Anti-CD160 transmembrane isoform antibodies

Abstract

The present invention relates to antibodies and antigen-binding fragments thereof that bind to CD160-TM isoforms, and their use to treat cancer and other diseases.

Description

[Technical field]

[0001] The present invention relates to antibodies and antigen-binding fragments thereof that bind to the CD160-TM transmembrane isoform, and their use to treat cancer and other diseases. [Background technology]

[0002] CD160 was first identified as a GPI-anchored (CD160-GPI) MHC class I activating receptor that is predominantly expressed on peripheral blood NK cells. Furthermore, the identification of a CD160 transmembrane isoform (CD160-TM) resulting from alternative splicing of the CD160 gene was reported. It was established that CD160-TM surface expression is highly restricted to NK cells and activation-dependent (Giustiniani J et al. J Immunol. 2009 Jan 1;182(1):63-71). Indeed, it is accepted in the art that CD160-TM is expressed exclusively by activated NK cells, whereas CD160-GPI is expressed by NK cells (activated or not) and various T cell subsets. Furthermore, evidence has been presented that CD160-TM is a novel activating receptor, as assessed by the increase in CD107a NK cell surface mobilization observed upon engagement (Giustiniani J et al. 2009).

[0003] Therefore, antibodies that bind to the CD160-TM isoform without binding to the CD160 GPI-anchored isoform (or to the CD160 soluble isoform that may result from proteolytic cleavage of the CD160-GPI isoform) may therefore be suitable for therapeutic purposes and may present the advantage of avoiding systemic toxicity such as the risk of cytokine storm. For example, such antibodies may be used to induce NK cell activation and thus amplify the effector functions of NK cells (cytotoxicity, cytokine secretion, etc.) or to deplete CD160-TM expressing cells in vivo (particularly activated NK cells).

[0004] In the present invention, the inventors have developed new antibodies that specifically bind to the CD160-TM isoform, which can be used as pharmaceuticals for human therapy. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International patent application WO2008155363 [Patent Document 2] International patent application WO2006015886 [Patent Document 3] EP0404097 [Patent Document 4] Patent application WO1993011161 [Patent Document 5] US4,816,567 [Patent Document 6] US5,939,598 [Patent Document 7] US6,075,181 [Patent Document 8] US6,114,598 [Patent Document 9] US6,150,584 [Patent Document 10] US6,162,963 [Patent Document 11] Patent application WO2002043478 [Patent Document 12] Patent application WO2008151081 [Patent Document 13] U.S. Patent Publication No. 20030153043 [Patent Document 14] U.S. Patent No. 6,194,551 [Patent Document 15] EP0154316 [Patent Document 16] EP0401384 [Non-patent literature]

[0006] Non-Patent Document 1 Giustiniani J et al. J Immunol. January 1, 2009;182(1):63-71 Non-Patent Document 2 Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988) Non-Patent Document 3 Coligan et al. (eds.), Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993) Non-Patent Document 4 Muller, Meth. Enzymol. 92:589-601 (1983) Non-Patent Document 5 ANUMANTHAN et al., 1998, J Immunol; 161:2780-2790 Non-Patent Document 6 MAIZA et al., J. Exp. Med., Vol. 178, 1121-1126, 1993 Non-Patent Document 7 Nikolova M. et al. (Int Immunol. May 2002;14(5):445-51) Non-Patent Document 8 Giustiniani J. et al. (J Immunol. February 1, 2007;178(3):1293-300) Non-Patent Document 9 Kabat et al. (1991), "Sequences of Proteins of Immunological Interest" 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD Non-Patent Document 10 Al-Lazikani et al., (1997) JMB 273,927-948

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[0007] The present invention relates to an isolated anti-human CD160-TM antibody or antigen-binding fragment thereof, the variable region (VL) of the light chain of the antibody or antigen-binding fragment having the following complementarity determining region 3 (CDR3): 1 QSX 2 SYPX 3 T (SEQ ID NO: 15) (wherein X 1 is G or L, and X 2 is Y or Q, and X 3 is Y or F).

[0008] In one embodiment, the variable region (VH) of the heavy chain of the isolated anti-human CD160-TM antibody or antigen-binding fragment thereof described above comprises the three following complementarity determining regions (CDRs): - CDR1: GYTFTSYW (SEQ ID NO: 1) - CDR2: IYPGSGFT (SEQ ID NO:2) - CDR3: SLDEGFAY (SEQ ID NO: 3) and the light chain variable region (VL) comprises the three following CDRs: - CDR1: QGINIW (SEQ ID NO: 4) - CDR2: KAS - CDR3: LQSQSYPFT (SEQ ID NO:5) Includes.

[0009] In one embodiment, the variable region (VH) of the heavy chain of the isolated anti-human CD160-TM antibody or antigen-binding fragment thereof described above comprises the three following complementarity determining regions (CDRs): - CDR1: GFSLTSYG (SEQ ID NO: 6) - CDR2: IWRGGNT (SEQ ID NO: 7) - CDR3: VKNGGNSKDYALDY (SEQ ID NO: 8) and the light chain variable region (VL) comprises the three following CDRs: - CDR1: ENVGIY (SEQ ID NO: 9) - CDR2: GSS - CDR3: GQSYSYPYT (SEQ ID NO: 10) Includes.

[0010] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising a sequence having at least 70% identity to SEQ ID NO:11, and a light chain variable region comprising a sequence having at least 70% identity to SEQ ID NO:12.

[0011] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising a sequence having at least 70% identity to SEQ ID NO: 13, and a light chain variable region comprising a sequence having at least 70% identity to SEQ ID NO: 14.

[0012] In one embodiment, the above-described isolated anti-human CD160-TM antibody or antigen-binding fragment thereof is chimeric or humanized, and preferably, said antibody or antigen-binding fragment thereof is monoclonal.

[0013] In one embodiment, the above-described isolated anti-human CD160-TM antibody or antigen-binding fragment thereof is a bispecific antibody.

[0014] In one embodiment, the above-described isolated anti-human CD160-TM antibody or antigen-binding fragment thereof is conjugated, preferably the antibody or fragment thereof is conjugated to a detectable label.

[0015] The present invention further relates to fusion proteins comprising the above-described isolated anti-human CD160-TM antibodies or antigen-binding fragments thereof.

[0016] The present invention further relates to nucleic acids encoding the above-described isolated anti-human CD160-TM antibodies or antigen-binding fragments thereof, or fusion proteins.

[0017] The present invention further relates to a pharmaceutical composition comprising the above-described isolated anti-human CD160-TM antibody or antigen-binding fragment thereof, or fusion protein, and at least one pharma- ceutically acceptable excipient.

[0018] The present invention further relates to the above-described isolated anti-human CD160-TM antibody or antigen-binding fragment thereof, fusion protein, or pharmaceutical composition for use as a medicament.

[0019] The present invention further relates to the above-described isolated anti-human CD160-TM antibody or antigen-binding fragment thereof, fusion protein, or pharmaceutical composition for use in treating cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or paroxysmal nocturnal hemoglobinuria in a subject in need thereof.

[0020] In one embodiment, the above-described isolated anti-human CD160-TM antibody or antigen-binding fragment thereof, fusion protein, or pharmaceutical composition is for the treatment of breast cancer in a subject in need thereof. In one embodiment, the above-described isolated anti-human CD160-TM antibody or antigen-binding fragment thereof, fusion protein, or pharmaceutical composition is for the treatment of breast cancer selected from the group including or consisting of luminal A breast cancer, luminal B breast cancer, triple-negative breast cancer (TNBC), and HER2-positive breast cancer.

[0021] In one embodiment, the above-described isolated anti-human CD160-TM antibody or antigen-binding fragment thereof, fusion protein, or pharmaceutical composition is for the treatment of NK leukemia or NK lymphoma, e.g., extranodal and non-extranodal NK / T lymphoma; NK cell-derived malignancies; acute NK leukemia, and peripheral T cell lymphoma (PTCL), e.g., peripheral T cell lymphoma not otherwise specified (PTCL-NOS).

[0022] The present invention further relates to an in vitro method for detecting CD160-TM in a sample, preferably a biological sample, comprising the step of contacting the sample with at least one isolated anti-human CD160-TM antibody or antigen-binding fragment thereof described above.

[0023] The present invention further relates to an isolated anti-human CD160-TM antibody, or antigen-binding fragment thereof, described above, for use in the in vivo detection of a tumor expressing CD160-TM in a subject.

[0024] definition For the purposes of the present invention, the following terms have the following meanings.

[0025] The word "about" preceding a number includes up to plus or minus 10% of the value of said number. It is to be understood that the value to which the term "about" refers is itself also specifically and preferably disclosed.

[0026] "Affinity" as used herein means the strength of binding of an antibody to an epitope. The affinity of an antibody is determined by the dissociation constant K, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. D The affinity constant K A is defined by 1 / KD. Preferred methods for determining the affinity of mAbs can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), all of which are incorporated by reference in their entireties. The binding characteristics of an antibody or antigen-binding fragment thereof to an antigen, cell or tissue can generally be determined and evaluated using immunodetection methods including, for example, ELISA, immunofluorescence-based assays such as immunohistochemistry (IHC) and / or fluorescence-activated cell sorting (FACS), or by surface plasmon resonance (SPR, e.g., using BIAcore®).

[0027] "Antibody" and "immunoglobulin" as used herein may be used interchangeably and refer to a protein having a combination of two heavy chains and two light chains with or without any associated specific immunoreactivity. "Antibody" refers to such an association having significant known specific immunoreactive activity against an antigen of interest (e.g., CD160 transmembrane (TM) isoform). The term "anti-CD160-TM isoform" is used herein to refer to an antibody exhibiting immunoglobulin specificity for the human CD160-TM isoform. As explained elsewhere herein, "specificity" for the human CD160-TM isoform does not exclude cross-reactivity with species homologs of the CD160-TM isoform, such as monkey CD160-TM isoform. Antibodies and immunoglobulins comprise light and heavy chains with or without interchain covalent bonds between them. The basic immunoglobulin structure of vertebrate systems is relatively well understood. The collective term "immunoglobulin" includes five different classes of antibodies that can be biochemically distinguished. Although the following discussion will be directed generally to the IgG class of immunoglobulin molecules, antibodies of all five classes are within the scope of the present invention. With respect to IgG, immunoglobulins contain two identical polypeptide light chains of molecular weight approximately 23 kDa, and two identical heavy chains of molecular weight approximately 53-70 kDa. The four chains are linked by disulfide bonds in a "Y" shaped configuration, with the light chains flanking the heavy chains, which begin at the opening of the "Y" and continue through the variable region. The light chains of antibodies are classified as either kappa (κ) or lambda (λ). Each heavy chain class may be associated with either a κ or λ light chain. Generally, the light and heavy chains are covalently linked to each other, and the "tail" regions of the two heavy chains are linked to each other by covalent disulfide bonds, or by non-covalent bonds when the immunoglobulins are produced by either hybridomas, B cells, or genetically modified host cells. In the heavy chains, the amino acid sequence runs from the N-terminus at the forked end of the Y to the C-terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε), with several subclasses within them (e.g., γ1-γ4).It is the nature of these chains that determine the "class" of an antibody, IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses or "isotypes" (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well characterized and are known to confer functional differentiation. Modified versions of each of these classes and isotypes are readily discernible to one of skill in the art in light of the present disclosure and are therefore within the scope of the present invention. As indicated above, the variable region of an antibody enables the antibody to selectively recognize and specifically bind to an epitope of an antigen. That is, the light chain variable domain (VL domain) and the heavy chain variable domain (VH domain) of an antibody combine to form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the "Y". More specifically, the antigen-binding site is defined by three complementarity determining regions (CDRs) of each VH and VL chain.

[0028] "Antigen-binding fragment" as used herein refers to a portion or region of an antibody that contains fewer amino acid residues than the whole antibody. An "antigen-binding fragment" binds to an antigen and / or competes for antigen binding (e.g., specific binding to a CD160-TM isoform) with the whole antibody from which the fragment is derived. Antigen-binding fragments include, but are not limited to, single chain antibodies, dimeric single chain antibodies, Fv, scFv, Fab, Fab', Fab'-SH, F(ab)'2, Fd, defucosylated antibodies, diabodies, triabodies, and tetrabodies. Antigen-binding fragments may also include unibodies, domain antibodies, and nanobodies.

[0029] "CD160" has its general meaning in the art and refers to the CD160 molecule. There are three CD160 isoforms, the CD160-TM isoform, the CD160 GPI-anchored isoform, and the soluble CD160 isoform. CD160-GPI is expressed by intestinal intraepithelial T lymphocytes, as well as NK cells, TCRγδ and the cytotoxic effector CD8 brightIt is expressed by a small subset of circulating lymphocytes, including CD28- T cells (ANUMANTHAN et al., 1998, J Immunol; 161:2780-2790; MAIZA et al., J. Exp. Med., vol. 178, pp. 1121-1126, 1993). The CD160 transmembrane isoform ("CD160-TM") is described in Giustiniani J et al. (J Immunol. 2009 Jan. 1; 182(1):63-71) and in international patent application WO2008155363, and is characterized by the amino acid sequence set forth in SEQ ID NO:21. The extracellular domain of the CD160-TM isoform may be defined by the amino acid sequence spanning from amino acid residue 26 to amino acid residue 189 of SEQ ID NO:21. The CD160 GPI-anchored isoform ("CD160-GPI") is described in Nikolova M. et al. (Int Immunol. 2002 May; 14(5): 445-51) and in international patent application WO2006015886 and is characterized by the amino acid sequence set forth in SEQ ID NO: 22 fused to a C-terminal GPI anchor. The CD160 soluble isoform is described in Giustiniani J. et al. (J Immunol. 2007 Feb. 1; 178(3): 1293-300) and is characterized by the amino acid sequence set forth in SEQ ID NO: 22. Amino acids 1-25 in SEQ ID NOs: 21-22 correspond to the signal peptide and may therefore not be present in the expressed protein. SEQ ID NO: 21: CD160-TM isoform

[0030] [ka]

[0031] SEQ ID NO: 22: CD160 soluble isoform

[0032] [ka]

[0033] "CDR" or "complementarity determining region" refers to the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. The exact amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), or a combination thereof, or the ImMunoGeneTics (IMGT) Information System® (Lefranc et al., Nucleic Acids Res. 27: 209-212 1999). IMGT is an integrated information system specialized in immunoglobulins (IG), T cell receptors (TR), and major histocompatibility complexes (MHC) of humans and other vertebrates. In this specification, CDRs are referred to both in terms of amino acid sequence and location within the light or heavy chain. Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved between species and occurs within structures called loops, CDR and framework residues can be easily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from an immunoglobulin of one species into an acceptor framework, typically from a human antibody. The correspondence between Kabat numbering and the IMGT specific numbering system is also well known to those skilled in the art (see, for example, Lefranc et al., supra). Thus, in one embodiment, CDR regions or CDRs are intended to refer to the hypervariable regions of the heavy and light chains of immunoglobulins as defined by the IMGT® numbering system.

[0034] "Diabody" as used herein refers to small antibody fragments prepared by constructing scFv fragments with a short linker (about 5-10 residues) between the VH and VL such that interchain but not intrachain pairing of the variable domains is achieved, resulting in a bivalent fragment, i.e. a fragment with two antigen binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments, in which the VH and VL of the two antibodies are present on different polypeptide chains. Diabodies are described, for example, in patent EP0404097 or patent application WO1993011161.

[0035] "Domain antibody" refers to the smallest functional binding unit of an antibody, corresponding to the variable region of either the heavy or light chain of an antibody.

[0036] "Epitope" refers to a specific sequence of amino acids located on a protein to which an antibody or antigen-binding fragment thereof binds. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes may be linear (or continuous) or conformational (i.e., comprising two or more amino acid sequences from different regions of an antigen, which may not necessarily be contiguous).

