Anti-CD160 transmembrane isoform antibodies

Abstract

The present invention relates to antibodies and antigen-binding fragments thereof that bind to the CD160-TM isoform, and their use for the treatment of 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 in treating cancer and other diseases. Background Art

[0002] CD160 was initially identified as a GPI-anchored (CD160-GPI) MHC-I class activating receptor, which is mainly expressed on peripheral blood NK cells. It has also been reported that a CD160 transmembrane isoform (CD160-TM) has been identified to be produced by alternative splicing of the CD160 gene. It has been determined that CD160-TM surface expression is highly confined to NK cells and depends on activation (Giustiniani J et al. J Immunol. 2009 Jan 1; 182 (1): 63-71). In fact, it is recognized in the art that CD160-TM is only expressed by activated NK cells, while CD160-GPI is expressed by NK cells (activated or unactivated) and different T cell subsets. In addition, there is evidence that CD160-TM represents a new type of activating receptor, which is evaluated by observing the increase in CD107a NK cell surface mobilization when it is involved (Giustiniani J et al. 2009).

[0003] Therefore, antibodies that bind to CD160-TM isoforms but not to CD160 GPI-anchored isoforms (nor to CD160 soluble isoforms that can be generated by proteolytic cleavage of CD160-GPI isoforms) may 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 amplify NK cell activation and thus the effector functions (cytotoxicity, cytokine secretion, etc.) of NK cells or to induce depletion of CD160-TM expressing cells (particularly activated NK cells) in vivo.

[0004] In the present invention, the inventors have developed novel antibodies that specifically bind to the CD160-TM isoform, which can be used as drugs for human therapy.

[0005] Brief description of the invention

[0006] The present invention relates to an isolated anti-human CD160-TM antibody or an antigen-binding fragment thereof, wherein the light chain variable region (VL) of the antibody or antigen-binding fragment comprises the following complementarity determining region 3 (CDR3): X1QSX2SYPX3T (SEQ ID NO: 15), wherein X1 is G or L, X2 is Y or Q, and X3 is Y or F.

[0007] In one embodiment, the heavy chain variable region (VH) of the isolated anti-human CD160-TM antibody or antigen-binding fragment thereof as described above comprises the following three complementarity determining regions (CDRs):

[0008] -CDR1:GYTFTSYW(SEQ ID NO:1),

[0009] -CDR2:IYPGSGFT(SEQ ID NO:2),

[0010] - CDR3: SLDEGFAY (SEQ ID NO: 3); and

[0011] The light chain variable region (VL) contains the following three CDRs:

[0012] -CDR1:QGINIW(SEQ ID NO:4),

[0013] -CDR2: KAS,

[0014] -CDR3: LQSQSYPFT (SEQ ID NO: 5).

[0015] In one embodiment, the heavy chain variable region (VH) of the isolated anti-human CD160-TM antibody or antigen-binding fragment thereof as described above comprises the following three complementarity determining regions (CDRs):

[0016] -CDR1:GFSLTSYG(SEQ ID NO:6),

[0017] -CDR2:IWRGGNT(SEQ ID NO:7),

[0018] - CDR3: VKNGGNSKDYALDY (SEQ ID NO: 8); and

[0019] The light chain variable region (VL) contains the following three CDRs:

[0020] -CDR1:ENVGIY(SEQ ID NO:9),

[0021] -CDR2: GSS,

[0022] -CDR3: GQSYSYPYT (SEQ ID NO: 10).

[0023] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence that is at least 70% identical to SEQ ID NO:11, and the light chain variable region comprises a sequence that is at least 70% identical to SEQ ID NO:12.

[0024] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising a sequence that is at least 70% identical to SEQ ID NO:13 and a light chain comprising a sequence that is at least 70% identical to SEQ ID NO:14.

[0025] In one embodiment, the isolated anti-human CD160-TM antibody or antigen-binding fragment thereof described above is chimeric or humanized, and preferably the antibody or antigen-binding fragment thereof is monoclonal.

[0026] In one embodiment, the isolated anti-human CD160-TM antibody or antigen-binding fragment thereof described above is a bispecific antibody.

[0027] In one embodiment, the isolated anti-human CD160-TM antibody or antigen-binding fragment thereof described above is conjugated, preferably the antibody or fragment thereof is conjugated to a detectable label.

[0028] The present invention also relates to a fusion protein comprising the isolated anti-human CD160-TM antibody or an antigen-binding fragment thereof as described above.

[0029] The present invention also relates to a nucleic acid encoding the isolated anti-human CD160-TM antibody or its antigen-binding fragment or fusion protein as described above.

[0030] The present invention also relates to a pharmaceutical composition comprising the isolated anti-human CD160-TM antibody or antigen-binding fragment or fusion protein described above, and at least one pharmaceutically acceptable excipient.

[0031] The present invention also relates to the isolated anti-human CD160-TM antibody or antigen-binding fragment, fusion protein or pharmaceutical composition described above, which is used as a medicament.

[0032] The present invention also relates to the isolated anti-human CD160-TM antibody or its antigen-binding fragment, fusion protein or pharmaceutical composition described above, which is used to treat cancer, infectious diseases, autoimmune diseases, inflammatory diseases or paroxysmal nocturnal hemoglobinuria in a subject in need thereof.

[0033] In one embodiment, the anti-human CD160-TM antibody or its antigen-binding fragment, fusion protein or pharmaceutical composition described above is used to treat breast cancer in a subject in need. In one embodiment, the anti-human CD160-TM antibody or its antigen-binding fragment, fusion protein or pharmaceutical composition described above is used to treat breast cancer selected from the group consisting of luminal A breast cancer, luminal B breast cancer, triple negative breast cancer (TNBC) and HER2 positive breast cancer.

[0034] In one embodiment, the isolated anti-human CD160-TM antibody or its antigen-binding fragment, fusion protein or pharmaceutical composition described above is used to treat NK leukemia or NK lymphoma, such as extranodal and non-extranodal NK / T lymphoma; NK cell-derived malignancies; acute NK cell leukemia and peripheral T-cell lymphoma (PTCL), such as peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS).

[0035] The present invention also relates to an in vitro detection method for detecting CD160-TM in a sample, preferably a biological sample, comprising the step of contacting the sample with at least one of the above-described isolated anti-human CD160-TM antibodies or antigen-binding fragments thereof.

[0036] The present invention also relates to the isolated anti-human CD160-TM antibody or antigen-binding fragment thereof as described above, which is used for in vivo detection of a tumor expressing CD160-TM in a subject.

[0037] definition

[0038] In the present invention, the following terms have the following meanings:

[0039] "About" preceding a number includes plus or minus 10% or less of the numerical value. It should be understood that the value referred to by the term "about" itself is also specifically and preferably disclosed.

[0040] As used herein, "affinity" refers to the strength with which an antibody binds to an epitope. The affinity of an antibody is determined by the dissociation constant, K D The affinity constant K is given by [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. A Defined as 1 / K DPreferred 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 herein by reference. The binding properties of antibodies or antigen-binding fragments thereof to antigens, cells or tissues can generally be determined and assessed using immunoassay 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, for example, using ).

[0041] As used herein, "antibody" and "immunoglobulin" are used interchangeably and refer to a protein having a combination of two heavy chains and two light chains, whether or not they have any associated specific immunoreactivity. "Antibody" refers to such assemblies that have significant known specific immunoreactivity to target antigens (e.g., CD160-transmembrane (TM) isoforms). The term "anti-CD160-TM isoform" used herein refers to antibodies that exhibit immunospecificity to human CD160-TM isoforms. As described elsewhere herein, "specificity" for human CD160-TM isoforms does not exclude cross-reactions with species homologs of CD160-TM isoforms, such as cross-reactions with ape CD160-TM isoforms. Antibodies and immunoglobulins contain light and heavy chains with or without interchain covalent bonds between them. The basic immunoglobulin structure in vertebrate systems is fairly clear. The general term "immunoglobulin" includes five different types of antibodies that can be distinguished biochemically. Although the discussion below generally relates to IgG class immunoglobulin molecules, all 5 classes of antibodies are within the scope of the present invention. In the case of IgG, the immunoglobulin comprises two identical light polypeptide chains with a molecular weight of approximately 23 kDa and two identical heavy chains with a molecular weight of approximately 53-70 kDa. The four chains are connected in a "Y" configuration by disulfide bonds, wherein the light chain is supported by the heavy chain, starting from the "Y" opening and extending to the variable region. The light chain of the antibody is classified as κappa (κ) or lambda (λ). Each heavy chain type can be combined with a κ or λ light chain. In general, the light chain and the heavy chain are covalently bound to each other, and when the immunoglobulin is produced by a hybridoma, a B cell or a genetically engineered host cell, the "tail" region of the two heavy chains is bound to each other by a covalent disulfide bond or a non-covalent bond. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. It is understood by those skilled in the art that heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε), with some subclasses (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, which is IgG, IgM, IgA IgD or IgE, respectively. Immunoglobulin subclasses or "isotypes" (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) have been fully characterized and are known to have functional specializations. In view of the present disclosure, modifications to each of these classes and isotypes are readily discernible to those skilled in the art, and are therefore within the scope of the present invention. As described above, the variable region of an antibody can selectively recognize and specifically bind to an epitope on an antigen with the antibody. That is, the light chain variable domain (VL domain) and the heavy chain variable domain (VH domain) of the antibody combine to form a variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms an 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) on each of the VH and VL chains.

[0042] As used herein, "antigen binding fragment" refers to a portion or region of an antibody that contains fewer amino acid residues than a complete antibody. An "antigen binding fragment" binds to an antigen and / or competes for antigen binding with the complete antibody from which it is derived (e.g., specifically binds to a CD160-TM isotype). 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, double antibodies, three antibodies, and four antibodies. It may also include single antibodies, domain antibodies, and nanobodies.

[0043] "CD160" has its ordinary meaning in the art and refers to the CD160 molecule. There are three CD160 isoforms: CD160-TM isoform, CD160 GPI-anchored isoform and soluble CD160 isoform. CD160-GPI is expressed by intestinal epithelial T lymphocytes and cytotoxic effectors including NK cells, TCRγδ and CD8 bright CD28-T lymphocytes are expressed by a small subset of circulating lymphocytes (ANUMANTHAN et al., 1998, J Immunol; 161: 2780-2790; MAIZA et al., J. Exp. Med., vol. 178, p: 1121-1126, 1993). The CD160 transmembrane isoform ("CD160-TM") has been described in Giustiniani J et al. (J Immunol. 2009 Jan 1; 182 (1): 63-71.) and in International Patent Application WO2008155363, characterized by the amino acid sequence shown in SEQ ID NO: 21. The extracellular domain of the CD160-TM isoform can be defined by the amino acid sequence of amino acid residue 26 to amino acid residue 189 in 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, characterized by the amino acid sequence as shown in SEQ ID NO: 22 fused to the GPI anchor at the C-terminus. The CD160 soluble isoform is described in Giustiniani J. et al. (J Immunol. 2007 Feb 1; 178(3): 1293-300), characterized by the amino acid sequence as shown in SEQ ID NO: 22. In SEQ ID NO: 21-22, amino acids 1-25 correspond to the signal peptide and therefore may not be present in the expressed protein.

[0044] SEQ ID NO:21: CD160-TM isotype

[0045] MLLEPGRGCCALAILLAIVDIQSGGCINITSSASQEGTRLNLICTVWHKKEE

[0046] AEGFVVFLCKDRSGDCSPETSLKQLRLKRDPGIDGVGEISSQLMFTISQVTP

[0047] LHSGTYQCCARSQKSGIRLQGHFFSILFTETGNYTVTGLKQRQHLEFSHNE

[0048] GTLSSGFLQEKVWVMLVTSLVALQGMSKRAVSTPSNEGAIIFLPPWLFSRR

[0049] RRLERMSRGREKCYSSPGYPQESSNQFH

[0050] SEQ ID NO:22: CD160 soluble isotype

[0051] MLLEPGRGCCALAILLAIVDIQSGGCINITSSASQEGTRLNLICTVWHKKEE

[0052] AEGFVVFLCKDRSGDCSPETSLKQLRLKRDPGIDGVGEISSQLMFTISQVTP

[0053] LHSGTYQCCARSQKSGIRLQGHFFSILFTETGNYTVTGLKQRQHLEFSHNE

[0054] GTLSS

[0055] "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites present within the variable regions of the heavy and light chain polypeptides. The precise 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 ImMunoGeneTics (IMGT) (Lefranc et al., Nucleic Acids Res. 27: 209-212 1999). IMGT is a comprehensive information system dedicated to the study of immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complexes (MHC) of humans and other vertebrates. In this article, CDR refers to the amino acid sequence and position within the light chain or heavy chain. Since the "position" of CDR within the structure of the immunoglobulin variable domain is conserved between species and exists in a structure called a loop, CDR and framework residues can be easily determined by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to transplant and replace CDR residues from immunoglobulins from one species into an acceptor framework. The acceptor framework is usually derived from human antibodies. The correspondence between Kabat numbering and the IMGT feature numbering system is also well known to those skilled in the art (e.g., Lefranc et al., supra). Therefore, in one embodiment, the CDR region or CDR is represented by The numbering system defines the hypervariable regions of the heavy and light chains of immunoglobulins.

