectin 9 induces the apoptosis of tumor cells, in particular in malignant or metastatic cells, but not normal cells. The application EP1586325 thus targets a drug comprising galectin 9 or molecules inducing the production and / or release of galectin 9, etc.
[0008] The objective was therefore to use galectin 9 or factors making it possible to increase galectin 9, while the present invention tends, on the contrary, to inhibit it. The present invention thus relates to an antibody directed against galectin 9 and an inhibitor of the suppressive activity of regulatory T cells. Indeed, the invention is based on the unexpected findings made by the inventors, who have observed (i) on the one hand, that galectin 9 is directly expressed by regulatory T cells and that its expression is increased during their activation and (ii) on the other hand, that galectin 9 is very weakly expressed by effector T lymphocytes and that this expression disappears during activation. Moreover, the inventors have observed that the inhibition of galectin 9 by an antibody allows the inhibition of the suppressive activity of regulatory T cells.
[0009] By "regulatory T cells" is meant the subregion of naturally occurring regulatory T cells, also called Treg, characterized by constitutive expression of CD25, CTLA-4, and GITR and by specific expression of the Foxp3 transcription factor. Regulatory T lymphocytes more particularly targeted by the present invention are therefore natural regulatory T cells, or nTreg.
[0010] The term "suppressive activity of regulatory T cells" is understood to mean the immunosuppressive activity that regulatory T cells exert on effector T lymphocytes, once activated, in a pathological situation and which particularly favors tumor growth. Preferably, the suppressive activity of regulatory T cells can be understood as the activity decreasing anti-tumor immune responses by inhibiting the activity of effector T cells. The suppressive activity of regulatory T cells can be analyzed according to various techniques known to those skilled in the art. For example, a MLR (Mixed Leukocyte Reaction) method can be performed, which implements a co-culture of Treg lymphocytes and immune cells (total PBMC or T CD4 +) autologous or heterologous. This can be carried out by proliferation tests based on (i) the incorporation of radioelements such as Tritiated Thymidine or (ii) the incorporation of EdU (5-ethynyl-2'-deoxyuridine) which is incorporated during the synthesis of the DNA and which following an enzymatic reaction will allow the emission of a fluorescence (Click-it EdU Proliferation test) or (iii) flow cytometry (CSFE). The inhibitory effect of an antibody according to the invention can thus for example be analyzed by such an MLR method, carried out in the presence of the antibody tested. Here we can refer to the "Examples", "Materials and methods" section for more details on the implementation of such a method of analysis. The terms "antibodies" and "immunoglobulin" are used interchangeably and refer to immunoglobulin molecules or immunologically active portions of immunoglobulin molecules, i.e. molecules comprising the specific binding sites of a given antigen. The term antibody covers not only whole antibody molecules but also antibody fragments and variants (including derivatives such as humanized antibodies) of antibodies and antibody fragments. Immunoglobulins are well known to those skilled in the art and consist of two heavy chains connected to each other by disulfide bridges, each heavy chain being connected to a light chain by a disulfide bridge. There are two kinds of light chains, the lambda (X) and kappa (K) strings. There are five major classes of heavy chains that determine the functional activity of the antibody: IgM, IgD, IgG, IgA and IgE.
[0011] Each string contains separate sequence domains. The light chain comprises two domains, a variable domain (or region) (VL) and a constant domain (CL). The heavy chain comprises four or five domains according to the classes of antibodies, a variable domain (VH) and three or even four constant domains (CH1, CH2, CH3 and optionally CH4). The variable regions of the light (VL) and heavy (VH) chains determine the specificity for the antigen and the binding site on this antigen. The constant domains of the light (CL) and heavy (CH) chains confer on the antibody important biological properties such as the association of the antibody chains with each other, the mobility through the placenta, the fixation of the complement and / or the attachment to the antibodies. Fc receptors (FcR). The Fv fragment corresponds to the V-terminal part of the Fab fragment, described below, of the immunoglobulin, and comprises the variable portions of a light chain and a heavy chain (VL and VH). The specificity of the antibody lies in the structural complementarity between the recognition site of the antibody and the antigenic determinant. The antibody recognition site consists essentially of residues from hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable regions or framework or framework regions influence the general structure of the domain and hence the recognition sites. The term "complementarity regions" (CDRs) refers to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of the native immunoglobulin binding site. Each of the light and heavy chains of an immunoglobulin has three CDR regions designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen binding site therefore comprises six CDRs. The framework regions (FR) refer to the amino acid sequences interposed between the CDRs, i.e., portions of the variable regions of the light and heavy immunoglobulin chains that are relatively conserved between different immunoglobulins of the same species. .
[0012] An antibody according to the invention may be a monoclonal or polyclonal antibody. Preferably, an antibody according to the invention is a monoclonal antibody. The term "monoclonal antibody" or "mAb" (for "monoclonal antibody") refers to an antibody of unique amino acid composition, which is directed against a specific antigen and can be produced by a single B cell clone, or hybridoma. Monoclonal antibodies can also be recombinant, i.e., be produced by protein engineering techniques.
[0013] The term "Fab" refers to an antibody fragment of about 50,000 dalton molecular weight and having antigen binding activity. It comprises about half of the N-terminal side of the heavy chain and the entire light chain bound by a disulfide bridge. The Fab can be obtained in particular by the treatment of immunoglobulin with a protease, papain. The term "F (ab ') 2" refers to a fragment of about 100,000 dalton and antigen binding activity. This fragment is slightly larger than two Fab fragments connected via a disulfide bridge in the hinge region. These fragments are obtained by treatment of an immunoglobulin with a protease, pepsin. The Fab fragment can be obtained from the F (ab ') 2 fragment by cleaving the disulfide bridge of the hinge region. A single Fv chain "scFv" corresponds to a VH: VL polypeptide synthesized using the genes encoding the VL and VH domains and a sequence encoding a peptide for binding these domains. An scFv according to the invention includes CDRs maintained in a suitable conformation, for example using genetic recombination techniques.
[0014] The dimers of "ScFv" correspond to two molecules of scFv linked together by a peptide bond. This Fv chain is frequently the result of the expression of a fusion gene including the genes encoding VH and VL linked by a linker sequence encoding a peptide. The human scFv fragment may include CDRs regions that are maintained in an appropriate conformation, preferably through the use of genetic recombination techniques. The "dsFv" fragment is a VH-VL heterodimer stabilized by a disulfide bridge; it can be divalent (dsFV2). Sc (Fv) 2 or multivalent divalent antibody fragments can be formed spontaneously by association of monovalent scFvs or produced by linking scFvs fragments by peptide linker sequences.
[0015] The Fc fragment supports the biological properties of the antibody, in particular its ability to be recognized by immunity effectors or to activate complement. It consists of the constant fragments of the heavy chains beyond the hinge region.
[0016] The term "diabodies" means small antibody fragments having two antigen binding sites. These fragments comprise in the same VHVL polypeptide chain a VH heavy chain variable domain connected to a VL light chain variable domain. By using a binding sequence that is too short to allow pairing of two domains of the same chain, pairing with two complementary domains of another chain necessarily occurs and thus two antigen binding sites are created.
[0017] An antibody of the invention may thus be an immunoglobulin consisting of two heavy chains and two complete light chains, or may be an immunoglobulin fragment according to the invention, for example F (ab ') 2, Fab, Fv, scFv or Fc. Preferably, such an antibody fragment is the Fab region of an immunoglobulin, in particular the Fv region of an IgG1 antibody.
[0018] The antibodies described in the invention are isolated and purified, and are different from natural antibodies. When subject to an antibody or a nucleotide sequence according to the invention, the terms "isolated" and "purified" indicate that the molecule is present in the major absence of other biological macromolecules of the same type.
[0019] The term "chimeric antibody" refers to an antibody in which the sequence of each light chain and / or heavy chain constituting it comprises or consists of a hybrid sequence derived from at least two distinct animals. Preferably, the chimeric antibodies of the invention are human / mouse hybrids. In particular, a chimeric antibody of the invention may comprise a VH domain and a VL domain of an antibody originating from a non-human animal, in particular a murine animal, and a CH domain and a CL domain of a human antibody. Thus, preferably, an antibody of the invention comprises a VH domain and a VL domain of an antibody derived from the 1G3 antibody, defined hereinafter, and a CH domain and a CL domain of a human antibody.
[0020] According to the invention, the term "humanized antibody" refers to an antibody derived from a non-human animal in which heavy and light chain sequences other than CDRs have been replaced by corresponding sequences of one or more antibodies of human origin. Preferably, the term "humanized antibody" refers to an antibody whose heavy chain and light chain sequences are of human origin, and whose CDRs are derived from the 1G3 antibody.