[0037] "Fab" refers to an antibody fragment produced by papain digestion of a whole IgG antibody to remove the entire Fc fragment, including the hinge region. These antibodies are monovalent and contain only a single antigen-binding site. In contrast, F(ab') 2 Antibody fragments are produced by pepsin digestion of a whole IgG antibody to remove most of the Fc region while leaving part of the hinge region intact. F(ab') 2 The fragment has two antigen-binding F(ab) portions linked by disulfide bonds. The term "Fab'" refers to the F(ab') 2Fab'-SH refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, which is obtained by cleaving the disulfide bond in the hinge region of Fab'. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues in the constant domains have a free thiol group.

[0038] The terms "Fc domain," "Fc portion," and "Fc region" refer to the C-terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of a human gamma heavy chain or its outpart sequences in other types of antibody heavy chains (e.g., α, δ, ε, and μ of human antibodies), or naturally occurring allotypes thereof.

[0039] "Fd fragment" refers to the heavy chain of a Fab fragment containing the VH and CH1 regions.

[0040] "Framework regions" or "FR regions" include amino acid residues that are part of the variable regions but not part of the CDRs (e.g., using the IMGT® numbering definition of CDRs). Similarly, the framework regions of the light chain are separated by each of the CDRs of the VL. In naturally occurring antibodies, the six CDRs present in each monomeric antibody are short non-contiguous sequences of amino acids that are specifically positioned to form an antigen-binding site as the antibody assumes its three-dimensional structure in an aqueous environment. The remainder of the heavy and light chain variable domains show less intermolecular variability of amino acid sequence and are referred to as framework regions. The framework regions largely adopt a β-sheet conformation, with the CDRs forming loops that connect, and in some cases form part of, the β-sheet structure. These framework regions thus act to form a scaffold that positions the six CDRs in the correct orientation by non-covalent interactions between the chains. The antigen-binding site formed by the positioned CDRs defines a surface complementarity to the epitope of the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. The locations of the CDRs can be readily identified by one of skill in the art.

[0041] "Fv" as used herein refers to the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one VH and one VL in tight non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the heavy and light chains) that contribute to antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0042] A "heavy chain region" comprises an amino acid sequence derived from the constant domain of an immunoglobulin heavy chain. A protein comprising a heavy chain region is H 1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, C H 2 domain, C H In one embodiment, the antibody or antigen-binding fragment thereof according to the invention may comprise an Fc region of an immunoglobulin heavy chain (e.g., a hinge portion, C H 2 domain, and C H In another embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises at least one region of a constant domain (e.g., C H In certain embodiments, at least one, and preferably all, of the constant domains are derived from a human immunoglobulin heavy chain. For example, in one embodiment, the heavy chain region includes a fully human hinge domain. In other embodiments, the heavy chain region includes a fully human Fc region (e.g., a hinge, C domain, or both from a human immunoglobulin). H 2, and C H In certain embodiments, the constituent constant domains of the heavy chain region are derived from different immunoglobulin molecules. For example, the heavy chain region of the protein comprises a C HIn other embodiments, the constant domain is a chimeric domain comprising regions of different immunoglobulin molecules. For example, the hinge may comprise a first region derived from an IgG1 molecule and a second region derived from an IgG3 or IgG4 molecule. In some embodiments, the constant domain of the heavy chain region may be modified to vary in amino acid sequence from a naturally occurring (wild type) immunoglobulin molecule. That is, an antibody or antigen-binding fragment thereof according to the invention may comprise a heavy chain constant domain (C H 1, hinge, C H 2 or C H 3) and / or a light chain constant domain (C L Exemplary modifications include the addition, deletion, or substitution of one or more amino acids in one or more domains.

[0043] The "hinge region" is C H 1 domain C H The hinge region comprises the region of the heavy chain molecule that connects the two N-terminal antigen-binding domains. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains, the upper, middle, and lower hinge domains (Roux et al., 1998. J Immunol. 161(8):4083-90).

[0044] As used herein in the context of two or more amino acid sequences, or two or more nucleic acid sequences, "identity" or "identical" refers to the degree of sequence relatedness between the amino acid or nucleic acid sequences, as determined by the number of matches between strings of two or more amino acid or nucleic acid residues. "Identity" measures the percentage of exact matches between the smaller of the two or more sequences, with gap alignments (if any) processed by a particular mathematical model or computer program (i.e., an "algorithm"). The identity of related amino acid or nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Lesk AM (1988). Computational molecular biology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; Smith DW (1993). Biocomputing: Informatics and genome projects. San Diego, CA: Academic Press; Griffin AM & Griffin HG (1994). Computer analysis of sequence data, Part 1. Totowa, NJ: Humana Press; von Heijne G. (1987). Sequence analysis in molecular biology: treasure trove or trivial pursuit. San Diego, CA: Academic press; Gribskov MR & Devereux J. (1991). Sequence analysis primer. New York, NY: Stockton Press; Carillo et al., 1988. SIAM J Appl Math. 48(5):1073-82. Preferred methods for determining identity are designed to give the largest match between the sequences examined.Methods for determining identity are described in published computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package including GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux et al., 1984. Nucleic Acids Res. 12(1 Pt 1):387-95), BLASTP, BLASTN, and FASTA (Altschul et al., 1990. J Mol Biol. 215(3):403-10). The BLASTX program is published by the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894). The well-known Smith Waterman algorithm can also be used to determine identity.

[0045] An "isolated protein," and in particular an "isolated antibody," as used herein, is intended to refer to a protein, particularly an antibody, that is substantially free of other proteins or antibodies with different antigenic specificities (e.g., an isolated protein or antibody that specifically binds to a CD160-TM isoform is substantially free of proteins or antibodies that specifically bind to antigens other than the CD160-TM isoform). However, an isolated protein, particularly an isolated antibody, that specifically binds to a CD160-TM isoform may have cross-reactivity with other antigens, such as CD160-TM molecules from other species. Furthermore, an isolated protein or antibody may be substantially free of other cellular material and / or chemicals, particularly materials that would interfere with therapeutic use of the protein or antibody, including, but not limited to, enzymes, hormones, and other proteinaceous or non-proteinaceous components.

[0046] "Mammal" refers to any mammal, including humans, non-human primates, domestic and farm animals, as well as zoo animals, sport animals, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, monkeys, etc. Preferably, the mammal is a human.

[0047] "Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each antibody in the population is identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized without contamination by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies or antigen-binding fragments thereof according to the invention may be prepared by the hybridoma methodology first described by Kohler et al., 1975. Nature. 256(5517):495-7, or may be made using recombinant DNA methods in bacterial, eukaryotic or plant cells (Patent US 4,816,567). The "monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., 1991. Nature. 352(6336):624-8 and Marks et al., 1991. J Mol Biol. 222(3):581-97.

[0048] "Nanobodies" refer to antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy chain antibodies (Muyldermans, 2013. Annu Rev Biochem. 82:775-97). These heavy chain antibodies consist of a single variable domain (VHH) and two constant domains (C H 2 and C H 3) may be contained.

[0049] "Single-chain antibody", as used herein, refers to any antibody or fragment thereof that is a protein having a primary structure that comprises or consists of a single uninterrupted sequence of contiguous amino acid residues, including, but not limited to, (1) a single-chain Fv molecule (scFv), (2) a single-chain protein containing only one light chain variable domain, or a fragment thereof containing the three CDRs of a light chain variable domain, without an associated heavy chain portion, and (3) a single-chain protein containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of a heavy chain variable region, without an associated light chain portion.

[0050] "Single-chain Fv," also abbreviated as "sFv" or "scFv," refers to a single Fv consisting of V linked in a single amino acid chain. H and V L scFv refers to an antibody fragment that contains an antibody domain. Preferably, the scFv amino acid sequence further comprises a peptide linker that enables the scFv to form the desired structure for antigen binding. H and V L Include between domains.

[0051] "Specificity" refers to the ability of an antibody to detectably bind to an epitope presented on an antigen, such as CD160-TM, while having relatively little detectable reactivity with non-CD160-TM proteins, such as CD160 GPI-anchored isoforms and CD160 soluble isoforms. Specificity can be determined relatively by binding or competitive binding assays, for example, using a Biacore instrument. Specificity can be demonstrated by an affinity / avidity ratio of, for example, about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or more, for binding to a specific antigen versus nonspecific binding to other unrelated molecules (in this case, the specific antigen is a CD160-TM polypeptide).

[0052] "Subject" refers to a warm-blooded animal, preferably a mammal (including humans, farm animals and livestock, as well as zoo animals, sport animals, or pet animals, e.g., dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc...), more preferably a human. In one embodiment, the subject may be a "patient", i.e. a warm-blooded animal, more preferably a human, who is waiting to receive medical care, or is receiving medical care, or has been / is / will be the subject of medical treatment, or is being monitored for disease progression. In one embodiment, the subject is an adult (e.g., a subject over 18 years of age). In another embodiment, the subject is a child (e.g., a subject under 18 years of age). In one embodiment, the subject is a male. In another embodiment, the subject is a female.

[0053] A "therapeutically effective amount" refers to a level or amount of an antibody or antigen-binding fragment thereof described herein that is intended to (1) delay or prevent the onset of a disease, disorder, or condition, (2) slow or halt the progression, progression, or worsening of one or more symptoms of a disease, disorder, or condition, (3) bring about amelioration of symptoms of a disease, disorder, or condition, (4) reduce the severity or incidence of a disease, disorder, or condition, or (5) cure a disease, disorder, or condition, without causing significant negative or deleterious side effects to the target. A therapeutically effective amount may be administered prior to the onset of a disease, disorder, or condition as a prophylactic or preventative measure. Alternatively, or in addition, a therapeutically effective amount may be administered after the initiation of a disease, disorder, or condition as a therapeutic measure.

[0054] "Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic or preventative measures, where the purpose is to prevent or slow (alleviate) the targeted pathological condition or disorder. Those in need of treatment include those who already have the disorder and those who are prone to have the disorder, or those in whom the disorder is to be prevented. In one embodiment, a subject is successfully "treated" for a disease described herein if, after receiving a therapeutic amount of an antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, or expression vector described herein, the subject exhibits at least one of the following: a reduction in the number of pathogenic cells, some alleviation of one or more symptoms associated with the disease being treated, a reduction in morbidity and mortality, and an improvement in quality of life issues. The above parameters for assessing the success of treatment and improvement of the disease are easily measurable by routine methods familiar to physicians.

[0055] "Unibody" refers to an antibody fragment that lacks the hinge region of an IgG4 antibody. The deletion of the hinge region results in a molecule that is essentially half the size of a traditional IgG4 antibody and has a univalent binding region rather than the bivalent binding region of IgG4 antibodies.

[0056] "Variable" refers to the variable domain V H and V L This refers to the fact that certain regions of the variable domains of antibodies differ widely in sequence among antibodies and are used in the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. The variability is found in the V domains, which form part of the antigen-binding site. L Domain and V H The first, second, and third hypervariable loops of a Vλ light chain domain are referred to herein as L1 (λ), L2 (λ), and L3 (λ), and the second, third, and third hypervariable loops of a Vλ light chain domain are referred to herein as L1 (λ), L2 (λ), and L3 (λ), and the third, ... LThe Vκ light chain domain can be defined as comprising residues 24-33 (L1(λ), ​​consisting of 9, 10 or 11 amino acid residues), 49-53 (L2(λ), consisting of 3 residues), and 90-96 (L3(λ), consisting of 6 residues) (Morea et al., 2000. Methods. 20(3):267-79). The first, second, and third hypervariable loops of the Vκ light chain domain are referred to herein as L1 (κ), L2 (κ), and L3 (κ), and the Vκ light chain domain is referred to herein as L1(κ), L2(κ), and L3(κ). L Domains can be defined as including residues 25-33 (L1(κ), consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2(κ), consisting of 3 residues), and 90-97 (L3(κ), consisting of 6 residues) (Morea et al., supra). H The first, second, and third hypervariable loops of a domain are referred to herein as H1, H2, and H3, and V H Domains can be defined as including residues 25-33 (H1, consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4 residues), and 91-105 (H3, highly variable in length) (Morea et al., supra). Unless otherwise indicated, the terms L1, L2, and L3 refer to V, respectively. L The terms H1, H2 and H3 refer to the first, second and third hypervariable loops of the V domain and include hypervariable loops from both Vκ and Vλ isotypes. H It refers to the first, second, and third hypervariable loops of a domain and includes hypervariable loops from any of the known heavy chain isotypes, including gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). Each hypervariable loop may comprise a portion of a "complementarity determining region" or "CDR" as defined above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] The present invention relates to antibodies or antigen-binding fragments thereof that bind to CD160-TM isoforms.

[0058] Examples of antibodies and antigen-binding fragments include, but are not limited to, whole antibodies, single chain antibodies, dimeric single chain antibodies, Fv, scFv, Fab, Fab', Fab'-SH, F(ab)' 2 , Fd, defucosylated antibodies, bispecific antibodies, diabodies, triabodies, tetrabodies, unibodies, domain antibodies, and nanobodies.

[0059] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is a whole antibody, a single chain antibody, a dimeric single chain antibody, an Fv, an scFv, an Fab, an Fab', an Fab'-SH, an F(ab)' 2 , Fd, defucosylated antibodies, bispecific antibodies, diabodies, triabodies, and tetrabodies.

[0060] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is a whole antibody, a single chain variable fragment (scFv), an Fv, an Fab, an Fab', an Fab'-SH, an F(ab)' 2 , defucosylated antibodies, bispecific antibodies, diabodies, triabodies, and tetrabodies.

[0061] In one embodiment, the isolated antibody or antigen-binding fragment according to the invention is selected from the group comprising or consisting of unibodies, domain antibodies, and nanobodies.

[0062] In one embodiment, the isolated antibody or antigen-binding fragment according to the invention comprises or is selected from the group consisting of a whole antibody, an Fv, an scFv, an Fab, and a unibody.

[0063] In one embodiment, an antibody or antigen-binding fragment thereof according to the invention specifically binds to the CD160-TM isoform. In one embodiment, an antibody or antigen-binding fragment thereof according to the invention specifically binds to the human CD160-TM isoform.

[0064] As used herein, "specifically binds" refers to an antibody or antigen-binding fragment thereof that recognizes and binds to a binding partner present in a sample, but which does not substantially recognize or bind to other molecules in the sample.

[0065] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention has low or no affinity for other CD160 isoforms, such as the CD160 GPI-anchored isoform and the CD160 soluble isoform.

[0066] The affinity of an antibody or antigen-binding fragment thereof can be readily determined using conventional techniques, such as those described by Scatchard, 1949. Ann NY Acad Sci. 51:660-672. The binding characteristics of an antibody or antigen-binding fragment thereof for an antigen, cell or tissue can generally be determined and evaluated using immunodetection methods including, for example, ELISA, immunofluorescence-based assays such as immunohistochemistry (IHC) and / or fluorescence activated cell sorting (FACS), or by surface plasmon resonance (SPR, e.g., using BIAcore®).

[0067] Below, and unless expressly stated otherwise, CDR numbering and definitions are according to the IMGT® numbering system.

[0068] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region (abbreviated herein as VH) comprising the following complementarity determining region 1 (CDR1): GX 1 X 2 X 3 TSYX 4 (SEQ ID NO: 16) (wherein X 1 is F or Y, and X 2 is S or T, and X 3 is L or F, and X 4 is G or W).

[0069] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises a light chain variable region (abbreviated herein as VL) comprising the following complementarity determining region 3 (CDR3): 1 QSX 2 SYPX 3 T (SEQ ID NO: 15) (wherein X 1 is G or L, and X 2 is Y or Q, and X 3 is Y or F).

[0070] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises at least one, preferably at least two, more preferably at least three of the following complementarity determining regions (CDRs): CDR1: GYTFTSYW (SEQ ID NO:1); CDR2: IYPGSGFT (SEQ ID NO:2); and / or CDR3: SLDEGFAY (SEQ ID NO:3) The heavy chain variable region comprises:

[0071] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises at least one, preferably at least two, more preferably at least three of the following complementarity determining regions (CDRs): CDR1: GFSLTSYG (SEQ ID NO:6); CDR2: IWRGGNT (SEQ ID NO: 7); and / or CDR3: VKNGGNSKDYALDY (SEQ ID NO:8) The heavy chain variable region comprises:

[0072] In one embodiment, any of CDR1, CDR2 and / or CDR3 of VH having SEQ ID NOs: 1 to 3, 6 to 8 can be characterized as having one, two, three or more amino acids substituted with different amino acids.