[0056] As used herein, "diabodies" refer to small antibody fragments prepared by constructing scFv fragments with a short linker (about 5-10 residues) between VH and VL, thereby achieving interchain pairing of variable domains rather than intrachain pairing to produce 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.

[0057] "Domain antibody" refers to the smallest functional binding unit of an antibody, corresponding to the variable region of an antibody heavy or light chain.

[0058] An "epitope" is a specific arrangement of amino acids located on one or more proteins to which an antibody or antigen-binding fragment thereof binds. An epitope generally consists of a chemically active surface combination of molecules such as amino acids or sugar side chains, and has specific three-dimensional structural characteristics as well as specific charge characteristics. An epitope may be linear (or continuous) or conformational, i.e., involving two or more amino acid sequences in different regions of an antigen, which sequences may not necessarily be continuous.

[0059] "Fab" refers to a fragment antibody produced by digesting an intact IgG antibody with papain to remove the intact Fc fragment (including the hinge region). These antibodies are monovalent and contain only a single antigen binding site. In contrast, F(ab')2 fragment antibodies are produced by digesting an intact IgG antibody with pepsin to remove most of the Fc region while retaining part of the hinge region. The F(ab')2 fragment has two antigen-binding F(ab) parts linked together by a disulfide bond. The term "Fab'" refers to an antibody fragment with a molecular weight of about 50,000 and antigen-binding activity, which is obtained by cleaving the disulfide bonds in the hinge region of F(ab')2. Fab'-SH is referred to herein as Fab', in which the cysteine ​​residues of the constant domains carry a free thiol group.

[0060] “Fc domain,” “Fc portion,” and “Fc region” refer to the C-terminal fragment of an antibody heavy chain, e.g., about amino acids (aa) 230 to about aa 450 of a human gamma heavy chain or its corresponding sequences in other types of antibody heavy chains (e.g., α, δ, ε, and μ of human antibodies), or a naturally occurring allotype thereof.

[0061] "Fd fragment" refers to the heavy chain of the Fab fragment, comprising the VH and CH1 regions.

[0062] A "framework region" or "FR region" includes a portion of a variable region other than a CDR (e.g., using The framework region of the light chain is also separated by each CDR of VL. In naturally occurring antibodies, the six CDRs present in each monomer antibody are short and non-continuous amino acid sequences, and when the antibody presents its three-dimensional configuration in an aqueous environment, the amino acid sequence is specifically positioned to form an antigen binding site. The rest of the heavy and light chain variable regions show less intermolecular variability in the amino acid sequence and are referred to as framework regions. Most of the framework regions adopt a β-folded conformation, and the CDRs form loops connecting the β-folded structure, forming a part of the β-folded structure in some cases. Therefore, these framework regions play a role in forming a scaffold, which positions the six CDRs in the correct direction through interchain non-covalent interactions. The antigen binding site formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. The complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. One of ordinary skill in the art can easily determine the position of the CDR.

[0063] As used herein, "Fv" refers to the smallest antibody fragment containing a complete antigen recognition and binding site. The fragment consists of a dimer of a VH and a VL that are tightly non-covalently bound. Six hypervariable loops (three per loop from a heavy chain and a light chain) are produced from the folding of these two domains, which contribute to antigen binding and give the antibody antigen binding specificity. 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 antigens, although the affinity is lower than that of the entire binding site.

[0064] A "heavy chain region" includes an amino acid sequence derived from the constant region of an immunoglobulin heavy chain. A protein comprising a heavy chain region includes C H 1 domain, hinge (e.g., upper hinge region, middle hinge region and / or lower hinge region) domain, C H 2 domains, C H In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention may comprise the Fc region of an immunoglobulin heavy chain (e.g., hinge portion, C H 2 domains and C H In another embodiment, an antibody or antigen-binding fragment thereof according to the invention lacks at least one region of a constant domain (e.g., all or part of a C H 2 domains). In certain embodiments, at least one, preferably all, constant regions are or are derived from a human immunoglobulin heavy chain. For example, in one embodiment, the heavy chain region comprises a fully human hinge domain. In other embodiments, the heavy chain region comprises a fully human Fc region (e.g., hinge, C from a human immunoglobulin). H 2 and C HIn some 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 may comprise C3 domains derived from an IgG1 molecule. H 2 domains and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domain is a chimeric domain comprising regions of different immunoglobulin molecules. For example, the hinge may comprise a first region from an IgG1 molecule and a second region from an IgG3 or IgG4 molecule. In some embodiments, the constant domain of the heavy chain region may be modified so that it differs from a naturally occurring (wild-type) immunoglobulin molecule in amino acid sequence. That is, the antibody or antigen-binding fragment thereof according to the present invention may comprise one or more heavy chain constant domains (C H 1. Hinge, C H 2 or C H 3) and / or light chain constant domain (C L ). Exemplary modifications include addition, deletion or substitution of one or more amino acids in one or more domains.

[0065] The "hinge region" includes the C H Domain 1 is connected to C H The hinge region is the region of the heavy chain molecule that contains the two domains. The hinge region contains about 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 different domains: the upper hinge region, the middle hinge region, and the lower hinge region (Roux et al., 1998. J Immunol. 161(8): 4083-90).

[0066] When used herein to describe the relationship between two or more amino acid sequences or two or more nucleic acid sequences, "identity" or "same" refers to the degree of sequence relatedness between amino acid sequences or nucleic acid sequences, as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. "Identity" measures the percentage of identical matches between the smaller sequences in two or more sequences, where gap comparisons (if any) are solved by a specific mathematical model or computer program (i.e., "algorithm"). The identity of related amino acid sequences or nucleic acid sequences can be easily calculated by known methods. Such methods include, but are not limited to, 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 test sequences. Methods to determine identity are described in publicly available computer programs.Preferred computer program methods for determining the 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 publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLASTManual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894). The well-known Smith Waterman algorithm can also be used to determine identity.

[0067] As used herein, "isolated protein", in particular "isolated antibody" refers to a protein, in particular 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, in particular an isolated antibody that specifically binds to a CD160-TM isoform may have cross-reactivity with other antigens, such as CD160-TM molecules of other species. In addition, an isolated protein or antibody may be substantially free of other cellular material and / or chemicals, in particular those that would interfere with the therapeutic use of the protein or antibody, including but not limited to enzymes, hormones, and other protein or non-protein components.

[0068] "Mammal" refers to any mammal, including humans, non-human primates, domestic and farm animals, and zoo, sport or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, monkeys, etc. Preferably, the mammal is a human.

[0069] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., each antibody contained in the population is identical, except for a possible naturally occurring mutation that may be present in a small amount. Monoclonal antibodies are highly specific for a single antigenic site. In addition, unlike polyclonal antibody preparations comprising different antibodies for different determinants (epitopes), each monoclonal antibody is directed to a single determinant on an antigen. In addition to its specificity, monoclonal antibodies are advantageous because they can be synthesized without being contaminated by other antibodies. The modifier "monoclonal" should not be interpreted as requiring antibodies to be produced by any ad hoc method. For example, monoclonal antibodies or antigen-binding fragments thereof according to the present invention can be prepared by the hybridoma method first described by Kohler et al., 1975. Nature. 256 (5517): 495-7, or can be prepared using recombinant DNA methods in bacteria, eukaryotic animals or plant cells (patent US4,816,567). "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.

[0070] "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 may contain a single variable region (VHH) and two constant regions (C H 2 and C H 3).

[0071] As used herein, "single-chain antibody" refers to any antibody or fragment thereof, which is a protein having a primary structure comprising or consisting of one uninterrupted sequence of consecutive 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 the light chain variable domain, without the 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 the heavy chain variable region, without the associated light chain portion.

[0072] "Single-chain Fv", also abbreviated as "sFv" or "scFv", refers to a fragment comprising a V linked to a single amino acid chain. H and V L Preferably, the scFv amino acid sequence further comprises V H and V LThe peptide linker between the domains enables the scFv to form the desired structure for antigen binding.

[0073] "Specificity" refers to the ability of an antibody to detectably bind to an epitope present 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 relatively determined by binding or competitive binding assays using, for example, a Biacore instrument. Specificity can be demonstrated, for example, by a ratio of affinity / avidity for binding to a specific antigen of about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1 or greater relative to nonspecific binding to other unrelated molecules (in this case, the specific antigen is a CD160-TM polypeptide).

[0074] "Subject" refers to a warm-blooded animal, preferably a mammal (including humans, livestock and farm animals, and zoo, sports or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc.), and more preferably a human. In one embodiment, the subject can be a "patient", i.e., a warm-blooded animal, more preferably a human, who is waiting to receive or is receiving medical care or has been / is / will be the subject of a medical procedure, or is being monitored for the development of a disease. 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.

[0075] 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 prevent the progression, aggravation, or worsening of one or more symptoms of the disease, disorder, or condition; (3) ameliorate the symptoms of the disease, disorder, or condition; (4) reduce the severity or incidence of the disease, disorder, or condition; or (5) treat the disease, disorder, or condition, without causing significant negative or adverse side effects to the target. A therapeutically effective amount can be administered prior to the onset of a disease, disorder, or condition for a prophylactic or preventive effect. Alternatively or additionally, a therapeutically effective amount can be administered after the onset of a disease, disorder, or condition for a therapeutic effect.

[0076] "Treating" or "relief" refers to both therapeutic treatment and prophylactic or preventative measures; wherein the purpose is to prevent or slow down (mitigate) the target pathological condition or disorder. Those in need of treatment include those already suffering from the condition as well as those susceptible to the condition or the condition 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 symptoms: a decrease in the number of pathogenic cells; one or more symptoms associated with the disease to be treated are alleviated to some extent; morbidity and mortality are reduced; and quality of life issues are improved. The above parameters for assessing successful treatment and improvement of the disease can be easily measured by routine procedures familiar to physicians.

[0077] "Unibody" refers to an antibody fragment lacking the hinge region of an IgG4 antibody. The absence of the hinge region results in a molecule that is essentially half the size of a traditional IgG4 antibody and has a monovalent binding region rather than the bivalent binding region of an IgG4 antibody.

[0078] "Variable" refers to the variable domain V H and V L Certain regions of the variable domain vary greatly in sequence between antibodies and are used for the binding and specificity of each particular antibody to its target antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in the V domains that form part of the antigen binding site. L Domain and V H Each of the Vλ light chain domains is comprised of three segments called “hypervariable loops”. The first, second, and third hypervariable loops of the Vλ light chain domain are referred to herein as L1(λ), ​​L2(λ), and L3(λ), and can be defined as the regions comprising the Vλ light chain domain. L 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 can be defined as the region comprising the Vκ light chain domain. L 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) of the V domain (Morea et al., supra). H The first, second and third hypervariable loops of the domain are referred to herein as H1, H2 and H3, and can be defined as comprising VH 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) in the domain (Morea et al., supra). Unless otherwise indicated, the terms L1, L2 and L3 refer to the first, second and third hypervariable loops of the VL domain, respectively, and include hypervariable loops from Vκ and Vλ isotypes. The terms H1, H2 and H3 refer to the first, second and third hypervariable loops of the VH domain, respectively, and include hypervariable loops from any known heavy chain isotype, including gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). The hypervariable loops may each include a portion of a "complementarity determining region" or "CDR" as defined above. DETAILED DESCRIPTION OF THE INVENTION

[0080] The present invention relates to antibodies or antigen-binding fragments thereof that bind to the CD160-TM isoform.

[0081] 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, monobodies, domain antibodies, and nanobodies.

[0082] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is selected from the group consisting of: whole antibody, single-chain antibody, dimeric single-chain antibody, Fv, scFv, Fab, Fab', Fab'-SH, F(ab)'2, Fd, defucosylated antibody, bispecific antibody, diabody, triabody and tetrabody.

[0083] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is selected from the group consisting of: a whole antibody, a single-chain variable fragment (scFv), Fv, Fab, Fab', Fab'-SH, F(ab)'2, a defucosylated antibody, a bispecific antibody, a diabody, a triabody and a tetrabody.

[0084] In one embodiment, the isolated antibody or antigen-binding fragment according to the present invention is selected from the group comprising or consisting of a monoclonal antibody, a domain antibody and a nanobody.

[0085] In one embodiment, the isolated antibody or antigen-binding fragment according to the present invention is selected from the group consisting of: whole antibody, Fv, scFv, Fab and single antibody.

