Antibodies that are against galectin-9 and are inhibitors of regulatory T cell suppressive activity

Abstract

The present invention relates to an antibody that is against galectin-9 and is an inhibitor of the inhibitory activity of regulatory T cells, and also relates to the use of the antibody in treating diseases associated with the inhibitory activity of regulatory T cells, especially cancer.

Description

[0001] The present invention relates to an anti-galectin-9 antibody and an inhibitor of the inhibitory activity of regulatory T cells, and also relates to using the antibody to treat diseases associated with the inhibitory 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 antigen-specific recognition. This antigen recognition by naive T lymphocytes leads to the activation of a primary immune response, as a result of which the phenotype and activity of the T lymphocytes are modified.

[0003] For example, each type of T lymphocyte population develops "effect" or effector T lymphocytes, thereby performing specific functions to protect the body. Thus, CD4+ T lymphocytes, also known as helper T lymphocytes, secrete the main cytokines that assist, in particular, the humoral functions of B lymphocytes (production of specific antibodies) and the cytotoxic activity of CD8+ T lymphocytes.

[0004] Another group of CD4+T lymphocytes is composed of natural regulatory T cells, which will be more simply referred to as "regulatory T cells" in the following. They structurally and continuously express the CD25 molecule (regulatory T lymphocytes can also be referred to as "CD4+CD25+") and the Foxp3 transcription factor. This small number of CD4+CD25+T lymphocytes has the specificity of negatively regulating immune response participants, which recognize various self-antigens through their TCR. Regulatory T lymphocytes also perform major tasks in the physiological functions of the immune system, especially protecting the body from autoimmune diseases.

[0005] However, it has been proposed that, in pathological situations, regulatory T cells can lead to inappropriate immunosuppression, which promotes tumor growth or assists the persistence of infectious pathogens (viruses, bacteria, parasites, etc.). Numerous studies have shown that regulatory T cells reduce anti-tumor or anti-viral immune responses, in particular by not locally suppressing the activity of effector T lymphocytes, thus assisting the persistence of most cancer viruses and tumor progression.

[0006] Little is known about the mechanism by which regulatory T cells exert their inhibitory effects on effector T lymphocytes. However, a number of studies have demonstrated various mechanisms by which regulatory T cells may suppress immune responses. Among the various possible explanations, studies have shown, for example, that Foxp3+ regulatory lymphocytes may destroy effector T lymphocytes by producing granzymes / perforins (1), or by depriving effector T lymphocytes of IL-2, or by inhibiting effector T lymphocyte proliferation, particularly by expressing surface molecules such as galectin 1, which interacts with receptors expressed on effector T lymphocytes and causes cell cycle arrest of effector T lymphocytes (2).

[0007] The pathophysiological role of regulatory T cells in cancer therefore encourages the emergence of a new anti-tumor therapeutic strategy that consists in neutralizing factors that suppress immune responses, especially regulatory T cells, or, in other words, in breaking tolerance to tumor antigens.

[0008] This is because, with the goal of reversing the balance between regulatory T cells and effector T lymphocytes under the control of anti-tumor immunity, many groups have attempted to develop therapeutic strategies aimed at inhibiting CD4+CD25+ regulatory T lymphocytes, especially by targeting surface molecules expressed by these regulatory T cells through monoclonal antibodies, and in particular those surface molecules that act on the inhibitory activity of regulatory T cells.

[0009] For example, analyses in rodents have shown that inhibition of CD4+CD25+ regulatory T cells by monoclonal antibodies against the IL-2 (CD25) α receptor promotes activation and expansion of effector T cells, thereby inhibiting tumor growth (3). However, CD25 is also expressed by activated effector T cells. Therefore, this strategy must be used with caution because it also promotes the elimination of effector T lymphocytes.

[0010] It has also been demonstrated that the inhibitory activity of regulatory T cells (4) can be inhibited by anti-GITR antibodies through GITR activation signals. In this regard, the use of anti-GITR antibodies in the treatment of murine tumors has the potential to increase the anti-tumor response of CD4+ and CD8+ T lymphocytes, and this is more effective in the presence of already established tumors. However, activated effector T lymphocytes also express GITR. Therefore, there is a risk of suppressing effector T lymphocytes during the use of anti-GITR antibodies.

[0011] Strategies aimed at inhibiting regulatory T cells have also been envisioned by using anti-CTLA4 antibodies, which are markers expressed by regulatory T lymphocytes. Thus, in mouse models in which CTLA-4 is KO (knockout) in regulatory T cells, or during the use of anti-CTLA-4 antibodies, it has been shown that the activity of effector T lymphocytes is increased, and the inhibition mediated by regulatory T lymphocytes is reduced, resulting in inhibition of tumor growth (5). However, activated effector T lymphocytes also express the CRLA-4 marker. Therefore, the use of anti-GITR antibodies also carries the risk of inhibiting effector T lymphocytes.

[0012] Therefore, although these molecules have good effects, one obstacle to the specific depletion of regulatory T cells is the lack of specificity of their surface markers. This is because the surface proteins CD25, CTLA-4 or GITR expressed by regulatory T cells are also markers of effector T lymphocyte activation. Therefore, the adverse effect of using these proteins as targets for regulatory T cell depletion is the elimination of many CD4+ and CD8+ effector T lymphocytes that are essential for tumor regression. In this regard, it is still difficult to specifically target regulatory T cells in treatment regimens.

[0013] Therefore, there remains a need for compounds that selectively and effectively inhibit the suppressive activity of regulatory T cells.

[0014] Therefore, one object of the present invention is to provide an antibody that is directed against a specific marker of regulatory T cells and is sufficient to inhibit the suppressive activity of regulatory T cells without altering the function of effector T lymphocytes.

[0015] The inventors have demonstrated the advantages of showing that a molecule, namely galectin-9, is specifically expressed by regulatory T cells during activation and that antibodies directed against this molecule make it possible to inhibit the suppressive activity of regulatory T cells and this in a specific manner, in other words without inhibiting effector T lymphocytes. Furthermore, it has been shown that this antibody is able to nullify the conversion of conventional CD4+ T lymphocytes into immunosuppressive CD4+ T lymphocytes induced by galectin-9, thus contributing to the maintenance of an anti-tumor immune response in the patient concerned according to the invention.

[0016] It has just been discovered that such antibodies that are directed against galectin-9 and inhibit the suppressive activity of regulatory T cells can be used to treat diseases associated with the suppressive activity of regulatory T cells.

[0017] Here, "diseases associated with the suppressive activity of regulatory T cells" refers to all diseases (non-autoimmune) in which the suppressive activity of regulatory T cells plays a role, in particular by promoting the development or persistence of the disease. In particular, it has been shown that the suppressive activity of regulatory T cells promotes the development of tumors. Therefore, more specifically, the present invention is aimed at cancers in which the suppressive activity of T lymphocytes plays a role.

[0018] Galectin 9, more succinctly referred to as "Gal9", forms part of the galectin family. Galectins, or S-type lectins, form a family that includes fifteen members in vertebrates, including ten in humans. Galectin 9 preferentially interacts with the beta-galactosidic residues of glycoproteins and glycolipids. In humans, Galectin 9 exists in three forms: long, medium and short.

[0019] Several studies have been conducted to determine the link between cancer development and galectins, especially galectin 9. However, most of these studies suggest that galectin 9 has cytotoxic activity against activated T lymphocytes (either CD4+ or CD8+), but does not have any cytotoxic activity against non-activated T lymphocytes.

[0020] For example, patent application EP 1586325 starts with the hypothesis that "in vitro", galectin 9 causes apoptosis of tumor cells, in particular malignant cells or metastatic cells, but not normal cells. Application EP 1586325 therefore relates to a drug comprising galectin 9 or a molecule that causes the production and / or release of galectin 9, etc.

[0021] Therefore, the aim is to utilize galectin-9 or factors that enable it to increase galectin-9, while the present invention conversely tends to inhibit it.

[0022] Therefore, the subject of the present invention is an antibody that is directed against galectin-9 and inhibits the suppressive activity of regulatory T cells.

[0023] In other words, the present invention relates to an anti-galectin-9 antibody characterized in that it is an inhibitor of the suppressive activity of regulatory T cells.

[0024] This is because the present invention is based on the unexpected findings of the inventors, who observed that (i) on the one hand, Galectin-9 is directly expressed by regulatory T cells and that its expression increases during their activation, and (ii) secondly, Galectin-9 is very weakly expressed by effector T lymphocytes and that this expression disappears during activation. Furthermore, the inventors observed that inhibition of Galectin-9 by antibodies makes it possible to suppress the suppressive activity of regulatory T cells.

[0025] "Regulatory T cells" means a subpopulation of natural regulatory T cells, also known as Tregs, characterized by the expression of CD25, CTLA-4 and GITR, and also characterized by the specific expression of the transcription factor Foxp3. More specifically, the regulatory T cells referred to in the present invention are therefore natural regulatory T cells, or nTregs.

[0026] "The suppressive activity of regulatory T cells" refers to the immunosuppressive activity of regulatory T cells on effector T lymphocytes, which, once activated, in pathological conditions, particularly promotes tumor growth. Preferably, the suppressive activity of regulatory T cells can be understood as reducing the activity of anti-tumor immune responses by suppressing the activity of effector T lymphocytes. The suppressive activity of regulatory T cells can be analyzed according to various techniques known to those skilled in the art, for example, the MLR (mixed lymphocyte reaction) method can be implemented, which uses the co-culture of Treg lymphocytes with autologous or heterologous immune cells (collectively referred to as PBMC or T CD4+). This method can be accomplished by a proliferation assay based on (i) the inclusion of radioactive elements such as tritiated thymidine or (ii) the inclusion of EdU (5-ethynyl-2'-deoxyuridine), which is included during the synthesis of DNA and, after the enzyme reaction, enables it to emit fluorescence (click EdU proliferation assay) or (iii) flow cytometry (CSFE).

[0027] For example, the inhibitory effect of the antibodies according to the invention can thus be analyzed by an MLR method which is performed in the presence of the test antibody.

[0028] Detailed information on applying this analytical approach can be found in the “Examples” and “Apparatus and Methods” sections.

[0029] The terms "antibody" and "immunoglobulin" are used indiscriminately to refer to immunoglobulin molecules or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain a specific binding site for a given antigen. The term antibody includes not only whole antibody molecules but also antibody fragments and variants of antibodies and antibody fragments (including derivatives such as humanized antibodies).

[0030] Immunoglobulins are well known to those skilled in the art and consist of two heavy chains connected to each other by a disulfide bond, each of which is connected to a light chain by a disulfide bond. There are two types of light chains, lambda (λ) chains and kappa (κ) chains. There are five major classes of heavy chains that determine the functional activity of antibodies: IgM, IgD, IgG, IgA and IgE.

[0031] Each chain contains different sequence domains. The light chain includes two domains, namely the variable domain (or region) (VL) and the constant domain (CR). The heavy chain includes four or five domains, namely a variable domain (VH) and three or four constant domains (CH1, CH2, CH3 and optional CH4), depending on the antibody class. The variable region of the light chain (VL) and the variable region of the heavy chain (VH) determine the specificity of the antigen and the fixed site of the antigen.

[0032] The constant domains of the light chain (CL) and the heavy chain (CH) give antibodies important biological characteristics, such as the association of antibody chains, mobility through the placenta, fixation of complement and / or fixation to Fc receptors (FcR). The Fv segment corresponds to the V-terminal portion of the Fab segment of the immunoglobulin described below, and includes the variable parts of the light chain and the heavy chain (VL and VH). The specificity of the antibody lies in the structural complementarity between the antibody recognition site and the antigenic determinant. The recognition site of the antibody is essentially composed of residues from the hypervariable region or the region that determines complementarity ("complementarity determining region" or CDR). Occasionally, residues from non-hypervariable regions or framework regions (FR) affect the overall structure of the domain and therefore affect the recognition site. The term "complementarity determining region" (CDR) refers to the amino acid sequence of the fixed and specific affinity of the natural Fv region that jointly defines the fixed site of the natural immunoglobulin. Each light chain and each heavy chain of an immunoglobulin has three CDR regions, which are called L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3. Therefore, the antigen binding site includes six CDRs. The framework region (FR) refers to the amino acid sequence placed between the CDRs, that is, the part of the light chain variable region and the heavy chain variable region of the immunoglobulin that are respectively preserved between the immunoglobulins of the same type.

[0033] An antibody according to the invention may be a monoclonal antibody or a polyclonal antibody. Preferably, the antibody according to the invention is a monoclonal antibody.

[0034] The term "monoclonal antibody" or "mAb" refers to an antibody with a 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, in other words, they can be produced by protein engineering techniques.

[0035] The term "Fab" refers to an antibody fragment with a molecular mass of about 50,000 daltons and has the activity of binding to an antigen. It includes approximately half of the n-terminal side of the heavy chain and the entire light chain connected by a disulfide bond. Specifically, Fab can be obtained by treating immunoglobulin with a protease, i.e., papain.

[0036] The term "F(ab')2" refers to a fragment of about 100,000 daltons and has the activity of binding to an antigen. This fragment is slightly larger than two Fab fragments connected by a disulfide bond in the hinge region. These fragments are obtained by treating immunoglobulins with a protease, i.e., pepsin. Fab fragments are obtained from F(ab')2 fragments by separating the disulfide bonds in the hinge region.

[0037] Single Fv chain "scFv" corresponds to a VH:VL polypeptide that is synthesized using genes encoding VL and VH domains and sequences encoding peptides intended to bind to these domains. The scFv according to the present invention includes, for example, CDRs maintained in an appropriate structure using genetic recombination technology.