[0021] The antibodies according to the invention are preferably monoclonal antibodies, that is to say that they recognize only one antigenic determinant in galectin 9, unlike polyclonal antibodies which correspond to a mixture of monoclonal antibodies, which therefore recognize several antigenic determinants in the same molecule. Monoclonal antibodies according to the invention can be obtained according to the techniques well known to those skilled in the art. For example, it is possible to use the cell fusion technique, the heavy and light chain sequence cloning technique, the display phage or ribosome technique, the immunization of mice with the human immunoglobulin repertoire and expression in a ad hoc cell or a transgenic animal. These techniques are well known to those skilled in the art. The present invention relates to antibodies directed against galectin 9 and inhibitors of regulatory T cell suppressive activity. In particular, the inventors have developed a hybridoma producing a murine IgG 1 Kappa antibody, 1G3, directed against galectin 9 and an inhibitor of the suppressive activity of regulatory T cells. The inventors have characterized the variable domains of light and heavy chains of the mAb 1G3 and thus determined the CDRs of this antibody shown in Table 1. Areas mAb 1G3 VH sequence MKCSWGIFFLLSVTAGVHSKVQLQQSGAELVKPGASVKLSCKAS GYTFTDYTIHWVKQRSGQGLEWIGWFYPGSHSIKYNEQFKDRAT LTADKSSSTVYMELSRLTSEDSAVYFCTRHGGYDGFDYWGQGTT LTVSSAKTTPPSVYPL (SEQ ID NO: 1) H-CDR1 GYTFTDYTIH (SEQ ID NO: 2) H-CDR2 WFYPGSHSIKYNEQFKDR (SEQ ID NO: 3) H-CDR3 HGGYDGFDY (SEQ ID NO: 4) VL LDGGKMDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLAVSVGEKI TMSCKSSQSLFYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESG VPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYYFPYTFGGGT KLEIKRADAAPTVSIFPPSS (SEQ ID NO: 5) L-CDR1 KSSQSLFYSTNQKNYLA ( SEQ ID NO: 6) L-CDR2 WASTRES (SEQ ID NO: 7) L-CDR3 QQYYYFPYT (SEQ ID NO: 8) Table 1 A particular embodiment of the invention therefore relates to an antibody directed against galectin 9 and inhibitor suppressive activity of regulatory T cells, having the same area of fix 1G3 antibody having for CDRs the six CDRs defined by: - the amino acid sequence SEQ ID NO: 2 in the H-CDR1 region, - the amino acid sequence SEQ ID NO: 3 in the H region -CDR2, - the amino acid sequence SEQ ID NO: 4 in the H-CDR3 region, 10 - the amino acid sequence SEQ ID NO: 6 in the L-CDR1 region, - the amino acid sequence SEQ ID NO: 7 in the L-CDR2 region, - the amino acid sequence SEQ ID NO: 8 in the L-CDR3 region. In particular, one subject of the invention relates to an antibody directed against galectin 9 and an inhibitor of the suppressive activity of regulatory T lymphocytes having for CDRs the six CDRS defined by: the amino acid sequence SEQ ID NO: 2 in the H-CDR1 region, - the amino acid sequence SEQ ID NO: 3 in the H-CDR2 region, - the amino acid sequence SEQ ID NO: 4 in the H-CDR3 region, - the sequence of amino acids SEQ ID NO: 6 in the L-CDR1 region, - the amino acid sequence SEQ ID NO: 7 in the L-CDR2 region, - the amino acid sequence SEQ ID NO: 8 in the L region -CDR3. In a particular embodiment, the heavy chain variable region of said antibody has the amino acid sequence SEQ ID NO: 1 and the light chain variable region of said antibody has the amino acid sequence SEQ ID NO: 5.
[0022] An antibody according to the invention therefore binds specifically to galectin 9 and is an inhibitor of the suppressive activity of regulatory T cells. According to another particular embodiment of the invention, an antibody according to the invention can bind specifically to an epitope of galectin 9. Advantageously, an antibody according to the invention is capable of binding in a specific manner. In particular, an antibody according to the invention can bind to the epitope recognized by the 1G3 antibody, defined above.
[0023] Thus, according to one particular embodiment of the invention, an antibody according to the invention can bind specifically to the epitope of amino acid sequence SEQ ID NO: 9, presented in Table 2. This epitope consisting of amino acid sequence SEQ ID NO: 9 corresponds to peptide P4 and covers the end of the binding peptide and the beginning of the C-terminal portion of galectin 9. This sequence exists in the three isoforms of galectin 9 (amino acids 166 to 178 of the isoform S, amino acids 178 to 190 of the isoform M, amino acids 210 to 222 of the isoform L). Such an antibody can thus react with all isoforms of galectin 9. Sequence of galectin 9 (epitope) TPAIPPMMYPHPA (SEQ ID NO: 9) Table 2 According to another particular embodiment, the antibody according to the invention is a chimeric antibody, preferably a murine / human chimeric antibody. In particular, this murine / human chimeric antibody may comprise the variable domains of the 1G3 antibody as defined above. According to another particular embodiment, the antibody according to the invention is a humanized antibody. In particular, the variable domain of this humanized antibody may comprise framework regions of the human acceptor, and possibly human constant domains, and the CDRs of the non-human donor, in particular the CDRs defined above.
[0024] Antibody production methods The antibodies of the invention may be produced by any technique known to those skilled in the art, for example but not limited to, by any chemical, biological, genetic or enzymatic technique alone or in combination. For example, it is possible to use the technique described below for the production of the hybridoma producing the monoclonal antibody against galectin 9, 1G3. The specific binding of the antibodies according to the invention directed against galectin 9 can be analyzed according to any known method of the state of the art. As immunoassays that may be used, mention may be made, for example, of Western blot techniques, radioimmunoassays, ELISA, sandwich immunoassays, immunoprecipitation tests, precipitin tests, and precipitin gel diffusion tests. , immunoradiometric tests, fluorescence immunoassays or complement fixation tests. Such tests are well known to those skilled in the art.
[0025] The inhibitory action of the suppressive activity of regulatory T cells of an antibody according to the invention thus generated can be analyzed according to various techniques known to those skilled in the art. For example, a cell proliferation assay can be performed. We can thus refer to the technique used below concerning the cell proliferation test performed with the antibody directed against galectin 9, 1G3.
[0026] When the amino acid sequence of the desired sequence is known, those skilled in the art can readily replicate the antibody by standard polypeptide production techniques.
[0027] For example, such antibodies can be synthesized by a well known solid phase method, preferably using a commercially available peptide synthesizer and following the recommendations of the supplier.
[0028] Alternatively, the antibodies of the invention may be obtained by techniques, well known to those skilled in the art, of recombinant DNA in a suitable expression system. The term "expression system" means a cellular host and a compatible vector under appropriate conditions, i.e., conditions allowing the expression of the protein encoded by the foreign DNA carried by the vector and introduced into the vector. the host cell. Typically, the nucleic acid sequence encoding an antibody may be inserted into an appropriate expression vector which will then be introduced into a suitable prokaryotic or eukaryotic host which will produce the desired antibody.
[0029] The terms "vector", "cloning vector" and "expression vector" refer to vehicles by which the DNA or RNA sequences encoding the antibody can be introduced into a host cell so as to transform it and to allow expression (i.e., transcription and translation) of the introduced sequence. An expression vector is typically a plasmid, a cosmid, an episome, an artificial chromosome, a phage or a viral vector. As viral vectors, mention may be made of adenoviruses, retroviruses, herpesvirus and vectors derived from adeno-associated virus (AAV). Such recombinant viruses can be produced by well-known techniques, such as transfection of cell lines for their encapsidation or by transient transfection with plasmids or complementation viruses expressing the necessary missing functions. Cell lines for encapsidation are for example PA317, PsiCRIP, GPenv +, 293, etc. Detailed protocols for producing such replication-defective recombinant viruses are available in patent applications WO 95/14785, WO 96/22378, US 5,882,877, and the like. The host cells are therefore transfected, infected or transformed with a nucleic acid or a suitable vector as described above.
[0030] The term "transformation" refers here to the introduction of a foreign gene (extrinsic or extracellular), a DNA or RNA sequence into a host cell such that this host cell expresses the gene or the sequence introduced to produce the desired substance, typically a protein encoded by the introduced gene or sequence.
[0031] Common expression systems include, but are not limited to, host cells and plasmid vectors of E. coli. coli, insect host cells and Baculovirus vectors and mammalian cells and vectors.
[0032] A method of production from a host cell expressing an antibody according to the invention may comprise the steps of: (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into the cell competent host, (ii) culturing in vitro or ex vivo the recombinant host cell thus obtained, (iii) optionally selecting the cells which express and / or secrete said antibody or polypeptide. Such host cells can be used for the production of antibodies according to the invention. According to a particular embodiment, a method of producing an antibody according to the invention may comprise the steps of: (i) culturing the transformed cell described above under conditions appropriate for the expression of the antibody; and (ii) recovering the antibody thus expressed. The antibodies can be separated from the culture medium by conventional immunoglobulin purification methods such as, for example, protein A-Sepharose purification, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. In a particular embodiment of the invention, a human chimeric antibody according to the invention can be produced by obtaining the nucleic sequences encoding the VL and VH domains as mentioned above, by constructing a human chimeric antibody expression vector by inserting the nucleic sequences into an animal cell expression vector having genes encoding the CH and CL domains, and expressing the encoded sequences by introducing the expression vector into the animal cell.
[0033] The CH domain of the human chimeric antibody can be from any region belonging to human immunoglobulin. Preferably, it is the IgG class, and more preferably IgG1. Similarly, the CL domain of the human chimeric antibody can be from any region belonging to human immunoglobulin. Preferably, it is the Kappa class. The chimeric or humanized antibodies according to the invention may in particular be obtained by genetic engineering of the antibodies. The construction of a chimeric antibody can for example be performed by a gene transfection technique or by a recombinant DNA technique.
[0034] A humanized antibody according to the invention can be produced by obtaining the CDR domains as mentioned above, by constructing a human antibody expression vector by inserting the nucleic sequences into an animal cell expression vector having genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expressing the coded sequences by introducing the expression vector into the animal cell. With regard to the humanized antibody expression vector, it may be a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exist in separate vectors, either of a type in which the two genes exist in the same vector (tandem type). Tandem-type vectors are preferred over the ease of construction of the expression vector, ease of introduction into animal cells etc. As an example of a tandem type humanized antibody expression vector, mention may be made of pKANTEX93 or pEE18.