[0086] In one embodiment, the antibody or antigen binding fragment thereof according to the present invention specifically binds to the CD160-TM isoform. In one embodiment, the antibody or antigen binding fragment thereof according to the present invention specifically binds to the human CD160-TM isoform.

[0087] As used herein, "specific binding" refers to an antibody or antigen-binding fragment thereof that recognizes and binds to a binding partner present in a sample, but the antibody or antigen-binding fragment thereof does not substantially recognize or bind other molecules in the sample.

[0088] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention has low or no affinity for other CD160 isoforms, such as CD160 GPI-anchored isoforms and CD160 soluble isoforms.

[0089] The affinity of an antibody or antigen-binding fragment thereof can be readily determined using conventional techniques, such as those described in Scatchard, 1949. Ann NY Acad Sci. 51: 660-672. The binding properties of an antibody or antigen-binding fragment thereof to an antigen, cell or tissue can generally be determined and assessed using immunoassay 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 ).

[0090] In the following, unless explicitly stated otherwise, CDR numbers and definitions are based on Numbering system.

[0091] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region (abbreviated herein as VH) comprising the following complementarity determining region 1 (CDR1): GX1X2X3TSYX4 (SEQ ID NO: 16), wherein X1 is F or Y, X2 is S or T, X3 is L or F, and X4 is G or W.

[0092] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a light chain variable region (abbreviated herein as VL) comprising the following complementarity determining region 3 (CDR3): X1QSX2SYPX3T (SEQ ID NO: 15), wherein X1 is G or L, X2 is Y or Q, and X3 is Y or F.

[0093] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region comprising at least one, preferably at least two, more preferably three of the following complementarity determining regions (CDRs):

[0094] CDR1:GYTFTSYW(SEQ ID NO:1);

[0095] CDR2: IYPGSGFT (SEQ ID NO: 2); and / or

[0096] CDR3:SLDEGFAY (SEQ ID NO:3).

[0097] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region comprising at least one, preferably at least two, more preferably three of the following complementarity determining regions (CDRs):

[0098] CDR1: GFSLTSYG (SEQ ID NO:6);

[0099] CDR2: IWRGGNT (SEQ ID NO: 7); and / or

[0100] CDR3: VKNGGNSKDYALDY (SEQ ID NO: 8).

[0101] In one embodiment, any of the CDR1, CDR2 and / or CDR3 of the VH having SEQ ID NOs 1-3, 6-8 may be characterized as having 1, 2, 3 or more amino acids substituted with different amino acids.

[0102] In one embodiment, any of the CDR1, CDR2 and / or CDR3 of the VH having SEQ ID NOs 1-3, 6-8 can be characterized as having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a specific CDR or group of CDRs listed in the corresponding SEQ ID NO.

[0103] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a light chain variable region comprising at least one, preferably at least two, more preferably three of the following complementarity determining regions (CDRs):

[0104] CDR1: QGINIW (SEQ ID NO: 4);

[0105] CDR2: KAS; and / or

[0106] CDR3: LQSQSYPFT (SEQ ID NO: 5).

[0107] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a light chain variable region comprising at least one, preferably at least two, more preferably three of the following complementarity determining regions (CDRs):

[0108] CDR1: ENVGIY (SEQ ID NO: 9);

[0109] CDR2: GSS; and / or

[0110] CDR3: GQSYSYPYT (SEQ ID NO: 10).

[0111] 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 1, 2, 3 or more amino acids substituted with different amino acids.

[0112] 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 is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a specific CDR or group of CDRs listed above.

[0113] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises:

[0114] - a VH comprising at least one, preferably at least two, more preferably three of the following CDRs:

[0115] CDR1:GYTFTSYW(SEQ ID NO:1);

[0116] CDR2: IYPGSGFT (SEQ ID NO: 2); and / or

[0117] CDR3: SLDEGFAY (SEQ ID NO: 3); and

[0118] - VL, which comprises at least one, preferably at least two, more preferably three of the following CDRs:

[0119] CDR1: QGINIW (SEQ ID NO: 4);

[0120] CDR2: KAS; and / or

[0121] CDR3: LQSQSYPFT (SEQ ID NO: 5).

[0122] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises:

[0123] - VH, which contains the following three CDRs:

[0124] CDR1:GYTFTSYW(SEQ ID NO:1);

[0125] CDR2: IYPGSGFT (SEQ ID NO: 2); and

[0126] CDR3: SLDEGFAY (SEQ ID NO: 3); and

[0127] -VL, which contains the following three CDRs:

[0128] CDR1: QGINIW (SEQ ID NO: 4);

[0129] CDR2: KAS; and

[0130] CDR3: LQSQSYPFT (SEQ ID NO: 5).

[0131] 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, can be characterized as having 1, 2, 3 or more amino acids substituted by different amino acids.

[0132] In one embodiment, any one 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, can be characterized as having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a specific CDR or CDR group listed above.

[0133] An example of an antibody comprising a heavy chain comprising CDR1, CDR2 and CDR3 having SEQ ID NOs: 1-3 and a light chain comprising CDR1, CDR2 and CDR3 having SEQ ID NO: 4, sequence KAS and SEQ ID NO: 5 is the 21C8 antibody.

[0134] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises:

[0135] - a VH comprising at least one, preferably at least two, more preferably three of the following CDRs:

[0136] CDR1: GFSLTSYG (SEQ ID NO:6);

[0137] CDR2: IWRGGNT (SEQ ID NO: 7); and / or

[0138] CDR3: VKNGGNSKDYALDY (SEQ ID NO: 8); and

[0139] - VL, which comprises at least one, preferably at least two, more preferably three of the following CDRs:

[0140] CDR1: ENVGIY (SEQ ID NO: 9);

[0141] CDR2: GSS; and / or

[0142] CDR3: GQSYSYPYT (SEQ ID NO: 10).

[0143] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises:

[0144] - VH, which contains the following three CDRs:

[0145] CDR1: GFSLTSYG (SEQ ID NO:6);

[0146] CDR2: IWRGGNT (SEQ ID NO: 7); and

[0147] CDR3: VKNGGNSKDYALDY (SEQ ID NO: 8); and

[0148] -VL, which contains the following three CDRs:

[0149] CDR1: ENVGIY (SEQ ID NO: 9);

[0150] CDR2: GSS; and

[0151] CDR3: GQSYSYPYT (SEQ ID NO: 10).

[0152] 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: 9, sequence GSS and SEQ ID NO: 10, respectively, can be characterized as having 1, 2, 3 or more amino acids substituted by different amino acids.

[0153] 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:9, sequence GSS and SEQ ID NO:10, respectively, can be characterized as having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a specific CDR or CDR group listed above.

[0154] An example of an antibody comprising a heavy chain having CDR1, CDR2 and CDR3 of SEQ ID NOs: 6-8, respectively, and a light chain comprising CDR1, CDR2 and CDR3 of SEQ ID NO: 9, sequence GSS and SEQ ID NO: 10, respectively, is the 22B12 antibody.

[0155] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises:

[0156] - a VH comprising at least one, preferably at least two, more preferably three of the following CDRs:

[0157] CDR1: GFSLTSYG (SEQ ID NO:6);

[0158] CDR2: IWRGGNT (SEQ ID NO: 7); and / or

[0159] CDR3: VKNGGNSKDYALDY (SEQ ID NO: 8); and

[0160] - VL, which comprises at least one, preferably at least two, more preferably three of the following CDRs:

[0161] CDR1: QGINIW (SEQ ID NO: 4);

[0162] CDR2: KAS; and / or

[0163] CDR3: LQSQSYPFT (SEQ ID NO: 5).

[0164] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises:

[0165] - VH, which contains the following three CDRs:

[0166] CDR1: GFSLTSYG (SEQ ID NO:6);

[0167] CDR2: IWRGGNT (SEQ ID NO: 7); and / or

[0168] CDR3: VKNGGNSKDYALDY (SEQ ID NO: 8); and

[0169] -VL, which contains the following three CDRs:

[0170] CDR1: QGINIW (SEQ ID NO: 4);

[0171] CDR2: KAS; and / or

[0172] CDR3: LQSQSYPFT (SEQ ID NO: 5).

[0173] In one embodiment, any of the CDR1, CDR2 and / or CDR3 of the VH having SEQ ID NOs: 6-8 and / or the VL having SEQ ID NOs: 4, sequence KAS and SEQ ID NO: 5, respectively, can be characterized as having 1, 2, 3 or more amino acids substituted by different amino acids.

[0174] 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, can be characterized as having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a specific CDR or CDR group listed above.

[0175] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises:

[0176] - a VH comprising at least one, preferably at least two, more preferably three of the following CDRs:

[0177] CDR1:GYTFTSYW(SEQ ID NO:1);

[0178] CDR2: IYPGSGFT (SEQ ID NO: 2); and / or

[0179] CDR3: SLDEGFAY (SEQ ID NO: 3); and

[0180] - VL, which comprises at least one, preferably at least two, more preferably three of the following CDRs:

[0181] CDR1: ENVGIY (SEQ ID NO: 9);

[0182] CDR2: GSS; and / or

[0183] CDR3: GQSYSYPYT (SEQ ID NO: 10).

[0184] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises:

[0185] - VH, which contains the following three CDRs:

[0186] CDR1:GYTFTSYW(SEQ ID NO:1);

[0187] CDR2: IYPGSGFT (SEQ ID NO: 2); and / or

[0188] CDR3: SLDEGFAY (SEQ ID NO: 3); and

[0189] -VL, which contains the following three CDRs:

[0190] CDR1: ENVGIY (SEQ ID NO: 9);

[0191] CDR2: GSS; and / or

[0192] CDR3: GQSYSYPYT (SEQ ID NO: 10).

[0193] 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: 9, sequence GSS and SEQ ID NO: 10, respectively, can be characterized as having 1, 2, 3 or more amino acids substituted by different amino acids.

[0194] In one embodiment, any one of the CDR1, CDR2 and / or CDR3 of the VH having SEQ ID NO: 1-3 and / or the VL having SEQ ID NO: 9, sequence GSS and SEQ ID NO: 10, respectively, can be characterized as having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a specific CDR or CDR group listed above.

[0195] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a VH comprising or consisting of the sequence SEQ ID NO: 11.

[0196] SEQ ID NO:11

[0197] QVQLQQPGSELVRPGASVRLSCKASGYTFTSYWMHWVRQRHGQGLEWI

[0198] GNIYPGSGFTNYDEKFKNKGTLTVDTSSSTAYMHLSSSLTSEDSAVYYCSLD

[0199] EGFAYWGQGTLVTVSA

[0200] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a VH comprising or consisting of the sequence SEQ ID NO: 13.

[0201] SEQ ID NO:13

[0202] QVQLKQSGPGLVQPSQSLSIMCTVSGFSLTSYGLHWVRQSPGKGLEWLGV

[0203] IWRGGNTDYNADFMSRLTITKDNSKSQVFFKMNSLQADDTAVYYCVKNG

[0204] GNSKDYALDYWGQGTSVTVSS

[0205] In one embodiment, the VH comprises or consists of the sequence of SEQ ID NO: 11 or 13, wherein 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 of the sequence are substituted by a different amino acid.

[0206] In one embodiment, the VH has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:11 or 13.

[0207] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a VL comprising or consisting of the sequence SEQ ID NO: 12.

[0208] SEQ ID NO:12

[0209] DIQMNQSPSSLSASLGDTITITCRASQGINIWLNWYQQKPGNIPKRLIYKAS

[0210] NLHTGVPPRFRGSGSGTTDFTLTISSLQPEDIATYYCLQSQSYPFTFGSGTKLE

[0211] IR

[0212] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a VL comprising the sequence SEQ ID NO: 14 or a composition thereof.

[0213] SEQ ID NO:14

[0214] NIVMTQSPKMSMSVGERVTLSCKASENVGIYVSWYQQKPEQSPKLLIYG

[0215] SSNRYTGVPDRFTGSGSATDFTLIISSVQAEDLADYHCGQSYSYPYTFGGG

[0216] TKLEIK

[0217] In one embodiment, the VL comprises or consists of the sequence of SEQ ID NO: 12 or 14, wherein 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 of the sequence are substituted by a different amino acid.

[0218] In one embodiment, the VH has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:12 or 14.

[0219] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises:

[0220] - VH comprising or consisting of the sequence SEQ ID NO: 11; and

[0221] - VL comprising or consisting of the sequence SEQ ID NO: 12.

[0222] In one embodiment, the VH comprises or consists of the sequence of SEQ ID NO: 11 and / or the VL comprises or consists of the sequence of SEQ ID NO: 12, wherein 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 of said sequence are substituted by a different amino acid.

[0223] In one embodiment, the VH and / or VL have an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 11 and / or SEQ ID NO: 12, respectively.

[0224] An example of such an antibody is the 21C8 antibody.

[0225] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises:

[0226] - VH comprising or consisting of the sequence SEQ ID NO: 13; and

[0227] - VL comprising or consisting of the sequence SEQ ID NO: 14.