[0038] A "scFv" dimer corresponds to two scFv molecules linked together by a peptide bond. The Fv chain is usually the result of expressing a fusion gene that includes genes encoding VH and VL connected by a linker sequence that encodes a peptide. Human scFv fragments may include CDR regions that are preferably maintained in an appropriate structure using genetic recombination techniques. A "dsFv" fragment is a VH-VL heterodimer stabilized by disulfide bonds; it can be divalent (dsFV2). Fragments of divalent Sc(Fv)2 or multivalent antibodies can be formed spontaneously by the combination of monovalent scFvs, or by connecting scFv fragments with peptide binding sequences.

[0039] The Fc fragment is the support for the biological characteristics of antibodies, especially their ability to be recognized by immune effectors or to activate complement. It includes the constant fragment of the heavy chain outside the hinge region.

[0040] The term "diabody" refers to small antibody fragments with two antigen-fixing sites. These fragments include a variable heavy domain VH connected to a variable light domain VL in the same VH-VL polypeptide chain. With too short a binding sequence to be able to match the two domains of the same chain, matching with the two complementary domains of the other chain must occur, and thus two antigen-fixing sites are formed.

[0041] The antibody of the present invention can therefore be an immunoglobulin composed of two heavy chains and two complete light chains, or can be a fragment of an immunoglobulin according to the present invention, such as F(ab')2, Fab, Fv, scFv or Fc. Preferably, this antibody fragment is the Fab region of an immunoglobulin, especially the Fv region of an IgG1 antibody.

[0042] The antibodies described in the present invention are isolated and purified antibodies, which are different from natural antibodies. In the case of antibodies or nucleotide sequences according to the present invention, the terms "isolated" and "purified" refer to the presence of molecules in the absence of other biological macromolecules of the same type.

[0043] The term "chimeric antibody" refers to an antibody in which the sequence of each light chain and / or heavy chain constituting the antibody comprises or includes a mixed sequence from at least two different animals. Preferably, the chimeric antibody of the present invention is a human / mouse hybrid antibody. Specifically, the chimeric antibody of the present invention may include the VH domain and VL domain of an antibody from a non-human animal, especially a murine animal, and the CH domain and CL domain of a human antibody. Therefore, preferably, the antibody of the present invention includes the VH domain and VL domain derived from the 1G3 antibody defined below and the CH domain and CL domain of a human antibody.

[0044] According to the present invention, the term "humanized antibody" refers to an antibody produced in a non-human animal, wherein the sequences of the heavy chain and the light chain except for the CDR are replaced by the corresponding sequences of one or more antibodies from humans. Preferably, the term "humanized antibody" refers to an antibody, wherein the sequences of the heavy chain and the light chain are from humans, and the CDR thereof is generated from the 1G3 antibody.

[0045] The antibody according to the invention is preferably a monoclonal antibody, in other words, it recognizes only one antigenic determinant in galectin-9, in contrast to a polyclonal antibody which corresponds to a mixture of monoclonal antibodies and thus recognizes multiple antigenic determinants in the same molecule.

[0046] Monoclonal antibodies according to the present invention can be obtained according to techniques well known to those skilled in the art. For example, cell fusion techniques, cloning heavy and light chain sequence techniques, phage or ribosome display techniques, mouse immunization with a human immunoglobulin list, and expression with specific cells or transgenic animals can be utilized. These techniques are well known to those skilled in the art.

[0047] The present invention relates to antibodies against galectin-9 and inhibitors of the suppressive activity of regulatory T cells. Specifically, the inventors developed a hybridoma that produces the mouse antibody IGg1, Kappa, 1G3, which is against galectin-9 and inhibits the suppressive activity of regulatory T cells. The inventors characterized the variable domains of the light chain and heavy chain of this monoclonal antibody mAb1G3 and thus determined the CDRs of the antibody, as shown in Table 1.

[0048]

[0049] Table 1

[0050] Therefore, a specific embodiment of the present invention relates to an antibody against galectin-9 and inhibiting the suppressive activity of regulatory T cells, having the same fixed regions as the 1G3 antibody, having six CDRs defined as follows:

[0051] - Amino acid sequence in the H-CDR1 region SEQ ID NO: 2

[0052] - Amino acid sequence in the H-CDR2 region SEQ ID NO: 3

[0053] - Amino acid sequence in the H-CDR3 region SEQ ID NO: 4

[0054] - Amino acid sequence in the L-CDR1 region SEQ ID NO: 6

[0055] - Amino acid sequence in the L-CDR2 region SEQ ID NO: 7

[0056] - Amino acid sequence in the L-CDR3 region SEQ ID NO: 8

[0057] In particular, a subject of the present invention relates to an antibody against galectin-9 and inhibiting the suppressive activity of regulatory T cells, having six CDRs defined as follows:

[0058] - Amino acid sequence in the H-CDR1 region SEQ ID NO: 2

[0059] - Amino acid sequence in the H-CDR2 region SEQ ID NO: 3

[0060] - Amino acid sequence in the H-CDR3 region SEQ ID NO: 4

[0061] - Amino acid sequence in the L-CDR1 region SEQ ID NO: 6

[0062] - Amino acid sequence in the L-CDR2 region SEQ ID NO: 7

[0063] - Amino acid sequence in the L-CDR3 region SEQ ID NO: 8

[0064] In a specific embodiment, the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 5.

[0065] Thus, an antibody according to the invention specifically binds to galectin-9 and is an inhibitor of the suppressive activity of regulatory T cells.

[0066] According to another specific embodiment of the present invention, an antibody according to the present invention can specifically bind to an epitope of galectin 9. Advantageously, an antibody according to the present invention can specifically bind to galectin 9 in a membrane or in a cell.

[0067] More specifically, an antibody according to the invention may bind to the epitope recognized by the 1G3 antibody as defined above.

[0068] Therefore, according to a specific embodiment of the present invention, an antibody according to the present invention can specifically bind to the epitope of the amino acid sequence SEQ ID NO: 9, as shown in Table 2. The epitope consisting of the amino acid sequence SEQ ID NO: 9 corresponds to the P4 peptide and covers the end of the binding peptide and the beginning of the C-terminal part of galectin 9. This sequence is present in three isomers of galectin 9 (amino acids 166 to 178 of the S isomer, amino acids 178 to 190 of the M isomer, and amino acids 210 to 222 of the L isomer). Therefore, this antibody can react with all isomers of galectin 9.

[0069] Sequence of Galectin-9 (epitope) TPAIPPMMYPHPA (SEQ ID NO: 9)

[0070] Table 2

[0071] According to another specific embodiment, the antibody according to the present invention is a chimeric antibody, preferably a mouse / human chimeric antibody. In particular, this mouse / human chimeric antibody may comprise the variable domains of the 1G3 antibody as described above.

[0072] According to another specific embodiment, the antibody according to the invention is a humanized antibody. In particular, the variable domain of such a humanized antibody may comprise a framework region of a human acceptor, and optionally a human constant domain and CDRs of a non-human donor, in particular the CDRs defined above.

[0073] The inventors also developed a hybridoma that produces the murine antibody IGg1, Kappa, 2E12, which is an inhibitor of anti-galectin 9 and regulatory T cell suppressive activity. The inventors characterized the variable domains of the light and heavy chains of this monoclonal antibody mAb 2E12 and thus determined the CDRs of the antibody, as shown in Table 3.

[0074]

[0075] Table 3

[0076] A specific embodiment of the present invention therefore relates to an antibody against galectin-9 and inhibiting the suppressive activity of regulatory T cells, having the same fixed regions as the 2E12 antibody, having six CDRs defined as follows:

[0077] - Amino acid sequence in the H-CDR1 region SEQ ID NO: 11

[0078] - Amino acid sequence in the H-CDR2 region SEQ ID NO: 12

[0079] - Amino acid sequence in the H-CDR3 region SEQ ID NO: 13

[0080] - Amino acid sequence in the L-CDR1 region SEQ ID NO: 15

[0081] - Amino acid sequence in the L-CDR2 region SEQ ID NO: 16

[0082] - Amino acid sequence in the L-CDR3 region SEQ ID NO: 17

[0083] In particular, a subject of the present invention relates to an antibody against galectin-9 and inhibiting the suppressive activity of regulatory T cells, having six CDRs defined as follows:

[0084] - Amino acid sequence in the H-CDR1 region SEQ ID NO: 11

[0085] - Amino acid sequence in the H-CDR2 region SEQ ID NO: 12

[0086] - Amino acid sequence in the H-CDR3 region SEQ ID NO: 13

[0087] - Amino acid sequence in the L-CDR1 region SEQ ID NO: 15

[0088] - Amino acid sequence in the L-CDR2 region SEQ ID NO: 16

[0089] - Amino acid sequence in the L-CDR3 region SEQ ID NO: 17

[0090] In a specific embodiment, the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 14.

[0091] As mentioned above, according to another specific embodiment of the present invention, an antibody according to the present invention specifically binds to an epitope of galectin 9. Advantageously, an antibody according to the present invention is capable of specifically binding to galectin 9 in a membrane or in a cell.

[0092] More specifically, an antibody according to the invention is capable of binding to the epitope recognized by the 2E12 antibody defined above.

[0093] According to another specific embodiment, the antibody according to the invention is a chimeric antibody, preferably a mouse / human chimeric antibody. In particular, the mouse / human chimeric antibody may comprise the variable domains of the 2E12 antibody as defined above.

[0094] According to another specific embodiment, the antibody according to the invention is a humanized antibody. In particular, the variable domain of the humanized antibody may comprise a framework region of a human acceptor, and optionally a human constant domain and CDRs of a non-human donor, in particular the CDRs defined above.

[0095] Methods for generating antibodies

[0096] The antibodies of the present invention can be produced by any technique known to those skilled in the art, for example, any chemical technique, biological technique, genetic technique or enzyme technique used alone or in combination, but not limited thereto.

[0097] For example, the techniques described below for producing hybridomas producing anti-galectin-9 monoclonal antibodies, 1G3 or 2E12, can be utilized.

[0098] According to the present invention, the specific binding of the anti-galectin-9 antibody can be analyzed according to any method known in the prior art. According to the immunoassay that can be used, for example, immunoblotting, radioimmunoassay test, ELISA, sandwich immunoassay, immunoprecipitation test, precipitin test, gel diffusion precipitin test, immunoradiometric assay test, fluorescent immunoassay or complement tuberculosis test can be set. These tests are well known to those skilled in the art.

[0099] The inhibitory activity of an antibody according to the present invention against regulatory T cells can be analyzed using various techniques known to those skilled in the art. For example, a cell proliferation assay can be performed. Reference can therefore be made to the techniques used below for cell proliferation assays performed with anti-galectin-9 antibodies, 1G3 or 2E12.

[0100] Once the amino acid sequence of the desired sequence is known, antibodies can be readily produced by those skilled in the art using standard polypeptide production techniques.

[0101] For example, such antibodies can be analyzed by well-known solid phase methods, preferably using a commercially available peptide synthesis apparatus and following the supplier's recommendations.

[0102] Alternatively, the antibodies of the present invention can be obtained by techniques well known to those skilled in the art for recombinant DNA in an appropriate expression system. The term "expression system" refers to a cell host and a compatible vector under appropriate conditions, in other words, conditions that allow expression of a protein encoded by an exogenous DNA, which is carried by a vector and introduced into a host cell. Typically, a nucleic acid sequence encoding an antibody can be inserted into an appropriate expression vector, which is then introduced into an appropriate prokaryotic or eukaryotic host that will produce the desired antibody.

[0103] The terms "vector", "cloning vector" and "expression vector" refer to a vehicle for introducing a DNA or RNA sequence encoding an antibody into a host cell, thereby transforming it and enabling it to express the introduced sequence (in other words, transcribe and translate it). The expression vector is usually a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0104] Viral vectors include adenovirus, retrovirus, herpes virus and vectors derived from adenovirus group (AAV). This recombinant virus can be manufactured by well-known techniques, such as by cell line transfection, so that it can be encapsidated, or by expressing a plasmid or complementary virus that has a necessary missing function for transient transfection. For example, cell lines that can be encapsidated are PA317, PsiCRIP, GPenv+, 293, etc. Detailed schemes for manufacturing this defective recombinant virus for replication are provided in patent applications such as WO 95 / 14785, WO 96 / 22378, and US 5.882.877.

[0105] Host cells are thus transfected, infected or transformed by the nucleic acid or an appropriate vector as described above.

[0106] The term "transformation" as used herein refers to the introduction of an exogenous (foreign or extracellular) gene or DNA or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence, thereby producing a desired substance, usually a protein encoded by the introduced gene or sequence.

[0107] Such common expression systems include, but are not limited to, E. coli host cells and plasmid vectors, insect host cells, baculovirus-type vectors, and mammalian cells and vectors.

[0108] A method of producing by a host cell expressing an antibody according to the present invention may comprise the steps of: (i) introducing a recombinant nucleic acid or vector as described above into a competent host cell in vivo or in vitro, (ii) culturing the recombinant host cell thus obtained in vivo or in vitro, (iii) randomly selecting cells expressing and / or secreting the antibody or polypeptide. Such host cells may be used to produce antibodies according to the present invention.

[0109] According to a particular embodiment, a method for producing an antibody according to the invention may comprise the steps of: (i) culturing the transformed cell as described above under conditions suitable for expressing the antibody; and (ii) recovering the antibody thus expressed.