[0073] In one embodiment, any of the CDR1, CDR2 and / or CDR3 of a VH having SEQ ID NO: 1-3, 6-8 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with a particular CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0074] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises at least one, preferably at least two, more preferably at least three of the following complementarity determining regions (CDRs): CDR1: QGINIW (SEQ ID NO:4); CDR2: KAS; and / or CDR3: LQSQSYPFT (SEQ ID NO:5) The light chain variable region comprises:

[0075] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises at least one, preferably at least two, more preferably at least three of the following complementarity determining regions (CDRs): CDR1: ENVGIY (SEQ ID NO:9); CDR2: GSS; and / or CDR3: GQSYSYPYT (SEQ ID NO: 10) The light chain variable region comprises:

[0076] In one embodiment, any of CDR1, CDR2 and / or CDR3 having SEQ ID NO: 4 or 9, sequences KAS or GSS, and SEQ ID NO: 5 or 10, respectively, can be characterized as having one, two, three or more amino acids substituted with different amino acids.

[0077] In one embodiment, any of the CDR1, CDR2 and / or CDR3 of the VL having SEQ ID NO: 4 or 9, sequence KAS or GSS, and SEQ ID NO: 5 or 10, respectively, can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with a particular CDR or set of CDRs listed above.

[0078] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: - a VH comprising at least one, preferably at least two, more preferably three of the following CDRs: CDR1: GYTFTSYW (SEQ ID NO:1); CDR2: IYPGSGFT (SEQ ID NO:2); and / or CDR3: SLDEGFAY (SEQ ID NO:3); and - a VL comprising at least one, preferably at least two, more preferably three of the following CDRs: CDR1: QGINIW (SEQ ID NO:4); CDR2: KAS; and / or CDR3: LQSQSYPFT (SEQ ID NO:5) Includes.

[0079] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: - a VH comprising the three following CDRs: CDR1: GYTFTSYW (SEQ ID NO:1); CDR2: IYPGSGFT (SEQ ID NO:2); and CDR3: SLDEGFAY (SEQ ID NO:3); and - A VL containing the three following CDRs: CDR1: QGINIW (SEQ ID NO:4); CDR2: KAS; and CDR3: LQSQSYPFT (SEQ ID NO:5) Includes.

[0080] In one embodiment, any of the CDR1, CDR2 and / or CDR3 of the VH having SEQ ID NO:1 to 3 and / or the VL having SEQ ID NO:4, SEQ ID NO:KAS, and SEQ ID NO:5, respectively, can be characterized as having one, two, three or more amino acids replaced by different amino acids.

[0081] In one embodiment, any of the CDR1, CDR2 and / or CDR3 of the VH having SEQ ID NO:1-3, and / or the VL having SEQ ID NO:4, sequence KAS, and SEQ ID NO:5, respectively, may be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with a particular CDR or set of CDRs listed above.

[0082] An example of an antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 having SEQ ID NO:1 to 3, and a light chain comprising CDR1, CDR2, and CDR3 having SEQ ID NO:4, sequence KAS, and SEQ ID NO:5, respectively, is the 21C8 antibody.

[0083] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: - a VH comprising at least one, preferably at least two, more preferably three of the following CDRs: CDR1: GFSLTSYG (SEQ ID NO:6); CDR2: IWRGGNT (SEQ ID NO: 7); and / or CDR3: VKNGGNSKDYALDY (SEQ ID NO:8); and - a VL comprising at least one, preferably at least two, more preferably three of the following CDRs: CDR1: ENVGIY (SEQ ID NO:9); CDR2: GSS; and / or CDR3: GQSYSYPYT (SEQ ID NO: 10) Includes.

[0084] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: - a VH comprising the three following CDRs: CDR1: GFSLTSYG (SEQ ID NO:6); CDR2: IWRGGNT (SEQ ID NO: 7); and CDR3: VKNGGNSKDYALDY (SEQ ID NO:8); and - A VL containing the three following CDRs: CDR1: ENVGIY (SEQ ID NO:9); CDR2: GSS; and CDR3: GQSYSYPYT (SEQ ID NO: 10) Includes.

[0085] In one embodiment, any of the CDR1, CDR2 and / or CDR3 of the VH having SEQ ID NO: 6 to 8 and / or the VL having SEQ ID NO: 9, sequence GSS, and SEQ ID NO: 10, respectively, can be characterized as having one, two, three or more amino acids replaced by different amino acids.

[0086] In one embodiment, any of the CDR1, CDR2 and / or CDR3 of the VH having SEQ ID NO:6-8 and / or of the VL having SEQ ID NO:9, sequence GSS, and SEQ ID NO:10, respectively, may be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with a particular CDR or set of CDRs listed above.

[0087] An example of an antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 having SEQ ID NO:6-8, and a light chain comprising CDR1, CDR2, and CDR3 having SEQ ID NO:9, sequence GSS, and SEQ ID NO:10, respectively, is the 22B12 antibody.

[0088] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: - a VH comprising at least one, preferably at least two, more preferably three of the following CDRs: CDR1: GFSLTSYG (SEQ ID NO:6); CDR2: IWRGGNT (SEQ ID NO: 7); and / or CDR3: VKNGGNSKDYALDY (SEQ ID NO:8); and - a VL comprising at least one, preferably at least two, more preferably three of the following CDRs: CDR1: QGINIW (SEQ ID NO:4); CDR2: KAS; and / or CDR3: LQSQSYPFT (SEQ ID NO:5) Includes.

[0089] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: - a VH comprising the three following CDRs: CDR1: GFSLTSYG (SEQ ID NO:6); CDR2: IWRGGNT (SEQ ID NO: 7); and / or CDR3: VKNGGNSKDYALDY (SEQ ID NO:8); and - A VL containing the three following CDRs: CDR1: QGINIW (SEQ ID NO:4); CDR2: KAS; and / or CDR3: LQSQSYPFT (SEQ ID NO:5) Includes.

[0090] In one embodiment, any of the CDR1, CDR2 and / or CDR3 of the VH having SEQ ID NO: 6 to 8 and / or the VL having SEQ ID NO: 4, sequence KAS, and SEQ ID NO: 5, respectively, can be characterized as having one, two, three or more amino acids replaced by different amino acids.

[0091] In one embodiment, any of the CDR1, CDR2 and / or CDR3 of the VH having SEQ ID NO:6-8 and / or the VL having SEQ ID NO:4, sequence KAS, and SEQ ID NO:5, respectively, may be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with a particular CDR or set of CDRs listed above.

[0092] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: - a VH comprising at least one, preferably at least two, more preferably three of the following CDRs: CDR1: GYTFTSYW (SEQ ID NO:1); CDR2: IYPGSGFT (SEQ ID NO:2); and / or CDR3: SLDEGFAY (SEQ ID NO:3); and - a VL comprising at least one, preferably at least two, more preferably three of the following CDRs: CDR1: ENVGIY (SEQ ID NO:9); CDR2: GSS; and / or CDR3: GQSYSYPYT (SEQ ID NO: 10) Includes.

[0093] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: - a VH comprising the three following CDRs: CDR1: GYTFTSYW (SEQ ID NO:1); CDR2: IYPGSGFT (SEQ ID NO:2); and / or CDR3: SLDEGFAY (SEQ ID NO:3); and - A VL containing the three following CDRs: CDR1: ENVGIY (SEQ ID NO:9); CDR2: GSS; and / or CDR3: GQSYSYPYT (SEQ ID NO: 10) Includes.

[0094] In one embodiment, any of the CDR1, CDR2 and / or CDR3 of the VH having SEQ ID NO: 1 to 3 and / or of the VL having SEQ ID NO: 9, sequence GSS, and SEQ ID NO: 10, respectively, can be characterized as having one, two, three or more amino acids substituted with different amino acids.

[0095] In one embodiment, any of the CDR1, CDR2 and / or CDR3 of the VH having SEQ ID NO: 1-3 and / or of the VL having SEQ ID NO: 9, sequence GSS, and SEQ ID NO: 10, respectively, may be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with a particular CDR or set of CDRs listed above.

[0096] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises a VH comprising or consisting of the sequence of SEQ ID NO:11. SEQ ID NO:11

[0097] [ka]

[0098] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises a VH comprising or consisting of the sequence of SEQ ID NO:13. SEQ ID NO:13

[0099] [ka]

[0100] In one embodiment, the VH comprises or consists of the sequence of SEQ ID NO: 11 or 13, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids are substituted by a different amino acid.

[0101] In one embodiment, the VH has an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO:11 or 13.

[0102] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises a VL comprising or consisting of the sequence of SEQ ID NO:12. SEQ ID NO:12

[0103] [ka]

[0104] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises a VL comprising or consisting of the sequence of SEQ ID NO:14. SEQ ID NO:14

[0105] [ka]

[0106] In one embodiment, the VL comprises or consists of the sequence of SEQ ID NO: 12 or 14, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids are substituted by a different amino acid.

[0107] In one embodiment, the VL has an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 12 or 14.

[0108] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: - a VH comprising or consisting of the sequence SEQ ID NO: 11; and - a VL comprising or consisting of the sequence SEQ ID NO: 12 Includes.

[0109] In one embodiment, the VH comprises or consists of the sequence of SEQ ID NO: 11, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids are substituted by different amino acids. and / or the VL comprises or consists of the sequence of SEQ ID NO: 12 in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids are replaced by different amino acids.

[0110] In one embodiment, the VH and / or VL have an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO:11 and / or SEQ ID NO:12, respectively.

[0111] An example of such an antibody is the 21C8 antibody.

[0112] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: - a VH comprising or consisting of the sequence SEQ ID NO: 13; and - a VL comprising or consisting of the sequence SEQ ID NO: 14 Includes.

[0113] In one embodiment, the VH comprises or consists of the sequence of SEQ ID NO: 13, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids are substituted by different amino acids. and / or the VL comprises or consists of the sequence of SEQ ID NO: 14 in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids are replaced by different amino acids.

[0114] In one embodiment, the VH and / or VL have an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO:13 and / or SEQ ID NO:14, respectively.

[0115] An example of such an antibody is the 22B12 antibody.

[0116] As used herein, the phrase "characterized as having [...] amino acids being substituted by a different amino acid" with respect to a given sequence refers to the occurrence of conservative amino acid modifications in said sequence.

[0117] As used herein, the phrase "conservative amino acid modifications" refers to modifications that do not significantly affect or alter the binding properties of an antibody or antigen-binding fragment thereof containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody or antigen-binding fragment thereof by standard techniques known in the art, such as site-directed mutagenesis and PCR mutagenesis.

[0118] Conservative amino acid substitutions are typically those in which an amino acid residue is replaced with an amino acid residue that has a side chain with similar physicochemical properties. A particular variable region and CDR sequence may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid insertions, deletions and / or substitutions. When substitutions are made, preferred substitutions are conservative modifications. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDRs and / or variable regions of an antibody or antigen-binding fragment thereof according to the invention can be replaced with other amino acid residues from the same side chain family, and the altered antibody can be tested for retained function (i.e., the properties described herein, e.g., binding to CD160-TM isoforms) using the assays described herein. In another embodiment, stretches of amino acids within the CDRs and / or variable regions of an antibody or antigen-binding fragment thereof according to the invention can be replaced with structurally similar stretches that differ in the order and / or composition of side chain family members.

[0119] The present invention also relates to 21C8-like antibodies, i.e., antibodies that bind to the same epitope as 21C8, or substantially the same epitope as 21C8. Accordingly, the present invention further relates to antibodies or antigen-binding fragments that compete with 21C8 for binding to CD160-TM isoforms.

[0120] The present invention also relates to 22B12-like antibodies, i.e., antibodies that bind to the same epitope as 22B12, or substantially the same epitope as 22B12. Accordingly, the present invention further relates to antibodies or antigen-binding fragments that compete with 22B12 for binding to CD160-TM isoforms.

[0121] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is polyclonal. In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is monoclonal.

[0122] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is monovalent. In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is bivalent.

[0123] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises a complete or substantially complete murine heavy chain constant region (referred to herein as C H ) and / or a light chain constant region (abbreviated herein as C L In one embodiment, the constant region is of murine origin.

[0124] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is a murine antibody or fragment thereof.

[0125] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is a chimeric antibody or fragment thereof.

[0126] "Chimeric antibody or fragment thereof," as used herein, refers to an antibody or antigen-binding fragment thereof that comprises a first amino acid sequence linked to a second amino acid sequence that is not naturally linked in nature. The amino acid sequences may normally be present in separate proteins that are joined in the fusion protein, or may normally be present in the same protein, but are placed in a new arrangement in the fusion protein. Chimeric proteins can be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.

[0127] The term "chimeric antibody or fragment thereof" is used herein to refer to (a) the constant region or a portion thereof is altered, replaced or exchanged such that the variable region is linked to a constant region of a different or altered class, effector function, and / or species, or to an entirely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or a portion thereof is modified, replaced or exchanged by a variable region or portion thereof having a different or altered antigenic specificity, or by a corresponding sequence from another species or another antibody class or subclass. It includes antibodies and antigen-binding fragments thereof.

[0128] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is a humanized antibody or fragment thereof.

[0129] "Humanized antibody or fragment thereof" as used herein refers to a chimeric antibody or antigen-binding fragment thereof that contains minimal sequence derived from a non-human immunoglobulin. Humanized antibodies or fragments thereof include antibodies made by non-human cells with altered variable and constant regions that more closely resemble antibodies made by human cells, e.g., by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies or antigen-binding fragments thereof according to the invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs.

[0130] The term "humanized antibody or fragment thereof" also includes antibodies and antigen-binding fragments thereof in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. In other words, the term "humanized antibody or fragment thereof" refers to an antibody or antigen-binding fragment thereof in which the CDRs of a recipient human antibody are replaced by CDRs from a donor non-human antibody. A humanized antibody or antigen-binding fragment thereof may also comprise residues of donor origin in the framework sequences. A humanized antibody or antigen-binding fragment thereof may also comprise at least a portion of a human immunoglobulin constant region. A humanized antibody or antigen-binding fragment thereof may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Humanization can be carried out using methods known in the art, including techniques such as "superhumanization" of antibodies (e.g., Tan et al., 2002. J Immunol. 169(2):1119-25) and "resurfacing" (e.g., Staelens et al., 2006. Mol Immunol. 43(8):1243-57; Roguska et al., 1994. Proc Natl Acad Sci USA. 91(3):969-73) (e.g., Jones et al., 1986. Nature. 321(6069):522-5; Riechmann et al., 1988. Nature. 332(6162):323-7; Verhoeyen et al., 1988. Science. 239(4847):1534-6; Presta, 1992. Curr Opin Biotechnol. 3(4):394-8; Patent US4,816,567).

[0131] A "humanized antibody or fragment thereof" may retain a similar antigen specificity as the original antibody or fragment. However, using certain methods of humanization, the binding affinity and / or specificity of the antibody may be increased.

[0132] Methods for humanizing the antibody or antigen-binding fragment thereof according to the invention are well known in the art. The choice of human variable domains, both light and heavy, used in the construction of the humanized antibody or antigen-binding fragment thereof is crucial to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of the antibody or antigen-binding fragment thereof according to the invention is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the mouse sequence is then accepted as the human framework (FR) of the humanized antibody or fragment (Sims et al., 1993. J Immunol. 151(4):2296-308; Chothia and Lesk, 1987. J Mol Biol. 196(4):901-17).