[0035] Methods of producing humanized antibodies based on recombinant DNA or gene transfection techniques are well known from the state of the art. The antibodies can be humanized according to various techniques known from the state of the art, for example by CDR grafting, veneering or resurfacing, or by chain shuffling. . The recombinant DNA-based technique for the preparation of such antibodies is also known. A Fab fragment according to the invention can be obtained by treatment of an antibody, in particular an antibody directed against galectin 9 and inhibitor of the suppressive activity of regulatory T cells, with a protease, papain. This Fab fragment may also be produced by insertion of a DNA encoding the Fab fragment of the antibody into a vector usable in a prokaryotic or eukaryotic expression system and the introduction of this vector into the prokaryote or eukaryote appropriate for express the Fab fragment. An F (ab ') 2 fragment according to the invention can be obtained by treatment of an antibody, in particular an antibody directed against galectin 9 and an inhibitor of the suppressive activity of regulatory T cells, by a protease, pepsin. The F (ab ') 2 fragment can also be obtained by joining together Fab' fragments as described below, by a thioether bond or a disulfide bridge. A Fab 'fragment according to the invention can be obtained by treatment of the F (ab') 2 complex of an antibody, in particular an antibody directed against galectin 9 and an inhibitor of the suppressive activity of regulatory T cells, by a reducing agent. , dithiothreitol. The Fab 'fragment may also be produced by insertion of a DNA encoding the Fab' fragment of the antibody into a vector usable in a prokaryotic or eukaryotic expression system and the introduction of this vector into the prokaryote or eukaryote suitable for expressing the Fab 'fragment.
[0036] The ScFv fragment according to the invention can be produced by obtaining a cDNA sequence encoding the previously described VH and VL domains followed by the insertion of this DNA into a vector that can be used in a eukaryotic or prokaryotic expression system. and introducing this vector into the eukaryotic or appropriate prokaryote to express the ScFv fragment. To obtain a humanized ScFv fragment, it is possible to use the CDR grafting technique. This technique involves the selection of complementarity regions (CDRs) of a donor ScFv fragment and their graft on the framework of a human ScFv fragment of known three-dimensional structure (see for example WO 98/45322, EP0173494).
[0037] Modifications of the amino acid sequences of the antibodies according to the invention can be carried out. For example, it may be desirable to improve the binding affinity and / or biological properties of the antibody. It is known that when a humanized antibody is produced by simply grafting only VH and VL CDRs of an antibody derived from a non-human animal into the frameworks (FR) of a human antibody, the binding power to the antigen is decreased in comparison with that of an antibody derived from a non-human animal. It is believed that some amino acid residues of the VH and VL of a non-human antibody, not only in CDRs but also in FRs, are directly or indirectly associated with the antigenic binding power. The substitution of these amino acid residues with different amino acid residues derived from the FRs of the VH and VL of the human antibody would therefore reduce the binding power. Therefore, in order to solve this problem, tests must be performed on the human CDRs grafted antibodies in order to identify, among the amino acid sequences of the FR of the VH and VL human antibodies, an amino acid residue that either directly associated with the antigen binding, or interacting with a CDR amino acid residue or maintaining the three-dimensional structure of the antibody and directly associated with the antigen binding. The binding power could be increased by replacing the amino acids identified by the amino acid residues of the original antibody derived from a non-human antibody. Modifications and changes can be made in the structure of the antibodies of the present invention and in the DNA sequences encoding them, while again obtaining a functional molecule that encodes an antibody with the desired characteristics.
[0038] Another aspect of the present invention relates to conservative functional variants of the antibodies of the present invention. "Functional conservative variants" are those in which a given amino acid residue in a protein has been changed without impairing the overall conformation and inhibitory function of the regulatory T cell suppressive activity. Thus, it is possible to replace one amino acid with another having similar properties (eg, polarity, hydrogen bonding potential, etc.) as long as the inhibitory function of the regulatory T-cell suppressive activity is maintained.
[0039] Thus, according to a particular embodiment of the invention, it is possible to have an antibody as defined above, directed against galectin 9 and inhibitor of the suppressive activity of regulatory T lymphocytes, comprising: an H-CDR1 having 1 or 2 amino acids of difference with the sequence defined by SEQ ID NO: 2, - an H-CDR2 having 1 or 2 amino acids of difference with the sequence defined by SEQ ID NO: 3, - an H-CDR3 having 1 or 2 acids amines of difference with the sequence defined by SEQ ID NO: 4, - L-CDR1 having 1 or 2 amino acids of difference with the sequence defined by SEQ ID NO: 6, - L-CDR2 having 1 or 2 amino acids of difference with the sequence defined by SEQ ID NO: 7, 10 - an L-CDR3 having 1 or 2 amino acids of difference with the sequence defined by SEQ ID NO: 8. According to another particular embodiment of the invention, an antibody as defined above, directed against galectin 9 and inhibits ur of the suppressive activity of regulatory T lymphocytes, with 1, 2 or 3 amino acids of difference with all six CDRs of sequences as defined above, that is to say the CDRs of sequences SEQ ID NO : 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. Therapeutic Uses As previously mentioned, it is known that in pathological conditions, Regulatory T cells can induce inappropriate immunosuppression, which then promotes tumor growth. Numerous studies have thus shown that regulatory T lymphocytes reduce anti-tumor immune responses, in particular by inappropriately inhibiting the activity of effector T lymphocytes, thus favoring the development of cancer-type pathologies. It has been demonstrated that, on the one hand, during activation, galectin 9 is directly expressed by regulatory T lymphocytes while it is only very slightly, if at all, by T lymphocytes. effectors, the targeting of galectin 9 to specifically inhibit regulatory T cells without the risk of causing depletion of effector T cells. On the other hand, it has been demonstrated that the inhibition of galectin 9 by an antibody allows the inhibition of the suppressive activity of regulatory T cells. The antibodies according to the invention, directed against galectin 9 and inhibitors of the suppressive activity of regulatory T lymphocytes, can therefore be used in the treatment of diseases associated with the suppressive activity of regulatory T cells, in particular the treatment of cancers.
[0040] An object of the invention thus relates to an antibody as described above for its use in the treatment of diseases associated with the suppressive activity of regulatory T cells. A particular embodiment of the invention relates to an antibody, as previously described, used in the treatment of cancer. By "cancer treatment" is meant any treatment that can, for example, suppress a tumor or metastases, reduce the risk of recurrence, slow tumor development or metastasis, and / or treat the symptoms of the disease. The cancers of the present invention are those in which the regulatory T cells exert their suppressive activity. Advantageously, the cancers targeted by the present invention are those in which the regulatory T cells are present in a large amount in the tumor tissue or in the circulation, the expansion of the regulatory T lymphocytes generally being correlated with the increase in their number. activation (6). The frequency of regulatory T lymphocytes can be evaluated by any method known to those skilled in the art, for example by flow cytometric analysis (FACS) of intra-tumor lymphocytes or circulating lymphocytes or by immunohistological staining of the tumor tissue. In general, an antibody as defined above can therefore be used in the treatment of all types of cancers in which regulatory T cells exert their suppressive activity. Many types of cancers in which regulatory T cells exert their suppressive activity have been studied and are known to those skilled in the art. It is thus known that high levels of regulatory T cells in tumors are clearly associated with poor prognosis in chronic myeloid leukemia (7), colon cancer (8), melanoma (9), cancer of the colon uterus (10), breast cancer (11), pancreatic cancer (12), gastric cancers (13), ovarian cancer (14), primary lymphoma of the central nervous system (15), myeloma multiple (16), prostate cancer (17), Hodgkin lymphoma or hepatocellular carcinoma (18, 19).
[0041] An antibody as defined above can therefore in particular be used in the treatment of cancer, the cancer being chosen from the group consisting of chronic myeloid leukemias, colon cancer, melanoma, uterine cancer, breast cancer, pancreatic cancer. , gastric cancers, ovarian cancer, primary lymphoma of the central nervous system, multiple myeloma, prostate cancer, Hodgkin lymphoma and hepatocellular carcinoma. Studies have also shown that some cancers produce significant amounts of exosomes carrying galectin 9 playing an immunosuppressive role, that is to say, inhibiting the immune response and potentially the anti-tumor response. Non-limiting examples of cancers producing large amounts of exosomes carrying galectin 9 include virally induced cancers, for example nasopharyngeal carcinomas associated with EBV virus (Epstein-Barr virus), or hepatocellular carcinoma (HCC) associated with HCV (hepatitis C virus) or HBV (hepatitis B virus) (20,21). An antibody as defined above can therefore in particular be used in the treatment of cancer, the cancer being a viro-induced cancer, preferably selected from the group consisting of nasopharyngeal carcinomas associated with Epstein-Barr Virus, hepatocellular carcinomas hepatitis C virus or hepatitis B virus. It has also been shown that an increase in the frequency of regulatory T cells is a predictor of the recurrence of hepatitis C-related fibrosis ( 22,23).
[0042] An antibody according to the invention can therefore be used to prevent a recurrence of fibrosis consecutive to hepatitis C.