[0110] Antibodies can be separated from the culture medium by conventional immunoglobulin purification procedures, for example, purification of A-Sepharose protein by hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0111] In a specific embodiment of the present invention, human chimeric antibodies according to the present invention can be generated by the following steps: obtaining nucleic acid sequences encoding the VL domain and VH domain as described above; constructing an expression vector for human chimeric antibodies by inserting the nucleic acid sequence into an expression vector of an animal cell, wherein the animal cell has genes encoding the CH domain and CL domain; and expressing the encoded sequence by introducing the expression vector into the animal cell.

[0112] The CH domain of the human chimeric antibody can be any region belonging to human immunoglobulin. Preferably, it is of the IgG class, and more preferably, it is of the IgG1 class. Similarly, the CL domain of the human chimeric antibody can be any region belonging to human immunoglobulin. Preferably, it is of the Kappa class.

[0113] In particular, the chimeric antibody or humanized antibody according to the present invention can be obtained by antibody genetic engineering.

[0114] For example, chimeric antibodies can be constructed by gene transfection technology or recombinant DNA technology.

[0115] The humanized antibodies according to the present invention can be generated by the following steps: obtaining the CDR domain as described above; constructing an expression vector for human antibodies by inserting the nucleic acid sequence into an expression vector of an animal cell, wherein the animal cell has genes encoding the following regions: (i) a constant heavy chain region identical to that of a human antibody and (ii) a constant light chain region identical to that of a human antibody; and expressing the encoded sequence by introducing the expression vector into an animal cell.

[0116] Regarding the expression vector of humanized antibody, it can be of the type in which the gene encoding the heavy chain of the antibody and the gene encoding the light chain of the antibody are present in separate vectors, in other words, the genes are present in the same vector (tandem). For the case of constructing an expression vector, the case of introducing into animal cells, etc., a tandem vector is preferred. pKANTEX93 or pEE18 can be cited as an example of an expression vector of tandem humanized antibody.

[0117] The methods for producing humanized antibodies based on gene or recombinant DNA technology are well known in the prior art. Antibodies can be humanized by various techniques known in the prior art, for example, by CDR transplantation, gluing or re-, or by chain replacement. The technology for preparing such antibodies based on recombinant DNA is also known.

[0118] The Fab fragment according to the present invention can be obtained by treating an antibody, especially an antibody that is anti-galectin-9 and inhibits the inhibitory activity of regulatory T cells, with a protease, i.e., papain. The FAB fragment can also be produced by inserting a DNA encoding an antibody FAB fragment into a vector that can be used in a prokaryotic or eukaryotic expression system and introducing the vector into a prokaryotic cell or eukaryotic cell suitable for expressing the FAB fragment.

[0119] The F(ab')2 fragment according to the present invention can be obtained by treating an antibody, especially an antibody that is anti-galectin-9 and inhibits the inhibitory activity of regulatory T cells, with a protease, i.e., pepsin. The Fab' fragments described above can also be linked together by a thioether bond or a disulfide bond to obtain a F(ab')2 fragment.

[0120] The Fab' fragment according to the present invention can be obtained by treating the F(ab')2 complex of an antibody, especially an antibody that is anti-galectin-9 and inhibits the inhibitory activity of regulatory T cells, with a reducing agent, i.e., dithiothreitol. The Fab' fragment can also be prepared by inserting a DNA encoding the Fab' fragment of an antibody into a vector that can be used in a prokaryotic or eukaryotic expression system and introducing the vector into a prokaryotic cell or eukaryotic cell suitable for expressing the Fab' fragment.

[0121] The ScFv fragment according to the present invention can be generated by the following steps: obtain the sequence of the DNA encoding the VH domain and VL domain as described above, then insert the DNA into a vector that can be used in a eukaryotic or prokaryotic expression system, and introduce the vector into a eukaryotic cell or prokaryotic cell suitable for expressing the ScFv fragment. In order to obtain a humanized ScFv fragment, CDR transplantation technology can be used. This technology involves selecting the complementary determining region (CDR) of the donor cFv fragment and transplanting it to the framework of a human ScFv fragment with a known three-dimensional structure (for example, see WO 98 / 45322; EP 0173494).

[0122] The amino acid sequence of the antibody according to the present invention can be modified. For example, it may be desirable to improve the affinity and / or biological characteristics of the antibody. As is well known, in the case of generating humanized antibodies by simply transplanting the CDRs of the VH and VL of antibodies of non-human animals derived from the human antibody framework (FR), the binding ability to the antigen is reduced compared to the original antibody derived from the non-human animal. It is believed that certain amino acid residues of the VH and VL of non-human antibodies not only in the CDR, but also in the FR are directly or indirectly related to the antigen binding ability. By replacing these amino acid residues with different amino acid residues derived from FR, the VH and VL of the human antibody will therefore reduce the binding ability. Therefore, in order to solve this problem, the transplanted antibodies of human CDR must be tested, so as to identify the amino acid residues that are directly related to the binding of the antigen, or that interact with the CDR amino acid residues, or that maintain the three-dimensional structure of the antibody and are directly related to the binding of the antigen in the FR, VH and VL amino acid sequences of the human antibody. Replacing the identified amino acids with the amino acid residues of the original antibody derived from the non-human antibody can increase the binding ability. The antibody structure of the present invention and the sequence encoding it are modified and altered, and functional molecules encoding antibodies with desired characteristics are again obtained.

[0123] Another aspect of the invention relates to variants of the antibodies of the invention which retain functionality.

[0124] A "variant retaining function" is one in which a specified amino acid residue in a protein has been changed without changing the overall structure and the function of inhibiting the suppressive activity of regulatory T cells. Thus, an amino acid may be replaced with another amino acid having similar properties (e.g., polarity, hydrogen bonding ability, etc.) as long as the function of inhibiting the suppressive activity of regulatory T lymphocytes is retained.

[0125] Therefore, according to a specific embodiment of the present invention, the antibody having the above-defined anti-galectin-9 and inhibiting the inhibitory activity of regulatory T cells comprises:

[0126] -H-CDR1, having 1 or 2 different amino acids, the amino acid sequence being defined as SEQ ID NO: 2,

[0127] -H-CDR2, having 1 or 2 different amino acids, the amino acid sequence being defined as SEQ ID NO: 3,

[0128] -H-CDR3, having 1 or 2 different amino acids, the amino acid sequence being defined as SEQ ID NO: 4,

[0129] - L-CDR1, having 1 or 2 different amino acids, the amino acid sequence being defined as SEQ ID NO: 6,

[0130] - L-CDR2, having 1 or 2 different amino acids, the amino acid sequence being defined as SEQ ID NO: 7,

[0131] - L-CDR3 having 1 or 2 different amino acids, said amino acid sequence being defined as SEQ ID NO: 8.

[0132] According to another specific embodiment of the present invention, the above-defined anti-galectin-9 antibody that inhibits the inhibitory activity of regulatory T cells has 1, 2 or 3 different amino acids, and the amino acids have sequences of all six CDRs defined above, in other words, CDRs with sequences of 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.

[0133] According to another specific embodiment of the present invention, the antibody having the above-defined anti-galectin-9 and inhibiting the inhibitory activity of regulatory T cells comprises:

[0134] -H-CDR1, having 1 or 2 different amino acids, the amino acid sequence being defined as SEQ ID NO: 11,

[0135] -H-CDR2, having 1 or 2 different amino acids, the amino acid sequence being defined as SEQ ID NO: 12,

[0136] -H-CDR3, having 1 or 2 different amino acids, the amino acid sequence being defined as SEQ ID NO: 13,

[0137] - L-CDR1, having 1 or 2 different amino acids, the amino acid sequence being defined as SEQ ID NO: 15,

[0138] - L-CDR2, having 1 or 2 different amino acids, the amino acid sequence being defined as SEQ ID NO: 16,

[0139] - L-CDR3 having 1 or 2 different amino acids, said amino acid sequence being defined as SEQ ID NO: 17.

[0140] According to another specific embodiment of the present invention, the above-defined anti-galectin-9 antibody that inhibits the inhibitory activity of regulatory T cells has 1, 2 or 3 different amino acids, and the amino acids have the sequences of all six CDRs defined above, in other words, the CDRs of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17.

[0141] Therapeutic applications

[0142] As mentioned above, it is well known that under pathological conditions, regulatory T cells can lead to inappropriate immunosuppression, thereby promoting tumor growth. Thus, numerous studies have shown that regulatory T cells reduce anti-tumor immune responses, especially by inappropriately suppressing the activity of effector T lymphocytes, thus promoting the pathological progression of cancer types.

[0143] It has been demonstrated here that, firstly, galectin-9 is directly expressed by regulatory T cells during activation, while effector T lymphocytes express it only very weakly or not at all, and that targeting galectin-9 makes it possible to specifically inhibit regulatory T cells without the risk of causing depletion of effector T lymphocytes. In addition, it has been demonstrated that inhibition of galectin-9 by antibodies makes it possible to inhibit the suppressive activity of regulatory T cells. Antibodies against galectin-9 and suppressing the suppressive activity of regulatory T cells according to the invention can therefore be used for the treatment of diseases associated with the suppressive activity of regulatory T cells, in particular for the treatment of cancer.

[0144] Therefore, one object of the present invention relates to an antibody as described above for use in the treatment of diseases associated with the suppressive activity of regulatory T cells.

[0145] A particular embodiment of the present invention relates to an antibody as described hereinbefore for use in the treatment of cancer.

[0146] "Cancer treatment" refers to any treatment that, for example, inhibits tumors or metastases, reduces the risk of recurrence, reduces the rate of progression of tumors or metastases, and / or treats symptoms of the disease.

[0147] The cancers targeted by the present invention are cancers in which regulatory T cells exert their inhibitory activity. Advantageously, the cancers targeted by the present invention are cancers in which regulatory T cells are present in large numbers in tumor tissue or circulation, and the expansion of regulatory T cells is generally associated with increased activation (6). The frequency of regulatory T cells can be assessed by any method known to those skilled in the art, for example, by flow cytometry (FACS) analysis of intratumoral lymphocytes or circulating lymphocytes or by immunohistochemical markers of tumor tissue.

[0148] In general, the antibodies described hereinbefore can therefore be used to treat all types of cancer in which regulatory T cells exert their suppressive activity.The treatment of various types of cancer in which regulatory T cells exert their suppressive activity has been the subject of research and is well known to those skilled in the art.

[0149] Thus, it is known that a higher level of regulatory T cells in tumors is clearly associated with a poor prognosis in chronic myeloid leukemia (7), colon cancer (8), melanoma (9), uterine cancer (10), breast cancer (11), pancreatic cancer (12), gastric cancer (13), ovarian cancer (14), primary lymphomas of the central nervous system (15), multiple myeloma (16), prostate cancer (17), Hodgkin lymphoma or liver cancer (18, 19).

[0150] Therefore, the antibodies described above are particularly useful for the treatment of cancer selected from chronic myeloid leukemia, colon cancer, melanoma, uterine cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, primary lymphoma of the central nervous system, multiple myeloma, prostate cancer, Hodgkin's lymphoma and liver cancer.

[0151] Studies have also shown that certain cancers produce large amounts of exosomes carrying galectin-9, which perform immunosuppressive actions, in other words, suppress immune responses and potentially anti-tumor responses. Non-limiting examples of cancers that produce large amounts of exosomes carrying galectin-9 are virus-induced cancers, such as nasopharyngeal carcinoma associated with EBV (Epstein-Barr virus) or hepatocellular carcinoma (CHC) associated with HCV (hepatitis C virus) or HBV (hepatitis B virus) (20, 21).

[0152] Thus, the antibodies defined hereinbefore are particularly useful in the treatment of cancer, which is a virus-induced cancer, preferably selected from nasopharyngeal carcinoma associated with Epstein-Barr virus and hepatocellular carcinoma associated with hepatitis C virus or hepatitis B virus.

[0153] It has also been shown that increased frequency of regulatory T cells is a factor predicting recurrence of fibrosis caused by hepatitis C ( 22 , 23 ).

[0154] Therefore, an antibody according to the present invention can be used to prevent the recurrence of fibrosis caused by hepatitis C.

[0155] In each of the foregoing embodiments, antibodies that are directed against galectin-9 and inhibit the suppressive activity of regulatory T cells are administered in a manner appropriate for a patient in need of such treatment.

[0156] The antibodies according to the present invention may be used alone or in combination with any other suitable mixtures.

[0157] An object of the present invention relates to a method for treating cancer associated with the expression of galectin-9 and the suppressive activity of regulatory T cells, comprising administering to a patient a therapeutically effective amount of an antibody according to the present invention.

[0158] The term "patient" means a person suffering from or afflicted with a disease associated with the suppressive activity of regulatory T cells, particularly cancer.

[0159] The term "therapeutically effective amount" of an antibody refers to an amount of the antibody sufficient to treat such cancer, with an acceptable benefit / risk ratio for drug therapy. The amount of the antibody according to the present invention and the composition and frequency of administration are determined by clinical studies, physicians or pharmacists. The "therapeutically effective" amount for each patient may depend on a number of factors, such as the nature and severity of the disease to be treated, the activity of the antibody employed, the composition employed, the patient's age, weight, general health, sex and diet, the method of administration, the duration of treatment (single or multiple doses), the drugs used in combination, and other factors well known to medical professionals.

[0160] According to a particular embodiment of the present invention, the above-defined anti-galectin-9 antibody that inhibits the suppressive activity of regulatory T cells is used in combination with a second agent for treating a disease associated with the suppressive activity of regulatory T cells, for example, the second agent is an anticancer agent.