[0133] Another method for humanizing an antibody or antigen-binding fragment thereof according to the invention uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies or fragments (Carter et al., 1992. Proc Natl Acad Sci USA. 89(10):4285-9; Presta et al., 1993. J Immunol. 151(5):2623-32). Furthermore, it is important that the antibody or fragment is humanized while retaining high affinity for the CD160-TM isoform and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies and antigen-binding fragments thereof are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display predicted three-dimensional structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind to an epitope. In this way, CDR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, for example increased affinity for the CD160-TM isoform, is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0134] Another method for humanizing the antibodies or antigen-binding fragments thereof according to the invention is to use transgenic or transchromosomic animals carrying parts of the human immune system for immunization. As hosts, these animals have their immunoglobulin genes replaced by functional human immunoglobulin genes. Thus, the antibodies produced by these animals or of hybridomas made from the B cells of these animals are already humanized. Examples of such transgenic or transchromosomic animals include, but are not limited to: - XenoMouse (Abgenix, Fremont, CA), as described in patents US5,939,598, US6,075,181, US6,114,598, US6,150,584, and US6,162,963; - HuMAb Mouse® (Medarex, Inc.), Lonberg et al., 1994. Nature. 368(6474):856-859; Lonberg and Huszar, 1995. Int Rev Immunol. 13(1):65-93; Harding and Lonberg, 1995. Ann NY Acad Sci. 764:536-46; Taylor et al., 1992. Nucleic Acids Res. 20(23):6287-95; Chen et al., 1993. Int Immunol. 5(6):647-56; Tuaillon et al., 1993. Proc Natl Acad Sci USA. 90(8):3720-4; Choi et al., 1993. Nat Genet. 4(2):117-23; Chen et al., 1993. EMBO J. 12(3):821-30; Tuaillon et al., 1994. J Immunol. 152(6):2912-20; Taylor et al., 1994. Int Immunol. 6(4):579-91; Fishwild et al., 1996. Nat Biotechnol. 14(7):845-51; - KM Mouse®, as described in patent application WO2002043478; - TC mice, as described in Tomizuka et al., 2000. Proc Natl Acad Sci USA. 97(2):722-7; and - OmniRat™ (OMT, Inc.), as described in patent application WO2008151081; Geurts et al., 2009. Science. 325(5939):433; Menoret et al., 2010. Eur J Immunol. 40(10):2932-41 Examples include:

[0135] Humanized antibodies and antigen-binding fragments thereof can also be produced according to various other techniques, for example by immunizing other transgenic animals engineered to express human antibody repertoires (Jakobovitz et al., 1993. Nature. 362(6417):255-8), or by selection of antibody repertoires using phage display techniques. Such techniques are known to those skilled in the art and can be performed starting from the monoclonal antibodies or antigen-binding fragments thereof disclosed in the present application.

[0136] In one embodiment, the antibody or antigen-binding fragment thereof is from the IgG class.

[0137] In one embodiment, the antibody or antigen-binding fragment thereof is from the human IgG1 subclass. In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is therefore an IgG1 antibody, preferably a human IgG1 antibody.

[0138] In another embodiment, the antibody or antigen-binding fragment thereof is from the human IgG2 subclass.

[0139] The Fc region of IgG antibodies interacts with cellular Fcγ receptors (FcγRs) to stimulate and regulate downstream effector mechanisms. There are five activating receptors, namely FcγRI (CD64), FcγRIIa (CD32a), FcγRIIc (CD32c), FcγRIIIa (CD16a), and FcγRIIIb (CD16b), and one inhibitory receptor, FcγRIIb (CD32b). Communication between IgG antibodies and the immune system is controlled and mediated by FcγRs. FcγRs relay information sensed and collected by antibodies to the immune system, providing a link between the innate and adaptive immune systems and especially in the context of biotherapeutics (Hayes J et al., 2016. J Inflamm Res 9: 209-219).

[0140] IgG subclasses differ in their ability to bind to FcγR, and this differential binding determines the ability of the IgG subclass to induce a range of functional responses. For example, in humans, FcγRIIIa is the primary receptor involved in the activation of antibody-dependent cellular cytotoxicity (ADCC), and IgG3 (closely followed by IgG1) exhibits the highest affinity for this receptor, reflecting the ability of IgG3 to potently induce ADCC. IgG2 has been shown to bind weakly to this receptor.

[0141] Thus, the constant region of an antibody is important in the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity and phagocytosis. Thus, as discussed herein, the isotype of the antibody can be selected based on whether it is desirable for the antibody to mediate cytotoxicity / phagocytosis.

[0142] In one embodiment, the antibodies or antigen-binding fragments thereof according to the invention bind with high affinity to FcγR, preferably to activating receptors.

[0143] In one embodiment, an antibody or antigen-binding fragment thereof according to the invention binds with high affinity to FcγRI and / or FcγRIIa and / or FcγRIIc and / or FcγRIIIa and / or FcγRIIIb.

[0144] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is an IgG1 antibody (preferably a human IgG1 antibody) or a fragment thereof and binds to at least one Fc activating receptor. For example, the antibody or antigen-binding fragment thereof may bind to one or more receptors selected from FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb. In one embodiment, the antibody or antigen-binding fragment thereof can bind to FcγRIIIa. In one embodiment, the antibody or antigen-binding fragment thereof can bind to FcγRIIa. In one embodiment, the antibody or antigen-binding fragment thereof can bind to FcγRIIIa, FcγRIIc, and optionally FcγRI. In one embodiment, the antibody or antigen-binding fragment thereof can bind to FcγRIIIa, FcγRIIa, and optionally FcγRI.

[0145] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention has a binding affinity of about 10 to at least one activating Fcγ receptor. -6 μ, 10 -7 M, 10 -8 μ, 10 -9 M or 10 -10 It binds with a dissociation constant less than M.

[0146] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is an IgG1 antibody (preferably a human IgG1 antibody) or fragment thereof, which binds to FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb with higher affinity than it binds to FcγRIIb with lower affinity.

[0147] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention induces antibody-dependent cellular cytotoxicity (ADCC). In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is from the IgG1 (preferably human IgG1) subclass and has ADCC activity.

[0148] The term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to cell-mediated cytotoxicity induced in an antibody-dependent manner upon binding of the Fc region of an antigen-bound antibody to Fc receptors of effector cells, such as natural killer cells, macrophages, neutrophils, eosinophils, and mononuclear cells (e.g., peripheral blood mononuclear cells), thereby resulting in lysis of the target cells. ADCC can be measured using assays known and available in the art (e.g., Clynes et al. (1998) Proc Natl Acad Sci USA 95, 652-6).

[0149] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention induces antibody-dependent cellular phagocytosis (ADCP). In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is from the IgG1 (preferably human IgG1) subclass and has ADCP activity.

[0150] The term "antibody-dependent cellular phagocytosis" (ADCP) or "opsonization" refers to a cellular reaction in which non-specific cytotoxic cells expressing Fc receptors (FcR) (e.g., phagocytes, macrophages) recognize an antibody bound to a target cell and induce phagocytosis of the target cell. ADCP can be measured using assays known and available in the art (e.g., Clynes et al. (1998) Proc Natl Acad Sci USA 95, 652-6).

[0151] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention induces complement-dependent cytotoxicity (CDC). In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is derived from the IgG1 (preferably human IgG1) subclass and has CDC activity.

[0152] The term "complement dependent cytotoxicity" (CDC) refers to the induction of lysis of cells expressing an antigen recognized by an antibody or antigen-binding fragment thereof of the invention in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. CDC can be measured using assays known and available in the art (e.g., Clynes et al. (1998) Proc Natl Acad Sci USA 95, 652-6; Gazzano-Santaro et al., J. Immunol. Methods, 202:163 (1996)).

[0153] In one embodiment, an antibody or antigen-binding fragment thereof according to the invention comprises an Fc region that mediates ADCC, ADCP and / or CDC.

[0154] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention does not induce ADCC, ADCP and / or CDC.

[0155] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention does not comprise an Fc region that mediates ADCC, ADCP and / or CDC.

[0156] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention lacks an Fc domain (e.g., H 2 and / or C H 3 domain), or comprises the Fc domain of an IgG2 or IgG4 isotype (preferably human IgG2 or IgG4).

[0157] Antibodies or antigen-binding fragments thereof according to the invention can be produced by any technique known in the art, including but not limited to any chemical, biological, genetic, or enzymatic technique (either alone or in combination).

[0158] Therefore, another object of the present invention is a method for producing and purifying the antibodies or antigen-binding fragments thereof described herein.

[0159] In one embodiment, the method comprises: - introducing a recombinant nucleic acid or vector described herein into a competent host cell in vitro or ex vivo; - culturing in vitro or ex vivo a host cell transformed with the nucleic acid or expression vector under conditions suitable for expression of the antibody or antigen-binding fragment thereof; - optionally selecting cells which express and / or secrete said antibody or fragment thereof; and - recovering the expressed antibody or antigen-binding fragment thereof. Includes.

[0160] This recombinant process can be used for large-scale production of antibodies or antigen-binding fragments thereof, including monoclonal antibodies, intended for therapeutic use in vitro, ex vivo and / or in vivo.

[0161] In one embodiment, the expressed antibody or antigen-binding fragment thereof is further purified.

[0162] Methods for purifying antibodies or antigen-binding fragments thereof are well known in the art and include, but are not limited to, protein A-sepharose, gel electrophoresis, chromatography (preferably by affinity chromatography, more preferably by affinity chromatography with protein L agarose).

[0163] It will also be appreciated that the antibodies or antigen-binding fragments thereof according to the invention can be modified using methods known in the art.

[0164] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is an engineered antibody or fragment thereof.

[0165] The modified antibodies or fragments thereof according to the invention may be modified, for example, by modifying the V H and / or V LThese framework modifications include those in which modifications have been made to framework residues within the framework region. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence, for example, by site-directed mutagenesis or PCR mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the present invention. Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in more detail in U.S. Patent Publication No. 20030153043 by Carr et al.

[0166] In one embodiment, an antibody or antigen-binding fragment thereof according to the invention is modified to elicit an enhanced, increased, or improved ADCC, ADCP, and / or CDC response.

[0167] Methods for increasing ADCC, ADCP and / or CDC are well known in the art. For example, ADCC can be increased by methods that eliminate fucose moieties from antibody glycans, such as producing antibodies in YB2 / 0 cell lines, or by introducing specific mutations in the Fc portion of human IgG1 (e.g., S298A / E333A / K334A, S239D / I332E / A330L, G236A / S239D / A330L / I332E) (Lazar et al. (2006) Proc Natl Acad Sci USA 103, 2005-2010; Smith et al. (2012) Proc Natl 25 Acad Sci USA 109, 6181-6). ADCP can also be increased by introducing specific mutations in the Fc portion of human IgG1 (Richards et al. (2008) Mol Cancer Ther 7, 2517-27). CDC responses can be increased by antibody mutations that increase the affinity of C1q binding (Idusogie et al. (2001) J Immunol 166, 2571-5).

[0168] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is modified to elicit a decreased ADCC, ADCP, and / or CDC response.

[0169] Methods for reducing or eliminating ADCC, ADCP and / or CDC are also well known in the art. For example, ADCC may be reduced or eliminated by methods that modify the glycosylation profile of the Fc domain of immunoglobulins. CDC can be reduced or eliminated by replacing one or more amino acids with other amino acids such that the antibody has altered C2q binding (Idusogie et al., U.S. Patent No. 6,194,551).

[0170] As used herein, the terms "enhanced, increased, or improved ADCC, ADCP, and / or CDC response" and "reduced ADCC, ADCP, and / or CDC response" refer to the ADCC, ADCP, and / or CDC response induced by a modified antibody or fragment thereof according to the present invention compared to the ADCC, ADCP, and / or CDC response induced using other anti-CD160-TM antibodies, such as unmodified anti-CD160-TM monoclonal antibodies.

[0171] In one embodiment, an antibody or antigen-binding fragment thereof according to the invention is modified to modify its glycosylation.

[0172] For example, the antibody or fragment thereof according to the invention is aglycosylated (i.e., the antibody or fragment thereof lacks glycosylation). Glycosylation may be altered, for example, to increase the affinity of the antibody for the antigen or to alter the ADCC activity of the antibody. Such glycosylation can be achieved, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions may be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such glycosylation may increase the affinity of the antibody for the antigen. Additionally or alternatively, antibodies may be generated that have an altered type of glycosylation, for example, hypofucosylated or non-fucosylated antibodies with reduced amounts or no fucosyl residues, or antibodies with increased bisecting GlcNac structures. Such alterations in fucosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such glycosylation can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. Alternatively, antibodies of the invention (preferably monoclonal antibodies) can be produced in yeast or filamentous fungi that have been engineered for a mammalian-like glycosylation pattern and are capable of producing antibodies lacking fucose as a glycosylation pattern.

[0173] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is a pegylated antibody or fragment thereof.

[0174] An antibody or fragment thereof can be PEGylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To PEGylate an antibody or fragment thereof, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), e.g., a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. PEGylation may be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, e.g., mono (DY12- DY120) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol maleimide. In certain embodiments, the antibody to be PEGylated is an aglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies or fragments according to the invention, e.g., methods described in EP0154316 and EP0401384 (herein incorporated by reference).

[0175] The present invention further relates to fusion proteins comprising the antibodies or antigen-binding fragments described above.

[0176] The present invention further relates to the antibodies or antigen-binding fragments described above which are conjugated.

[0177] In one embodiment, the antibody or antigen-binding fragment according to the invention is conjugated to a therapeutic moiety, i.e., a drug.

[0178] The therapeutic moiety may be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as "antibody-drug conjugates" or "ADCs."

[0179] Examples of radioisotopes include: 90 Y, 131 I, or67 These include, but are not limited to, Cu.

[0180] In one embodiment, the antibody or antigen-binding fragment according to the invention is conjugated to a cytotoxic moiety.

[0181] Cytotoxic moieties include, for example, taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxyanthracin dione; tubulin inhibitors, such as maytansine or an analogue or derivative thereof; mitosis inhibitors, such as monomethyl auristatin E or F or an analogue or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; calichease. antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; alkylating agents such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; platinum derivatives such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, rachelmycin. (CC-1065);Antibiotics such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC);Pyrrolo[2,1-c][1,4]benzodiazepines (PDB);Diphtheria toxin and related molecules, such as diphtheria A chain and active fragments and hybrid molecules thereof, ricin toxin, such as ricin A or deglycosylated ricin A chain toxin, cholera toxin, shiga-like toxins, such as SLT I, SLT II, ​​SLT IIV, LT toxin, C3 toxin, shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas aeruginosa exotoxin, alorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (e.g., PAPI, PAPII, and PAP-S), momordica charantia inhibitors, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins; ribonuclease (RNase); DNase I, Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtherin toxin; and Pseudomonas aeruginosa endotoxin.

[0182] In one embodiment, the antibody or antigen-binding fragment according to the invention is conjugated to a cell-killing cytotoxic drug (payload) selected from the group including or consisting of ozogamicin, vedotin, emtansine, deruxtecan, govitecan, mafodotin, pasudotox, and tesirin.

[0183] In one embodiment, the antibody or antigen-binding fragment according to the invention is conjugated to a detectable label.

[0184] Labels that can be used with the anti-CD160-TM antibodies, or antigen-binding fragments thereof, of the invention include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials.

[0185] Examples of such enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase.

[0186] Examples of prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin.

[0187] Examples of fluorescent substances include, but are not limited to, organic molecules, such as fluorescent proteins or fluorescent small organic molecules, and inorganic molecules, including, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyne chloride, phycoerythrin, green fluorescent protein (GFP) and its derivatives, or quantum dots.

[0188] Examples of luminescent materials include, but are not limited to, luminal.

[0189] An example of a magnetic agent is gadolinium, and an example of a suitable radioactive material is 125 I, 131 I, 35 S, or 3 Examples include H.

[0190] Techniques for conjugating molecules to antibodies or antigen-binding fragments thereof are well known in the art. Typically, nucleic acid molecules are covalently linked to lysines or cysteines of antibodies or fragments thereof through N-hydroxysuccinimide esters or maleimide functional groups, respectively. Conjugation methods using engineered cysteines or the incorporation of unnatural amino acids have been reported to improve the homogeneity of conjugates.