[0228] In one embodiment, the VH comprises or consists of the sequence of SEQ ID NO: 13 and / or the VL comprises or consists of the sequence of SEQ ID NO: 14, wherein 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 of said sequence are substituted by a different amino acid.

[0229] In one embodiment, the VH and / or VL have an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 13 and / or SEQ ID NO: 14, respectively.

[0230] An example of such an antibody is the 22B12 antibody.

[0231] As used herein, the phrase "characterized as having an amino acid [ . . . ] substituted with a different amino acid" with respect to a given sequence means that conservative amino acid modifications occur in the sequence.

[0232] As used herein, the expression "conservative amino acid modification" refers to a modification that does not significantly affect or change the binding properties of the 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-mediated mutagenesis.

[0233] Conservative amino acid substitutions are typically amino acid residues wherein the amino acid residue is replaced by an amino acid residue with a side chain having similar physicochemical properties. The variable region and CDR sequences specified can comprise 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 replacements. When replacing, conservative modifications are preferred replacements. Amino acid residue families with similar side chains have been defined in the art. These families include amino acids with the following side chains: 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), non-polar 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 the antibodies or antigen-binding fragments thereof according to the invention can be replaced with other amino acid residues from the same side chain family, and the retained function of the altered antibodies (i.e., the properties described herein, e.g., binding to CD160-TM isoforms) can be tested using the assays described herein. In another embodiment, the amino acid string within the CDR and / or variable region of the antibody or antigen-binding fragment thereof according to the invention may be replaced with an amino acid string that is structurally similar but differs in the order and / or composition of side chain family members.

[0234] The present invention also relates to 21C8-like antibodies, ie, antibodies that bind to the same epitope as 21C8 or bind to substantially the same epitope as 21C8. Thus, the present invention also relates to antibodies or antigen-binding fragments that compete with 21C8 for binding to the CD160-TM isotype.

[0235] The present invention also relates to 22B12-like antibodies, i.e. antibodies that bind to the same epitope as 22B12 or bind to substantially the same epitope as 22B12. Thus, the present invention also relates to antibodies or antigen-binding fragments that compete with 22B12 for binding to the CD160-TM isotype.

[0236] 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.

[0237] 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.

[0238] In one embodiment, the antibodies or antigen-binding fragments thereof according to the present invention comprise a complete or substantially complete murine heavy chain constant region (abbreviated herein as C H ) and / or light chain constant region (abbreviated herein as C L ). In one embodiment, the constant region is of murine origin.

[0239] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a murine antibody or fragment thereof.

[0240] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a chimeric antibody or fragment thereof.

[0241] As used herein, "chimeric antibody or fragment thereof" refers to an antibody or antigen-binding fragment thereof comprising a first amino acid sequence linked to a second amino acid sequence, which are not naturally linked in nature. The amino acid sequences may normally exist in separate proteins, which are brought together in a fusion protein, or may normally exist in the same protein, but are placed in a new arrangement in the fusion protein. Chimeric proteins can be produced, for example, by chemical synthesis or by generating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.

[0242] The term "chimeric antibody or fragment thereof" herein encompasses antibodies and antigen-binding fragments thereof, wherein

[0243] (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the variable region is linked to a different constant region or a constant region of a changed type, effector function and / or species, or to an entirely different molecule, such as an enzyme, toxin, hormone, growth factor, drug, etc., that confers new properties to the chimeric antibody; or

[0244] (b) the variable region, or portion thereof, is altered, replaced or exchanged with a variable region, or portion thereof, having a different or altered antigenic specificity; or with a corresponding sequence from another species or from another antibody type or subclass.

[0245] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a humanized antibody or fragment thereof.

[0246] As used herein, "humanized antibody or its fragment" refers to a chimeric antibody or its antigen-binding fragment containing a minimal sequence derived from a non-human immunoglobulin. It includes antibodies prepared by non-human cells having variable regions and constant regions, which have been changed to more closely resemble antibodies prepared by human cells, such as by changing the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibody or its antigen-binding fragment according to the present invention may include amino acid residues (e.g., mutations, which are introduced by random or site-specific mutagenesis in vitro, or by somatic mutations in vivo), such as in CDRs.

[0247] The term "humanized antibody or its fragment" also includes antibodies and antigen-binding fragments thereof, wherein CDR sequences derived from another mammalian species (such as mice) germline are transplanted onto human framework sequences. In other words, the term "humanized antibody or its fragment" refers to antibodies or its antigen-binding fragments in which the CDRs of the recipient human antibody are replaced by CDRs from donor non-human antibodies. Humanized antibodies or their antigen-binding fragments may also include donor-derived residues in the framework sequence. Humanized antibodies or their antigen-binding fragments may also include at least a portion of human immunoglobulin constant regions. Humanized antibodies or their antigen-binding fragments may also include residues that are neither present in the recipient antibody nor in the imported CDR or framework sequences. Humanization can be performed using methods known in the art (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; US Pat. No. 4,816,567), including techniques such as "superhumanization" of antibodies (e.g., Tan et al., 2002. J Immunol. 169(2):1119-25) and "surface remodeling" (e.g., Staelens et al., 2006. Mol Immunol. 43(8):1243-57; Roguska et al., 1994. Proc Natl Acad. Sci. USA. 1994:1243-57). Sci USA.91(3):969-73).

[0248] A "humanized antibody or fragment thereof" may retain antigenic specificity similar to the original antibody or fragment. However, using certain humanization methods, the affinity and / or specificity of antibody binding may be increased.

[0249] The humanization method of the antibody or its antigen-binding fragment according to the present invention is well known in the art. The human variable domains selected for preparing humanized antibodies or their antigen-binding fragments, including light chain and heavy chain variable domains, are very important for reducing antigenicity. According to the "best fit" method, the sequence of the variable domain of the antibody or its antigen-binding fragment according to the present invention is screened according to the complete library of known human variable domain sequences. The human sequence closest to the mouse sequence is thus accepted as the human framework region (FR) of the humanized antibody or fragment (Sims et al., 1993. J Immunol. 151 (4): 2296-308; Chothia & Lesk, 1987. J Mol Biol. 196 (4): 901-17.

[0250] Another method of humanizing the antibody or antigen-binding fragment thereof according to the present invention uses a specific framework of the consensus sequence of all human antibodies from a specific 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). More importantly, the humanization of the antibody or fragment retains high affinity for CD160-TM isotypes and other favorable biological properties. To achieve this goal, according to a preferred method, a humanized antibody and its antigen-binding fragment are prepared by analyzing the parental sequence and various conceptual humanized products using a three-dimensional model of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs that illustrate and display the possible three-dimensional structures of selected candidate immunoglobulin sequences are available. Inspection of these displays allows analysis of the possible role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind to an epitope. Thus, CDR residues can be selected and combined from the consensus sequence and the input sequence to obtain the desired antibody characteristics, such as increased affinity for the CD160-TM isotype. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0251] Another method of humanizing the antibodies or antigen-binding fragments thereof according to the invention includes immunization with transgenic or transchromosomal animals carrying parts of the human immune system. As hosts, the immunoglobulin genes of these animals are replaced by functional human immunoglobulin genes. Thus, the antibodies produced by these animals or in hybridomas prepared from B cells of these animals have been humanized. Examples of such transgenic or transchromosomal animals include, but are not limited to:

[0252] - XenoMouse (Abgenix, Fremont, CA), as described in US Pat. Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963;

[0253] -HuMAb (Medarex, Inc.), such as Lonberg et al., 1994. Nature.

[0254] 368(6474):856-859; Lonberg&Huszar, 1995. Int Rev Immunol.

[0255] 13(1):65-93; Harding&Lonberg,1995. Ann NY Acad Sci.

[0256] 764:536-46; Taylor et al., 1992. Nucleic Acids Res. 20(23):6287-95;

[0257] Chen et al., 1993. Int Immunol. 5(6):647-56; Tuaillon et al., 1993.

[0258] Proc Natl Acad Sci USA.90(8):3720-4; Choi et al., 1993. Nat Genet.

[0259] 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.

[0260] 14(7):845-51;

[0261] -KM As described in patent application WO2002043478;

[0262] -TC mice, as described in Tomizuka et al., 2000. Proc Natl Acad Sci USA.

[0263] 97(2):722-7; and

[0264] -OmniRat TM (OMT, Inc.), such as patent application WO2008151081; Geurts et al., 2009. Science. 325(5939):433; Menoret et al., 2010. Eur J Immunol.

[0265] 40(10):2932-41.

[0266] Humanized antibodies and antigen-binding fragments thereof can also be produced according to various other techniques, such as by using other transgenic animals engineered to express human antibody repertoires for immunization (Jakobovitz et al., 1993. Nature. 362(6417): 255-8), or by using phage display methods to screen antibody repertoires. These techniques are known to those skilled in the art and can be implemented starting from the monoclonal antibodies or antigen-binding fragments thereof disclosed in the present application.

[0267] In one embodiment, the antibody or antigen-binding fragment thereof is from the IgG class.

[0268] In one embodiment, the antibody or antigen binding fragment thereof is from the human IgGl subclass.In one embodiment, the antibody or antigen binding fragment thereof according to the invention is thus an IgGl antibody, preferably a human IgGl antibody.

[0269] In another embodiment, the antibody or antigen-binding fragment thereof is from the human IgG2 subclass.

[0270] 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), as well as an inhibitory receptor FcγRIIb (CD32b). The communication between IgG antibodies and the immune system is controlled and mediated by FcγRs, which transmit information sensed and collected by antibodies of the immune system, providing a connection between the innate and adaptive immune systems, especially in the case of biological therapy (Hayes J et al., 2016. J Inflamm Res 9: 209-219).

[0271] IgG subclasses differ in their ability to bind to FcγRs, and this differential binding determines their ability to elicit a range of functional responses. For example, in humans, FcγRIIIa is the primary receptor involved in activating antibody-dependent cell-mediated cytotoxicity (ADCC), and IgG3 (followed closely by IgG1) displays the highest affinity for this receptor, reflecting their ability to effectively induce ADCC. IgG2 has been shown to have weak binding to this receptor.

[0272] Therefore, the constant region of an antibody is important for the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity and phagocytosis. Thus, as described herein, the isotype of an antibody can be selected based on whether the antibody is required to mediate cytotoxicity / phagocytosis.

[0273] In one embodiment, the antibodies or antigen-binding fragments thereof according to the present invention bind with high affinity to FcγR, preferably to an activating receptor with high affinity.

[0274] In one embodiment, the antibody or antigen binding fragment thereof according to the present invention binds to FcγRI and / or FcγRIIa and / or FcγRIIc and / or FcγRIIIa and / or FcγRIIIb with high affinity.

[0275] In one embodiment, the antibody or antigen binding fragment thereof according to the present invention is an IgG1 antibody (preferably a human IgG1 antibody) or a fragment thereof, and binds to at least one Fc activation receptor. For example, the antibody or antigen binding fragment thereof may bind to one or more receptors selected from the group consisting of FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and FcγRIIIb. In one embodiment, the antibody or antigen binding fragment thereof is capable of binding to FcγRIIIa. In one embodiment, the antibody or antigen binding fragment thereof is capable of binding to FcγRIIa. In one embodiment, the antibody or antigen binding fragment thereof is capable of binding to FcγRIIIa, FcγRIIc, and, optionally, FcγRI. In one embodiment, the antibody or antigen binding fragment thereof is capable of binding to FcγRIIIa, FcγRIIa and, optionally, FcγRI.

[0276] In one embodiment, the antibodies or antigen-binding fragments thereof according to the present invention are present in an amount of less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The dissociation constant of M binds to at least one activating Fcγ receptor.

[0277] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is an IgG1 antibody (preferably a human IgG1 antibody) or a fragment thereof, and binds to FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb with higher affinity, and binds to FcyRIIb with lower affinity.

[0278] In one embodiment, the antibody or antigen binding fragment thereof according to the present invention induces antibody-dependent cellular cytotoxicity (ADCC). In one embodiment, the antibody or antigen binding fragment thereof according to the present invention is from the IgG1 (preferably human IgG1) subclass and has ADCC activity.

[0279] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to cell-mediated cytotoxicity induced in an antibody-dependent manner when the Fc region of the antibody bound to its antigen binds to Fc receptors on effector cells such as natural killer cells, macrophages, neutrophils, eosinophils, and monocytes (e.g., peripheral blood mononuclear cells), resulting in lysis of the target cell. 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).

[0280] In one embodiment, the antibody or antigen binding fragment thereof according to the present invention induces antibody-dependent cell-mediated phagocytosis (ADCP). In one embodiment, the antibody or antigen binding fragment thereof according to the present invention is from the IgG1 (preferably human IgG1) subclass and has ADCP activity.

[0281] The term "antibody-dependent cell-mediated phagocytosis" (ADCP) or "opsonization" refers to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., phagocytes, macrophages) expressing Fc receptors (FcRs) recognize bound antibodies on target cells and induce phagocytosis of the target cells. 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).