[0161] Thus, where the use is to treat cancer, the antibody may be used in combination with known cancer treatment methods, such as surgery, radiotherapy, chemotherapy, or a combination thereof. For example, the antibody may be used in combination with adoptive immunotherapy, including one or more injections of effector lymphocytes directed against tumor antigens, particularly EBV antigens. According to certain aspects, other anticancer agents used for cancer treatment in combination with the anti-galectin-9 antibody according to the invention include anti-angiogenic anticancer agents. According to certain aspects, the antibody is administered in combination with a cytokine, such as a cytokine that stimulates an anti-tumor immune response.

[0162] A subject of the present invention therefore relates to a combination product comprising an antibody as defined above and an anticancer agent.

[0163] A particular embodiment relates to such a combination product for use simultaneously, separately or spread over time in the treatment of cancer.

[0164] Drug ingredients

[0165] Regarding the mode of administration, the antibody is usually formulated in the form of a pharmaceutical composition. A pharmaceutical composition comprising an antibody according to the invention can be formulated by methods known in the art, wherein the therapeutic molecule is combined with at least one excipient.

[0166] A subject of the present invention is therefore a pharmaceutical composition comprising an antibody as described above and at least one pharmaceutically acceptable carrier.

[0167] "Pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier that can be accepted by patients in a manner of administration. For example, sterile phosphate buffered saline solution is pharmaceutically acceptable.

[0168] The pharmaceutically acceptable carrier may generally include one or two compounds, for example, selected from excipients, preservatives, solubilizers, buffers, albumin, etc. For example, known excipients are starch, gelatin, stearic acid, calcium stearate or magnesium stearate, etc. Those skilled in the art can determine the mixture suitable for the current components.

[0169] The form of the pharmaceutical composition, the mode of administration, the dosage and dosing will of course depend on, among other things, the disease to be treated, its symptoms, its severity, and the age, weight and sex of the patient.

[0170] Without limitation, the pharmaceutical composition according to the present invention may be formulated so as to be administered topically, parenterally, nasally, intravenously, subcutaneously / intradermally, conjunctivally, or by intramuscular or intraocular means.

[0171] The pharmaceutical composition according to the present invention may optionally comprise a pharmaceutically acceptable excipient suitable for injection. In particular, the excipient may be an isotonic sterile saline solution, sodium dihydrogen phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or a mixture of these salts. These components may also be dry components, in particular frozen, lyophilized or freeze-dried components, which, depending on the environment, after adding sterile water or physiological water, constitute an injectable solution.

[0172] The dose employed can be adjusted according to various parameters, such as, inter alia, the pathology or, alternatively, the intended duration of treatment, the mode of administration.

[0173] To prepare the pharmaceutical composition, a sufficient amount of the antibody can be dissolved or dispersed in a pharmaceutically acceptable solvent or aqueous medium.

[0174] Dosage forms suitable for use by injection include sterile aqueous solutions, dispersions, formulations containing sesame oils or aqueous propylene glycol, and sterile powders for the extemporaneous preparation of sterile injectable solutions. In all cases, the form employed must be sterile and must be sufficiently fluid to permit easy injection by syringe. The dosage form must be stable during manufacture and storage and protected from contamination by microorganisms such as bacteria or fungi.

[0175] Solutions of the active ingredient, either in the free state or as a pharmaceutically acceptable salt, can be prepared in water mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can be made in glycerol, in liquid polyethylene glycol, in a mixture of both, or in oils. These preparations usually contain a preservative to prevent the growth of microorganisms under ordinary conditions of storage and use.

[0176] An antibody according to the present invention can be formulated with components in neutral form or in salt form. Pharmaceutically acceptable salts include salts of acid salts formed by free amino groups of proteins, and by inorganic or organic acids, for example, the inorganic acid is hydrochloric acid or phosphoric acid, such as acetic acid, oxalic acid, mandelic acid, etc. Salts formed by free hydroxyl groups can also be derived from inorganic and organic bases, such as inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, such as organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0177] Once in pharmaceutical form, the drug can be administered in a therapeutically effective amount in a dosage form matched to the dosage form. Administration can be as described above, but it can also be administered in the form of a capsule that releases the drug.

[0178] Attached photos

[0179] Figure 1 : Phenotypic analysis by multiparameter flow cytometry of natural regulatory T cells isolated from human blood samples.

[0180] Figure 2 : Analysis of the suppressive function of natural regulatory T cells isolated in vitro. (A) Analysis of the proliferation inhibition of PBMCs activated by autologous regulatory T cells in terms of counts per minute. (B) Analysis of the cytolysis ratio of PBMCs activated by autologous regulatory T cells.

[0181] Figure 3: Analysis by qPCR of the expression of the gene encoding galectin-9 in human regulatory T cells (n=12). 1) 8, D1, D2, A1, A2 represent blood bags. CT represents cycle threshold. It is the average threshold from which galectin-9 gene amplification is detected. The results are expressed as ΔCT after normalization with four housekeeping genes (β-actin, glyceraldehyde phosphate dehydrogenase, hypoxanthine guanine phosphoribosyltransferase, ubiquitin).

[0182] [ΔCT = CT (sample) - CT (average number of housekeeping genes)]

[0183] Figure 4 : Analysis by Western Blot of the expression of the three isoforms of galectin-9 in human regulatory T cells. HeLa cells were used as negative control.

[0184] Figure 5 : Analysis by qPCR of the expression of the gene encoding galectin-9 in conventional T lymphocytes during activation (n=4).

[0185] Figure 6 : Analysis by qPCR of the expression of the gene encoding galectin-9 in human regulatory T cells during activation (n=4).

[0186] Figure 7 : Analysis of the inhibition of the suppressive activity of regulatory T cells by anti-Gal9 1G3 antibody by analyzing the proliferation of PBMCs in the presence of irradiated C15, in the presence or absence of regulatory T cells, and in the presence or absence of 1G3 antibody at a concentration of 1 μg / ml.

[0187] Figure 8 : Analysis of the inhibition of Jurkat cell apoptosis by galectin-9.

[0188] Galectin 9S at a concentration of 1 μg / mL and antibodies (9M1 antibody (anti-galectin 9), 9S2-3 (anti-galectin 9), 1G3 (anti-galectin 9), anti-TIM3, 2E12 antibody (anti-galectin 9)) at a concentration of 5 μg / mL were pre-incubated for 1 hour.

[0189] Fig. 9: Analysis of the restoration of proliferation of cells activated ("A", by anti-CD3 and anti-CD28) and not activated (NA), after treatment with Galectin-9 (antibodies tested = ECA-42 (anti-Galectin-9), 1G3 (anti-Galectin-9), 2E2 (anti-TIM3) and 2E12 (anti-Galectin-9)). Control corresponds to the medium.

[0190] Fig.10 : Analysis of galectin-9 expression by flow cytometry (FACS).

[0191] A: Expression of Galectin-9 from regulatory T cells and freshly isolated CD4+ Tconv cells (n=2 donors).

[0192] B: Expression of Galectin-9 extracted from regulatory T cells and CD4+ Tconv cells 24 hours after activation (n=5 donors).

[0193] C: Expression of Galectin-9 extracted from regulatory T cells and CD4+ Tconv cells 48 hours after activation (n=5 donors).

[0194] D: Expression of Galectin-9 extracted from regulatory T cells and CD4+ Tconv cells 72 hours after activation (n=5 donors).

[0195] Fig.11 : Relative proliferation of Tconv lymphocytes in different conditions after 3 days of culture (cells activated or not, in the presence of galectin-9, and with 1G3 ("anti-X"), control isotype (IgG1), inhibitor (lactose) and reference inhibitor (sucrose)).

[0196] Fig.12 : Proliferation of PBMCs under different conditions (activated or non-activated cells, in the presence of 1G3 or control isotype (IgG1)) after 5 days of culture.

[0197] Fig.13 : Viability of PBMCs under different conditions (activated or unactivated cells, presence of 1G3 or control isotype (IgG1)) after 5 days of culture.

[0198] Fig.14 : Measurement of Galectin-9 secretion by conventional CD4+ T cells ("Tconv") and regulatory T cells ("Treg") under non-activated and activated conditions (assays performed after 48 hours of culture).

[0199] Fig.15: Relative proliferation of conventional T lymphocytes by co-culturing with autologous regulatory T cells under different conditions (cell activation, presence of 1G3 ("anti-X"), control isotype (IgG1), inhibitor (lactose) and / or reference inhibitor (sucrose)).

[0200] Fig.16 : Relative proliferation of PBMCs in the presence of different concentrations of 1G3 (1μ / mL, 3μ / mL, 5μ / mL).

[0201] Fig.17 : Tumor volume under different treatments: PBMC alone (control); PBMC+Treg+1G3 (activated); PBMC+Treg+IgG1 (activated); without PBMC+1G3 (activated); without PBMC+IgG1 (activated).

[0202] Fig.18 : Graphical representation of mouse body weight according to day.

[0203] A and B: Xenografted SCID mice (50.10 6 Separate experiments were performed on PBMC+6% Treg) and treated with 1G3 or control isotype IgG1 (20 μg / mouse).

[0204] Fig.19 : Graphical representation of mouse body weight according to day.

[0205] A: Xenografted SCID mice (20.10 6 PBMC+10% Treg) and treated with 1G3 or control isotype IgG1 (2 μg / mouse).

[0206] B: Xenografted SCID mice (50.10 6 PBMC+6% Treg) and treated with 1G3 or control isotype IgG1 (20 μg / mouse).

[0207] C: Xenografted SCID mice (50.10 6 PBMC+6% Treg) and treated with 1G3 or control isotype IgG1 (20 μg / mouse).

[0208] D: Xenografted SCID mice (50.10 6 PBMC+6% Treg) and treated with 1G3 or control isotype IgG1 (200 μg / mouse).

[0209] Fig. 20 : Tumor volume (mm) measured manually in 6 independent experiments 3) average (20 micrograms of 1G3 per mouse, 50.10 6 PBMC + 6 to 8% Tregs compared to control isotype IgG1) (manual measurements). (*p<=0.05, **p<=0.001).

[0210] Fig.21 : Measurements of tumor volume measured by bioluminescence.

[0211] A: Xenografted SCID mice (50.10 6 PBMC+6% Treg) and treated with 1G3 or control isotype IgG1 (2 μg / mouse).

[0212] B: Xenografted SCID mice (50.10 6 PBMC+6% Treg) and treated with 1G3 or control isotype IgG1 (20 μg / mouse).

[0213] C: Xenografted SCID mice (40.10 6 PBMC+6% Treg) and treated with 1G3 or control isotype IgG1 (20 μg / mouse).

[0214] D: Xenografted SCID mice (50.10 6 PBMC+6% Treg) and treated with 1G3 or control isotype IgG1 (200 μg / mouse).

[0215] Fig. 22 : Mean values ​​of tumor volumes measured by bioluminescence in 6 independent experiments (20 μg of 1G3 or IgG1 per mouse, 50.10 6 PBMC+6% to 8% Treg) (measurement of total flux) (*p<=0.05, **p<=0.001).

[0216] Fig.23 : Tumor mass in grams measured in 6 independent experiments (20 μg of 1G3 or IgG1 per mouse, 50.10 6 PBMC+6% to 8% Treg). (*p<=0.05, **p<=0.001).

[0217] Fig.24: Comparative cytometry analysis of the expression of different markers after 5 days of conditioning. A: Expression of CD4 between activated Tconv (light grey), Tconv activated with Gal-9M (medium grey) or Tconv activated with Gal-9-S (black). B: Expression of CD4 between activated Tconv (light grey), Tconv activated with Gal-9M (medium grey) or Tconv activated with Gal-9-S (black). C: Expression of CD4 between activated Tconv (light grey), Tconv activated with Gal-9M (medium grey) or Tconv activated with Gal-9-S (black).

[0218] Fig.25 : Relative proliferation of PBMCs under different conditions after 3 days of culture with Tconv treated with M or S type Gal-9 for 5 days (*p<=0.05, **p<=0.001).

[0219] Fig.26 : Analysis of the inhibition of the suppressive activity of regulatory T cells by anti-Gal9 antibody 1G3 and anti-Gal9 antibody 2E12 (at a concentration of 1 μg / ml) by analyzing the proliferation of PBMCs co-cultured with regulatory T cells.

[0220] Examples

[0221] 1. Equipment and Methods

[0222] Donors, cell lines, and culture conditions

[0223] Donor cells

[0224] Healthy donor cells were isolated from blood from the French National Blood Service (EFS) in accordance with the official ethical agreement between the EFS and the CNRS. The study was approved by the Pasteur Institute of Lille (Centre Nationale Scientifique) and the CNRS committee, and each donor had signed an explained consent in advance.

[0225] NPC cell lines

[0226] The C15 tumor cell line is derived from EBV-positive NPC (nasopharyngeal carcinoma) serially xenografted and propagated subcutaneously in mice every 6-7 weeks. All animal experiments were performed by qualified personnel in accordance with French and European regulations at the facility of the Pasteur Institute in Lille (France). C15 cells were recovered from xenografted tumors and then irradiated (5000 rads) before pre-cultivation with immune cells that stimulate the tumor environment.

[0227] Jurkat human T lymphocyte cell line

[0228] The Jurkat cell line was established from human T lymphocyte leukemia. Its phenotype is CD4+ lymphocyte.