[0191] The present invention further relates to the above described bispecific antibodies or antigen-binding fragments.

[0192] The present invention further relates to the above described antibodies or antigen-binding fragments which are not conjugated.

[0193] Another object of the invention is an isolated nucleic acid encoding the above described antibody or antigen-binding fragment thereof, or a fusion protein.

[0194] "Isolated nucleic acid", as used herein, is intended to refer to a nucleic acid that is substantially separated from other genomic DNA sequences and from proteins or complexes, such as ribosomes and polymerases, that naturally accompany the native sequence. The term encompasses a nucleic acid sequence that has been removed from its naturally occurring environment, including recombinant or cloned DNA isolates, and chemically synthesized analogs or biologically synthesized analogs by heterologous systems. A substantially pure nucleic acid includes a nucleic acid in an isolated form. Of course, this refers to the nucleic acid as originally isolated, and does not exclude genes or sequences that are later added to the isolated nucleic acid by the hand of man.

[0195] In one embodiment, the isolated nucleic acid is purified.

[0196] In one embodiment, the isolated nucleic acid comprises: (1) By absorbance or fluorescence method (for example, the ratio of absorbance at 260 and 280 nm (A 260 / 280 ), greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95% or more, most preferably greater than 96%, greater than 97%, greater than 98%, or greater than 99% by weight of the nucleic acids; (2) homogeneity as demonstrated by agarose gel electrophoresis and using intercalating agents such as ethidium bromide, SYBR Green, GelGreen, etc. It is refined up to

[0197] In one embodiment, the nucleic acid encodes at least the heavy chain variable region or the light chain variable region of an antibody or antigen-binding fragment thereof according to the invention.

[0198] In one embodiment, the nucleic acid encodes the variable and constant regions of an antibody or antigen-binding fragment thereof according to the invention.

[0199] In one embodiment, the nucleic acid may encode the heavy and light chains of the antibody or antigen-binding fragment thereof on separate nucleic acids or on the same nucleic acid molecule.

[0200] In one embodiment, the nucleic acid comprises or consists of a sequence encoding the VH of an antibody or antigen-binding fragment thereof according to the invention.

[0201] In one embodiment, the nucleic acid comprises or consists of a sequence encoding the VH of an antibody or antigen-binding fragment thereof according to the invention, said sequence comprising or consisting of SEQ ID NO: 17, SEQ ID NO: 19, and any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NOs: 17 and 19. SEQ ID NO:17

[0202] [ka]

[0203] SEQ ID NO:19

[0204] [ka]

[0205] In one embodiment, the nucleic acid comprises or consists of a sequence encoding the VL of an antibody or antigen-binding fragment thereof according to the invention.

[0206] In one embodiment, the nucleic acid comprises or consists of a sequence encoding the VL of an antibody or antigen-binding fragment thereof according to the invention, said sequence comprising or consisting of SEQ ID NO: 18, SEQ ID NO: 20, and any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NOs: 18 and 20. SEQ ID NO:18

[0207] [ka]

[0208] SEQ ID NO:20

[0209] [ka]

[0210] In one embodiment, the nucleic acid according to the invention comprises - a sequence encoding the VH of an antibody or antigen-binding fragment thereof according to the invention; and a sequence encoding the VL of an antibody or antigen-binding fragment thereof according to the invention; It comprises or consists of.

[0211] In one embodiment, the nucleic acid according to the invention comprises - a VH coding sequence comprising or consisting of the sequence SEQ ID NO: 17; and - a sequence encoding a VL comprising or consisting of the sequence SEQ ID NO: 18 It comprises or consists of.

[0212] In one embodiment, the nucleic acid encodes the VH and VL of the 21C8 antibody.

[0213] In one embodiment, the nucleic acid according to the invention comprises - a VH coding sequence comprising or consisting of the sequence SEQ ID NO: 19; and - a sequence encoding a VL comprising or consisting of the sequence SEQ ID NO: 20 It comprises or consists of.

[0214] In one embodiment, the nucleic acid encodes the VH and VL of the 22B12 antibody.

[0215] In one embodiment, the nucleic acid according to the invention comprises a fully or substantially fully human CDNA encoding an antibody or antigen-binding fragment thereof according to the invention. H and / or C. L In such embodiments, the constant region may be derived from any human antibody constant region.

[0216] In one embodiment, the nucleic acid according to the invention comprises a complete or substantially complete mouse CDNA clone of an antibody or antigen-binding fragment thereof according to the invention. H and / or C. L In such embodiments, the constant region may be derived from any murine antibody constant region.

[0217] In one embodiment, the nucleic acid according to the invention comprises or consists of a sequence encoding the heavy chain of a chimeric antibody or antigen-binding fragment thereof according to the invention. In one embodiment, the nucleic acid according to the invention comprises or consists of a sequence encoding the light chain of a chimeric antibody or antigen-binding fragment thereof according to the invention.

[0218] In one embodiment, the nucleic acid according to the invention comprises or consists of a sequence encoding the heavy chain of a humanized antibody or antigen-binding fragment thereof according to the invention. In one embodiment, the nucleic acid according to the invention comprises or consists of a sequence encoding the light chain of a humanized antibody or antigen-binding fragment thereof according to the invention.

[0219] Typically, the nucleic acid is a DNA or RNA molecule that may be comprised in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0220] Therefore, another object of the invention is an expression vector comprising a nucleic acid as described above.

[0221] The terms "vector", "cloning vector" and "expression vector" refer to vehicles by which DNA or RNA sequences (e.g., foreign genes) can be introduced into a host cell to transform the host and promote expression (e.g., transcription and translation) of the introduced sequences. Such vectors can include regulatory elements, such as promoters, enhancers, terminators, etc., that result in or drive expression of the protein or antibody or antigen-binding fragment thereof when administered to a host. Examples of promoters and enhancers used in expression vectors for animal cells include SV40 early promoter and enhancer, Moloney murine leukemia virus LTR promoter and enhancer, immunoglobulin H chain promoter and enhancer, etc. Any expression vector for animal cells can be used as long as a gene encoding a protein, antibody or fragment thereof can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSG1 beta d2-4, etc. Other examples of plasmids include replicating plasmids containing an origin of replication, or integrating plasmids, such as pUC, pcDNA, pBR, etc. Other examples of viral vectors include adenovirus, retrovirus, herpes virus, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found in the art.

[0222] In one embodiment, an expression vector according to the invention comprises a sequence encoding the VH of an antibody or antigen-binding fragment thereof according to the invention operably linked to a regulatory element.

[0223] In one embodiment, an expression vector according to the invention comprises a sequence selected from the group consisting of or including SEQ ID NO:17, SEQ ID NO:19, and any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NOs:17 and 19, operably linked to a regulatory element.

[0224] In one embodiment, an expression vector according to the invention comprises a sequence encoding the VL of an antibody or antigen-binding fragment thereof according to the invention, operably linked to a regulatory element.

[0225] In one embodiment, an expression vector according to the invention comprises a sequence selected from the group consisting of or including SEQ ID NO:18, SEQ ID NO:20, and any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NOs:18 and 20, operably linked to a regulatory element.

[0226] In one embodiment, the expression vector according to the invention comprises: - a sequence encoding the VH of an antibody or antigen-binding fragment thereof according to the invention, operably linked to a regulatory element; and - a sequence encoding the VL of an antibody or antigen-binding fragment thereof according to the invention, operably linked to a regulatory element; Includes.

[0227] In one embodiment, the expression vector according to the invention comprises: - a VH coding sequence comprising or consisting of a sequence of SEQ ID NO: 17 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 17, and - a sequence encoding a VL comprising or consisting of the sequence of SEQ ID NO: 18 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 18, operably linked to a regulatory element; Includes.

[0228] In one embodiment, the expression vector according to the invention comprises: - a VH coding sequence comprising or consisting of a sequence of SEQ ID NO: 19 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 19, operably linked to a regulatory element; and - a sequence encoding a VL comprising or consisting of a sequence of SEQ ID NO: 20 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 20, operably linked to a regulatory element; Includes.

[0229] In one embodiment, the expression vector according to the invention comprises a C511 gene encoding an antibody or antigen-binding fragment thereof according to the invention, operably linked to a regulatory element. H The C H is any human antibody C H It can be derived from.

[0230] In one embodiment, the expression vector according to the invention comprises a C511 gene encoding an antibody or antigen-binding fragment thereof according to the invention, operably linked to a regulatory element. L The C L is any human antibody C L It can be derived from.

[0231] In one embodiment, the expression vector according to the invention comprises a C511 gene encoding an antibody or antigen-binding fragment thereof according to the invention, operably linked to a regulatory element. H The C H is any mouse antibody CH It can be derived from.

[0232] In one embodiment, the expression vector according to the invention comprises a C511 gene encoding an antibody or antigen-binding fragment thereof according to the invention, operably linked to a regulatory element. L The C L is any mouse antibody C L It can be derived from.

[0233] In one embodiment, the expression vector according to the invention comprises a sequence encoding the heavy chain of a chimeric antibody or antigen-binding fragment thereof according to the invention, operably linked to a regulatory element.

[0234] In one embodiment, the expression vector according to the invention comprises a sequence encoding the light chain of a chimeric antibody or antigen-binding fragment thereof according to the invention, operably linked to a regulatory element.

[0235] In one embodiment, an expression vector according to the invention comprises a sequence encoding the heavy chain of a humanized antibody or antigen-binding fragment thereof according to the invention, operably linked to a regulatory element.

[0236] In one embodiment, an expression vector according to the invention comprises a sequence encoding the light chain of a humanized antibody or antigen-binding fragment thereof according to the invention, operably linked to a regulatory element.

[0237] In one embodiment, the expression vector according to the invention is monocistronic.

[0238] "Monocistronic" means that a single nucleic acid is expressed on a single expression vector.

[0239] In one embodiment, the expression vector according to the invention is polycistronic.

[0240] By "polycistronic" is meant that at least two or more nucleic acids are expressed in a single expression vector.

[0241] Another object of the present invention is an isolated host cell comprising an expression vector as described above.

[0242] Said host cells can be used for the recombinant production of an antibody or antigen-binding fragment thereof according to the invention.

[0243] In one embodiment, the host cell is a prokaryotic, yeast, or eukaryotic cell, preferably a mammalian cell, such as monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), mouse myeloma cells SP2 / 0-AG14 (ATCC CRL 1581 ; ATCC CRL 8287) or NSO (HPA culture collections number 85110503), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), dog kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)), MRC 5 cells, FS4 cells, and a human hepatocellular carcinoma line (Hep G2), as well as DSM's The host cell may be a PERC-6 cell line. Suitable expression vectors for use in each of these host cells are also generally known in the art. It should be noted that the term "host cell" generally refers to a cultured cell line. In one embodiment, any human into which an expression vector encoding an antibody or antigen-binding fragment thereof according to the invention has been introduced is excluded from the definition of "host cell".

[0244] Another object of the invention is a composition comprising, consisting essentially of, or consisting of the antibody or antigen-binding fragment, nucleic acid, expression vector, or fusion protein described above.

[0245] Another object of the invention is a pharmaceutical composition comprising, consisting essentially of, or consisting of an antibody or antigen-binding fragment, a nucleic acid, an expression vector, or a fusion protein as described above, and at least one pharma- ceutically acceptable excipient.

[0246] As used herein, "consisting essentially of" with respect to a composition means that the antibody or antigen-binding fragment, nucleic acid, expression vector, or fusion protein is the only biologically active therapeutic or pharmaceutical agent within the composition.

[0247] The term "pharmaceutical acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The excipients do not produce adverse, allergic, or other untoward reactions when administered to animals, preferably mammals, more preferably humans. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by regulatory agencies, such as the FDA Office or EMA.

[0248] Examples of pharma- ceutically acceptable excipients that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (e.g. sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0249] In one embodiment, the pharmaceutical compositions according to the invention comprise pharma- ceutical acceptable vehicles for formulations that can be injected into a subject. These may be in particular isotonic sterile saline (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of such salts) or may be dry, in particular lyophilized, compositions that allow reconstitution of the injection, optionally upon addition of sterile water or saline.

[0250] Another object of the invention is a medicament comprising, consisting essentially of or consisting of an antibody or antigen-binding fragment, a nucleic acid, an expression vector, or a fusion protein as described above.

[0251] In one embodiment, the composition, pharmaceutical composition, or medicament according to the invention is formulated for administration to a subject.

[0252] In one embodiment, a composition, pharmaceutical composition, or medicament according to the invention is administered parenterally by inhalation spray, rectally, nasally, or via an implanted reservoir.

[0253] In one embodiment, the composition, pharmaceutical composition, or medicament is administered by injection, including but not limited to subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0254] Examples of forms suitable for injection include, but are not limited to, liquid solutions, such as sterile aqueous solutions, gels, dispersions, emulsions, suspensions, and the like, solid forms suitable for use in preparing a solution or suspension upon the addition of a liquid prior to use, such as powders, liposomal forms, and the like.

[0255] Sterile injectable forms of the compositions, pharmaceutical compositions, or medicaments of the present invention may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil (especially in their polyoxyethylated forms). These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose, or similar dispersants commonly used in the formulation of pharmaceutically acceptable forms, including emulsions and suspensions. Other commonly used surfactants such as Tweens, Spans, and other emulsifiers, or bioavailability enhancers commonly used in the manufacture of pharma- ceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes.

[0256] In one embodiment, an antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition, or medicament according to the invention is administered in a therapeutically effective amount to a subject in need thereof.

[0257] However, it will be understood that the total daily usage of the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition, or medicament according to the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disease and severity of the disease under treatment; the activity of the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, or expression vector used; the age, weight, general health, sex, and diet of the subject; the timing, route of administration, and excretion rate of the specific antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, or expression vector used; the duration of treatment; drugs used in conjunction with or simultaneously with the specific antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, or expression vector used; and similar factors well known in the medical art. For example, it is well within the skill of the artisan to start the dose of the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. The total dose required for each treatment may be administered by multiple doses or in a single dose.

[0258] The daily dosage of the antibody or antigen-binding fragment thereof according to the invention may vary over a wide range from 10 to 1000 mg per adult per day.

[0259] The present invention further relates to an antibody or antigen-binding fragment, a nucleic acid, an expression vector, or a fusion protein as described above for use as a medicament, i.e. for use in the treatment of a disease, disorder, or condition in a subject in need thereof.

[0260] The present invention further relates to a composition, pharmaceutical composition or medicament as described above for use as a medicament, i.e. for use in the treatment of a disease, disorder or condition in a subject in need thereof.

[0261] The present invention further relates to a method for treating a disease, disorder, or condition in a subject in need thereof, comprising administering to the subject the antibody or antigen-binding fragment, nucleic acid, expression vector, or fusion protein described above.

[0262] The present invention further relates to a method for treating a disease, disorder, or condition in a subject in need thereof, comprising administering to the subject the composition, pharmaceutical composition, or medicament described above.

[0263] The present invention further relates to the use of an antibody or antigen-binding fragment, nucleic acid, expression vector, or fusion protein as described above for the manufacture of a medicament for treating a disease, disorder, or condition in a subject in need thereof.

[0264] Diseases, disorders, or conditions that can be treated in the present invention include, but are not limited to, cancer, infectious diseases, inflammatory diseases, and / or autoimmune diseases. Another example of a disease, disorder, or condition that can be treated in the present invention is paroxysmal nocturnal hemoglobinuria.

[0265] As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer includes, but is not limited to, diseases of the skin, tissues, organs, bones, joints, blood, and blood vessels. The term "cancer" further encompasses both primary and metastatic cancers.

[0266] Examples of cancers that can be treated in the present invention include, but are not limited to, cancer cells originating from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gingiva, head, kidney, liver, lung, nasopharyngeal, cervical, ovary, prostate, skin, stomach, testis, tongue, endometrium, pancreas, or uterus.