[0043] In each of the previously described embodiments, the anti-galectin-9 antibody and inhibitor of regulatory T-cell suppressive activity is suitably administered to a patient in need of such treatment.
[0044] The antibodies according to the invention may be used alone or in combination with any other suitable compound. An object of the invention relates to a method of treating cancer, associated with the expression of galectin 9 and the suppressive activity of regulatory T cells, comprising administering a therapeutically active amount of an antibody according to the invention to a patient. The term "patient" is intended to mean a human affected, or caused to be affected by, a disease associated with the suppressive activity of regulatory T cells, including cancer. The term "therapeutically active amount" of an antibody means an amount of antibody sufficient to treat such a cancer, having an acceptable benefit / risk ratio for drug treatment. The amount of antibodies and compositions according to the present invention as well as the frequency of administration will be determined by clinical studies, by the physician or by the pharmacist. The amount "therapeutically active" specific to each patient may depend on a number of factors such as the nature and severity of the disorder to be treated, the activity of the antibody used, the composition used, the age, the weight , the general state of health, the sex and the diet of the patient, the mode of administration, the duration of the treatment (in one dose or in several doses), the drugs used in combination and other factors well known to specialists medical. According to a particular embodiment of the invention, an antibody directed against galectin 9 and an inhibitor of the regulatory T cell suppressive activity as previously defined is used in combination with a second agent for the treatment of an associated disease. the suppressive activity of regulatory T cells, for example an anti-cancer agent.
[0045] Thus, when the use is the treatment of cancer, the antibody can be used in combination with known therapies against cancer such as, for example, surgery, radiotherapy, chemotherapy or combinations thereof. For example, the antibody may be used in combination with adoptive immunotherapy, consisting of one or more effector lymphocyte injections against tumor antigens including EBV antigens. In certain aspects, other anti-cancer agents used in combination with the anti-galectin antibody 9 of the invention for cancer therapy include anti-angiogenic agents. In some aspects, said antibody is co-administered with a cytokine, for example a cytokine that stimulates an anti-tumor immune response. An object of the invention therefore relates to a combination product comprising an antibody, as defined above, and an anticancer agent. One particular embodiment relates to such a combination product for simultaneous, separate or spread over time use in cancer treatment. Pharmaceutical Compositions For administration, the antibody is generally formulated as a pharmaceutical composition. The pharmaceutical composition comprising an antibody according to the invention may be formulated by methods known from the state of the art, in which the therapeutic molecule is in combination with at least one excipient. An object of the invention thus relates to a pharmaceutical composition comprising an antibody as described above and at least one pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" means any standard pharmaceutical carrier, its administration being tolerable by a patient. For example, sterile phosphate buffered saline solutions are pharmaceutically acceptable. The pharmaceutically acceptable carriers can usually comprise one or more compounds, for example selected from excipients, preservatives, solubilizers, buffering agents, albumin, etc. Known excipients are, for example, starch, gelatin, stearic acid, calcium or magnesium stearate and the like. Those skilled in the art will be able to determine the compounds suitable for the present composition. The form of the pharmaceutical composition, the mode of administration, the dosage and the dosage may, of course, depend on, among other things, the disease to be treated, its symptoms, severity, age, weight and sex. of the patient. In a nonlimiting manner, the pharmaceutical composition according to the invention may be formulated so as to be administered topically, parenterally, nasally, intravenously, subcutaneously / intradermally, conjunctivally, intramuscularly or intraocularly. The pharmaceutical compositions according to the invention may optionally contain pharmaceutically acceptable excipients suitable for injection. In particular these may be isotonic and sterile saline solutions, monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or a mixture of these salts. These compositions may also be dry compositions, in particular dry and frozen, freeze-dried or refrigerated compositions which, after addition, according to the case, of sterile water or physiological saline, constitute injectable solutions. The doses used may be adapted according to various parameters, such as, in particular, the mode of administration according to the pathology or, alternatively, the duration of the treatment envisaged. To prepare the pharmaceutical compositions, a sufficient amount of antibody can be dissolved or dispersed in a pharmaceutically acceptable carrier or an aqueous medium. Pharmaceutical forms suitable for injection use include sterile water solutions, dispersions, formulations including sesame oil, or aqueous propylene glycol, as well as sterile powders for the extemporaneous preparation of sterile injectable solutions. In any case, the form used must be sterile and must be sufficiently fluid to be easily injected by means of a syringe. It must be stable under the conditions of production and storage and be protected from contamination by microorganisms, such as bacteria or fungi. Solutions of the active compounds, whether in free form or as pharmaceutically acceptable salts, may be prepared with water mixed with a surfactant such as hydroxypropylcellulose. The dispersions can be made in glycerol, in liquid polyethylene glycols, in a mixture of both or in oils. These preparations generally contain a preservative to prevent the growth of microorganisms under normal conditions of storage and use.
[0046] An antibody according to the invention may be formulated in a composition in neutral form or in salt form. Pharmaceutically acceptable salts include acid addition salts, formed with free amino groups of the protein, and formed with inorganic acids such as hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, mandelic, etc. . The salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. After formulation as a drug, the solutions may be administered in a manner compatible with the dosage of the formulation and in a therapeutically active amount. The drugs can be administered as described above but also in the form of capsules that release them. FIGURES Figure 1: Phenotypic analysis by multiparametric flow cytometry of natural regulatory T cells isolated from human blood.
[0047] Figure 2: Analysis of the suppressive function of natural regulatory T cells isolated ex vivo. (A) Analysis of inhibition of proliferation of PBMCs activated by autologous regulatory T cells in cpm. (B) Cytolytic percentage analysis of PBMCs activated by autologous regulatory T cells.
[0048] Figure 3: Q-PCR analysis of gene expression encoding galectin 9 in human regulatory T cells (n = 12). 1) 8, D1, D2, A1, A2 represent the blood pockets. CT (Cycle Threshold) represents the cycle threshold. This is the average threshold at which the amplification of the Galectin 9 gene begins to be detected. The results are expressed in ACT following normalization with four housekeeping genes 03-Actin, GAPDH, HPRT, Ubiquitin) [ ACT = CT (the sample) - CT (household gene mean)] Figure 4: Western blot analysis of the expression of the three isoforms of galectin 9 in human regulatory T cells. Hela cells are used as a negative control. Figure 5: Q-PCR analysis of expression of the gene encoding galectin 9 in conventional T cells during activation (n = 4).
[0049] Figure 6: Q-PCR analysis of gene expression encoding galectin 9 in human regulatory T cells during activation (n = 4). FIG. 7: Analysis of the inhibition of the suppressive activity of regulatory T lymphocytes by the anti-gal9 1G3 antibody by the analysis of the proliferation of PBMCs in the presence of irradiated C15, in the presence or absence of regulatory T lymphocytes, and in the presence or absence of 1G3 antibodies at a concentration of 1 μg / mL. FIG. 8: Galectin-induced Jurkat Apoptosis Inhibition Assay 9 With Galectin-9 S pre-incubation lh at 1 μg / ml and antibodies at 1. μg / ml (9M1 antibody (anti-galectin 9), 9S2-3 (anti-galectin 9), 1G3 (anti-galectin 9), anti-TIM3 antibody, 2E12 (anti-galectin 9)).
[0050] FIG. 9: Analysis of the Restoration of Proliferation on Activated ("A", with Anti-CD3 and Anti-CD28) and Non-Activated (NA) Cells after Treatment with Galectin-9 (Antibody) tested = ECA-42 (anti-galectin 9), 1G3 (anti-galectin 9), 2E2 (antiTIM3) and 2E12 (anti-galectin 9) The control corresponds to the culture medium.
[0051] EXAMPLES 1. Materials and methods Donors, cell lines and culture conditions Donor cells Healthy donor cells were isolated from blood from the French Blood Establishment - Nord de France (EFS), according to an official agreement between the latter and the National Center for Scientific Research (CNRS) - Delegation Nord Pas-de-Calais and Picardie. The study was approved by the Institute of Biology of Lille (CNRS) and the institutional committee of the EFS, and each of the donors had previously signed an informed consent. CNP Cell Lines C15 tumor cell lines are derived from a xenotransplanted EBV-positive CNP (Nasopharyngeal Carcinoma) and continuously propagated in SCID mice subcutaneously every 6-7 weeks. All animal experiments were carried out by qualified personnel, in accordance with the French and European regulations, in the pet shop of the Pasteur Institute of Lille (France). C15 cells were recovered from xenotransplanted tumors and irradiated (5000 rads) before being preincubated with immune cells simulating a tumor context. Jurkat Human T Lymphocyte Lineage The Jurkat line was derived from human T lymphocyte leukemia. It has the phenotype of CD4 + lymphocytes. Cell culture conditions The standard culture medium used is RPMI 1640 (Invitrogen, Paisley, UK) supplemented with 10% human AB serum (BioWest, Nuaillé, France), 2 mM L Glutamine, 1 mM sodium pyruvate, 10 mM non-essential amino acids, 10 mM HEPES, 50 U / mL streptomycin, 501..tg / mL gentamycin and 50 μM f3 mercaptoethanol. The cells were incubated at 37 ° C under a controlled atmosphere (5% CO2 and 95% humidity) in a Hera Cell 150 incubator (Thermo Electron, Cergy Pontoise, France). Where appropriate, PBMCs and CD4 + T cells were activated with anti-CD3 antibody (1 μg / mL) (Clinisciences, Montrouge, France), which binds to the plate after incubation for 2 hours at 37 ° C. ° C before culture, and soluble anti-CD28 antibody (100 ng / mL) (Clinisciences) added extemporaneously. Isolation of Human Immune Cells Isolation of PBMCs The peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by standard density gradient centrifugation using Ficoll Easter PLUS (Amersham Biosciences, Uppsala, Sweden). Isolation of CD4 + T Cells CD4 + T cells were isolated from PBMCs using a negative selection protocol according to the manufacturer's instructions (Miltenyi Biotec, Berlin, Germany). Briefly, the PBMCs are incubated for 10 minutes with a cocktail of biotinylated antibodies directed against CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRy / 15 and glycophorin A. Anti-biotin magnetic beads are then added during 15 minutes. The cells to be removed are magnetically retained in a Magnetic Activated Cell Sorting (MACS®) column placed in a MACS® separator. The cells to be isolated pass through the column, they are collected and enriched in unmarked cells, depleted of non-targeted cells. Flow cytometry analysis shows that more than 98% of the isolated cells are CD4 + cells. Isolation of regulatory T cells The isolation of human regulatory T cells from PBMCs of adult donors was achieved using a CD4 + CD25 + regulatory T cell isolation kit (Miltenyi Biotech, Germany) according to the manufacturer's instructions. . The CD4 + CD25 + T cell fraction was preserved for flow cytometry and chemoattraction experiments. The flow cytometry analysis consistently shows an enrichment greater than 95% of the CD4 + CD25 + fraction.