[0229] Cell culture conditions

[0230] The standard culture medium used was RPMI 1640 (Invitrogen, Paesdale, UK) supplemented with 10% AB human serum (BioWest, Nielles, France), 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM non-essential amino acids, 10 mM hydroxyethylpiperazineethanesulfonic acid, 50 μ / mL streptomycin, 50 μg / mL gentamicin and 50 μM beta-mercaptoethanol. Cells were cultured at 37°C in a Hera cell 150 incubator (Thermo Electron, Cergy-Pontoise, France) in a controlled atmosphere (5% CO2 and 95% humidity). Where applicable, PBMCs and CD4+ T cells were activated by anti-CD3 antibodies (1 μg / ml) (Clinisciences, Montrouge, France), which were fixed to the plates after incubation for 2 h at 37°C and soluble antibodies against CD28 (100 ng / ml) (Clinisciences) were added extemporaneously before culture.

[0231] Isolation of human immune cells

[0232] Isolation of PBMCs

[0233] Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by standard density gradient centrifugation using Ficoll Paque PLUS (Amersham Biosciences, Uppsala, Sweden).

[0234] Isolation of CD4+ T cells

[0235] CD4+ T cells were isolated from PBMC using a negative selection protocol according to the manufacturer's instructions (Miltenyi Biotec, Berlin, Germany). Briefly, PBMC were incubated for 10 minutes with a cocktail of biotinylated antibodies against CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRγ / δ, and glycophorin A. Anti-biotin magnetic beads were then added for 15 minutes. The cells to be eliminated were magnetically retained in a Magnetic activated cell separation columns in separators The cells to be separated pass through the column and are then collected and enriched for label-free cells with little to no non-target cells. Flow cytometric analysis showed that more than 98% of the separated cells were CD4+ cells.

[0236] Isolation of regulatory T cells

[0237] Human regulatory T cells were isolated from adult donor PBMC using a kit for the isolation of CD4+CD25+ regulatory T cells (Miltenyi Biotech, Germany) according to the manufacturer's instructions. The CD4+CD25- T cell fraction was retained for flow cytometry experiments and chemotaxis experiments. Flow cytometric analysis showed that the CD4+CD25+ fraction was consistently enriched for more than 95%.

[0238] Cell proliferation assay

[0239] During the last 18 hours of the culture period, the cells were incubated with [3H-methyl]thymidine at 1.10 5Cells (PBMC or CD4+T cells) were collected on glass fiber filters (Printed Filtermat A, Wallac, Turku, Finland) using a Tomtec collector (Wallac). After drying and adding scintillation fluid (Beckman Coulter, USA), the filters were sealed in bags. Proliferation was measured after culturing in the presence of [3H] thymidine (1 μCi / well) (PerkinElmer, Courtaboeuf, France) for the last 18 hours before collection. Radioactivity was measured using a β-meter (1450Trilux, Wallac, Finland). Each proliferation experiment was performed in three instances and estimated by counts per minute (cpm). Proliferation assays were performed in the presence of 1 μg / mL of the short form of recombinant galectin-9 (Gal9S) provided by Dr. Toshiro Niki (Galpharma, Japan), 1 μg / mL or a range of anti-galectin-9 antibody 1G3, an irrelevant anti-IGg1 antibody used as a negative control (eBioscience, UK), 10 μg / mL of C15 exosomes, and 5 mM lactose or sucrose (Sigma Aldrich), depending on the experiment.

[0240] Cell lysis

[0241] The cell lysis measurement technique is based on the use of a cytotoxicity assay kit (CytoTox-Glo assay, Promega, USA) which measures the activity of luciferase proportional to the cellular proteases released after cell lysis. 5 CD4+CD25+ and 2.10 5 The test was performed on autologous PBMC. The cells were cultured in 200 μl of culture medium (RPMI-1640, 1% 2 mM L-glutamine, 0.02 mM sodium pyruvate, 100 μ / ml penicillin, 100 μg / ml streptomycin, 10% decomplemented AB human serum) (GIBCO BRLTM, The cells were cultured in round-bottom 96-well plates (Maxisorb Nunc, Denmark) in a 24-well plate (UK). The cells were activated with 1 μg / ml of anti-CD3 and 100 ng / ml of anti-CD28 coated on the plate in advance (2 hours at 37°C). After 48 hours of culture, 50 μl of reagent (amino luciferin-Glo) was precipitated in each well. After slight stirring, the culture plate was incubated in the dark for 15 minutes at room temperature. The first luminescence measurement was measured with a photometer (Centro LB960, C Berthold Technologies, France) and was proportional to the number of cells that were lysed by regulatory T cells. Then, 50 μl of digitonin solution was precipitated in each well to cause all cells to dissolve. Then, before the second luminescence detection, the plate was stirred and incubated in the dark for 15 minutes at room temperature. Triple tests were performed and the results were expressed as dissolution ratios.

[0242] Lysis ratio = cell viability / average of total lysis - background noise

[0243] Cell viability = average total lysis - cell lysis

[0244] Cell lysis = average number of lysis caused by Treg lymphocytes - background noise

[0245] Induction test of apoptosis in Jurket cells

[0246] Jurkat cells cultured in 5% fetal bovine serum in RPMI were transferred to serum-free culture dishes (Hybridoma SFM - Life Technologies) and then incubated in the presence of 30 nM galectin-9 for 24 h in one well of a 96-well plate (100,000 cells / well). Apoptotic cells were counted by flow cytometry after labeling with V-APC annexin (allophycocyanin) and propidium iodide. To evaluate the protective effect of the monoclonal antibody, galectin was pre-incubated in the presence of the antibody for 30 min, and after 24-h incubation, its final concentration was 10 μg / ml.

[0247] Western Blot

[0248] Exosomes were lysed (10 min, paused) in PY buffer consisting 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 eliminated and the supernatant was collected. Protein concentration was determined using the Bio Rad protein concentration assay according to the manufacturer's instructions (BioRad, Marnesla Coquette, France). Exosomes were then analyzed by Western blotting. Briefly, proteins were separated by SDS PAGE electrophoresis under standard conditions using precast gels according to a gradient (gradient 4, 12%, Bis Tris, Invitrogen). Proteins were then transferred to nitrocellulose membranes (Hybon dTM-C Extra, Amersham Biosciences, UK). The nitrocellulose membrane was blocked for 1 hour at room temperature in a blocking buffer containing 0.2% AuroraTN blocking solution (MP Biomedicals, Illkirch Graffenstaden, France), 0.1% Tween 20 (Sigma Aldrich) and PBS (1X), and then incubated overnight at 4°C with a primary antibody against Galectin-9: Galectin-9-CT-L1 1:100 (provided by Galpharma, Japan). The membrane was rinsed with blocking buffer and then incubated with a secondary antibody conjugated to peroxidase (anti-mouse, 1:10000) (General Electric Healthcare, Wauwatosa, USA) for 1 hour at room temperature and rinsed again with blocking buffer. The membrane was purified by Western Light Ni Plus ECL and LAS3000 luminescence image analyzer (Fuji Film) showed protein-specific signals. Plus ECL is an amplification matrix chemiluminescence kit (PerkinElmer, Boston, MA, USA).

[0249] FACS analysis

[0250] Cell immunophenotype was performed by flow cytometry using the instrument "FACS Calibur flow cytometer". After being collected, the cells were rinsed with "phosphate buffered saline" (PBS) (GIBCO-Life Technologies) and labeled with a monoclonal antibody (1: 10) conjugated to a fluorescent agent. For each test, the marker was adjusted using an appropriate control isotype (monoclonal antibody). Finally, the data was analyzed using FlowJo software. In order to detect the surface antigens of the cells, anti-human mouse antibodies were used according to the manufacturer's instructions: CD4-phycoerythrin (PE)-anthocyanin (Cy) 5 (BD Pharmingen, San Diego, USA), -CD25-PE (Miltenyi Biotech, Germany) and -CD127-FITC (1: 20) (Clinisciences, Montrouge, France).

[0251] Real-time quantitative PCR

[0252] Total RNA from regulatory T cells was isolated using the "RNeasy Minikit II" kit (Qiagen) according to the manufacturer's instructions. The concentration and purity of RNA were determined spectrophotometrically (Ultrospec 3000, Pharmacia Biotec). Total RNA was stored at -80°C before subsequent use.

[0253] Reverse transcription of mRNA was performed as follows: 2 μg of total RNA was mixed with 5 μl of a master mix consisting of 1 μl of dT (Roche Diagnostic, Meylan, France) and 0.1 μl of RNase inhibitor (40 U / μl, Promega, Charbonnieres, France), and then incubated at 70°C for 5 to 10 minutes. After 5 minutes at room temperature, 10 μl of a second mix was added: 6 μl of 5X buffer (Invitrogen) + 1 μl of 0.1 M DTT (Invitrogen) + 2 μl of 10 mM dNTP (Amersham) + 0.1 μl of RNase inhibitor 40 U / μl (Promega) + 1 μl of Super Reverse Transcriptase (Invitrogen). After the reaction, a primary incubation was performed at 45°C for 45 to 60 minutes, a secondary incubation was performed at 95°C for 5 minutes, and then a 20-minute treatment with RNase H (Promega). Finally, ultrapure distilled water (GIBCO-Life Technologies) was added to give a final concentration of 10 ng total DNA / μl. The DNA was stored at -20°C until subsequent use.

[0254] Transcription was quantified by real-time PCR (RT-PCR) in 96-well optical plates (Eurogentec S.A., Belgium) using the Mx3005PTM sequence monitoring system (Agilent Technologies, France). In each well, 10 μl of a specific primer set designed for RT-PCR purchased from MWG-Biotech (Germany) was treated at a final concentration of 10 μg / ml and then stored at -20°C. β-actin, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), ubiquitin, and hypoxanthine guanine phosphoribosyltransferase (HPRT) housekeeping genes were used as controls in each plate. PCR reactions were performed in a final volume of 20 μl against 1 μl of cDNA (equivalent to 10 ng of total RNA / μl) according to the manufacturer's instructions using 258 assay (Eurogentech) was performed using 2X MESA GREEN qPCR MasterMix Plus, which contained Meteor Taq DNA polymerase, magnesium chloride (MgCl2) (final concentration of 4 mM), dNTPs (containing dUTP), 260Green I, stabilizers and passive references required for signal normalization, and buffers for optimized components.

[0255] The PCR program resulted in an initial denaturation and activation of Meteor Taq at 95°C for 5 minutes, 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 end of each cycle. Fusion curves were analyzed immediately after amplification according to the manufacturer's instructions.

[0256] Quantitative PCR reactions were used to quantify the amount of galectin-9 gene expressed by regulatory T cells. β-actin, G3PDH, ubiquitin, and hypoxanthine guanine phosphoribosyltransferase (HPRT) housekeeping genes were used as controls. All primers were designed for RT-PCR and purchased from MWG-Biotech (Germany). Quantitative analysis was performed based on the cycle threshold (CT) of each well and was calculated using MxPro software. ΔCT was calculated according to standard methods: ΔCT = C T -C T管家基因 , each value was normalized using the mean of the four housekeeping genes. For comparisons between groups, the relative expression of genes was given an arbitrary value of 1 for the reference sample. express.

[0257] Production of hybridomas producing monoclonal antibodies against galectin-9, 1G3 and 2E12

[0258] A recombinant protein representing the C-terminal portion of human galectin-9 was used as the 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 size exclusion chromatography.

[0259] Immunization was performed by the company PX Therapeutics (Grenoble, France). Five eight-week-old female BALB-c mice were immunized with the C-terminal portion of Galectin-9 mentioned above. For immunization, 40 micrograms of protein were injected intraperitoneally on days 0, 22, 37 and 54, either in combination with complete Freund's adjuvant for the first injection or with incomplete Freund's adjuvant for the subsequent injections. The quality of the immunization was evaluated by the ELISA test described below on serum samples from the immunized mice. The same formulation of recombinant C-terminal Galectin-9 was used for immunization on the one hand and for the ELISA test on the other hand. These tests showed that the five treated mice were well immunized.

[0260] Three days after the last repetition, the two mice showing the best response were sacrificed and their spleen cells were obtained. These spleen cells were used to fuse with Sp2 / O mouse myeloma cells in liquid medium or in semi-solid medium at a ratio of 5:1 and 2:1, respectively. The hybridoma supernatants were then evaluated by ELISA assays against the recombinant galectin-9 preparations mentioned above, which were performed as described above and below.

[0261] Semi-solid fusion was successful, resulting in 39 hybridomas that secreted antibodies that reacted with galectin-9 in ELISA. Seven of them were selected because of particularly abundant immunoglobulin secretion and high activity in ELISA. These seven hybridomas were then subjected to a new functional screening to investigate the anti-galectin-9 neutralizing properties of the generated antibodies.

[0262] The ELISA assay was performed as follows. The recombinant protein representing the C-terminal part of galectin-9 was adsorbed in the wells of a 96-well microtiter plate (50 ng / well) (Greiner Bio-One, Courtaboeuf, France) in the following fusion process: dissolved in 0.05 M carbonate / bicarbonate buffer, pH 9.6, and then incubated in the wells for 1 mouse at room temperature. After washing with PBS containing 0.1% Tween-20, the wells were immersed in 3% bovine serum albumin (BSA) dissolved in PBS for 1 hour at room temperature. They were then incubated with the mouse serum or hybridoma supernatant to be tested. The serum and hybridoma supernatant were diluted with 1% BSA in PBS and then incubated in the wells for 2 hours at room temperature. After a washing step with 0.1% PBS-Tween-20, the plate was treated with a secondary antibody (anti-mouse and sheep) labeled with peroxidase. Final revelation occurred after addition of substrate (3,3',5,5'-tetramethylbenzidine or TMB; Thermo Fisher Scientific) and measurement of absorbance at 405 and 620 nm on a Multiskan Ex microplate reader (Thermo Fisher Scientific).