[0282] 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 from the IgG1 (preferably human IgG1) subclass and has CDC activity.

[0283] The term "complement dependent cytotoxicity" (CDC) refers to the induction of lysis of antigen-expressing cells recognized by the antibodies or antigen-binding fragments thereof of the present 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., 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)).

[0284] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises an Fc region that mediates ADCC, ADCP and / or CDC.

[0285] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention does not induce ADCC, ADCP and / or CDC.

[0286] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention does not comprise an Fc region that mediates ADCC, ADCP and / or CDC.

[0287] In one embodiment, the antibodies or antigen-binding fragments thereof according to the present invention lack an Fc domain (e.g., lack a C H 2 and / or C H 3 domain) or an Fc domain comprising an IgG2 or IgG4 isotype (preferably human IgG2 or IgG4).

[0288] The antibodies or antigen-binding fragments thereof according to the present invention may be produced by any technique known in the art, such as but not limited to any chemical, biological, genetic or enzymatic technique, alone or in combination.

[0289] Therefore, another object of the present invention is a method for producing and purifying the antibodies or antigen-binding fragments thereof described herein.

[0290] In one embodiment, the method comprises:

[0291] - introducing the recombinant nucleic acid or vector described herein into a competent host cell in vitro or ex vivo;

[0292] - culturing in vitro or ex vivo a host cell transformed with the nucleic acid or expression vector under conditions suitable for expressing the antibody or antigen-binding fragment thereof;

[0293] -optionally, selecting cells that express and / or secrete said antibody or fragment thereof; and

[0294] - Recovering the expressed antibody or antigen-binding fragment thereof.

[0295] The recombinant methods can be used for large-scale production of antibodies or antigen-binding fragments thereof, including monoclonal antibodies for in vitro, ex vivo and / or in vivo therapeutic uses.

[0296] In one embodiment, the expressed antibody or antigen-binding fragment thereof is further purified.

[0297] Methods for purifying antibodies or antigen-binding fragments thereof are well known in the art and include, but are not limited to, protein A-agarose, gel electrophoresis, chromatography, preferably by affinity chromatography, more preferably by affinity chromatography on protein L agarose.

[0298] It will also be appreciated that the antibodies or antigen-binding fragments thereof according to the present invention may be modified using methods known in the art.

[0299] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is an engineered antibody or fragment thereof.

[0300] The engineered antibodies or fragments thereof according to the present invention include those in which the framework residues of VH and / or VL have been modified, for example, to improve the properties of the antibody. Typically, such framework modifications are performed to reduce the immunogenicity of the antibody. For example, one method is to "revert" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that are different from the germline sequence of the antibody source. Such residues can be identified by comparing the antibody framework sequence with the germline sequence of the antibody source. In order to return the framework region sequence to its germline configuration, the somatic mutation can be "reverted" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Such "reverted" antibodies are also intended to be encompassed in the present invention. Another type of framework modification includes 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 method is also referred to as "deimmunization" and is further described in detail in U.S. Patent Publication No. 20030153043 of Carr et al.

[0301] In one embodiment, the antibodies or antigen-binding fragments thereof according to the present invention are engineered to elicit an enhanced, increased or improved ADCC, ADCP and / or CDC response.

[0302] Methods for increasing ADCC, ADCP and / or CDC are well known in the art. For example, ADCC can be increased by eliminating the fucose moiety in the antibody polysaccharide, for example, by producing antibodies in the YB2 / 0 cell line, or by introducing specific mutations (e.g., S298A / E333A / K334A, S239D / I332E / A330L, G236A / S239D / A330L / I332E) on the Fc part of human IgG1 (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 on the Fc part of human IgG1 (Richards et al. (2008) Mol Cancer Ther 7, 2517-27). The CDC response can be increased with mutations in the antibody that increase the Clq binding affinity (Idusogie et al. (2001) J Immunol 166, 2571-5).

[0303] In one embodiment, the antibodies or antigen-binding fragments thereof according to the present invention are engineered to elicit a reduced ADCC, ADCP and / or CDC response.

[0304] Methods for reducing or eliminating ADCC, ADCP and / or CDC are also well known in the art. For example, ADCC can be reduced or eliminated by modifying the glycosylation distribution of the immunoglobulin Fc domain. CDC can be reduced or eliminated by replacing one or more amino acids with other amino acids, thereby changing the C2q binding of the antibody (Idusogie et al., U.S. Patent No. 6,194,551).

[0305] As used herein, the terms "enhanced, increased or improved ADCC, ADCP and / or CDC response" and "reduced ADCC, ADCP and / or CDC response" are relative to the ADCC, ADCP and / or CDC response induced by the modified antibodies or fragments thereof according to the present invention, compared to the ADCC, ADCP and / or CDC response induced by other anti-CD160-TM antibodies, e.g., unmodified anti-CD160-TM monoclonal antibodies.

[0306] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is engineered to modify its glycosylation.

[0307] For example, antibodies or fragments thereof according to the present invention are aglycosylated (i.e., the antibodies or fragments thereof lack glycosylation). For example, glycosylation can be changed to increase the affinity of the antibody to the antigen or to change the ADCC activity of the antibody. This carbohydrate modification can be achieved by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating the glycosylation at the site. This aglycosylation can increase the affinity of the antibody to the antigen. Additionally or alternatively, antibodies with altered glycosylation types can be prepared, such as low fucosylation or non-fucosylated antibodies with reduced amounts of fucosyl residues or no fucosyl residues, or antibodies with increased bisecting GlcNac structures. It has been confirmed that this altered fucosylation pattern can increase the ADCC ability of the antibody. This carbohydrate modification can be achieved by, for example, expressing the antibody in a host cell with an altered glycosylation mechanism. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells in which the recombinant antibodies of the present invention are expressed, thereby producing antibodies with altered glycosylation. Alternatively, antibodies (preferably monoclonal antibodies) of the invention can be produced in yeast or filamentous fungi engineered to have a mammalian-like glycosylation pattern and capable of producing antibodies that lack fucose as a glycosylation pattern.

[0308] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a pegylated antibody or fragment thereof.

[0309] For example, an antibody or a fragment thereof can be pegylated to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody or a fragment thereof, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG) (such as a reactive ester or aldehyde derivative of PEG) under conditions where one or more PEG groups are attached to the antibody or antibody fragment. PEGylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derive other proteins, such as mono (DY12-DY120) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a non-glycosylated antibody. Methods for protein pegylation are known in the art and can be applied to antibodies or fragments according to the present invention, for example, as described in EP0154316 and EP0401384 (incorporated herein by reference).

[0310] The present invention also relates to a fusion protein comprising the above antibody or antigen-binding fragment.

[0311] The present invention also relates to the conjugated antibodies or antigen-binding fragments described above.

[0312] In one embodiment, the antibody or antigen-binding fragment according to the invention is conjugated to a therapeutic moiety, ie, a drug.

[0313] The therapeutic moiety can be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulatory protein, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as "antibody-drug conjugates" or "ADCs."

[0314] Examples of radioisotopes include, but are not limited to 90 Y. 131 I or 67 Cu.

[0315] In one embodiment, the antibodies or antigen-binding fragments of the invention are conjugated to a cytotoxic moiety.

[0316] The cytotoxic moiety can be selected from the group consisting of paclitaxel; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; teniposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxyanthracenedione; microtubule inhibitors such as maytansine or its analogs or derivatives; antimitotic agents such as monomethyl auristatin E or F or its analogs or derivatives; dolastatin 10 or 15 or its analogs; irinotecan or its analogs; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or its analogs or derivatives; antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine , 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, razithromycin (CC-1065); antibiotics such as actinomycin D, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC); pyrrolo[2,1-c][1,4]-benzodiazepine (PDB); diphtheria toxin and related molecules such as diphtheria toxin A chain and its active fragments and hybrid molecules, ricin toxins such as ricin A or deglycosylated ricin A chain toxins, cholera toxins, 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 exotoxin, alorin, saporin, modulin, gelatin, abrin A chain, modulin A chain, α-sarcin, Aleurites fordii proteins, Dianthus caryophyllus proteins, Phytolacca americana proteins such as PAPI, PAPII and PAP-S, Momordica charantia inhibitor, curcumin, crotin, saponin, gelonin, mitogellin, restrictocin, phenomycin and enomycin toxins; ribonuclease (RNase); DNase I, Staphylococcus enterotoxin A; Phytolacca americana antiviral proteins; diphtheria toxin toxin; and Pseudomonas endotoxin.

[0317] In one embodiment, the antibody or antigen-binding fragment according to the present invention is conjugated to a cell-killing cytotoxic drug (payload), wherein the cell-killing cytotoxic drug is selected from the group consisting of: omamicin, vedotin, emtansine, deruxtecan, govitecan, mafodotin, pasudotox and tesirine.

[0318] In one embodiment, the antibody or antigen-binding fragment according to the invention is conjugated to a detectable label.

[0319] The labels of the anti-CD160-TM antibodies or antigen-binding fragments thereof according to the present invention that can be used include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials.

[0320] Examples of such enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase peroxidase.

[0321] Examples of prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin.

[0322] Examples of fluorescent materials include, but are not limited to, organic molecules, such as fluorescent proteins or fluorescent small organic molecules, and inorganic molecules. Examples of fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, phycoerythrin, green fluorescent protein (GFP) and its derivatives or quantum dots.

[0323] Examples of luminescent materials include, but are not limited to, luminol.

[0324] Examples of magnetic agents include gadolinium; and examples of suitable radioactive materials include 125 I. 131 I. 35 S or 3 H.

[0325] Techniques for coupling molecules to antibodies or antigen-binding fragments thereof are well known in the art. Typically, nucleic acid molecules are covalently linked to lysine or cysteine ​​on antibodies or fragments thereof via N-hydroxysuccinimide ester or maleimide functional groups, respectively. Coupling methods using engineered cysteine ​​or non-natural amino acid incorporation have been reported to improve the homogeneity of the conjugate.

[0326] The present invention also relates to the bispecific antibodies or antigen-binding fragments described above.

[0327] The present invention also relates to the unconjugated antibodies or antigen-binding fragments described above.

[0328] Another object of the present invention is an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof or fusion protein as described above.

[0329] As used herein, "isolated nucleic acid" is intended to refer to a nucleic acid that is substantially separated from other genomic DNA sequences and proteins or complexes (such as ribosomes and polymerases) that naturally accompany native sequences. The term includes nucleic acid sequences removed from their natural environment, including recombinant or cloned DNA isolates and chemically synthesized analogs, or analogs biosynthesized by heterologous systems. Substantially pure nucleic acids include nucleic acids in isolated form. Of course, this refers to the originally isolated nucleic acid and does not exclude genes or sequences that were later artificially added to the isolated nucleic acid.

[0330] In one embodiment, the isolated nucleic acid is purified.

[0331] In one embodiment, the isolated nucleic acid is purified by:

[0332] (1) Greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% by weight

[0333] or more nucleic acid, such as by absorbance or fluorescence methods (e.g., by measuring the ratio of absorbance at 260 and 280 nm (A 260 / 280 )), and most preferably greater than 96%, 97%, 98% or 99% by weight; or

[0334] (2) Homogeneity as demonstrated by agarose gel electrophoresis and the use of intercalating agents such as ethidium bromide, SYBR Green, GelGreen, etc.

[0335] In one embodiment, the nucleic acid encodes at least the heavy chain variable region or the light chain variable region of the antibody or antigen-binding fragment thereof according to the present invention.

[0336] In one embodiment, the nucleic acid encodes the variable region and the constant region of an antibody or antigen-binding fragment thereof according to the invention.

[0337] In one embodiment, the nucleic acid can 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.

[0338] 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.

[0339] In one embodiment, the nucleic acid comprises a sequence encoding the VH of an antibody or antigen-binding fragment thereof according to the present invention, or consists of it, wherein the sequence is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 19, and any sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 17 and 19, or consists of it.

[0340] SEQ ID NO:17

[0341] CAGGTCCAACTGCAGCAACCTGGGTCTGAGCTGGTGAGGCCTGGAGCT

[0342] TCAGTGAGGCTGTCCTGCAAGGCTTCTGGCTACACATTCACCAGCTACT

[0343] GGATGCACTGGGTGAGGCAGAGGCATGGACAAGGCCTTGAGTGGATTG

[0344] GAAATATTTATCCTGGTAGTGGTTTTACTAATTACGATGAGAAATTCAAG

[0345] AACAAGGGCACACTGACTGTAGACACATCCTCCAGTACAGCCTACATGC

[0346] ACCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTTCATTG

[0347] GACGAAGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCT

[0348] GCA

[0349] SEQ ID NO:19

[0350] CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCA

[0351] CAGAGCCTGTCCATAATGTGCACAGTCTCTGGTTTCTCATTAACTA

[0352] GCTATGGTTTACACTGGGTTCCGCCAGTCTCCAGGAAAGGGTCTGG

[0353] AGTGGCTGGGAGTGATATGGAGAGGTGGAAACACAGACTACAATG

[0354] CAGATTTCATGTCCAGACTGACCATCACCAAGGACAATTCCAAGA

[0355] GCCAAGTTTTCTTAAATGAACAGTCTGCAAGCTGATGACACTGC

[0356] CGTTTACTACTGTGTCAAAAATGGCGGTAACTCCAAGGACTATGCT

[0357] TTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0358] 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.