[0267] Further, the cancer may be selected from the following non-limiting list: malignant neoplasm, undifferentiated carcinoma, giant cell and spindle cell carcinoma, small cell carcinoma, papillary carcinoma, squamous cell carcinoma, lymphoepithelial carcinoma, basal cell carcinoma, pilomatrix carcinoma, transitional cell carcinoma, papillary transitional cell carcinoma, adenocarcinoma, malignant gastrinoma, cholangiocarcinoma, hepatocellular carcinoma, mixed hepatocellular carcinoma and cholangiocarcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma in adenomatous polyps, adenocarcinoma associated with familial adenomatous polyposis. coli), solid tumor, malignant carcinoid tumor, bronchiolo alveolar adenocarcinoma, papillary adenocarcinoma, chromophobe carcinoma, eosinophilic carcinoma, eosinophilic adenocarcinoma, basophilic carcinoma, clear cell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma, papillary follicular adenocarcinoma, non-encapsulating sclerosing carcinoma carcinoma, adrenocortical carcinoma, endometroid carcinoma, skin adnexal carcinoma, apocrine gland carcinoma, sebaceous gland carcinoma, ear canal adenocarcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, invasive ductal carcinoma, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, Paget's disease of the breast, acinic cell carcinoma, adenosquamous carcinoma, adenocarcinoma with squamous metaplasia, malignant thymoma, malignant ovarian stromal tumor, malignant theca cell tumor, malignant granulosa cell tumor, malignant neuroblastomaroblastoma), Sertoli cell carcinoma, malignant Leydig cell tumor, malignant lipocytoma, malignant paraganglioma, malignant extramammary paraganglioma, pheochromocytoma, hemangiosarcoma, malignant melanoma, amelanotic melanoma, superficial spreading melanoma, giant pigmented intranevus malignant melanoma, epithelioid cell melanoma, malignant blue nevus, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma tumor, malignant mixed tumor, mixed Müllerian tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, malignant mesenchymoma, malignant Brenner tumor, malignant phyllodes tumor, synovial sarcoma, malignant mesothelioma, dysgerminoma, embryonal carcinoma, malignant teratoma, malignant ovarian thyroid tumor, choriocarcinoma, malignant mesothelioma, angiosarcoma, malignant hemangioendothelioma, Kaposi's sarcoma, malignant hemangiopericytoma, lymphangiosarcoma, osteosarcoma, parosteal osteosarcoma, chondrosarcoma, malignant chondroblastoma, mesenchymal chondrosarcoma, giant cell tumor of bone, Ewing's sarcoma, malignant odontogenic tumor, ameloblastoma Epithelial sarcoma, malignant ameloblastoma, ameloblastic fibrosarcoma, malignant pinealoma, chordoma, malignant glioma, ependymoma, astrocytoma, protoplasmic astrocytoma, fibrous astrocytoma, astroblastoma, glioblastoma, oligodendroglioma, oligodendroglioma, primitive neuroectodermal tumor, cerebellar sarcoma, ganglioblastoma, neuroblastoma, retinoblastoma, olfactory neurogenic tumor, malignant meningioma, neurofibrosarcoma, malignant neurilemmoma, malignant granular cell tumor, malignant lymphoma, Hodgkin's disease, Hodgkin's lymphoma, parametrial tumor blastoma, malignant small lymphocytic lymphoma, malignant diffuse large cell lymphoma, malignant follicular lymphoma, mycosis fungoides, other specified non-Hodgkin's lymphomas, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, and hairy cell leukemia.

[0268] In one embodiment, the cancer is breast cancer. Examples of subtypes of breast cancer that may be treated by the antibodies or antigen-binding fragments of the invention include, but are not limited to, luminal A breast cancer, luminal B breast cancer, triple-negative breast cancer (TNBC), and HER2-positive breast cancer.

[0269] As used herein, luminal A breast cancer is positive for estrogen receptor and / or progesterone receptor, negative for HER2, and has low levels of protein Ki-67 (also known as MKI67 or antigen KI-67). As used herein, luminal B breast cancer is positive for estrogen receptor and / or progesterone receptor, positive for HER2, and has high levels of Ki-67. As used herein, TNBC is negative for estrogen receptor and progesterone receptor, and negative for HER2. As used herein, HER2-positive breast cancer is negative for estrogen receptor and progesterone receptor, and positive for HER2.

[0270] In one embodiment, the cancer is a breast cancer selected from the group including or consisting of luminal A breast cancer, luminal B breast cancer, triple-negative breast cancer (TNBC), and HER2-positive breast cancer.

[0271] In one embodiment, the cancer is luminal A breast cancer. In one embodiment, the cancer is luminal B breast cancer. In one embodiment, the cancer is TNBC. In one embodiment, the cancer is HER2 positive breast cancer.

[0272] As used herein, the term "infectious disease" includes any infection caused by a virus, bacteria, protozoa, mold, or fungus.

[0273] In some embodiments, the viral infection comprises, but is not limited to, an infection with one or more viruses selected from the group including: Arenaviridae, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepevirus, Leviviridae, Luteoviridae, Mononegavirales, Mosaicvirus, Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, Tymoviridae, Hepadnaviridae, Herpesviridae, Paramyxoviridae, or Papillomaviridae viruses. Relevant taxonomic families of RNA viruses include, but are not limited to, Astroviridae, Birnaviridae, Bromoviridae, Caliciviridae, Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepeviruses, Leviviridae, Luteoviridae, Mononegavirales, Mosaicviruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnaviruses, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae, Tenuiviruses, Togaviridae, Tombusviridae, Totiviridae, and Tymoviridae viruses.

[0274] In some embodiments, the viral infection is caused by a virus, such as Adenovirus, Alfuy virus, Banzi virus, Bovine diarrhea virus, Coronavirus, Coxsackie virus, Crimean-Congo virus, Dengue virus, Ebola virus, Encephalitis virus (including Japanese encephalitis virus, California encephalitis virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, Eastern equine encephalitis virus, St. Louis encephalitis virus, Tick-borne encephalitis virus), Gunarito virus, Hantavirus, Hepatitis virus, Irjeus virus, Immunodeficiency virus, Influenza virus (including human, avian, and porcine) including Influenza A and Influenza B viruses, and Parainfluenza virus, Junin virus, Cocobera virus, Kunjin virus, Kyasanur Forest virus, La Crosse virus, Lassa virus, Louping ill virus, Lymphocytic choriomeningitis virus, Measles virus, Machupo virus, Marburg virus, Murray Valley virus, Patindae virus, The present invention relates to infection with one or more viruses, including, but not limited to, infection with one or more viruses selected from the group including: HIV, Pichinde virus, poliovirus, Powassan virus, Punta Toro virus, Respiratory syncytial virus, rhinovirus, Rift Valley fever virus, Rocio virus, Severe Acute Respiratory Syndrome (SARS), smallpox virus, Tacaribe virus, West Nile virus, and Yellow Fever virus.

[0275] Examples of bacterial infections that may be treated with the present invention include, but are not limited to, infections caused by Staphylococcus, Streptococcus (including S. pyogenes), Enterococcus, Bacillus (such as, for example, Bacillus anthracis), Lactobacillus, Listeria, Corynebacterium diphtheriae, Gardnerella (such as, for example, G. vaginalis, Nocardia, Streptomyces, Thermoactinomyces vulgaris, Treponema, Campylobacter, Pseudomonas (such as, for example, P. aeruginosa), Legionella, Neisseria (such as, for example, N. gonorrhoeae, and N. meningitidis). meningitides), Flavobacterium (e.g., Flavobacterium meningosepticum and Flavobacterium odoraturn), Brucella, Bordetella (e.g., B. pertussis and B. bronchiseptica), Escherichia (e.g., E. coli), Klebsiella, Enterobacter, Serratia (e.g., S. marcescens and S. liquefaciens), Edwardsiella, Proteus (e.g., P. mirabilis and P. vulgaris), Streptobacillus, Rickettsiaceae (e.g., R. fickettsfii), fickettsfi), Chlamydia (e.g., C. psittaci and C. trachomatis), Mycobacterium (e.g., M. tuberculosis, M. intracellulare, M. folluiturn, Mycobacterium leprae),laprae, Mycobacterium avium, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium kansasii, and Mycobacterium lepraernurium, and Nocardia.

[0276] Examples of protozoal infections that can be treated with the present invention include, but are not limited to, infections caused by leishmania, kokzidioa, and trypanosoma.

[0277] A complete list of infectious diseases can be found on the website of the National Center for Infectious Diseases (NCID) of the Centers for Disease Control and Prevention (CDC) (World Wide Web (www) at cdc.gov / ncidod / diseases / ), which list is incorporated herein by reference.

[0278] As used herein, the term "inflammatory disease" includes numerous disorders and conditions characterized by inflammation.

[0279] Examples of inflammatory diseases include arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, arthritis uratica, gout, chronic polyarthritis, periarthritis of the shoulder, cervical arthritis, lumbosacral arthritis, enteropathic arthritis, and ankylosing spondylitis, asthma, dermatitis, psoriasis, scleroderma, polymyositis, dermatomyositis, juvenile dermatomyositis, primary biliary cirrhosis, fibrosis, cystic fibrosis, pulmonary fibrosis, liver cirrhosis, endomyocardial fibrosis, dediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, nephrogenic fibrosis, and the like. fibrosis), keloids, scleroderma, arthrofibrosis, late post-transplant and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, pemphigus, pemphigus vulgaris, pemphigus herpetiformis, pemphigus vegetans, IgA pemphigus, pemphigus erythematosus, bullous pemphigoid, gestational pemphigoid, mucocutaneous diseases, pemphigoid nodularis, linear IgA bullous dermatosis, bullous lichen planus, epidermolysis bullosa acquisita, autoimmune diabetes, diabetic retinopathy, diabetic nephropathy, diabetic vasculopathy, ocular inflammation, uveitis, rhinitis, ischemia-reperfusion injury, restenosis after angioplasty, chronic obstructive pulmonary disease (COPD), glomerulonephritis, Graves' disease, gastrointestinal allergies, conjunctivitis, atherosclerosis, coronary artery disease, angina pectoris, arteriolar disease, acute These include, but are not limited to, disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, multiple sclerosis, systemic sclerosis, antiphospholipid syndrome, Sjogren's syndrome, autoimmune hemolytic anemia, colitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease (IBD), embolism, pulmonary embolism, arterial embolism, venous embolism, allergic inflammation, cardiovascular disease, graft-associated disease, graft-versus-host disease (GVHD), graft rejection, chronic rejection, and disorders associated with tissue or cell allografts or xenografts, autoimmune diseases, post-traumatic degeneration, stroke, transplant rejection, allergic conditions and hypersensitivity disorders, such as allergic rhinitis, allergic eczema, and the like, skin diseases, skin inflammatory disorders, or any combination thereof.

[0280] As used herein, the term "autoimmune disease" refers to a disease involving a defect in the immune system that leads to an immune response against one's own tissues.

[0281] Examples of autoimmune diseases include lupus (e.g., lupus erythematosus, lupus nephritis), Hashimoto's thyroiditis, Wegener's disease; primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, diabetes (e.g., insulin-dependent diabetes mellitus, type I diabetes, type II diabetes), Goodpasture's syndrome, myasthenia gravis, pemphigus, inflammatory conditions of the intestine (such as Crohn's disease and ulcerative colitis); sympathetic ophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, and chronic pulmonary hypertension. hepatitis), ulcerative colitis, Sjogren's syndrome, arthritic conditions (such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and juvenile idiopathic arthritis); polymyositis, scleroderma, psoriasis, primary sclerosing cholangitis; asthma, transplant rejection (host-versus-graft disease); graft-versus-host disease, and mixed connective tissue disease.

[0282] As used herein, the term "paroxysmal nocturnal hemoglobinuria" or "PNH" has its common meaning in the art and refers to an acquired clonal hematopoietic stem cell disorder characterized by corpuscular hemolytic anemia, bone marrow failure, and frequent thrombotic events. In some embodiments, the subject does not have a mutation in the PIGA gene (phosphatidylinositol glycan anchor biosynthesis class A, gene ID: 5277).

[0283] It will be appreciated that the treatment of a disease, disorder, or condition may be mediated by different mechanisms, depending on the effector functions of the antibody or antigen-binding fragment thereof according to the invention.

[0284] In one embodiment, the antibodies or antigen-binding fragments thereof according to the invention mediate ADCC, ADCP and / or CDC, allowing them to target and deplete CD160-TM expressing cells to which they are bound.

[0285] In one embodiment, administration of the antibody or fragment thereof may result in depletion of cells expressing CD160-TM, e.g., CD160-TM-expressing tumor cells (e.g., resulting in elimination or reduction in the number of CD160-TM+ NK cells by 10%, 20%, 30%, 40%, 50%, 60% or more).

[0286] As used herein, the term "deplete" refers to a measurable reduction in the number of cells in a subject with respect to a cell or population of cells. The reduction may be at least about 10%, e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the term refers to a reduction in the number of cells in a subject or in a sample to an amount below the detectable limit.

[0287] Thus, a further object of the present invention relates to a method for depleting a population of cells expressing the CD160-TM isoform in a subject in need thereof, comprising the step of delivering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, wherein said antibody or fragment thereof mediates ADCC, ADCP and / or CDC.

[0288] A further object of the present invention relates to a method for depleting a population of malignant NK cells expressing the CD160-TM isoform in a subject in need thereof, comprising the step of delivering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, wherein said antibody or fragment thereof mediates ADCC, ADCP and / or CDC.

[0289] A further object of the present invention relates to a method for depleting a population of cells expressing the epitope recognized by the 21C8 or 22B12 antibody in a subject in need thereof, comprising the step of delivering to the subject a therapeutically effective amount of an antibody or an antigen-binding fragment thereof as described above, wherein said antibody or fragment thereof mediates ADCC, ADCP and / or CDC.

[0290] A further object of the invention relates to a method for treating cancer in a subject in need thereof, in which the cancer cells express CD160-TM, comprising the step of delivering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, wherein said antibody or fragment thereof mediates ADCC, ADCP and / or CDC.

[0291] A further object of the present invention relates to an antibody or antigen-binding fragment thereof as described above for use in the treatment of cancer in which the cancer cells express CD160-TM in a subject in need thereof, said antibody or antigen-binding fragment thereof mediating ADCC, ADCP and / or CDC.

[0292] In one embodiment, the cancer is a solid tumor. In one embodiment, the cancer is a liquid tumor, i.e., cancer cells are present in bodily fluids such as blood or bone marrow.

[0293] Examples of cancers in which cancer cells express CD160-TM include, but are not limited to, NK leukemia or NK lymphoma, such as extranodal and non-extranodal NK / T lymphoma; NK cell-derived malignancies; acute NK leukemia, and peripheral T cell lymphoma (PTCL), such as peripheral T cell lymphoma not otherwise specified (PTCL-NOS).

[0294] Examples of cancers in which the cancer cells express CD160-TM include, but are not limited to, breast cancer, such as luminal A breast cancer, luminal B breast cancer, triple-negative breast cancer (TNBC), and HER2-positive breast cancer.

[0295] Thus, in one embodiment, the cancer is breast cancer. In one embodiment, the cancer is a breast cancer selected from the group including or consisting of luminal A breast cancer, luminal B breast cancer, triple-negative breast cancer (TNBC), and HER2-positive breast cancer.

[0296] In one embodiment, the cancer is luminal A breast cancer. In one embodiment, the cancer is luminal B breast cancer. In one embodiment, the cancer is TNBC. In one embodiment, the cancer is HER2 positive breast cancer.

[0297] Another object of the invention is an antibody or fragment thereof that binds to human CD160-TM for use in the treatment of PTCL, such as peripheral T-cell lymphoma non-specific (PTCL-NOS).

[0298] In one embodiment, the antibodies or antigen-binding fragments thereof according to the invention do not mediate ADCC, ADCP and / or CDC, allowing them to target and activate CD160-TM expressing cells to which they are bound.

[0299] In one embodiment, administration of the antibody or fragment thereof does not directly or indirectly result in depletion of NK cells that express CD160-TM polypeptides (e.g., does not result in elimination or reduction in the number of CD160-TM+ NK cells by 10%, 20%, 30%, 40%, 50%, 60% or more).