[0052] Cell Proliferation Assay 1.105 cells (PBMCs or CD4 + T cells) were incubated with [methyl 3H] thymidine for the last 18 hours of culture and collected on a fiberglass filter (Printed Filtermat A, Wallac, Turku, Finland) in using a Tomtec collector (Wallac). The filter was then sealed in a bag after drying and addition of the scintillation liquid (Beckman Coulter, USA). Proliferation was measured after incubation in the presence of [3H] thymidine (1 1.t.Ci / well) (PerkinElmer, Courtaboeuf, France) for the last 18 hours before collection. The radioactivity was measured using a 3-meter (1450 Trilux, Wallac, Finland). Each proliferation test was performed in triplicate and estimated in "count per minute" (cpm). According to the experiments, the proliferation tests were performed in the presence of 1 μg / mL of the short isoform of recombinant Galectin 9 (Ga19S) provided by Dr. Toshiro Niki (Galpharma, Japan), of 1 μg / mL or a range anti-Galectin 9 1G3 antibody, irrelevant anti-IGg1 antibody as a negative control (ebiosciences, United Kingdom), 10 μg / ml C15 exosomes, 5 mM lactose or of sucrose (Sigma Aldrich).
[0053] Cell Lysis The cell lysis measurement technique is based on the use of a cytotoxicity measurement kit (CytoTox-Glo Assay, Promega, USA) which measures a luciferase activity proportional to cellular proteases released after cytolysis. The tests are performed by co-cultivating 6.105 CD4 + CD25 + and 2.105 autologous PBMCs. The cells are cultured in 96-well round bottom plates (Maxisorb Nunc, Denmark), in 200 μl of culture medium (RPMI-1640, 2 mM l-Glutamine 1%, 0.02 mM sodium-pyruvate, 100 U / mL penicillin, 100 μg / mL streptomycin, 10% AB Decomplemented Human Serum) (GIBCO BRL ™, Invitrogen®, GB). The cells are activated with 11 μg / ml of anti-CD3, previously coated on the plates (2 h at 37 ° C.) and 100 μg / ml of anti-CD28. After 48h of culture, 50 1..IL reagent (aminoluciferin-Glo) are deposited in each well. After gentle stirring, the culture plates are incubated for 15 min at room temperature and protected from light. A first luminescence measurement is made at the luminometer (Centro LB960, C Berthold Technologies, France), and is proportional to the amount of cells lysed by regulatory T cells. Then, 500% of a digitonin solution is deposited in each well in order to induce total lysis of the cells. The plates are then shaken and incubated for 15 min, at room temperature and in the dark, before making the second luminescence measurement. The tests are performed in triplicate and the results are expressed as a percentage of lysis. Percent Lysis = Cell Viability / Average Total Lysis - Background Cell Viability = Total Lysis Average - Cytolyse Cytolyse = Mean Treg Cell Induced Lysis - Background Noise Test for Induction of Apoptosis in Jurkat Cells Jurkat Cells grown in RPMI 5% fetal calf serum is transferred to serum-free medium (Hybridoma SFM - Life Technologies) and then incubated in the presence of 30 nM galectin-9 for 24 h in a 96 well plate well (100,000 cells / well). ). The apoptotic count of cells is done by flow cytometry after labeling with annexin V-APC (allophycocyanin) and propidium iodide. To evaluate the protective action of the monoclonal antibodies, the galectin is preincubated for 30 min in the presence of the antibody whose final concentration for the incubation of 24 hours is 10 μg / ml.
[0054] Western blot The exosomes were lysed (10 min on ice) in PY buffer composed of 20 mM Tris HCl, 50 mM NaCl, 5 mM EDTA, 1% Triton X 100, 0.02% sodium azide and a cocktail of protease inhibitors (Roche, Basel, Switzerland). After centrifugation (20,000 g, 15 min, + 4 ° C), cell debris was removed and the supernatants collected. Protein concentrations were measured using Bio Rad Protein Assay according to the manufacturer's instructions (BioRad, Marnes la Coquette, France). The exosomes were then analyzed by Western Blot. Briefly, the proteins were separated by SDS PAGE electrophoresis using precolumn gradient gels (12% gradient, Bis Tris, Invitrogen) under standard conditions. The proteins were then transferred to a nitrocellulose membrane (Hybon dTM-C Extra, Amersham Biosciences, UK). The latter was blocked for 1 hour at room temperature in a blocking buffer containing 0.2% AuroraTM blocking reagent (MP Biomedicals, Illkirch Graffenstaden, France), 0.1% Tween20 (Sigma Aldrich) and PBS (1X), and then it was incubated overnight at 4 ° C with a primary antibody against Galectin 9: Galectin-9CT-L1 1: 100 (supplied by Galpharma, Japan). The membrane was washed with blocking buffer, then incubated for 1 hour at room temperature with a peroxidase-conjugated secondary antibody (anti mouse, 1: 10000) (GE Healthcare, Wauwatosa, USA) and washed again with the blocking buffer. Protein-specific signals were visualized using Western Lightning® Plus ECL, a chemiluminescence enhancement kit for the substrate (Perkin Elmer, Boston, MA, USA) and a LAS3000 luminescent image analyzer (Fujifilm).
[0055] FACS Analysis Immunophenotyping of cells by flow cytometry was performed using the FACSCalibur flow cytometer. After collection, cells were washed with phosphate buffered saline (PBS) (GIBCO-Life technologies) and labeled with monoclonal antibodies conjugated to fluorochromes (1:10). For each assay, the appropriate control isotypes (monoclonal antibodies) were used for the marker settings. Finally, the data was analyzed with the FlowJo software. In order to detect cell surface antigens, anti-human mouse antibodies were used: CD4-phycoerythrin (PE) -cyanin (Cy) (BD Pharmingen, San Diego, USA), -CD25-PE (Miltenyi Biotech, Germany) and -CD127-FITC (1:20) (Clinisciences, Montrouge, France) according to the manufacturer's instructions. Real-time quantitative PCR: The total RNAs of the regulatory T cells were isolated using the kit "RNeasy minikit II" (Qiagen) according to the manufacturer's instructions. The concentration and purity of the RNAs were measured by the spectrophotometric method (Ultrospec 3000, Pharmacia Biotec). Total RNA was stored at -80 ° C until further use. The reverse transcription of the mRNAs was carried out as follows: 2 [tg of the total RNA were mixed with 5 [IL of the master mix composed of 1 [oligo dT (Roche Diagnostic, Meylan, France) and 0.1 pt RNAsin (40 UffiL, Promega, Charbonnières, France) and then incubated at 70 ° C for 5 to 10 minutes. After 5 minutes at room temperature, 10 [IL of a second mix were added: 6 [IL buffer 5X (Invitrogen) + 1 [IL DTT 0.1M (Invitrogen) + 2 [IL dNTPs 10mM (Amersham) + 0.1pt RNAsin 40 UffiL (Promega) + 1 [IL Superscript (Invitrogen). The reaction was followed by a first incubation of 45 to 60 minutes at 45 ° C, a second incubation of 5 minutes at 95 ° C followed by a 20-minute treatment with RNase H (Promega). Finally, ultra-pure distilled water (GIBCO-Life Technologies) was added in order to obtain a final concentration of 10 ng of total DNA / μl. The DNA was stored at -20 ° C until further use.
[0056] The transcripts were quantified using real-time PCR (RT-PCR) with the Mx3005PTM sequence detection system (Agilent technologies, France) in 96-well optical plates (Eurogentec S.A., Belgium). In each well, 10 1..IL of specific pair of primers, designed for RT-PCR and purchased from MWG-Biotech (Germany), were placed at a final concentration of 10 μg / mL and then stored at -20 °. vs. The household genes f3-actin, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), ubiquitin and hypoxanthine guanine phosphoRibosyl transferase (HPRT) were used as controls in each plate. The PCR reactions were carried out according to the manufacturer's instructions, to a final volume of 1.l.IL and for a 1 .IL cDNA (equivalent to 10 ng total RNA / pt), using 2X MESA. GREEN qPCR MasterMix Plus for SYBR® 258 Assay (Eurogentech) containing Meteor Taq DNA polymerase, MgC12 (final concentration of 4 mM), dNTPs (including dUTP), SYBR® 260 Green I, stabilizers and passive references required for signal normalization and buffer with optimized components The PCR program included initial denaturation and activation of the Meteor Taq for 5 minutes at 95 ° C, followed by 40 standard amplification cycles as follows: 15 seconds at 95 ° C ( denaturation), 1 minute at 60 ° C (synthesis and elongation). Fluorescent products were detected at the last step of each cycle. An analysis of the melting curves was carried out immediately after amplification, according to the manufacturer's instructions. Quantitative PCR reactions were used to quantify the expression of the galectin 9 gene by regulatory T cells. The household genes f3-actin, G3PDH, ubiquitin and hypoxanthine guanine PhosphoRibosyl Transferase (HPRT) were used as controls. All primers were designed for RT-PCR and purchased from MWGBiotech (Germany). A quantitative analysis was performed based on the "cycle threshold" (CT) value or cycle threshold for each well and calculated using the MxPro software.
[0057] Each individual value was normalized using the average of the 4 housekeeping genes according to the standard ACT method: ACT = 7 - rig z. For the comparison between the groups, the relative expression of the genes was expressed by giving an arbitrary value of 1 for the reference sample.