[0359] In one embodiment, the nucleic acid comprises a sequence encoding the VL of an antibody or antigen-binding fragment thereof according to the present invention, or consists of it, wherein the sequence is selected from the group consisting of SEQ ID NO:18, SEQ ID NO:20, and any sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO:18 and 20, or consists of it.

[0360] SEQ ID NO:18

[0361] GACATCCAAATGAACCAGTCTCCATCCAGTCTGTCTGCATCCCTCGGAG

[0362] ACACAATTACCATCACTTGCCGTGCCAGTCAGGGCATTAATATTTGGTTA

[0363] AACTGGTACCAGCAGAAACCAGGAAATATTCCTAAACGATTGATCTATA

[0364] AGGCTTCCAACTTGCACACAGGAGTCCCACCAAGGTTTAGAGGCAGTG

[0365] GATCTGGAACAGATTTCACATTAACTATCAGCAGCCTACAGCCTGAAGA

[0366] CATTGCCACTTACTACTGTCTACAGAGTCAAAGTTATCCATTCACGTTCG

[0367] GCTCGGGGACAAAGTTGGAAATTAGA

[0368] SEQ ID NO:20

[0369] AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAG

[0370] AGAGGGTCACCTTGAGCTGCAAGGCCAGTGAGAATGTGGGTATTTATGT

[0371] ATCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATAC

[0372] GGGTCATCCAACCGGTACACTGGGGTCCCTGATCGCTTCACAGGCAGTG

[0373] GATCTGCAACAGATTTCACTCTGATCATCAGCAGTGTTCAGGCTGAAGA

[0374] CCTTGCAGATTATCACTGTGGACAGAGTTACAGTTATCCGTACACGTTCG

[0375] GAGGGGGGACCAAGCTGGAAATAAAA

[0376] In one embodiment, the nucleic acid according to the invention comprises or consists of:

[0377] - a sequence encoding the VH of an antibody or antigen-binding fragment thereof according to the invention; and

[0378] - a sequence encoding the VL of the antibody or antigen-binding fragment thereof according to the invention.

[0379] In one embodiment, the nucleic acid according to the invention comprises or consists of:

[0380] - a sequence encoding a VH comprising or consisting of the sequence SEQ ID NO: 17; and

[0381] - a sequence encoding a VL comprising or consisting of the sequence SEQ ID NO: 18.

[0382] In one embodiment, the nucleic acid encodes the VH and VL of the 21C8 antibody.

[0383] In one embodiment, the nucleic acid according to the invention comprises or consists of:

[0384] - a sequence encoding a VH comprising or consisting of the sequence SEQ ID NO: 19; and

[0385] - a sequence encoding a VL comprising or consisting of the sequence SEQ ID NO: 20.

[0386] In one embodiment, the nucleic acid encodes the VH and VL of the 22B12 antibody.

[0387] In one embodiment, the nucleic acid according to the invention comprises a fully or substantially fully human C-terminal fragment encoding an antibody or antigen-binding fragment thereof according to the invention. H and / or C L In such embodiments, the constant region can be derived from any human antibody constant region.

[0388] In one embodiment, the nucleic acid according to the invention comprises a complete or substantially complete murine C-terminal fragment encoding an antibody or antigen-binding fragment thereof according to the invention. H and / or C L In such embodiments, the constant region can be derived from any murine antibody constant region.

[0389] In one embodiment, the nucleic acid according to the present invention comprises a sequence encoding a heavy chain of a chimeric antibody or its antigen-binding fragment according to the present invention, or consists of it. In one embodiment, the nucleic acid according to the present invention comprises a sequence encoding a light chain of a chimeric antibody or its antigen-binding fragment according to the present invention, or consists of it.

[0390] In one embodiment, the nucleic acid according to the present invention comprises a sequence encoding a heavy chain of a humanized antibody or its antigen-binding fragment according to the present invention, or consists of it. In one embodiment, the nucleic acid according to the present invention comprises a sequence encoding a light chain of a humanized antibody or its antigen-binding fragment according to the present invention, or consists of it.

[0391] Typically, the nucleic acid is a DNA or RNA molecule, which may be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0392] Therefore, another object of the present invention is an expression vector comprising the nucleic acid described above.

[0393] The terms "vector", "cloning vector" and "expression vector" refer to vectors that can introduce DNA or RNA sequences (e.g., foreign genes) into host cells, thereby transforming the host and promoting the expression (e.g., transcription and translation) of the introduced sequence. Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, etc., to cause or direct the expression of the protein or antibody or its antigen-binding fragment when applied to the host. Examples of promoters and enhancers for animal cell expression vectors include early promoters and enhancers of SV40, LTR promoters and enhancers of Moloney murine leukemia virus, promoters and enhancers of immunoglobulin H chains, etc. Any expression vector for animal cells can be used as long as the gene encoding the protein, antibody or its fragment can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSG1βd2-4, etc. Other examples of plasmids include replication plasmids containing a replication origin, or integration 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 exist in the art.

[0394] In one embodiment, the expression vector according to the present invention comprises a sequence encoding the VH of the antibody or antigen-binding fragment thereof according to the present invention, which is operably linked to a regulatory element.

[0395] In one embodiment, the expression vector according to the present invention comprises a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 19, and any sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO: 17 and 19, or consists of the same, which is operably linked to a regulatory element.

[0396] In one embodiment, the expression vector according to the present invention comprises a sequence encoding the VL of the antibody or antigen-binding fragment thereof according to the present invention, which is operably linked to a regulatory element.

[0397] In one embodiment, the expression vector according to the present invention comprises a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 20, and any sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO: 18 and 20, or consists of the same, which is operably linked to a regulatory element.

[0398] In one embodiment, the expression vector according to the present invention comprises:

[0399] - a sequence encoding the VH of an antibody or antigen-binding fragment thereof according to the invention, operably linked to a regulatory element; and

[0400] - a sequence encoding the VL of the antibody or antigen-binding fragment thereof according to the invention, operably linked to a regulatory element.

[0401] In one embodiment, the expression vector according to the present invention comprises:

[0402] a sequence encoding a VH operably linked to a regulatory element, said VH comprising or consisting of the sequence SEQ ID NO: 17 or any sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 17, and

[0403] - a sequence encoding a VL, which is operably linked to a regulatory element, wherein the VL comprises or consists of the sequence SEQ ID NO: 18 or any sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 18.

[0404] In one embodiment, the expression vector according to the present invention comprises:

[0405] a sequence encoding a VH comprising or consisting of the sequence SEQ ID NO: 19 or any sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto, operably linked to a regulatory element, and

[0406] - a sequence encoding a VL, which is operably linked to a regulatory element, wherein the VL comprises or consists of the sequence SEQ ID NO: 20 or any sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 20.

[0407] In one embodiment, the expression vector according to the present invention comprises a C-terminal fragment encoding an antibody or antigen-binding fragment thereof according to the present invention. H A sequence operably linked to a regulatory element, wherein the C H Derived from any human antibody C H .

[0408] In one embodiment, the expression vector according to the present invention comprises a C-terminal fragment encoding an antibody or antigen-binding fragment thereof according to the present invention. L A sequence operably linked to a regulatory element, wherein the C L Can be derived from any human antibody C L .

[0409] In one embodiment, the expression vector according to the present invention comprises a C-terminal fragment encoding an antibody or antigen-binding fragment thereof according to the present invention. H A sequence operably linked to a regulatory element, wherein the C H Can be derived from any mouse antibody C H .

[0410] In one embodiment, the expression vector according to the present invention comprises a C-terminal fragment encoding an antibody or antigen-binding fragment thereof according to the present invention. L A sequence operably linked to a regulatory element, wherein the C L Can be derived from any mouse antibody C L .

[0411] In one embodiment, the expression vector according to the present invention comprises a sequence encoding the heavy chain of the chimeric antibody or antigen-binding fragment thereof according to the present invention, which is operably linked to a regulatory element.

[0412] In one embodiment, the expression vector according to the present invention comprises a sequence encoding the light chain of the chimeric antibody or antigen-binding fragment thereof according to the present invention, which is operably linked to a regulatory element.

[0413] In one embodiment, the expression vector according to the present invention comprises a sequence encoding the heavy chain of the humanized antibody or antigen-binding fragment thereof according to the present invention, which is operably linked to a regulatory element.

[0414] In one embodiment, the expression vector according to the present invention comprises a sequence encoding the light chain of the humanized antibody or antigen-binding fragment thereof according to the present invention, which is operably linked to a regulatory element.

[0415] In one embodiment, the expression vector according to the present invention is monocistronic.

[0416] "Monocistronic" refers to the expression of a single nucleic acid in a single expression vector.

[0417] In one embodiment, the expression vector according to the present invention is polycistronic.

[0418] "Polycistronic" refers to the expression of at least two or more nucleic acids in a single expression vector.

[0419] Another object of the present invention is an isolated host cell comprising the expression vector described above.

[0420] The host cells can be used for recombinant production of the antibodies or antigen-binding fragments thereof according to the present invention.

[0421] In one embodiment, the host cell can be a prokaryotic cell, a yeast or a eukaryotic cell, preferably a mammalian cell, for example: a monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); a human embryonic kidney cell line (293 or a 293 cell subclone for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); a baby hamster kidney cell (BHK, ATCC CCL10); a Chinese hamster ovary cell / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); a mouse podocyte (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); a mouse myeloma cell SP2 / 0-AG14 (ATCC CRL1581; ATCC CRL8287) or NSO (HPA Culture Collection no. 85110503); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2), as well as DSM PERC-6 cell line. Expression vectors suitable for 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, all human cells into which an expression vector encoding an antibody or antigen-binding fragment thereof according to the present invention is introduced are excluded from the definition of "host cell".

[0422] Another object of the present 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.

[0423] Another object of the present invention is a pharmaceutical composition comprising, consisting essentially of, or consisting of the antibody or antigen-binding fragment, nucleic acid, expression vector or fusion protein described above, and at least one pharmaceutically acceptable excipient.

[0424] As used herein, "consisting essentially of" with respect to a composition means that the antibody or antigen-binding fragment thereof, nucleic acid, expression vector or fusion protein is the only biologically active therapeutic agent or agent in the composition.

[0425] The term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. When administered to animals, preferably mammals, more preferably humans, the excipients do not produce adverse, allergic or other untoward reactions. For human administration, the formulation should meet the sterility, pyrogenicity and general safety and purity standards required by regulatory agencies (e.g., FDA office or EMA).

[0426] Examples of pharmaceutically acceptable excipients that can 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, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

[0427] In one embodiment, the pharmaceutical composition according to the invention comprises a pharmaceutically acceptable carrier for a preparation that can be injected into a subject. These can be particularly isotonic, sterile saline solutions (monosodium phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of these salts), or dry, particularly lyophilized compositions, after adding sterile water or physiological saline as appropriate, injectable solutions can be prepared.

[0428] Another object of the present invention is a medicament comprising, essentially consisting of, or consisting of an antibody or antigen-binding fragment, a nucleic acid, an expression vector or a fusion protein as described above.

[0429] In one embodiment, a composition, pharmaceutical composition or medicament according to the invention is formulated for administration to a subject.

[0430] In one embodiment, the composition, pharmaceutical composition or medicament according to the invention is administered parenterally, by inhalation spray, rectally, nasally or via an implanted reservoir.

[0431] In one embodiment, the composition, pharmaceutical composition or medicament is administered by injection, including but not limited to subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0432] Examples of forms suitable for injection include, but are not limited to, solutions, such as sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms suitable for preparing solutions or suspensions by adding liquids before use, such as powders, liposome forms, and the like.

[0433] The sterile injectable form of the composition, pharmaceutical composition or medicine of the present invention can be an aqueous or oily suspension. These suspensions can be prepared using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenteral acceptable diluent or solvent. Acceptable carriers and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspension media. For this reason, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives can be used to prepare injections, natural pharmaceutically acceptable oils, such as olive oil or castor oil, particularly its polyoxyethylated form. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants, which are generally used to prepare pharmaceutically acceptable dosage forms (including emulsions and suspensions). Other commonly used surfactants, such as Tweens, sorbitan fatty acid esters and other emulsifiers or bioavailability enhancers, which are commonly used in preparing pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for formulation purposes.