[0300] In one embodiment, administration of the antibody or fragment thereof results in enhanced or improved NK cell activity or NK cell effector function.

[0301] Therefore, a further object of the present invention relates to a method for enhancing NK cell activity or NK cell effector function, in particular NK cell killing activity, in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, wherein said antibody or fragment thereof does not mediate ADCC, ADCP and / or CDC.

[0302] As used herein, "NK cells" refers to a subpopulation of lymphocytes involved in innate or non-conventional immunity. NK cells can be identified by certain characteristics and biological properties, such as the expression of specific surface antigens, including CD56 and / or CD16 for human NK cells, the absence of alpha / beta or gamma / delta TCR complexes on the cell surface, the ability to bind to and kill cells that do not express "self" MHC / HLA antigens by activation of specific cytolytic mechanisms, the ability to kill tumor cells or other pathological cells that express ligands for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or suppress immune responses ("NK cell activity"). Any subpopulation of NK cells is encompassed by the term NK cells. Within the context of the present invention, "active" NK cells refer to biologically active NK cells, including NK cells that have the ability to lyse target cells or enhance the immune function of other cells. For example, "active" NK cells can kill cells that express ligands for activating NK receptors and / or cells that do not express MHC / HLA antigens recognized by the KIR of the NK cells.

[0303] The ability of an antibody or antigen-binding fragment according to the invention to enhance NK cell activity, in particular NK cell killing activity, may be determined by any assay known in the art. Typically, said assay is an in vitro assay in which NK cells are contacted with target cells (e.g., target cells to be recognized and / or lysed by NK cells). For example, an antibody or antigen-binding fragment may be selected for its ability to increase specific lysis by NK cells by more than about 20%, preferably at least about 30%, at least about 40%, at least about 50% or more of the specific lysis obtained at the same effector:target cell ratio with an NK cell or NK cell line contacted by an antibody or fragment thereof according to the invention. Typically, NK cell cytotoxicity may be determined by any of the assays described in the Examples part, such as i) an in vitro assay evaluating NK cell-induced lysis of target cells using viability dye staining, or ii) an in vitro assay evaluating the expression of NK cell activation markers, such as CD107a expression. NK cell cytotoxicity may also be measured by a classical in vitro chromium release test of cytotoxicity. Effector cells may be fresh PB-NK or NK92 cell lines from healthy donors. Target cells may be mouse mastocytoma P815 cells, EBV-infected B cell lines, or other lines such as K562 cells.

[0304] Thus, an antibody or antigen-binding fragment according to the invention is selected if it results in increased NK cell reactivity or cytotoxicity against target cells (infected cells, tumor cells, pro-inflammatory cells, etc.), increased activation, activation markers (e.g., CD107 expression) and / or IFN-gamma production in NK cells, and / or an increase in the in vivo frequency of such activated reactivity, cytotoxicity and / or activated NK cells.

[0305] A further object of the present invention relates to an antibody or antigen-binding fragment thereof as described above, for use in the treatment of a cancer in a subject in need thereof, whose cancer cells do not express CD160-TM, wherein said antibody or antigen-binding fragment thereof does not mediate ADCC, ADCP and / or CDC.

[0306] A further object of the present invention relates to a method for treating cancer in a subject in need thereof, whose cancer cells do not express CD160-TM, comprising the step of delivering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, wherein said antibody or fragment thereof does not mediate ADCC, ADCP and / or CDC.

[0307] Examples of cancer are provided above.

[0308] A further object of the present invention relates to an antibody or an antigen-binding fragment thereof as described above, which does not mediate ADCC, ADCP and / or CDC, for use in the treatment of an infection in a subject in need thereof.

[0309] Examples of infections are provided above.

[0310] A further object of the present invention relates to an antibody or an antigen-binding fragment thereof as described above, which does not mediate ADCC, ADCP and / or CDC, for use in the treatment of an inflammatory disease in a subject in need thereof.

[0311] Examples of inflammatory diseases are provided above.

[0312] A further object of the present invention relates to an antibody or an antigen-binding fragment thereof as described above, which does not mediate ADCC, ADCP and / or CDC, for use in the treatment of an autoimmune disease in a subject in need thereof.

[0313] Examples of autoimmune diseases are provided above.

[0314] A further object of the present invention relates to a method for treating an infectious, inflammatory and / or autoimmune disease in a subject in need thereof, comprising the step of delivering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, wherein said antibody or fragment thereof does not mediate ADCC, ADCP and / or CDC.

[0315] A further object of the present invention relates to an antibody or an antigen-binding fragment thereof as described above, which does not mediate ADCC, ADCP and / or CDC, for use in the treatment of paroxysmal nocturnal hemoglobinuria (PNH) in a subject in need thereof.

[0316] A further object of the present invention relates to a method for treating PNH in a subject in need thereof, comprising the step of delivering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, wherein said antibody or fragment thereof does not mediate ADCC, ADCP and / or CDC.

[0317] In one embodiment, the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, or expression vector according to the invention is the only active or therapeutic agent administered to a subject in need thereof.

[0318] In one embodiment, the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, or expression vector according to the invention is used in combination with at least one additional therapeutic agent.

[0319] In one embodiment, the administration of at least one further therapeutic agent and the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, or expression vector according to the invention is simultaneous, separate or sequential.

[0320] In one embodiment, for simultaneous administration, at least one additional therapeutic agent and the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, or expression vector according to the invention are administered as one composition or as separate compositions, as appropriate.

[0321] In one embodiment, the at least one additional therapeutic agent is a therapeutic agent useful in the treatment of a disease, disorder, or condition described above.

[0322] Examples of additional therapeutic agents include, but are not limited to, other immunosuppressants and / or anti-inflammatory agents selected from chemotherapeutic agents, targeted cancer therapies, radiation therapy, immunotherapeutic agents or anti-cancer immunogens, anti-cancer antibodies, cytotoxic agents, anti-angiogenic agents, cell cycle control / apoptosis modulating agents, hormone modulating agents, and corticoids, e.g., glucocorticoids.

[0323] In one embodiment, the at least one additional therapeutic agent is a natural ligand of an NK cell activating receptor or an antibody that binds to and activates an NK cell activating receptor other than CD160-TM. In one embodiment, the agent is an agent that increases the presence of a natural ligand of the NK cell activating receptor on the surface of a target cell (e.g., an infected cell or a tumor cell).

[0324] In one embodiment, in which the antibody or antigen-binding fragment thereof does not mediate ADCC, CDC and / or ADCP, the antibody or fragment thereof is used in combination with (i) an additional therapeutic agent that is a natural ligand of an NK cell activating receptor other than CD160-TM or an antibody that binds to and activates an NK cell activating receptor other than CD160-TM, and / or (ii) an additional therapeutic agent that increases the presence of a natural ligand of an NK cell activating receptor on the surface of target cells.

[0325] NK cell activating receptors include, for example, NKG2D or activating KIR receptors (KIR2DS receptors, KIR2DS2, KIR2DS4).

[0326] As used herein, the term "activating NK receptor" refers to any molecule on the surface of an NK cell that, upon stimulation, results in any property or activity known in the art to be associated with NK activity, such as a measurable increase in cytokine (e.g., IFN-γ and TNF-α) production, an increase in intracellular free calcium levels, an increased ability to target cells in a redirect killing assay as described (e.g., elsewhere herein), or an increased ability to stimulate NK cell proliferation. The term "activating NK receptor" includes, but is not limited to, activated forms or KIR proteins (e.g., KIR2DS proteins), NKG2D, IL-2R, IL-12R, IL-15R, IL-18R, and IL-21R. Examples of ligands that act as agonists at activating receptors include, for example, IL-2, IL-15, IL-21 polypeptides.

[0327] In one embodiment, the at least one additional therapeutic agent is an antibody specific for CD137. As used herein, the term "CD137" has its general meaning in the art and may also be referred to as Ly63, ILA, or 4-1BB. CD137 is a member of the tumor necrosis factor (TNF) receptor family. Members of this receptor family and their structurally related ligands are important regulators of a wide variety of physiological processes and play a key role in regulating immune responses. CD137 is expressed by activated NK cells, T and B lymphocytes, and monocytes / macrophages. The gene encodes a 255 amino acid protein that includes three cysteine-rich motifs in the extracellular domain (characteristic of this receptor family), a transmembrane region, and a short N-terminal cytoplasmic portion that contains potential phosphorylation sites. Expression in primary cells is entirely activation-dependent. The ligand for the receptor is TNFSF9. Human CD137 has been reported to bind only to its ligand. Agonists include natural ligands (TNFSF9), aptamers (see McNamara et al. (2008) J. Clin. Invest. 1 18: 376-386), and antibodies.

[0328] In one embodiment, the at least one additional therapeutic agent is an antibody that induces death by ADCC of cells expressing the antigen to which said antibody binds.

[0329] In one embodiment in which an antibody or antigen-binding fragment thereof according to the invention does not mediate ADCC, ADCP and / or CDC, said antibody or fragment thereof is used in combination with an antibody that induces, by ADCC, the death of a cell expressing the antigen to which said antibody binds.

[0330] In one embodiment, the second antibody is specific for a cell surface antigen, for example a membrane antigen.

[0331] In some embodiments, the second antibody is specific for a tumor antigen (e.g., a molecule specifically expressed by a tumor cell), such as CD20, CD52, ErbB2 (or HER2 / Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, αVβ3, etc., in particular a lymphoma antigen (e.g., CD20).

[0332] Thus, the present invention also provides a method for enhancing the antitumor effect of a monoclonal antibody against a tumor antigen, in which sequential administration of an antibody against one or more tumor antigens and an antibody or antigen-binding fragment according to the invention specifically enhances ADCC function, which in turn enhances target cell killing.

[0333] Therefore, a further object of the present invention relates to a method for enhancing NK cell ADCC of an antibody in a subject in need thereof, comprising the steps of administering said antibody to the subject, and administering to the subject an antibody or an antigen-binding fragment according to the invention, preferably wherein the antibody or fragment thereof according to the invention does not mediate ADCC, ADCP and / or CDC.

[0334] A further object of the present invention relates to a method for treating cancer in a subject in need thereof, comprising the steps of administering to the subject a first antibody selective for a cancer cell antigen and administering to the subject an antibody or antigen-binding fragment according to the invention, preferably wherein the antibody or fragment thereof according to the invention does not mediate ADCC, ADCP and / or CDC.

[0335] Many antibodies are currently in clinical use for the treatment of cancer, and others are in various stages of clinical development. Although the antibodies of interest for the methods of the present invention act through ADCC and are typically selective for tumor cells, those skilled in the art will recognize that some clinically useful antibodies act on non-tumor cells, such as CD20. There are many antigens and corresponding monoclonal antibodies for treating B-cell malignancies. One common target antigen is CD20, which is found in B-cell malignancies. Rituximab is a chimeric, non-conjugated monoclonal antibody against the CD20 antigen. CD20 has an important functional role in B-cell activation, proliferation, and differentiation. The CD52 antigen is targeted by the monoclonal antibody alemtuzumab, which is indicated for the treatment of chronic lymphocytic leukemia. CD22 is targeted by many antibodies, and has recently demonstrated efficacy in combination with toxins in chemotherapy-resistant hairy cell leukemia. Monoclonal antibodies targeting CD20 also include tositumomab and ibritumomab. Monoclonal antibodies useful in the methods of the invention that have been used in solid tumors include, but are not limited to, edrecolomab and trastuzumab (Herceptin). Edrecolomab targets the 17-1 A antigen found in colon and rectal cancers and is approved for use in Europe for these indications. Its antitumor effects are mediated through induction of ADCC, CDC, and anti-idiotypic networks. Trastuzumab targets the HER-2 / neu antigen. This antigen is found in 25%-35% of breast cancers. Trastuzumab is believed to function in a variety of ways, namely, downregulating HER-2 receptor expression, inhibiting the growth of human tumor cells that overexpress the HER-2 protein, enhancing immune recruitment and ADCC against tumor cells that overexpress the HER-2 protein, and downregulating angiogenic factors. Alemtuzumab (Campus) is used in the treatment of chronic lymphocytic leukemia, colon cancer and lung cancer, gemtuzumab (Mylotarg) finds use in the treatment of acute myeloid leukemia, ibritumomab (Zevalin) finds use in the treatment of non-Hodgkin's lymphoma, and panitumumab (Vectibix) finds use in the treatment of colon cancer.Cetuximab (Erbitux) is also an antibody of interest for use in the methods of the invention. The antibody binds to the EGF receptor (EGFR) and has been used in the treatment of solid tumors, including colon cancer and squamous cell carcinoma of the head and neck (SCCHN).

[0336] In one embodiment, the at least one additional therapeutic agent is an immune checkpoint inhibitor (ICI).

[0337] Various tumors can express molecular factors that protect them from attacks by the immune system, and thus successfully evade the surveillance control of the immune system. This "tumor immune evasion" is mainly due to the competitive blockade of receptors and binding sites targeted by immune cell ligands. Immune checkpoint inhibitors are molecules that specifically target this type of suppressive mechanism generated by tumor cells.

[0338] Examples of ICIs include, but are not limited to, inhibitors of CTLA-4 (e.g., ipilimumab and tremelimumab), inhibitors of PD-1 (e.g., pembrolizumab, pidilizumab, nivolumab, and AMP-224), inhibitors of PD-L1 (e.g., atezolizumab, avelumab, durvalumab, and BMS-936559), inhibitors of LAG3 (e.g., IMP321), and inhibitors of B7-H3 (e.g., MGA271).

[0339] In one embodiment, where the antibody or antigen-binding fragment thereof according to the invention does not mediate ADCC, ADCP and / or CDC, said antibody or fragment thereof is used in combination with at least one immune checkpoint inhibitor (ICI).

[0340] The present invention also relates to the use of at least one antibody according to the invention or an antigen-binding fragment thereof in the in vitro or in vivo detection of CD160-TM in a sample, preferably in a biological sample.The present invention also relates to the use of at least one antibody according to the invention or an antigen-binding fragment thereof in the in vitro or in vivo detection and / or quantification of CD160-TM in a sample, preferably in a biological sample.

[0341] The present invention further relates to an antibody or antigen-binding fragment thereof according to the invention, preferably conjugated to a detectable label, for use in the in vivo detection of tumors expressing CD160-TM in a subject. Examples of cancers in which the cancer cells express CD160-TM are provided above. Examples of detectable labels are provided above.

[0342] The present invention further relates to an in vitro method for detecting CD160-TM in a sample, preferably a biological sample, comprising the step of contacting the sample with at least one antibody or antigen-binding fragment thereof as described above.

[0343] Examples of assays in which the antibodies or antigen-binding fragments thereof according to the invention can be used include, but are not limited to, ELISA, sandwich ELISA, radioimmunoassay (RIA), fluorescence activated cell sorting (FACS), tissue immunohistochemistry, Western blot, and immunoprecipitation.

[0344] The present invention further relates to a method for detecting CD160-TM in a sample, comprising contacting the sample with an antibody, or antigen-binding fragment thereof, according to the invention, and detecting an anti-CD160-TM antibody, or fragment thereof, bound to CD160-TM, thereby indicating the presence of CD160-TM in the sample, hi one embodiment, the method is an in vitro method.

[0345] In one embodiment, the sample is biological sample.The example of biological sample includes but is not limited to body fluid, preferably blood, more preferably serum, plasma, synovial fluid, bronchoalveolar lavage fluid, sputum, lymphatic fluid, ascites, urine, amniotic fluid, peritoneal fluid, cerebrospinal fluid, pleural fluid, pericardial fluid, and alveolar macrophage, tissue lysate, and the extract prepared from diseased tissue.

[0346] In one embodiment, the sample is breast tumor tissue. In one embodiment, the breast tumor tissue is from luminal A breast cancer, luminal B breast cancer, HER2 positive breast cancer, and TNBC.

[0347] In one embodiment, an antibody or antigen-binding fragment thereof according to the invention may be conjugated to a detectable label and directly detected. Examples of detectable labels are provided above.