[0058] Fabrication of the hybridoma producing the monoclonal antibody against galectin-9, 1G3. A recombinant protein representing the C-terminal portion of human galectin was used as an immunogen (residues 191 to 355 of the long isoform of galectin-9). It was produced in E. coli as a GST fusion protein. After separation of the GST tag, the protein was purified by exclusion chromatography. The immunizations were carried out by the company PX'Therapeutics (Grenoble, France). Five female BALB-c mice, eight weeks old, were immunized with the C-terminal portion of galectin 9 mentioned above. For immunizations, 40 micrograms of protein were injected intraperitoneally at day 0, 22, 37 and 54 in combination with Freund's complete adjuvant for the first injection, or with incomplete Freund's adjuvant for injections. following. The quality of the immunization was evaluated by ELISA, described below, on serum samples from the immunized mice. The same recombinant C-terminal galectin preparation was used for the immunizations on the one hand and for the ELISA tests on the other hand. These tests showed good immunization in the five treated mice. Three days after the last booster, the two mice that gave the best response were sacrificed and their splenocytes were collected. These splenocytes were used for fusion with Sp2 / O murine myeloma cells, either in liquid medium or in semi-solid medium, with respective ratios of 5: 1 and 2: 1. Hybridoma supernatants were then assayed by an ELISA assay, performed as above and as described below, on the recombinant galectin 9 preparation mentioned above. Semi-solid fusion was successful and 39 hybridomas secreting antibodies reactive with galectin 9 in ELISA were obtained. Seven of them were selected because of a particularly abundant immunoglobulin secretion and high ELISA reactivity. These seven hybridomas were then subjected to new functional screens to study the anti-galectin 9 neutralizing properties of the antibodies produced. The ELISA test was performed as follows. The recombinant protein representing the C-terminal portion of galectin 9 was adsorbed in wells of 96-well microtiter plates (50 ng / well) (Greiner Bio-One, Courtaboeuf, France) as follows: solution in 0.05 M carbonate / bicarbonate buffer at pH 9.6 and incubation in the wells for 1h at room temperature. After washing with PBS containing 0.1% Tween-20, the wells were saturated with 3% bovine serum albumin (BSA) in solution in PBS at room temperature for 1h. They were then incubated with mouse sera or hybridoma supernatants to be tested. Sera and hybridoma supernatants were diluted in PBS with 1% BSA and then incubated in wells at room temperature for 2h. After a washing step with 0.1% PBS-Tween-20, the plates were treated with a peroxidase-labeled secondary antibody (goat anti-mouse). The final revelation takes place after addition of substrate (3,3 ', 5,5'Tetramethylbenzidine or TMB, Thermo Fisher Scientific) and absorbance measurement at 405 and 620 nm on a Multiskan Ex microplate reader (Thermo Fisher Scientific) . Results Phenotypic analysis of PBMCs and regulatory T lymphocytes Flow cytometric analysis (FACS) of PBMCs shows that the CD4 + CD25 + CD127 regulatory T cells represent 1% of total PBMCs (results not shown).
[0059] Furthermore, FACS analysis of phenotypic markers of autologous regulatory T lymphocytes isolated ex vivo indicates that 95% of regulatory T cells are CD4 + CD25 + and that of these cells, 90% are CD127- or CD127low and more than 86% FoxP3 + ( Figure 1).
[0060] Activated regulatory T cells have suppressive activity The suppressive activity of human regulatory T lymphocytes isolated ex vivo from the blood of healthy donors has been characterized by two complementary functional assays: a proliferation suppression test of activated PBMCs by autologous regulatory T cells and a cytolysis test of PBMCs activated by autologous regulatory T cells (Figure 2). Figure 2A shows that single activated PBMCs proliferate well in vitro while ex vivo isolated regulatory T cells are anergic, even after activation.
[0061] However, the proliferation of activated PBMCs decreases by more than 24% in the presence of activated autologous regulatory T lymphocytes at a ratio of 4: 2 (see Fig. 2A). The proliferation test (MLR) thus clearly demonstrates that the regulatory T lymphocytes isolated ex vivo and under activation conditions have immunosuppressive activity.
[0062] The results obtained by the proliferation test are supported by the results of the cytolysis test. It is indeed demonstrated in FIG. 2B that the lower the ratio PBMC: regulatory T cells, the higher the percentage of lysis of the activated PBMCs. Regulatory T cells induce lysis of autologous PBMCs at different ratios and in a dose-dependent manner. Galectin 9 is present on and expressed by the regulatory T lymphocytes Real-time (Figure 3) and Western-Blot (Figure 4) quantitative PCR analyzes show that galectin 9 is present on isolated human regulatory T cells. ex vivo and that these express galectin 9, suggesting that regulatory T cells use the galectin 9 pathway to inhibit the proliferation of effector T cells. Expression of galectin 9 by effector T cells decreases upon activation; the expression of galectin 9 by regulatory T cells increases upon activation. FIG. 5 demonstrates that the ratio of conventional CD4 + T lymphocytes to regulatory T cells decreases considerably during activation. Activated conventional CD4 + T cells hardly express the gene encoding galectin 9 and this expression decreases significantly during their activation. On the other hand, the expression of the gene encoding galectin 9 by regulatory T cells increases during their activation (see FIGS. 5 and 6). Thus, constitutively activated effector T lymphocytes, having an antitumour action, will not be not the target of the anti-gal9 antibody. On the other hand, the activated and therefore functional regulatory T lymphocytes will be a preferred target of the anti-gal9 antibody.
[0063] The suppressive activity of regulatory T cells is inhibited by the anti-galectin 9 1G3 antibody. In vitro evaluation of the impact of the 1G3 antibody on the activity of regulatory T cells, a cell proliferation assay based on the incorporation of tritiated thymidine was used. FIG. 7 presents the results of the proliferation test of PBMCs in the presence of irradiated C15s in the presence or absence of regulatory T lymphocytes and in the presence or absence of 1G3 antibodies at the concentration of 11 μg / mL. First, it is confirmed by the positive controls of the test that the activation of PBMCs leads to an increase in their proliferation and that the presence of regulatory T cells induces a decrease in cell proliferation of PBMCs. It is also demonstrated that the irradiation of the C15 tumor cells indeed leads to a stop of their proliferation. C15 cells are anergic. The presence of C15 tumor cells does induce a decrease in cellular proliferation of human PBMCs. It is then demonstrated that the presence of regulatory T cells in the coculture of PBMCs and C15 induces a significant additional decrease in proliferation by about 56%. Figure 7 clearly shows that, unexpectedly, the presence of the 1G3 antibody can restore the proliferation of PBMCs. It is thus suggested that the 1G3 antibody neutralizes galectin 9 present on, and expressed by, regulatory T cells. Therefore, it is emphasized that the 1G3 antibody inhibits the suppressive activity of regulatory T cells. Anti-galectin 9 1G3 antibody has superior efficacy to other anti-galectin 9 antibodies. To compare the effect of 1G3 antibody with other anti-galectin 9 antibodies, or even an anti-Tim 3 antibody , on the inhibition of the suppressive activity of regulatory T lymphocytes, several tests were carried out.
[0064] Thus, the effect of different anti-galectin 9 antibodies on the inhibition of apoptosis induced by galectin 9, as well as the effect of anti-galectin antibodies 9 on the restoration of proliferation after treatment of human PBMCs with of galectin 9 were analyzed. The anti-galectin antibodies tested in comparison to 1G3 do not recognize the same epitope of galectin 9 as the 1G3 antibody.
[0065] An anti-Tim 3 antibody has also been tested. Indeed, some theories have been raised concerning a link between the Tim-3 receptor, which would be present on T lymphocytes, and the pro-apoptic effect of galectin 9.
[0066] Inhibition of galectin-induced apoptosis FIG. 8 shows the results of the test on the effect of the antibodies tested on the Jurkat apoptosis induced by recombinant galectin-9. The antibodies tested correspond to the antibody 9M1 (anti-galectin 9), 9S2-3 (anti-galectin 9), 1G3 (anti-galectin 9), an anti-TIM3 antibody, 2E12 (anti-galectin 9). As shown in FIG. 8, the protection against apoptosis of Jurkat is better with the 1G3 antibody than with the other anti-galectin 9 9S2-3 and 9M1 antibodies or the anti-TIM3 antibody.
[0067] Restoration of proliferation after treatment with galectin 9 FIG. 9 presents the results of the test on the effect of the antibodies tested on the proliferation of human PBMCs previously treated with galectin 9.
[0068] The antibodies tested correspond to the antibody ECA-42 (anti-galectin 9), 1G3 (anti-galectin 9), 2E2 (anti-TIM3) and 2E12 (anti-galectin 9).
[0069] As seen in FIG. 9, the proliferation of human PBMCS is restored more efficiently with the 1G3 antibody than with the other anti-galectin antibodies 9 ECA42 and 2E12 or the anti-TIM3 antibody (2E2).
[0070] REFERENCES (1) Grossman WJ, Verbsky JW, Barchet W, Colonna M, JP Atkinson, Ley TJ. "Human T regulatory cells can use the perforin pathway to cause autologous target cell death." Immunity.
[0071] Oct 2004; 21 (4): 589-601. (2) Garin MI, Chu CC, Golshayan D, Cernuda-Morollon E, Wait R, Lechler RI. "Galectin-1: a key effector of regulation mediated by CD4 + CD25 + T cells". Blood.
[0072] 2007 Mar 1; 109 (5): 2058-65 (3) BD Johnson, Jing W, Orentas RJ. "CD25 + regulatory T cell inhibition enhances vaccine-induced immunity to neuroblastoma." Immunother.
[0073] 2007 Feb-Mar; 30 (2): 203-14. (4) McHugh RS, Whitters MJ, Piccirillo CA, Young DA, Shevach EM, Collins M, Byrne MC. "CD4 (+) CD25 (+) Immunoregulatory T cells: Gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor". Immunity.
[0074] 2002 Feb; 16 (2): 311-23. (5) Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, Nomura T, Sakaguchi S. "CTLA-4 control over foxp3 + regulatory T cell function". Science.
[0075] 2008 Oct 10; 322 (5899): 271-5. (5) Zahran AM et al., Int J Clin Oncol.
[0076] 2013 Sep 26. (6) Fiss Sl, Darrasse-Jèze G, Litvinova E, Septier F, Klatzmann D, Liblau R, Solomon BL. Continuous activation of autoreactive CD4 + CD25 + regulatory T cells in the steady state. J Exp Med.
[0077] 2003 Sep 1; 198 (5): 737-46. Epub 2003 Aug 25. (7) Xu W et al, J Cancer Res Clin Oncol.
[0078] 2013 Nov; 139 (11): 1845-52 (8) Ladanyi A, Magy Onkol.
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[0080] 2014 Jan 2.Pi: S0090-8258 (13) 01427-3. (10) Faghih Z et al, Immunol Lett.
[0081] 2013 Dec 8; 158 (1-2): 57-65. (11) Aida K et al, Cancer Sci.
[0082] 2013 Nov 30 (12) Huang XM et al, Cancer Sci.
[0083] 2013 Nov (13) Preston CC et al, PLoS One.
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[0087] 2013 Feb; 34 (1): 531-42 (19) Delhem et al., Expert Opin Biol Ther. 2010; (11): 1563-1572 (20) Ouaguia et al. ISRN Hepatology. Volume 2013 (2013), Article ID 928485 (21) Carpentier et al., Am J Transplant. 2009; 9 (9): 2102-2112. (22) Moralès et al, BRMI, Volume 2014 (2014), Article ID 290878 (23) Baumforth et al., Am J Pathol.
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[0089] 2013 Sep 30; 66 (9-10): 343-8.