[0434] In one embodiment, the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is administered to a subject in need thereof in a therapeutically effective amount.

[0435] However, it should be understood that the total daily dosage of the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or drug according to the present invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dosage level of any particular patient will depend on a variety of factors, including the disease to be treated and the severity of the disease; 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 administration time, 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 combination or simultaneously with the specific antibody or antigen-binding fragment thereof, fusion protein, nucleic acid or expression vector used; and similar factors known in the medical field. For example, those skilled in the art can start the dosage of the compound at a level lower than that required to achieve the desired therapeutic effect, and then gradually increase the dosage until the desired effect is achieved. The total dose required for each treatment can be administered in multiple doses or in a single dose.

[0436] The daily dosage of the antibody or antigen-binding fragment thereof according to the present invention may be within a wide range of 10-1000 mg / adult / day.

[0437] The present invention also relates to the antibodies or antigen-binding fragments, nucleic acids, expression vectors or fusion proteins described above for use as a medicament, ie for treating a disease, disorder or condition in a subject in need thereof.

[0438] The present invention also relates to a composition, a pharmaceutical composition or a medicament as described above for use as a medicament, ie for use in treating a disease, disorder or condition in a subject in need thereof.

[0439] The present invention also 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.

[0440] The present invention also relates to a method for treating a disease, disorder or condition in a subject in need thereof, comprising administering to the subject a composition, pharmaceutical composition or medicament as described above.

[0441] The present invention also relates to the use of the above-described antibodies or antigen-binding fragments, nucleic acids, expression vectors or fusion proteins in the preparation of a medicament for treating a disease, disorder or condition in a subject in need thereof.

[0442] Diseases, disorders or conditions treatable by 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 treatable by the present invention is paroxysmal nocturnal hemoglobinuria.

[0443] As used herein, the term "cancer" has its ordinary 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, cartilage, blood, and blood vessels. The term "cancer" also includes primary and metastatic cancers.

[0444] Examples of cancers that can be treated in the present invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicles, tongue, endometrium, pancreas, or uterus.

[0445] In addition, the cancer may be selected from the following non-limiting list: malignant neoplasms; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; trichomonuclear carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma associated with familial polyposis coli; solid carcinoma; malignant carcinoid tumor; branchial-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; oncocytic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrial carcinoma; carcinoma of the skin appendages; apocrine gland carcinoma; sebaceous gland carcinoma; ceroid 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; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant stromal tumor of the ovary; malignant thecoma; malignant granulosa cell tumor; malignant fibroblastoma; podocyte carcinoma; malignant stromal cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; angiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma tumor; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mixed Müllerian tumor; Wilms' tumor; hepatoblastoma; carcinosarcoma; malignant stromal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant goiter of ovary; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontoma malignant tumors of origin; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma-small lymphocytic; malignant diffuse large cell lymphoma; malignant follicular lymphoma; mycosis fungoides;Other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative intestinal disease; leukemias; lymphoid leukemias; plasma cell leukemias; erythroleukemias; lymphosarcoma cell leukemias; myeloid leukemias; basophilic leukemias; eosinophilic leukemias; monocytic leukemias; mast cell leukemias; megakaryocyte leukemias; myeloid sarcoma; and hairy cell leukemias.

[0446] In one embodiment, the cancer is breast cancer. Examples of breast cancer subtypes that can be treated with 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), HER2 positive breast cancer.

[0447] As used herein, luminal A breast cancer is positive for estrogen receptor and / or progesterone receptor, negative for HER2, and has low protein Ki-67 (also referred to as MKI67 or antigen KI-67) levels. As used herein, luminal B breast cancer is positive for estrogen receptor and / or progesterone receptor, positive for HER2, and has high Ki-67 levels. 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.

[0448] In one embodiment, the cancer is breast cancer selected from the group consisting of luminal A breast cancer, luminal B breast cancer, triple negative breast cancer (TNBC), and HER2 positive breast cancer.

[0449] 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.

[0450] As used herein, the term "infectious disease" includes any infection caused by a virus, bacteria, protozoa, mold, or fungus.

[0451] In some embodiments, the viral infection comprises an infection with one or more viruses selected from the group consisting of, but not limited to, Arenaviridae, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Cowpea Mosavirus, Cystoviridae, Flaviviridae, Lineoviridae, Hepaciviridae, Glabraviridae, Flavoviridae, Mononegavirales, Mosaviruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Picornaviridae, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Associaviridae, Tenuiviridae, Togaviridae, Tomato Bushy Stunt Viridae, Entomaviridae, Turnip Yellow Mosaic Viridae, Hepadnaviridae, Herpesviridae, Paramyxoviridae, or Papillomaviridae. Related taxonomic families of RNA viruses include, but are not limited to, Astroviridae, Birnaviridae, Bromoviridae, Caliciviridae, Closteroviridae, Cowpea Mosaicaviridae, Cystoviridae, Flaviviridae, Lineoviridae, Hepaciviridae, Glabraviridae, Flavoviridae, Mononegavirales, Mosaicaviridae, Nidovirales, Nodaviridae, Orthomyxoviridae, Picornaviridae, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Associaviridae, Tenuiviridae, Togaviridae, Tomato Bushy Viridae, Entomaviridae, and Turnip Yellows Mosaicaviridae.

[0452] In some embodiments, the viral infection comprises infection with one or more viruses selected from the group consisting of, but not limited to, 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), Guanarito virus, Hantavirus, hepatitis virus, Ilheus virus, immunodeficiency virus, influenza virus including type A and influenza B viruses (including human, avian and swine), as well as parainfluenza viruses, Junin virus, Kokobera virus, Kunjin virus, Kisanur Forest disease virus, La Crosse virus, Lassa virus, sheep jumping virus, lymphocytic choriomeningitis virus, measles virus, Machupo virus, Marburg virus, Murray Valley virus, Pachinda virus, Pichinde virus, polio virus, Powassan virus, Punta Toro virus, respiratory syncytial virus, rhinovirus, Rift Valley fever virus, Rossi virus, severe acute respiratory syndrome (SARS), smallpox virus, Tacaribe virus, West Nile virus and yellow fever virus.

[0453] Examples of bacterial infections that can be treated in the present invention include, but are not limited to, infections caused by: Staphylococcus; Streptococcus, including S. pyogenes; Enterococci; Bacillus, such as Bacillus anthracis, and Lactobacillus; Listeria; Corynebacterium diphtheriae; Gardnerella, such as G. vaginalis; Nocardia; Streptomyces; Thermoactinomyces common. vulgaris; Treponema; Campylobacter, Pseudomonas, for example, P. aeruginosa; Legionella; Neisseria, for example, N. gonorrhoeae and N. meningitides; Flavobacterium, for example, F. meningosepticum and F. odoraturn; Brucella; Bordetella, for example, B. pertussis and B. bronchiseptica; Escherichia, for example, E. coli, Klebsiella; Enterobacteriaceae, Serratia, for example, Serratia marcescens and Serratia liquefaciens; s); Edwardsiella; Proteus, such as P. mirabilis and P. vulgaris; Streptobacillus; Rickettsia, such as R. fickettsfi, Chlamydia, such as C. psittaci and C. trachornatis; Mycobacteria, such as M. tuberculosis, M. intracellulare, M. folluiturn, M. laprae, M. avium, M. bovis, M. africanum, M. kansasii, and M. lepraernurium; and Nocardia.

[0454] Examples of protozoan infections that may be treated in the present invention include, but are not limited to, infections caused by leishmania, kokzidioa, and trypanosoma.

[0455] A complete listing of infectious diseases can be found on the website of the National Center for Infectious Disease (NCID) of the Centers for Disease Control (CDC) at the World Wide Web (www) atcdc.gov / ncidod / diseases / , which listing is incorporated herein by reference.

[0456] As used herein, the term "inflammatory disease" includes a wide variety of disorders and conditions characterized by inflammation.

[0457] Examples of inflammatory diseases include, but are not limited to, arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, urticarial arthritis, gout, chronic polyarthritis, frozen 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, cirrhosis, endocardial fibrosis , mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, nephrogenic fibrosis, keloid, scleroderma, arthrofibrosis, late and chronic solid organ rejection after transplantation, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, pemphigus, pemphigus vulgaris, pemphigus herpetiformis, pemphigus vegetative, IgA pemphigus, pemphigus erythematosus, bullous pemphigus, pemphigus gestationis, mucocutaneous diseases, pemphigus nodularis, linear IgA bullous dermatosis, bullous lichen planus, epidermolysis bullosa acquisita, autoimmune diabetes 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 allergy, conjunctivitis, atherosclerosis, coronary artery disease, angina pectoris, arteriole disease, acute disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, multiple sclerosis, systemic sclerosis, antiphospholipid syndrome, Sjögren's syndrome, autoimmune hemolytic anemia, colon inflammation, Crohn's disease, ulcerative colitis, inflammatory bowel disease (IBD), embolism, pulmonary embolism, arterial embolism, venous embolism, allergic inflammation, cardiovascular disease, transplant-related disease, graft-versus-host disease (GVHD) and transplant rejection, chronic rejection and conditions associated with tissue or cell allogeneic or xenografts, autoimmune diseases, post-traumatic degeneration, stroke, transplant rejection, allergic conditions and hypersensitivity reactions, such as allergic rhinitis, allergic eczema, etc., skin diseases, inflammatory diseases of the skin, or any combination thereof.

[0458] As used herein, the term "autoimmune disease" refers to a disease in which there is a defect in the immune system, resulting in an immune response to one's own tissues.

[0459] Examples of autoimmune diseases include, but are not limited to, 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, type I diabetes, type II diabetes), pulmonary hypertension syndrome, myasthenia gravis, pemphigus, intestinal inflammatory disorders such as Crohn's disease and ulcerative colitis; sympathetic ophthalmia, auto Immune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic active hepatitis, ulcerative colitis, Sjögren'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.

[0460] As used herein, the term "paroxysmal nocturnal hemoglobinuria" or "PNH" has its general meaning in the art and refers to an acquired clonal hematopoietic stem cell disorder characterized by erythrocyte hemolytic anemia, bone marrow failure, and frequent thrombosis. In some embodiments, the subject does not have a PIGA gene (phosphatidylinositol glycan anchor biosynthesis class A, GeneID: 5277) mutation.

[0461] It will be appreciated that treatment of a disease, disorder or condition may be mediated by different mechanisms, depending on the effector function of the antibodies or antigen-binding fragments thereof of the invention.

[0462] In one embodiment, the antibodies or antigen-binding fragments thereof according to the present invention mediate ADCC, ADCP and / or CDC and can target and deplete the CD160-TM expressing cells to which they bind.

[0463] In one embodiment, administration of the antibody or fragment thereof can result in depletion of cells expressing CD160-TM (e.g., resulting in the elimination or reduction of 10%, 20%, 30%, 40%, 50%, 60% or more of CD160-TM+ NK cells), such as tumor cells expressing CD160-TM.

[0464] As used herein, the term "depletion" with respect to a cell or cell population refers to a measurable reduction in the number of cells in a subject. The reduction can be at least about 10%, such as 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 sample to below a detectable limit.

[0465] Therefore, another object of the present invention relates to a method for depleting a cell population expressing a CD160-TM isoform in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, wherein the antibody or fragment thereof mediates ADCC, ADCP and / or CDC.

[0466] Another object of the present invention relates to a method for depleting a population of malignant NK cells expressing the CD160-TM isotype in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, wherein the antibody or fragment thereof mediates ADCC, ADCP and / or CDC.

[0467] Another object of the present invention relates to a method for depleting a cell population expressing an epitope recognized by the 21C8 or 22B12 antibody in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, wherein the antibody or fragment thereof mediates ADCC, ADCP and / or CDC.

[0468] Another object of the present invention relates to a method for treating cancer in which cancer cells express CD160-TM in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, wherein the antibody or fragment thereof mediates ADCC, ADCP and / or CDC.

[0469] Another object of the present invention relates to the antibody or antigen-binding fragment thereof as described above, for use in treating cancer in a subject in need thereof, wherein the cancer cells express CD160-TM, wherein the antibody or fragment thereof mediates ADCC, ADCP and / or CDC.

[0470] In one embodiment, the cancer is a solid tumor. In one embodiment, the cancer is a hematological tumor, ie, one in which cancer cells are present in a body fluid, such as the blood or bone marrow.

[0471] 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 cell leukemia and peripheral T-cell lymphoma (PTCL), such as peripheral T-cell lymphoma not otherwise specified (PTCL-NOS).

[0472] Examples of cancers in which 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.

[0473] Thus, in one embodiment, the cancer is breast cancer.In one embodiment, the cancer is breast cancer selected from the group consisting of luminal A breast cancer, luminal B breast cancer, triple negative breast cancer (TNBC) and HER2-positive breast cancer.

[0474] 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.