[0263] In vivo studies in transgenic mice

[0264] Immunodeficient mice were first spleened and then xenografted with the C666-1 cell line modified from CNP to express luciferase, which allowed monitoring of tumor growth during bioluminescent imaging in awake animals. The murine immune system was reconstituted and humanized after the injection of human PBMCs, which were more or less enriched in regulatory T cells (2% of regulatory T cells in the original PBMCs and 6% to 10% of regulatory T cells added to the PBMCs). It has been shown that PBMCs are able to limit tumor growth to a certain extent and that enrichment of regulatory T cells has no effect on this effect (Moralès et al., "Parvovirus activation of helper but not regulatory human CD4+ T cell responses by oncolytic H-1"; PLoS One. 2012; 7(2): e32197). The 1G3 antibody against Gal-9 was then injected and its effect on tumor growth was evaluated.

[0265] Then the following experimental scheme:

[0266] SCID mice aged 6 to 8 weeks were subjected to complete splenectomy. After 7 days, these mice were subjected to subcutaneous xenografting by cells (expressing luciferase) of the C666-luc tumor line produced by nasopharyngeal carcinoma (NPC). On the same day, mice received 30 to 50 million PBMCs in the peritoneal cavity to reconstruct the immune system, enriched or not enriched with 10% regulatory T cells, and, according to the animal, received 2 micrograms, 20 micrograms or 200 micrograms of IgG1 antibodies or 1G3 antibodies (3 mice / group) subcutaneously according to the following scheme:

[0267] - Group 1: IgG1 isotype (non-recombinant)

[0268] - Group 2: 1G3 (unrecombinant)

[0269] - Group 3: Recombinant + 10% Treg + IgG1 isotype

[0270] - Group 4: Recombinant (untreated)

[0271] - Group 5: Recombinant + 10% Treg + 1G3

[0272] All mice received 3 μg of CPG-ODN2216 (labeled by Miltenyi Biotech) to stimulate the immune response.

[0273] Within four weeks, the small animal imaging platform ( Lumina XRMS, PerkinElmer) and manual measurements of the volume and mass of the tumor were monitored three times a week. On the 7th, 14th and 21st days after transplantation, mice received repeated CPG-ODN2216 (labeled with Miltenyi Biotech) and, according to the groups shown above, repeated 1G3 or IgG1 antibodies. Before the first repetition, blood samples were taken to verify the reconstruction by flow cytometry (see treatment plan page 58). After 28 days of measurement, mice were sacrificed, tumors recovered and stored at low temperatures in order to prepare immunohistochemical plates or immunofluorescence plates, and blood samples were taken again for analysis by flow cytometry.

[0274] The effects of different concentrations [2 μg / mL, 20 μg / mL, and 200 μg / mL] of 1G3 and IgG1 isotype on (i) mouse body weight, (ii) tumor volume, and (iii) tumor mass at sacrifice were evaluated.

[0275] (i) Analysis of the effect of 1G3 antibody injection on mouse body weight

[0276] The recipient mice were weighed regularly [from day 5 to day 28].

[0277] (ii) Analysis of the effect of 1G3 on tumor volume (manual measurement)

[0278] Several independent experiments were performed in humanized SCID mice following the treatment protocol described above and varying the concentrations of 1G3 and 1G1 injected. Tumor volumes were measured manually.

[0279] In addition, to generate graphs showing the effect of 1G3 on tumor volume, the average results of 6 independent in vivo experiments were generated with 1G3 antibody or control isotype (20 μg).

[0280] Statistical analysis was performed by Rank Sum Mann Whitney test (*p<=0.05, **p<=0.001).

[0281] Tumor volume was also measured by analyzing bioluminescence emitted by tumor cells expressing the gene for luciferase after injection of luciferin. Bioluminescence was measured using a mouse bioluminescence measurement system (IVISLUMINA). Six independent experiments were performed on humanized SCID mice following the treatment regimen described above and varying the concentrations of injected 1G3 and 1G1.

[0282] In addition, to generate graphs showing the effect of 1G3 on tumor volume, the average results of 6 independent in vivo experiments were generated with 1G3 antibody or control isotype (20 μg).

[0283] Finally, tumors from mice treated with 1G3 or control isotype IgG1 were photographed on millimeter paper to measure tumor size at the time of euthanasia.

[0284] (iii) Analysis of the effect of 1G3 on tumor mass

[0285] After sacrifice, tumors in mice treated with 1G3 or control isotype IgG1 were weighed. Mass is expressed in grams.

[0286] Statistical analysis was performed by Rank Sum Mann Whitney test (*p<=0.05, **p<=0.001) ( Fig. 22 ).

[0287] Analysis of the ability of 1G3 to inhibit the induction of regulatory T cells by galectin-9

[0288] It has been shown that galectin-9 is able to induce the differentiation of naive CD4 T cells into Treg lymphocytes (Seki, Oomizu et al., 2008), thereby increasing the importance of this lectin in the phenomenon of exhaustion of antitumor immune responses.

[0289] First, we examined whether Galectin-9 could convert Tconv cells into regulatory T cells. Tconv cells were cultured for 3 or 5 days in the presence or absence of Galectin-9 isoforms (Gal-9S or Gal-9M) under activating or non-activating conditions, with or without 1G3. After these conditioning phases, cells were recovered, washed, and analyzed by flow cytometry, and co-cultured with PBMCs or autologous Tconv cells. For ELISA analysis, the culture medium was also recovered.

[0290] The treatment regimen was as follows: PBMCs were isolated by purification according to a Ficoll gradient, and conventional T lymphocytes were isolated by a magnesium column according to the treatment regimen of the supplier (Miltenyi Biotech, T cell isolation kit). Some PBMCs were retained under non-activated conditions, and autologous Tconv cells were cultured for 5 days under activated and non-activated conditions. Activation was performed by anti-CD3 (1 μg / ml) (provided by Anne Tsicopoulos team, CIIL) and anti-CD28 (100 nm / ml) (Clinisciences, France) coated on the plate in advance (2 hours, at 37°C).

[0291] These T cells were conditioned or not conditioned with 2 μg / ml of Gal-9 S or M isoforms in the presence or absence of 3 μg / ml 1G3 antibody. After 5 days, conditioned Tconv were washed, viability was determined by counting with trypan blue* (see below), supernatants were recovered for ELISA and analyzed by flow cytometry for 10 5 The remaining conventional cells were contacted with autologous PBMCs reserved for the MLR experiment.

[0292] By co-cultivating 10 5 The adjusted Tconv and 10 5 The inhibition test was performed by MLR (mixed lymphocyte reaction) with or without 1G3 or 2E12 antibodies, using autologous PBMC (ratio 1:1). The cells were activated with 1 μg / ml anti-CD3 and anti-CD28 (100 ng / ml) pre-coated on the plates (2 hours at 37°C).

[0293] After 3 days of co-culture, 18 hours before the end of the culture, 1 μCi / well of tritiated thymidine ( 3Proliferation was assessed using H Th (GE Healthcare, France). After 3 days, the plates were filtered using glass fiber filters (PerkinElmer, France). The filters were incubated in scintillation fluid (Beckman Instruments, Ready Safe, USA) and read by a scintillation counter (1450 Trilux, Wallac, Finland). Results were finally expressed in counts per minute (cpm). Statistical analysis was performed by Rank Sum Mann Whitney test (*p <= 0.05, **p <= 0.001).

[0294] Counting with trypan blue

[0295] The vital azo dye (Trypan blue) is used to stain dead cells. The counts are assessed by adding one volume of 0.4% Trypan blue solution (Sigma, USA) to one volume of the cell suspension. After 3 minutes, the mixture is precipitated on a Thomas plate. The number of blue dead cells and refracting live cells is counted. The measurements are performed in triplicate and the results are expressed as a percentage of live cells.

[0296] Measurement of Galectin-9 secretion by ELISA

[0297] 50 μl of a 0.2 μg / ml solution of anti-Gal-9 antibody (clone: ​​SEA309Hu-Uscn Life Sciences, USA) was fixed in a 96-well plate (NUNC, Denmark) overnight at 4°C. After washing four times with 0.05% 1X PBS (Euromedex, France)-Tween (Sigma Aldrich, USA), the plate was immersed in 3% PBS-BSA (Sigma Aldrich, USA) at room temperature. USA) for 2 hours. Then rinse it 3 times with 0.05% PBS-Tween. In the case of activation or non-activation, the culture supernatant of Treg lymphocytes of 100 μl / well was precipitated in duplicate and incubated for 2 hours at room temperature. Treg lymphocytes are usually activated by anti-CD3 (1 μg / ml) (provided by Anne Tsicopoulos team, CIIL) and anti-CD28 (100 ng / ml (Clinisciences, France) coated on the plate in advance (2 hours, at 37°C).

[0298] On each plate, a range of recombinant galectin-9 (Uscn Life Sciences, USA) was performed at 2.5 ng / ml in 1% PBS-BSA.

[0299] After washing twice with PBS-Tween, 100 μl / well of biotinylated secondary antibody at a concentration of 1 μg / ml was incubated for 1 hour at room temperature. The plate was washed three more times and the reaction was amplified by adding 100 μl / well of streptavidin-peroxidase at a ratio of 1 / 10000 for 30 minutes at room temperature. After washing four times, the plate was incubated with 100 μl / well of OPD (o-phenylenediamine dihydrochloride) (Sigma-Aldrich) containing 1 mg / ml. The plate was displayed for 10 minutes in the dark with 100 μl / well of a developing solution of VWR (USA). The reaction was terminated by adding 50 μl / well of 2N hydrochloric acid (VWR, USA).

[0300] The plates were then read using a spectrophotometer at a wavelength of 492 nm (Multiskan Ex, ThermoLabsystems, France).

[0301] Determination of PBMC viability

[0302] Viability assays were performed directly using the CellTiter-Glo assay, which allows for the measurement of mitochondrial metabolism. The same treatment regimen was used to assess the viability of Tconv lymphocytes.

[0303] 2.10 Precipitation by Co-culture 5 Autologous PBMCs were activated or not according to the conditions [activated by anti-CD3 (1 μg / ml) (provided by Anne Tsicopoulos team, CIIL) and anti-CD28 (100 ng / ml) (Clinisciences, France) coated on the plate in advance (2 hours at 37°C)] and incubated with 1G3 antibody or control isotype 1G1 at a concentration of 3 μg / ml, 6 μg / ml or 12 μg / ml. The cells were cultured in 100 μl of culture medium (DMEM + 4.5 g / l glucose + L-glutamine, 100 U / mmol penicillin, 100 μg / ml streptomycin, 10% decomplemented human AB serum) (GibcoBRLTM, Life Technologies, UK) in a flat-bottom 96-well plate with transparent "wells" (Corning 3610, Corning, USA).

[0304] The test uses the Promega CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, USA), which uses luciferase activity to measure cell metabolism (ATP) as a cell viability indicator under aerobic conditions. After 48 hours of culture, 100 microliters of reagents (luciferin, luciferase, and magnesium-containing buffer) are added to each well; the plate is agitated for 2 minutes and then incubated at room temperature in the dark for 15 minutes. The test is performed in triplicate and the plate is read using a luminometer (Centro LB960, C Berthold Technologies, France). Read at a speed of 1 second / well.

[0305] Membrane labeling of Tconv lymphocytes by flow cytometry (FACS)

[0306] Antibodies used (Ac)

[0307] Anti-human monoclonal primary antibodies conjugated with fluorescent dyes, anti-CD4-PE (phycoerythrin)-C (cyanine) 5 (BD Pharmingen TM, USA), anti-CD25-PE (Miltenyi Biotech, France), anti-CD127-FITC (fluorescein isothiocyanate) (Clinisciences, France).

[0308] As compensation, control isotypes of different monoclonal antibodies were used.

[0309] Direct Marking Protocol

[0310] In a volume of 100 μl sterile PBS (GIBCO BRLTM, UK) cells (2.10 5 ), and in 10 μl anti-CD4-PC, 10 μl anti-CD25-PE and 4 μl anti-CD127-FITC, the cells were incubated for 30 minutes at room temperature and in the dark. 400 μl PBS was then absorbed with the labeled cells and analyzed for fluorescence by flow cytometry using Facscalibur (FACS flow supply system, Becton Dickinson, USA).

[0311] Blood cell counting results were analyzed by Flow Jo software (Tree Star, USA). For some experiments, after adding labeled antibodies, cells were fixed by adding 100 μl 4% FBA (Sigma-Aldrich, USA) for 10 min and then absorbed by 200 μl PBS before analysis by blood cytometer.

[0312] Proliferation and suppression (MLR) (2E12)

[0313] Growth test

[0314] In a 96-well round-bottom plate (Nunc, Denmark), cells were grown in 200 μl of culture medium (RPMI-1640, 1% 2 mM L-glutamine, 0.02 mM sodium pyruvate, 100 U / mmol penicillin, 100 μg / ml streptomycin, 10% decomplemented human AB serum) (GibcoBRL TM , Invitrogen, UK) were used to culture 10 5 PBMCs were used for proliferation experiments. The cells were activated by anti-CD3 (1 μg / ml) (provided by Ann Tsicopoulos team, CIIL) and anti-CD28 (100 ng / ml) (Clinisciences, France) coated on the plates in advance (2 hours at 37°C) in the presence or absence of 1G3 or 2E12 antibodies.