权利要求:
Claims (12)
[0001]
REVENDICATIONS1. Antibody directed against galectin 9 and inhibitor of regulatory T cell suppressive activity.
[0002]
An antibody according to claim 1 which specifically binds to an epitope of amino acid sequence SEQ ID NO: 9.
[0003]
An antibody according to any of claims 1 or 2 having the same binding zone as the antibody having for CDRs the six CDRs defined by: the amino acid sequence SEQ ID NO: 2 in the H-CDR1 region, the amino acid sequence SEQ ID NO: 3 in the H-CDR2 region, the amino acid sequence SEQ ID NO: 4 in the H-CDR3 region, the amino acid sequence SEQ ID NO: 6 in the region L-CDR1, the amino acid sequence SEQ ID NO: 7 in the L-CDR2 region, the amino acid sequence SEQ ID NO: 8 in the L-CDR3 region.
[0004]
4. Antibody according to any one of claims 1 or 2 having for CDRs the six CDRS defined by: the amino acid sequence SEQ ID NO: 2 in the H-CDR1 region, the amino acid sequence SEQ ID NO: 3 in the H-CDR2 region, the amino acid sequence SEQ ID NO: 4 in the H-CDR3 region, the amino acid sequence SEQ ID NO: 6 in the L-CDR1 region, the amino acid sequence SEQ ID NO: 7 in the L-CDR2 region, the amino acid sequence SEQ ID NO: 8 in the L-CDR3 region.
[0005]
An antibody according to claims 1 to 4, characterized in that the heavy chain variable region of said antibody has the amino acid sequence SEQ ID NO: 1 and in that the light chain variable region of said antibody has the sequence of amino acids SEQ ID NO: 5.
[0006]
An antibody according to any one of claims 1 to 5 for use in the treatment of diseases associated with the suppressive activity of regulatory T cells.
[0007]
An antibody for use according to claim 6, characterized in that said antibody is used in the treatment of cancer.
[0008]
8. Antibody for use according to claim 7, characterized in that the cancer is selected from the group consisting of chronic myeloid leukemias, colon cancer, melanoma, uterine cancer, breast cancer, pancreatic cancer, gastric cancers. , ovarian cancer, primary lymphoma of the central nervous system, multiple myeloma, prostate cancer, Hodgkin lymphoma and hepatocellular carcinoma.
[0009]
9. An antibody for use according to claim 7, characterized in that the cancer is a virally induced cancer, preferably selected from the group consisting of nasopharyngeal carcinomas associated with Epstein-Barr Virus, hepatocellular carcinomas related to the virus. hepatitis C or hepatitis B.
[0010]
A pharmaceutical composition comprising an antibody according to any one of claims 1 to 5 and at least one pharmaceutically acceptable carrier.
[0011]
A combination product comprising: an antibody as defined in any one of claims 1 to 5, and an anticancer agent.
[0012]
12. Combination product as defined in claim 11 for simultaneous, separate or spread over time use in the treatment of cancer.