[0348] In one embodiment, an antibody or antigen-binding fragment thereof according to the invention is unlabeled (and referred to as the first antibody / primary antibody) and is used in combination with a secondary antibody or other molecule that is capable of binding to the anti-CD160-TM antibody and is labeled. Examples of detectable labels that may be bound to said secondary antibody or other molecule include, but are not limited to, the detectable labels provided above.

[0349] Compared to prior art anti-CD160-TM antibodies, the antibodies or antigen-binding fragments according to the invention may offer one or several of the following advantages: - in some embodiments, the antibody or antigen-binding fragment according to the invention does not require a secondary antibody to induce ADCC, CDC, and / or ADCP; - in some embodiments, the antibodies or antigen-binding fragments according to the invention exhibit increased ADCC, CDC, and / or ADCP compared to prior art CD160-TM antibodies; - in some embodiments, the antibodies or antigen-binding fragments according to the invention exhibit an increased capacity for NK cell activation or induce increased NK cell-induced lysis compared to prior art CD160-TM antibodies; - in some embodiments, the antibody or antigen-binding fragment according to the invention is suitable for therapeutic use, such as in humans; - In some embodiments, the antibodies or antigen-binding fragments thereof according to the invention exhibit increased affinity for CD160-TM, in particular increased affinity for CD160-TM in an ELISA test, compared to prior art CD160-TM antibodies. - In some embodiments, the antibodies or antigen-binding fragments according to the invention allow the detection of CD160-TM in non-native forms, such as fixed samples or tissues, and may therefore be used for immunohistochemistry or immunofluorescence applications. [Brief description of the drawings]

[0350] [Figure 1A] 1 is a combination of two graphs showing K562 target cell lysis, as indicated by the percentage of 7AAD positive cells, induced by NK92 cells in the presence of control mouse immunoglobulin G isotype 2a (muIgG2a) or anti-CD160-TM antibodies of the invention (22B12 and 21C8 antibodies) at an effector (E) and target (T) cell ratio of 8 / 1. The percentage of 7AAD positive cells among K562 cells is shown in the presence of control mouse IgG2a antibody or 21C8 antibody at an E / T cell ratio of 8 / 1. n=4 per group, *p<0.05 vs muIgG2a, paired t-test. [Figure 1B] 1 is a combination of two graphs showing K562 target cell lysis, as indicated by the percentage of 7AAD positive cells, induced by NK92 cells in the presence of control mouse immunoglobulin G isotype 2a (muIgG2a) or anti-CD160-TM antibodies of the invention (22B12 and 21C8 antibodies) at an effector (E) and target (T) cell ratio of 8 / 1. The percentage of 7AAD positive cells among K562 cells is shown in the presence of control mouse IgG2a antibody or 22B12 antibody at an E / T cell ratio of 8 / 1. n=4 per group, *p<0.05 vs muIgG2a, paired t-test. [Diagram 2]1 is a histogram showing the percentage of NK cells expressing CD107a, a marker of NK cell degranulation, in the presence of anti-CD160-TM antibodies of the invention (22B12 and 21C8) and Raji cells at different E / T ratios (2.5 / 1, 5 / 1, 10 / 1, 20 / 1). Results are presented as mean ± SEM (Ab - control isotype) (n=3 donors). [Diagram 3] Histogram showing NK cell degranulation during ADCC against CD160 negative target cells (Raji cells) or CD160 positive target cells (Raji cells expressing CD160-TM) in the presence of 21C8 antibody. Results are expressed as mean ± SEM (Ab - control isotype) (n=3), *p<0.05 vs Raji, unpaired t-test. [Figure 4] 1 is a graph showing induction of ADCC in spheroids of the IJG1731 breast cancer cell line expressing GFP, as monitored by the surface of green areas, upon addition of an anti-CD160-TM antibody of the invention (22B12 antibody) or an isotype control antibody (mouse IgG2a). [Diagram 5] A combination of three histograms showing induction of ADCP, as indicated by the percentage of phagocytosis, in three breast cancer cell lines: IJG1731 (A), MCF-7 (B), and MDA-MB-231 (C) in the presence of 1 or 10 μg / mL of an anti-CD160-TM antibody of the present invention (22B12 antibody) or an isotype control antibody (mouse IgG2a antibody). [Figure 6] A combination of immunofluorescence images with anti-CD160-TM antibody 22B12 or isotype control antibody (mouse IgG2a antibody) in three TNBC cell lines: IJG1731, MDA-MB-231, and BT20, and one luminal A cancer cell line: MCF7. [Figure 7] A combination of two histograms showing the percentage of CD160-TM expressing tumor cells (A) and the staining intensity (B) by the anti-CD160-TM antibody of the present invention (22B12 antibody) in breast tumor tissues from various cancers: HER2-positive cancer, TNBC, Luminal A and Luminal B. [Figure 8] 8A is a combination of a diagram (A) and a graph (B) showing the effect on tumor volume of administration of an anti-CD160-TM antibody of the invention (21C8 antibody) in a mammary tumor mouse model compared to control murine IgG2a (muIgG2a). FIG. 8A shows a schematic of the protocol used. SCID mice are injected intramammarily with BT-20 cells and treated on days 10, 13, 17 and 19 with either control murine IgG2a (muIgG2a) or anti-CD160-TM 21C8 antibody. FIG. 8B shows tumor volumes monitored on days 10-20 in mice treated with either muIgG2a or anti-CD160-TM 21C8 antibody. n=6 per group *p<0,05, **p<0,01, ***p<0,001, ****p<0,0001 vs isotype control, two-way ANOVA EXAMPLES

[0351] The invention is further illustrated by the following examples.

[0352] Materials and Methods NK cell activation NK92 cells (effector cells) were pre-incubated with either mouse IgG2a control isotype, CD160-TM 21C8 antibody or CD160-TM 22B12 antibody (10 μg / ml).The cells were then incubated with K562 cells (target cells) at an E / T cell ratio of 8 / 1 for 4 hours and 30 minutes at 37°C.

[0353] Labeling was performed with CD56-PE and 7AAD apoptosis dye staining. Cells were analyzed by flow cytometry. Results are expressed as % of 7AAD+ cells among K562; mean ± SEM (n=4 experiments).

[0354] CD107a analysis PBMCs were used as a source of NK cells. To assess the increased degranulation induced by CD160-TM engagement in NK cells, PBMCs were mixed with Raji cells at E / T ratios of 2.5 / 1, 5 / 1, 10 / 1, or 20 / 1 in the presence of either mouse IgG2a control isotype, CD160-TM 21C8 antibody, or CD160-TM 22B12 antibody (10 μg / ml) (to induce CD160-TM expression on NK cells). After 24 hours of incubation at 37°C, CD3 cells were labeled with a mix of anti-CD3, anti-CD19, anti-CD56, and anti-CD107a and gated. - CD56 + Expression of CD107a on NK cells was assessed by flow cytometry. Similar experiments were performed using Raji cells expressing CD160-TM (Raji-CD160-TM) to then assess NK cell degranulation as a manifestation of ADCC.

[0355] The results in Figure 2 are expressed as the increase in degranulation induced by CD160-TM antibodies (21C8 or 22B12) as a function of the E / T ratio compared to degranulation induced by an IgG2a control isotype (% by Ab - % by control).

[0356] Immunofluorescence in breast cancer cell lines After attachment to glass slides, breast cancer cell lines were fixed with PBS / 4% paraformaldehyde. After a blocking step with PBS / 3% goat serum / 3% BSA, anti-CD160-TM antibody 22B12 or a control isotype (muIgG2a) was added. After washing, an AlexaFluor 594-conjugated anti-mouse Ig antibody (Invitrogen) was added. Slides were mounted with DAPI Fluoromount solution (Southern Biotech).

[0357] Antibody-dependent cellular cytotoxicity (ADCC) TNBC IJG1731 cell line was stably transfected with GFP. For spheroid formation, cells were seeded in culture medium supplemented with 2.5% Matrigel in ultra-low attachment 96-well plates. After 96 hours, PBMCs as a source of NK cells were added together with a mixture of IL2 and IL15 (to promote activation) and either anti-CD160-TM antibody 22B12 or control isotype (muIgG2a). Live cell imaging was performed to monitor green fluorescence bound to the spheroids (6 hours per acquisition of one image). Results are expressed as the mean ± SD of the green fluorescent surface from triplicates.

[0358] Antibody-dependent cellular phagocytosis (ADCP) Breast cancer cell lines were loaded with CFSE and then incubated with CD33+ THP-1 monocytic cell line in the presence of anti-CD160-TM or control Ab (1 or 10 mg / mL) at various E / T ratios for 2 h 30 min at 37° C. ADCP was assessed by flow cytometry by analyzing the transfer of CFSE staining to THP-1 cells (detection of CD33+ CFSE+ cells).

[0359] In vivo effects of CD160-TM depletion 5 × 10 6 CD160-TM positive triple negative breast cancer cell line BT-20 was injected intramammarially. 3 When tumor size reached 10 days (day 10), mice were randomized into two groups of 6 and antibody treatment was initiated the next day. 21C8 or an isotype-matched negative control (muIgG2a) was administered intraperitoneally (100 μg) twice weekly. Tumor sizes were measured on each day of antibody injection (days 10, 13, 17, and 19) and at the time of sacrifice (day 20).

[0360] result CD160-TM Antibody Functionality The ability of the anti-CD160-TM antibodies of the invention to increase NK cell natural cytotoxicity was first evaluated. Figures 1A-1B show that, compared to a control murine antibody, antibodies 21C8 and 22B12 of the invention both target CD160-TM and, as a result, can promote increased NK cell killing activity against target cells.

[0361] Additionally, expression of CD107a, a marker of NK cell degranulation, was assessed in the presence of the CD160-TM antibodies of the invention (21C8 and 22B12 antibodies). As shown in Figures 2 and 3, the anti-CD160-TM antibodies of the invention can positively modulate NK cell degranulation when used as agonistic antibodies on effector cells (Figure 2) or as depleting antibodies on CD160-TM expressing target cells (Figure 3).

[0362] The ability of the anti-CD160-TM antibody of the present invention (22B12 antibody) to induce ADCC was further evaluated in spheroids derived from the GFP-expressing IJG1731 cell line. As shown in Figure 4, addition of 22B12 antibody induces a decrease in fluorescence binding to the spheroids (indicating induction of ADCC by 22B12 antibody), while a control isotype antibody fails to promote the disappearance of GFP-positive cells.

[0363] The ability of the anti-CD160-TM antibody of the invention (22B12 antibody) to induce ADCP was also evaluated in three breast cancer cell lines. As shown in Figures 5A-5C, the presence of 22B12 antibody increases phagocytosis of breast cancer cell lines compared to a control isotype antibody.

[0364] Taken together, these results clearly demonstrate that the anti-CD160-TM antibodies of the present invention can increase NK cell cytotoxicity and promote both ADCC and ADCP, supporting the fact that these antibodies can be used in human therapy.

[0365] Detection of CD160-TM in cancer The ability of the CD160-TM antibodies of the invention to detect cancer cells expressing CD160-TM was then evaluated.

[0366] FIG. 6 shows that the anti-CD160-TM antibody of the present invention (22B12 antibody) can detect CD160-TM in various breast cancer cell lines, including TNBC and luminal A cancer cell lines.

[0367] As shown in Figures 7A and 7B, CD160-TM expressing tumor cells were detected by the anti-CD160-TM antibody of the present invention (22B12 antibody) in breast cancer tissues, including tissues derived from luminal A breast cancer, luminal B breast cancer, HER2-positive breast cancer, and TNBC.

[0368] Overall, these data indicate that various subtypes of breast cancer are composed of CD160-TM positive tumor cells, and that the CD160-TM antibodies of the invention can be used to detect those CD160-TM positive tumor cells.

[0369] In vivo effects of CD160-TM depletion The ability of the anti-CD160-TM antibodies of the invention to treat cancer was evaluated in vivo in a mouse model of breast cancer. Figures 8A-8B demonstrate that administration of an anti-CD160-TM antibody of the invention, i.e., 21C8 antibody, reduces tumor growth compared to a mouse IgG2a control isotype.

[0370] These data clearly support the use of the anti-CD160-TM antibodies of the invention to treat cancer, eg, breast cancer.

Claims

1. 1. An isolated anti-human CD160-TM antibody or antigen-binding fragment thereof, comprising a light chain variable region (VL) of the antibody or antigen-binding fragment having the following complementarity determining region 3 (CDR3): 1 QSX 2 SYPX 3 T (SEQ ID NO: 15) (wherein X 1 is G or L, and X 2 is Y or Q, and X 3 is Y or F).

2. The heavy chain variable region (VH) has three complementarity determining regions (CDRs): - CDR1: GYTFTSYW (SEQ ID NO: 1), - CDR2: IYPGSGFT (SEQ ID NO: 2), - CDR3: SLDEGFAY (SEQ ID NO: 3) Including, The light chain variable region (VL) has three CDRs: - CDR1: QGINIW (SEQ ID NO: 4), - CDR2: KAS, - CDR3: LQSQSYPFT (SEQ ID NO:5) The isolated anti-human CD160-TM antibody or antigen-binding fragment thereof of claim 1, comprising:

3. The heavy chain variable region (VH) has three complementarity determining regions (CDRs): - CDR1: GFSLTSYG (SEQ ID NO: 6), - CDR2: IWRGGNT (SEQ ID NO: 7), - CDR3: VKNGGNSKDYALDY (SEQ ID NO: 8) Including, The light chain variable region (VL) has three CDRs: - CDR1: ENVGIY (SEQ ID NO: 9), - CDR2: GSS, - CDR3: GQSYSYPYT (SEQ ID NO: 10) The isolated anti-human CD160-TM antibody or antigen-binding fragment thereof of claim 1, comprising:

4. 3. An isolated anti-human CD160-TM antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising a sequence having at least 70% identity to SEQ ID NO:11, and a light chain variable region comprising a sequence having at least 70% identity to SEQ ID NO:

12.

5. 4. An isolated anti-human CD160-TM antibody or antigen-binding fragment thereof according to any one of claims 1 or 3, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising a sequence having at least 70% identity to SEQ ID NO: 13, and a light chain variable region comprising a sequence having at least 70% identity to SEQ ID NO:

14.

6. 6. An isolated anti-human CD160-TM antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which is chimeric or humanized, preferably said antibody or antigen-binding fragment thereof is monoclonal.

7. 7. The isolated anti-human CD160-TM antibody or antigen-binding fragment thereof of any one of claims 1 to 6, which is a bispecific antibody.

8. 8. An isolated anti-human CD160-TM antibody or antigen-binding fragment thereof described in any one of claims 1 to 7, which is conjugated, preferably wherein the antibody or fragment thereof is conjugated to a detectable label.

9. A fusion protein comprising an isolated anti-human CD160-TM antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A nucleic acid encoding the isolated anti-human CD160-TM antibody or antigen-binding fragment thereof of any one of claims 1 to 8, or the fusion protein of claim 9.

11. A pharmaceutical composition comprising an isolated anti-human CD160-TM antibody or antigen-binding fragment thereof described in any one of claims 1 to 8, or a fusion protein described in claim 9, and at least one pharma- ceutically acceptable excipient.

12. An isolated anti-human CD160-TM antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, a fusion protein according to claim 9, or a pharmaceutical composition according to claim 11, for use as a medicament.

13. An isolated anti-human CD160-TM antibody or antigen-binding fragment thereof described in any one of claims 1 to 8, a fusion protein described in claim 9, or a pharmaceutical composition described in claim 11, for use in treating cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or paroxysmal nocturnal hemoglobinuria in a subject in need thereof.

14. An in vitro method for detecting CD160-TM in a sample, preferably a biological sample, comprising contacting the sample with at least one isolated anti-human CD160-TM antibody or antigen-binding fragment thereof described in any one of claims 1 to 8.

15. 9. An isolated anti-human CD160-TM antibody or antigen-binding fragment thereof described in any one of claims 1 to 8 for use in in vivo detection of a tumor expressing CD160-TM in a subject.

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