[0315] inhibition

[0316] By co-cultivating 6.10 4 Treg and 10 5 The inhibition test was performed by MLR (mixed lymphocyte reaction) with or without 1G3 or 2E12 antibodies using autologous PBMCs. The cells were activated with 1 μg / ml anti-CD3 and anti-CD28 (100 ng / ml) pre-coated on the plates (2 hours at 37°C).

[0317] 18 hours before the end of the culture, 1 uCi / well of tritiated thymidine ( 3 Proliferation was assessed using H Th (GE Healthcare, France). After 48 hours, the plates were filtered using glass fiber filters (PerkinElmer, France). The filters were incubated in scintillation fluid (Beckman Instruments, Ready Safe, USA) and read by a scintillation counter (1450 Trilux, Wallac, Finland). Finally, the results were expressed in counts per minute (cpm).

[0318] result

[0319] Phenotypic analysis of PBMCs and regulatory T cells

[0320] Analysis of PBMCs by flow cytometry (FACS) showed that regulatory T cells CD4+CD25+CD127- accounted for 1% of total PBMCs (results not shown).

[0321] In addition, FACS analysis of phenotypic markers of autologous regulatory T cells isolated in vitro showed that 95% of regulatory T cells were CD4+CD25+, and among these cells, 90% were CD127; or CD127 was less, and FoxP3+ accounted for more than 86% ( Figure 1 ).

[0322] Activated regulatory T cells have suppressive activity

[0323] The suppressive activity of human Tregs isolated in vitro from healthy donor blood was characterized by two functional assays: an assay for the inhibition of proliferation of PBMCs activated by autologous Tregs and an assay for the cytolysis of PBMCs activated by autologous Tregs ( Figure 2 ).

[0324] Figure 2 A shows that activated PBMCs proliferate well alone in vivo, whereas isolated regulatory T cells in vitro are not immunogenic, even after activation. However, in the presence of activated autologous regulatory T cells at a ratio of 4:2, activated PBMC proliferation was reduced by more than 24% (ref. Figure 2 A) The proliferation assay (MLR) thus clearly demonstrated that under activation conditions, regulatory T cells isolated in vitro have immunosuppressive activity.

[0325] The results of the cell lysis assays reinforced those obtained by the proliferation assays. Figure 2 As shown in B, the lower the PBMC: Treg ratio, the higher the lysis rate of activated PBMC. Tregs thus induce lysis of autologous PBMCs in a dose-dependent manner at different ratios.

[0326] Galectin-9 is distributed in and expressed by regulatory T cells

[0327] By real-time quantitative PCR ( Figure 3 ) and Western Blot( Figure 4 ) analysis showed that galectin-9 was distributed in human regulatory T cells isolated in vitro, and the human regulatory T cells expressed galectin-9, indicating that regulatory T cells use galectin-9 meridians to inhibit the proliferation of effector T lymphocytes.

[0328] When activated, the expression of galectin-9 by effector T lymphocytes is reduced; when activated, the expression of galectin-9 by regulatory T lymphocytes is increased. Increased expression of galectin-9

[0329] Fig.14It was shown that Tconv lymphocytes and unactivated regulatory T cells instead produced very small amounts of galectin-9 (<10 pg / ml: in other words, below the detection threshold of the ELISA test), and that activated human regulatory T cells were able to synthesize and secrete galectin-9 in the extracellular environment. In addition, this secretion of galectin-9 was significantly stronger when regulatory T cells were activated, linking this secretion function to its inhibitory function.

[0330] Figure 5 It showed that the ratio of conventional CD4+ T lymphocytes to 5 regulatory T cells was greatly reduced during activation.

[0331] Activated conventional CD4+ T lymphocytes express only a small amount of the gene encoding galectin-9, and this expression is greatly reduced during activation.

[0332] On the other hand, expression of the gene encoding galectin-9 by regulatory T cells increases during activation (ref. Figure 5 and Figure 6 ).

[0333] Therefore, the constitutive activation of T lymphocytes with anti-tumor effects is not a target of anti-Gal-9 antibodies. On the other hand, regulatory T cells that are activated and therefore functional are ideal targets of anti-Gal-9 antibodies.

[0334] The analysis of differential protein expression of galectin-9 in regulatory T cells and CD4+ T cells was enhanced by flow cytometry. Fig.10 As can be seen in the figure, the basal expression is low and almost identical between regulatory T cells and freshly isolated CD4+ T cells. However, it was found that after activation of the TCR (anti-CD3 / anti-CD28), the expression profile changed and a population that very strongly expressed galectin-9 appeared in regulatory T cells. This overexpression persisted and increased proportionally with time, while the basal expression of galectin-9 remained low in CD4+ Tconv cells, even after activation ( Fig.10 ).

[0335] It is thus shown that the overexpression of Galectin-9 is targeted to activated regulatory T cells and that the expression of Galectin-9 in CD4+Tconv cells after activation under basal conditions is very low. The risk of targeting effector CD4+T lymphocytes is thus eliminated, thereby enabling the patients concerned according to the present invention to maintain their immune function.

[0336] The suppressive activity of regulatory T cells is inhibited by the anti-galectin-9 1G3 antibody

[0337] To evaluate the effect of 1G3 antibody on the activity of regulatory T cells in vivo, a cell proliferation assay based on tritiated thymidine incorporation was performed.

[0338] Figure 7 Shown are the results of tests on PBMC proliferation in the presence of irradiated C15, in the presence or absence of regulatory T cells, and in the presence or absence of 1G3 antibody at a concentration of 1 μg / mL.

[0339] First, the positive controls of the experiment confirmed that activation of PBMCs indeed led to an increase in their proliferation and that the presence of regulatory T cells indeed led to a decrease in PBMC cell proliferation.

[0340] It was also shown that irradiation of C15 tumor cells indeed resulted in cessation of their proliferation. C15 cells are non-immune.

[0341] The presence of C15 tumor cells indeed caused a decrease in cell proliferation of human PBMCs.

[0342] It was then shown that the emergence of regulatory T cells during co-culture of PBMC and C15 resulted in a significant additional reduction in proliferation of approximately 56%.

[0343] Figure 7 It is clear that, unexpectedly, the presence of the 1G3 antibody enables the restoration of PBMC proliferation. It is therefore suggested that the 1G3 antibody neutralizes galectin-9 present on and expressed by regulatory T cells. It is therefore suggested that the 1G3 antibody inhibits the suppressive activity of regulatory T cells.

[0344] 1G3 anti-galectin-9 antibody has better efficacy than other anti-galectin-9 antibodies

[0345] In order to compare the effects of 1G3 antibody with other anti-galectin-9 antibodies and even anti-TIM3 antibodies on the inhibitory activity of regulatory T cells, several tests were performed.

[0346] Therefore, the effects of different anti-Galectin-9 antibodies on the inhibition of apoptosis caused by Galectin-9 and the effects of anti-Galectin-9 antibodies on the restoration of proliferation after treatment of human PBMCs with Galectin-9 were analyzed. In contrast to 1G3, the anti-Galectin-9 antibodies tested did not recognize the same epitope of Galectin-9 as the 1G3 antibody.

[0347] Antibodies against TIM3 were also tested because some theories have been proposed regarding the link between the Tim-3 receptor present on T lymphocytes and the pro-apoptotic effects of galectin-9.

[0348] Inhibits apoptosis induced by galectin-9

[0349] Figure 8 Results of testing the effects of the tested antibodies on apoptosis of Jurkat cells caused by recombinant galectin-9 are shown.

[0350] The antibodies tested were 9M1 (anti-galectin 9), 9S2-3 (anti-galectin 9), and 1G3 (anti-galectin 9) antibodies, and antibodies against TIM3, 2E12 (anti-galectin 9).

[0351] like Figure 8 As can be seen in the figure, 1G3 antibody prevents Jurkat cell apoptosis better than other anti-galectin-9 antibodies 9S2-3 and 9M1 or anti-TIM3 antibody.

[0352] Restoration of proliferation after treatment with galectin-9

[0353] Fig. 9 The results of testing the effects of the tested antibodies on the proliferation of human PBMCs previously treated with galectin-9 are shown.

[0354] The antibodies tested were ECA-42 (anti-Galectin 9), 1G3 (anti-Galectin 9), 2E2 (anti-TIM3), and 2E12 (anti-Galectin 9) antibodies.

[0355] like Fig. 9 As can be seen in the Figure, the proliferation of human PBMCs was restored more effectively by the 1G3 antibody than by other anti-galectin-9 antibodies ECA-42 and 2E12 or the anti-TIM3 antibody (2E2).

[0356] Effects on Tconv lymphocytes (T CD4+)

[0357] Fig.11 Results of an experiment on the effect of galectin-9 and 1G3 on Tconv lymphocytes (T CD4+) measured by incorporation of tritiated thymidine during the last 18 hours of culture, are presented. Results are also presented in counts per minute (cpm).

[0358] As PBMCs ( Fig. 9 ), Tconv lymphocytes isolated in vitro proliferated under activation conditions. It was also shown that galectin-9 significantly inhibited the proliferation of Tconv lymphocytes in the same manner as PBMCs.

[0359] Finally, it should be noted that in this Fig.11 In the experiment, anti-galectin-9 (1G3) antibody had no effect on the proliferation of Tconv lymphocytes.

[0360] Analysis of cytotoxic effects on PBMC proliferation and its control isotype

[0361] The effect of increasing doses of 1G3 antibody or its control isotype (IgG1) on PBMCs was analyzed by measuring proliferation of freshly isolated cells extracted from the blood of three donors by incorporation of tritiated thymidine during the last 18 hours of culture. Results are reported in CPM.

[0362] exist Fig.12 It was observed in the Figure 1 that PBMCs isolated in vitro proliferated in vivo under conditions of activation. It was also noted that the 1G3 antibody and its control isotype IgG1 did not affect the proliferation of PBMCs even at high doses of the monoclonal antibody (3 to 12 μg / ml).

[0363] Analysis of the cytotoxic effect of 1G3 antibody and its control isotype on PBMC viability

[0364] Dose-increasing doses of mAb1G3 and its control isoform (1G1) were analyzed by chemiluminescence analysis by measuring the viability of freshly isolated PBMCs from three donors. Mitochondrial metabolism was measured during the 5-day activation. Results are reported in RLU.

[0365] Fig.13 It was shown that PBMC isolated in vitro maintained and increased their in vivo viability under activation conditions. It was also shown that 1G3 and 1G1 isotypes did not alter the viability of PBMC even in the presence of high doses of antibody (3 to 12 μg / ml).

[0366] Therefore, it is shown that the 1G3 antibody does not affect the proliferation of PBMC and human T CD4+, nor does it affect their viability. Therefore, the risk of side effects such as inducing immunosuppression that is conducive to cancer progression is reduced. The effect can better maintain the immune function of the patient concerned according to the present invention, especially the anti-tumor immune function.

[0367] Analysis of the neutralization effect of 1G3 on the inhibitory activity of regulatory T cells

[0368] The potential of the 1G3 antibody to inhibit the suppressive function of regulatory T cells was analyzed by testing the proliferation of mixed leukocyte reactions of regulatory T cells with conventional T lymphocytes.

[0369] As expected, the results showed that regulatory T cells inhibited cell proliferation during co-culture ( Fig.15 ). Moreover, a 2-hour pre-incubation with 1G3 was sufficient to reverse this inhibitory effect. The use of a control isoform of 1G3 that has no effect on Treg lymphocytes made it possible to conclude that the reversal of the inhibitory effect produced by regulatory T cells is specific and is mediated by galectin 9.

[0370] To determine whether 1G3 inhibits Tregs by acting on soluble galectin-9 or directly on Tregs, an experiment was performed using an inhibitor that blocks soluble galectin-9 and the results were compared with those obtained with the 1G3 antibody.

[0371] The results clearly show that chemical inhibitors have no significant effect on the suppressive activity of regulatory T cells. The use of the 1G3 antibody again leads to a reversal of the suppressive effect induced by regulatory T cells. The results therefore confirm that galectin-9 acts on the suppressive effect induced by regulatory T cells and that 1G3 is able to reverse this suppressive effect.

[0372] Neutralization of suppressive activity of regulatory T cells in vivo

[0373] The effects of different concentrations [2 μg / mL, 20 μg / mL, and 200 μg / mL] of 1G3 and IgG1 isotype on (i) mouse body weight, (ii) tumor volume, and (iii) tumor mass at sacrifice were evaluated.

[0374] (i) Analysis of the effect of 1G3 antibody injection on mouse body weight

[0375] like Fig.18 As can be seen in panels A and B, mice treated with 1G3 were lighter than mice treated with the control isotype. This result suggests that the weight loss in treated mice may be related to tumor shrinkage.

[0376] (ii) Analysis of the effect of 1G3 on tumor volume (manual measurement)

[0377] Fig.19 The results shown in AD demonstrate a positive effect of 1G3, which significantly limited tumor growth compared to the control isotype.

[0378] also, Fig. 20 The results shown in clearly demonstrate a significant difference between the immunological effects of 1G3 and its control isotype, with tumor growth being greatly restricted by 1G3.

[0379] These results were confirmed by bioluminescence measurements ( Fig.21 AD, Fig. 22 ), 1G3 induced restricted tumor growth compared to its control isoform.

[0380] Finally, tumor photographs (not shown) also demonstrated the positive effect of 1G3, resulting in limited tumor growth compared to its control isotype.

[0381] (iii) Analysis of the effect of 1G3 on tumor size

[0382] Results regarding tumor mass ( Fig.23) in turn confirmed the beneficial effects of 1G3 compared to its control isotype.