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LT3152234T|2020-12-28|

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CN107074936B|2021-11-02|

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EP3152234B1|2020-07-08|

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PT3152234T|2020-10-09|

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US20210107984A1|2021-04-15|

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HUE050905T2|2021-01-28|

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HUE050905T4|2021-01-28|

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WO2015185875A9|2016-04-14|

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FR3021970B1|2018-01-26|

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US20170283499A1|2017-10-05|

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法律状态:
2015-06-30| PLFP| Fee payment|Year of fee payment: 2 |

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2015-12-11| PLSC| Publication of the preliminary search report|Effective date: 20151211 |

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2016-07-08| PLFP| Fee payment|Year of fee payment: 3 |

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2017-06-30| PLFP| Fee payment|Year of fee payment: 4 |

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2018-06-27| PLFP| Fee payment|Year of fee payment: 5 |

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2019-07-01| PLFP| Fee payment|Year of fee payment: 6 |

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2020-06-30| PLFP| Fee payment|Year of fee payment: 7 |

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2021-06-30| PLFP| Fee payment|Year of fee payment: 8 |

优先权:

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申请号 | 申请日 | 专利标题

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FR1455177A|FR3021970B1|2014-06-06|2014-06-06|ANTIBODY AGAINST GALECTIN 9 AND INHIBITOR OF THE SUPPRESSIVE ACTIVITY OF T REGULATORY LYMPHOCYTES|

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FR1455177|2014-06-06|FR1455177A| FR3021970B1|2014-06-06|2014-06-06|ANTIBODY AGAINST GALECTIN 9 AND INHIBITOR OF THE SUPPRESSIVE ACTIVITY OF T REGULATORY LYMPHOCYTES|

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EP15732846.9A| EP3152234B1|2014-06-06|2015-06-05|Antibody which is directed against galectin-9 and is an inhibitor of the suppressor activity of regulatory t lymphocytes|

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LTEP15732846.9T| LT3152234T|2014-06-06|2015-06-05|Antibody which is directed against galectin-9 and is an inhibitor of the suppressor activity of regulatory t lymphocytes|

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CN201580035348.7A| CN107074936B|2014-06-06|2015-06-05|Antibodies against galectin 9 and inhibitors of regulatory T cell inhibitory activity|

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PT157328469T| PT3152234T|2014-06-06|2015-06-05|Antibody which is directed against galectin-9 and is an inhibitor of the suppressor activity of regulatory t lymphocytes|

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US15/316,074| US10899838B2|2014-06-06|2015-06-05|Antibody which is directed against galectin-9 and is an inhibitor of the suppressor activity of regulatory T lymphocytes|

---

HUE15732846A| HUE050905T2|2014-06-06|2015-06-05|Antibody which is directed against galectin-9 and is an inhibitor of the suppressor activity of regulatory t lymphocytes|

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SI201531368T| SI3152234T1|2014-06-06|2015-06-05|Antibody which is directed against galectin-9 and is an inhibitor of the suppressor activity of regulatory t lymphocytes|

---

ES15732846T| ES2822426T3|2014-06-06|2015-06-05|Antibody directed against galectin 9 and inhibitor of the suppressive activity of regulatory T lymphocytes|

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CA2950413A| CA2950413A1|2014-06-06|2015-06-05|Antibody which is directed against galectin-9 and is an inhibitor of the suppressor activity of regulatory t lymphocytes|

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PCT/FR2015/051498| WO2015185875A2|2014-06-06|2015-06-05|Antibody which is directed against galectin-9 and is an inhibitor of the suppressor activity of regulatory t lymphocytes|

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EP20184087.3A| EP3747908A1|2014-06-06|2015-06-05|Antibody directed against galectin-9 and inhibitor of the suppressive activity of t-lymphocyte regulators|

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JP2017516216A| JP2017521485A|2014-06-06|2015-06-05|Antibodies directed against galectin-9 and being inhibitors of regulatory T lymphocyte inhibitory activity|

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HRP20201542TT| HRP20201542T1|2014-06-06|2020-09-28|Antibody which is directed against galectin-9 and is an inhibitor of the suppressor activity of regulatory t lymphocytes|

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US17/131,946| US20210107984A1|2014-06-06|2020-12-23|Antibody which is Directed Against Galectin-9 and is an Inhibitor of the Suppressor Activity of Regulatory T Lymphocytes|