[0383] Analysis of the ability of 1G3 to inhibit the induction of regulatory T cells by galectin-9

[0384] It has been shown that galectin-9 is able to induce the differentiation of naive CD4 T lymphocytes into Treg lymphocytes (Seki et al., Galectin-9 inhibits the generation of Th17, promotes the induction of regulatory T cells, and modulates experimental autoimmune arthritis; Clin Immunol, 2008 Apr;127(1):78-88. doi:10.1016 / j.clim.2008.01.006, epub 2006 Feb), thus increasing the importance of this lectin in the phenomenon of exhaustion of antitumor immune responses.

[0385] Fig.25 The results shown in clearly show that CD4+Tconv lymphocytes conditioned by each isoform of galectin-9 acquire a suppressive phenotype and inhibit PBMC proliferation. This inhibitory effect of the S (short) form of galectin-9 is stronger than that of the M (medium) form. It was also found that the addition of 1G3 during conditioning inhibited the establishment of this suppressive phenotype, and proliferation was significantly restored by neutralizing galectin-9.

[0386] In addition, if Fig.24 As shown in , FACS analysis of cells at the end of the experiment showed that each isoform of galectin-9 resulted in increased expression of CD25. Fig.24 As shown, activated cells normally overexpress CD25.

[0387] The results clearly showed that the addition of galectin-9 to Tconv lymphocytes (i) resulted in a small increase in the expression of CD4 by S-type or M-type galectin-9 compared to activated Tconv lymphocytes; (ii) had no effect on the expression of CD127 except at the stage regulated by M-type or S-type galectin-9; and (iii) resulted in a significant increase in the expression of CD25 by M-type and S-type galectin-9 compared to activated Tconv lymphocytes.

[0388] Fig.24 It is therefore suggested that pre-incubation of Tconv lymphocytes with M or S from galectin 9 increases the expression of CD4 and CD25 markers, possibly resulting in a more suppressive phenotype. However, this hypothesis can only be accepted if it is associated with the suppressive activity of these conditioned Tconv lymphocytes, which would be the case if it were able to validate the suppressive phenotype (ref. Fig.25 ).

[0389] In summary, it is shown that galectin-9 causes the conversion of conventional CD4+ T lymphocytes into immunosuppressive CD4+ T lymphocytes. The 1G3 antibody is able to abrogate the induction of this conversion and thus contribute to the maintenance of an anti-tumor immune response in the patient concerned according to the present invention.

[0390] Analysis of the neutralization effect of 2E12 on the suppressive activity of Treg lymphocytes

[0391] The potential of the suppressive function of regulatory T cells inhibited by the 2E12 antibody was analyzed by testing their proliferation in mixed leukocyte reactions with autologous PBMCs (2 independent donors).

[0392] Fig.26 The results shown in Figure 2 show that pre-incubation of regulatory T cells with 2E12 for 2 hours is sufficient to reverse the inhibitory effect of cell proliferation by regulatory T lymphocytes. The use of this control isotype of 2E12, which has no effect on regulatory T cells, allows us to conclude again that such reversal of the induced inhibition of regulatory T cells is specific and through galectin 9.

[0393] Furthermore, it was observed that the 2E12 antibody was able to reverse this inhibitory effect, just like 1G3. 2E12 was not nearly as potent as 1G3, but, unlike 1G3, a slight reduction in PBMC proliferation was observed.

[0394] References

[0395] (1)Grossman WJ, Verbsky JW, Barchet W, Colonna M, Atkinson JP, Ley TJ.< <Human T regulatory cells can use the perforin pathway to cause autologoustarget cell death”.Immunity.2004 Oct;21(4):589-601.

[0396] (2)Garin MI, Chu CC, Golshayan D, Cernuda-Morollón E, Wait R, Lechler RI. "Galectin-1: a key effector of regulation mediated by CD4+CD25+T cells". Blood. 2007 Mar 1; 109(5): 2058-65

[0397] (3)Johnson BD,Jing W,Orentas RJ.<<CD25+regulatory T cell inhibitionenhances vaccine-induced immunity to neuroblastoma”.J Immunother.2007 Feb-Mar;30(2):203-14.

[0398] (4)McHugh RS,Whitters MJ,Piccirillo CA,Young DA,Shevach EM,Collins M,Byrne MC.“CD4(+)CD25(+)immunoregulatory T cells:gene expression analysisreveals a functional role for the glucocorticoid-induced TNF receptor”.Immunity.2002 Feb;16(2):311-23.

[0399] (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.2008 Oct 10;322(5899):271-5.(5)Zahran AM et al,Int J Clin Oncol.2013Sep 26.

[0400] (6)Fisson S1,Darrasse-Jèze G,Litvinova E,Septier F,Klatzmann D,LiblauR,Salomon BL.Continuous activation of autoreactive CD4+CD25+regulatory Tcells in the steady state.J Exp Med.2003 Sep 1;198(5):737-46.Epub 2003 Aug25.

[0401] (7)Xu W et al,J Cancer Res Clin Oncol.2013 Nov;139(11):1845-52

[0402] (8)Ladanyi A,Magy Onkol.2013 Jun;57(2):85-95

[0403] (9)Zhang W et al,Gynecol Oncol.2014 Jan 2.pii:S0090-8258(13)01427-3.

[0404] (10)Faghih Z et al,Immunol Lett,2013 Dec 8;158(1-2):57-65.

[0405] (11)Aida K et al,Cancer Sci.2013 Nov 30

[0406] (12)Huang XM et al,Cancer Sci.2013 Nov

[0407] (13)Preston CC et al,PLoS One.2013 Nov 14;8(11):e80063

[0408] (14)He M et al,Neuro Oncol.2013 Jun;15(6):727-34

[0409] (15)Muthu Raja KR et al,PLoS One.2012;7(10):e47077

[0410] (16)Davidson S et al,Mod Pathol.2013 Mar;26(3):448-55

[0411] (17)Huang Yet al,Digestion.2012;86(4):329-37

[0412] (18)Memarian A,Tumour Biol.2013 Feb;34(1):531-42

[0413] (19)Delhem et al,Expert Opin Biol Ther.2010;10(11):1563-1572

[0414] (20)Ouaguia et al,ISRN Hepatology.Volume 2013(2013),Article ID 928485

[0415] (21)Carpentier et al,Am J Transplant.2009;9(9):2102-2112.

[0416] (22)Moralès et al,BRMI,Volume 2014(2014),Article ID 290878

[0417] (23)Baumforth et al,Am J Pathol.2008 Jul;173(1):195-204

[0418] (24)Moralès et al,PlosOne,In press 2014

[0419] (25)Krausz LT et al,Ideggyogy Sz.2013 Sep 30;66(9-10):343-8.

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439] SEQUENCE LISTING <110> University of Lille National Center for Scientific Research Gustave Roussy Institute Servax Paris-Saclay University <120> A Antibody that is against galectin-9 and is an inhibitor of the suppressive activity of regulatory T cells <130> CN201580035348.7.1 <150> PCT / FR2015 / 051498 <151> 2015-06-05 <150> FR 14 55177 <151> 2014-06-06 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 148 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthetic Peptides <220> <223> Antibody fragments <400> 1 Met Lys Cys Ser Trp Gly Ile Phe Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His Ser Lys Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Thr Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Trp Phe Tyr Pro Gly Ser His Ser Ile Lys Tyr Asn 65 70 75 80 Glu Gln Phe Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Val Tyr Met Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Phe Cys Thr Arg His Gly Gly Tyr Asp Gly Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser 130 135 140 Val Tyr Pro Leu 145 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic Peptide <220> <223> Antibody fragments <400> 2 Gly Tyr Thr Phe Thr Asp Tyr Thr Ile His 1 5 10 <210> 3 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis Peptides <220> <223> Antibody fragments <400> 3 Trp Phe Tyr Pro Gly Ser His Ser Ile Lys Tyr Asn Glu Gln Phe Lys 1 5 10 15 Asp Arg <210> 4 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis Peptides <220> <223> Antibody fragments <400> 4 His Gly Gly Tyr Asp Gly Phe Asp Tyr 1 5 <210> 5 <211> 153 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis Peptides <220> <223> Antibody fragments <400> 5 Leu Asp Gly Gly Lys Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu 1 5 10 15 Leu Leu Trp Val Ser Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser 20 25 30 Pro Ser Ser Leu Ala Val Ser Val Gly Glu Lys Ile Thr Met Ser Cys 35 40 45 Lys Ser Ser Gln Ser Leu Phe Tyr Ser Thr Asn Gln Lys Asn Tyr Leu 50 55 60 Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr 65 70 75 80 Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser 85 90 95 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu 100 105 110 Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Tyr Phe Pro Tyr Thr 115 120 125 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro 130 135 140 Thr Val Ser Ile Phe Pro Pro Ser Ser 145 150 <210> 6 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis Peptides <220> <223> Antibody fragments <400> 6 Lys Ser Ser Gln Ser Leu Phe Tyr Ser Thr Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 7 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis Peptides <220> <223> Antibody fragments <400> 7 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis Peptides <220> <223> Antibody <400> 8 Gln Gln Tyr Tyr Tyr Phe Pro Tyr Thr 1 5 <210> 9 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis Peptides <220> <223> Galectin 9 fragment <400> 9 Thr Pro Ala Ile Pro Pro Met Met Tyr Pro His Pro Ala 1 5 10 <210> 10 <211> 175 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic Polypeptide <220> <223> VH 2E12 <400> 10 Met Gly Trp Ser Phe Ile Ile Leu Leu Ser Val Thr Ala Gly Val His 1 5 10 15 Ser Lys Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly 20 25 30 Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu 35 40 45 Tyr Thr Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp 50 55 60 Ile Gly Trp Phe Tyr Pro Gly Ser Gly Ser Met Glu Tyr Asn Glu Lys 65 70 75 80 Phe Asp Lys Ala Thr Leu Thr Ala Asp Asn Ser Ser Ser Thr Val Tyr 85 90 95 Met Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 100 105 110 Glu Arg His Gly Gly Tyr Asp Gly Phe Asp Tyr Trp Gly Gln Gly Thr 115 120 125 Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 130 135 140 Leu Ile Phe Leu Glu Asp Leu Leu Gln Tyr Ser Gln Leu Pro Trp Lys 145 150 155 160 Ile Asp Val Leu Leu Leu Phe Ser Gln Asp Phe Gln Ala Val Tyr 165 170 175 <210> 11 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis Peptides <220> <223> H-CDR1 2E12 <400> 11 Gly Tyr Thr Phe Thr Glu Tyr Thr Ile His 1 5 10 <210> 12 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis Peptides <220> <223> H-CDR2 2E12 <400> 12 Trp Phe Tyr Pro Gly Ser Gly Ser Met Glu Tyr Asn Glu Lys Phe Asp 1 5 10 15 <210> 13 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis Peptides <220> <223> H-CDR3 2E12 <400> 13 His Gly Gly Tyr Asp Gly Phe Asp Tyr 1 5 <210> 14 <211> 153 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis Peptides <220> <223> VL2E12 <400> 14 Leu Asp Gly Gly Lys Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu 1 5 10 15 Leu Leu Trp Val Ser Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser 20 25 30 Pro Ser Ser Leu Ala Val Ser Val Gly Glu Lys Val Thr Met Ser Cys 35 40 45 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Asn Asn Gln Lys Asn Tyr Leu 50 55 60 Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr 65 70 75 80 Trp Ala Ser Thr Arg Gly Ser Gly Val Pro Asp Arg Phe Thr Gly Ser 85 90 95 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu 100 105 110 Asp Leu Ala Ile Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Phe Thr 115 120 125 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro 130 135 140 Thr Val Ser Ile Phe Pro Pro Ser Ser 145 150 <210> 15 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis Peptides <220> <223> L-CDR1 2E12 <400> 15 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Asn Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 16 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis Peptides <220> <223> LCDR2 2E12 <400> 16 Trp Ala Ser Thr Arg Gly Ser 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis Peptides <220> <223> L-CDR3 2E12 <400> 17 Gln Gln Tyr Tyr Ser Tyr Pro Phe Thr 1 5

Claims

1. Antibodies that are anti-galectin-9 and inhibit the suppressive activity of regulatory T cells, in, The sequence of the variable heavy chain region of the antibody is the amino acid sequence shown in SEQ ID NO: 1, and the sequence of the variable light chain region of the antibody is the amino acid sequence shown in SEQ ID NO: 5, or The sequence of the variable heavy chain region of the antibody is the amino acid sequence shown in SEQ ID NO: 10, and the sequence of the variable light chain region of the antibody is the amino acid sequence shown in SEQ ID NO:

14. 2 . The antibody according to claim 1 , which specifically binds to the epitope whose amino acid sequence is shown in SEQ ID NO:

9.

3. Use of the antibody according to claim 1 in the manufacture of a medicament for treating cancer associated with the inhibitory activity of regulatory T cells, characterized in that: The cancer is selected from chronic myeloid leukemia, colon cancer, melanoma, uterine cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, primary lymphoma of the central nervous system, multiple myeloma, prostate cancer, Hodgkin's lymphoma or liver cancer.

4. Use of the antibody according to claim 1 in the manufacture of a medicament for treating cancer associated with the inhibitory activity of regulatory T cells, characterized in that: The cancer is a virus-induced cancer selected from nasopharyngeal carcinoma associated with Epstein-Barr virus and hepatocellular carcinoma associated with hepatitis C virus or hepatitis B virus.

5. A pharmaceutical composition comprising the antibody according to claim 1, and at least one pharmaceutically acceptable carrier.

6. Combination products comprising: - The antibody according to claim 1, and - Anticancer agent.

Images