[0475] Another object of the present invention is an antibody or a fragment thereof that binds to human CD160-TM for use in treating PTCL, such as peripheral T-cell lymphoma not otherwise specified (PTCL-NOS).

[0476] In one embodiment, the antibodies or antigen-binding fragments thereof according to the present invention do not mediate ADCC, ADCP and / or CDC, and can target and activate cells expressing CD160-TM to which they bind.

[0477] In one embodiment, administration of the antibody or fragment thereof does not directly or indirectly result in depletion of NK cells expressing the CD160-TM polypeptide (e.g., does not result in a 10%, 20%, 30%, 40%, 50%, 60% or greater elimination or reduction in the number of CD160-TM+ NK cells).

[0478] In one embodiment, administration of the antibody or fragment thereof results in enhancement or improvement of NK cell activity or NK cell effector function.

[0479] Therefore, another 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 administering to the subject a therapeutically effective amount of an antibody or an antigen-binding fragment thereof as described above, wherein the antibody or fragment thereof does not mediate ADCC, ADCP and / or CDC.

[0480] As used herein, "NK cell" refers to a lymphocyte subgroup that participates in innate or unconventional immunity. NK cells can be identified by certain features and biological characteristics, such as the expression of specific surface antigens of human NK cells (including CD56 and / or CD16), the absence of α / β or γ / δTCR complexes on the cell surface, the ability to bind and kill cells that cannot express "self" MHC / HLA antigens through the activation of specific cell lysis mechanisms, the ability to kill tumor cells or other diseased cells that express the ligands of NK activation receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit immune responses ("NK cell activity") . The term NK cell also includes any subgroup of NK cells. In the context of the present invention, "active" NK cells refer to biologically active NK cells, including NK cells with the ability to lyse target cells or enhance the immune function of other cells. For example, "active" NK cells can kill cells expressing the ligands of activated NK receptors and / or cells that cannot express the MHC / HLA antigens identified by the KIR on NK cells.

[0481] According to the antibody or antigen binding fragment of the present invention, the ability of enhancing NK cell activity, particularly NK cell killing activity can be determined by any assay method well known in the art. Generally, the assay is an in vitro assay, wherein NK cells are contacted with target cells (for example, target cells recognized and / or cracked by NK cells). For example, the antibody or antigen binding fragment with the ability to increase NK cell specific cracking can be selected, and its increase is greater than the effector of the NK cell or NK cell line contacted with the antibody or its fragment according to the present invention: the specific cracking amount obtained under the ratio of the target cell increases by more than about 20%, preferably at least about 30%, at least about 40%, at least about 50% or more. Generally, NK cell cytotoxicity can be determined by any assay described in the embodiment part, such as i) using the activity dye chromosome extrachromosomal assay to assess the target cell cracking induced by NK cells, or ii) in vitro assay to assess the expression of NK cell activation markers (such as CD107a expression). The cytotoxicity of NK cells can also be measured by the classical in vitro chromium release test of cytotoxicity. The effector cell can be a fresh PB-NK or NK92 cell line from a healthy donor. The target cells may be murine mastocytoma P815 cells, EBV-infected B cell lines, or other cell lines, such as K562 cells.

[0482] Therefore, the antibodies or antigen-binding fragments according to the present invention are selected because they cause increased reactivity or cytotoxicity of NK cells towards target cells (infected cells, tumor cells, proinflammatory cells, etc.), activation in NK cells, increased activation markers (e.g. CD107 expression) and / or IFNγ production, and / or an increase in the in vivo frequency of such activated, reactive, cytotoxic and / or activated NK cells.

[0483] Another object of the present invention relates to an antibody or an antigen-binding fragment thereof as described above, for use in treating cancer in a subject in need thereof, wherein the cancer cells do not express CD160-TM, wherein the antibody or fragment thereof does not mediate ADCC, ADCP and / or CDC.

[0484] Another object of the present invention relates to a method for treating cancer in which cancer cells do not express CD160-TM in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, wherein the antibody or fragment thereof does not mediate ADCC, ADCP and / or CDC.

[0485] Examples of cancer are provided above.

[0486] Another object of the present invention relates to an antibody or an antigen-binding fragment thereof as described above, for use in treating an infectious disease in a subject in need thereof, wherein the antibody or fragment thereof does not mediate ADCC, ADCP and / or CDC.

[0487] Examples of infectious diseases are provided below.

[0488] Another object of the present invention relates to an antibody or an antigen-binding fragment thereof as described above, for use in treating an inflammatory disease in a subject in need thereof, wherein the antibody or fragment thereof does not mediate ADCC, ADCP and / or CDC.

[0489] Examples of inflammatory diseases are provided below.

[0490] Another object of the present invention relates to an antibody or an antigen-binding fragment thereof as described above, for use in treating an autoimmune disease in a subject in need thereof, wherein the antibody or fragment thereof does not mediate ADCC, ADCP and / or CDC.

[0491] Examples of autoimmune diseases are provided below.

[0492] Another object of the present invention relates to a method for treating an infectious disease, an inflammatory disease and / or an autoimmune disease in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of an antibody or an antigen-binding fragment thereof as described above, wherein the antibody or fragment thereof does not mediate ADCC, ADCP and / or CDC.

[0493] Another object of the present invention relates to the antibody or antigen-binding fragment thereof as described above, for use in treating paroxysmal nocturnal hemoglobinuria (PNH) in a subject in need thereof, wherein the antibody or fragment thereof does not mediate ADCC, ADCP and / or CDC.

[0494] Another object of the present invention relates to a method for treating PNH in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof described above, wherein the antibody or fragment thereof does not mediate ADCC, ADCP and / or CDC.

[0495] 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 agent or therapeutic agent administered to a subject in need thereof.

[0496] In one embodiment, the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid or expression vector according to the present invention is used in combination with at least one other therapeutic agent.

[0497] In one embodiment, the administration of at least one other therapeutic agent and the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid or expression vector according to the present invention is performed simultaneously, separately or sequentially.

[0498] In one embodiment, the simultaneous administration of at least one other therapeutic agent and the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid or expression vector according to the invention is optionally administered as one composition, or as separate compositions.

[0499] In one embodiment, the at least one additional therapeutic agent is a therapeutic agent useful for treating a disease, disorder, or condition as described above.

[0500] Examples of other therapeutic agents include, but are not limited to, chemotherapeutic agents, targeted cancer therapies, radiotherapy, immunotherapeutic agents or anti-cancer immunogens, anti-cancer antibodies, cytotoxic agents, anti-angiogenic agents, cell cycle control / apoptosis regulators, hormone regulators and other immunosuppressive and / or anti-inflammatory drugs (selected from corticosteroids, such as glucocorticoids).

[0501] In one embodiment, the at least one other therapeutic agent is a natural ligand of an NK cell activation receptor, or an antibody that binds and activates an NK cell activation receptor other than CD160-TM. In one embodiment, the reagent is a reagent that increases the presence of a natural ligand of an NK cell activation receptor on the surface of a target cell (e.g., an infected cell or a tumor cell).

[0502] In one embodiment, wherein 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 other therapeutic agents, wherein the other therapeutic agents (i) are natural ligands for NK cell activation or antibodies that bind to and activate NK cell activation receptors other than CD160-TM, and / or (ii) increase the presence of natural ligands for NK cell activation receptors on the surface of target cells.

[0503] NK cell activating receptors include, for example, NKG2D or activating KIR receptors (KIR2DS receptor, KIR2DS2, KIR2DS4).

[0504] As used herein, the term "activated NK receptor" refers to any molecule on the surface of NK cells, when it is stimulated, causing any characteristic known in the art related to NK activity or a measurable increase in activity, such as the production of cytokines (e.g., IFN-γ and TNF-α), an increase in intracellular free calcium levels, such as the ability of targeting cells in the redirected killing assay described elsewhere in this specification, or the ability to stimulate NK cell proliferation. The term "activated 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 as activated receptor agonists include, for example, IL-2, IL-15, and IL-21 polypeptides.

[0505] In one embodiment, the at least one other therapeutic agent is an antibody specific to 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 the receptor family and their structurally related ligands are important regulators of a variety of physiological processes and play an important role in the regulation of immune responses. CD137 is expressed by activated NK cells, T and B lymphocytes, and monocytes / macrophages. The gene encodes a protein of 255 amino acids with 3 cysteine-rich motifs in the extracellular domain (a feature of the receptor family), a transmembrane region, and a short N-terminal cytoplasmic portion containing potential phosphorylation sites. Expression in primary cells is strictly activation-dependent. The ligand of the receptor is TNFSF9. It is reported that human CD137 binds only to its ligand. Agonists include natural ligands (TNFSF9), aptamers (see McNamara et al. (2008) J. Clin. Invest. 1 18:376-386), and antibodies.

[0506] In one embodiment, at least one additional therapeutic agent is an antibody that induces death via ADCC of cells expressing the antigen to which the antibody binds.

[0507] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention does not mediate ADCC, ADCP and / or CDC and is used in combination with an antibody that induces cell death by ADCC that expresses the antigen to which the antibody binds.

[0508] In one embodiment, the second antibody is specific for a cell surface antigen, such as a membrane antigen.

[0509] In some embodiments, the second antibody is specific to a tumor antigen (e.g., a molecule specifically expressed by tumor cells), 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).

[0510] Therefore, the present invention also provides a method for enhancing the anti-tumor effect of monoclonal antibodies against tumor antigens. In the method of the present invention, by sequentially administering antibodies against one or more tumor antigens and antibodies or antigen-binding fragments according to the present invention, ADCC function is specifically enhanced, thereby enhancing target cell killing.

[0511] Therefore, another object of the present invention relates to a method for enhancing NK cell ADCC of an antibody in a subject in need thereof, comprising administering the antibody to the subject, and administering the antibody or antigen-binding fragment according to the present invention to the subject, wherein preferably the antibody or fragment thereof according to the present invention does not mediate ADCC, ADCP and / or CDC.

[0512] Another object of the present invention relates to a method for treating cancer in a subject in need thereof, comprising administering to the subject a first antibody that is selective for a cancer cell antigen, and administering to the subject an antibody or antigen-binding fragment according to the present invention, wherein preferably the antibody or fragment thereof according to the present invention does not mediate ADCC, ADCP and / or CDC.

[0513] Many antibodies are currently used clinically to treat cancer, while other antibodies are at different stages of clinical development. The target antibodies used in the method of the present invention act through ADCC and are generally selective for tumor cells, although those skilled in the art recognize that some clinically useful antibodies do act on non-tumor cells, such as CD20. There are many antigens and corresponding monoclonal antibodies for the treatment of B cell malignancies. A popular target antigen is CD20, which is present 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 monoclonal antibody alentuzumab targets the CD52 antigen, which can be used to treat chronic lymphocytic leukemia. Many antibodies target CD22, and recently it has been demonstrated that it is effective in combination with toxins in chemotherapy-resistant hairy cell leukemia. Monoclonal antibodies targeting CD20 also include tositumomab and ibritumomab tiuxetan. Monoclonal antibodies that have been used for solid tumors that can be used in the method of the present invention include, but are not limited to, edrecolomab and trastuzumab (Herceptin). Edrecolomab targets the 17-1A antigen seen in colon and rectal cancer and has been approved in Europe for these indications. Its anti-tumor effects are mediated by the induction of ADCC, CDC, and anti-idiotypic networks. Trastuzumab targets the HER-2 / neu antigen. This antigen is present in 25%-35% of breast cancers. Trastuzumab is believed to work in multiple ways: downregulating HER-2 receptor expression, inhibiting the proliferation of human tumor cells that overexpress HER-2 protein, enhancing immune recruitment and ADCC against tumor cells that overexpress HER-2 protein, and downregulating angiogenic factors. Alentuzumab (Campath) can be used to treat chronic lymphocytic leukemia; colon cancer and lung cancer; gemtuzumab (Mylotarg) can be used to treat acute myeloid leukemia; ibritumomab tiuxetan (Zevalin) can be used to treat non-Hodgkin's lymphoma; and pertuzumab (Vectibix) can be used to treat colon cancer. Cetuximab (Erbitux) is also used in the methods of the present invention. The antibody binds to the EGF receptor (EGFR) and has been used to treat solid tumors, including colon cancer and squamous cell carcinoma of the head and neck (SCCHN).

[0514] In one embodiment, the at least one additional therapeutic agent is an immune checkpoint inhibitor (ICI).

[0515] Various tumors are able to express molecular factors that protect them from immune system attacks and are therefore able to successfully evade immune system surveillance. This "tumor immune escape" is mainly due to antagonistic blockade of receptors and binding sites targeted by immune cell ligands. Immune checkpoint inhibitors are molecules that specifically target such inhibitory mechanisms developed by tumor cells.

[0516] Examples of ICI include, but are not limited to, inhibitors of CTLA-4 (e.g., ipilimumab and tremelimumab), inhibitors of PD-1 (e.g., pembrolizumab, sintilimab, 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).

[0517] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention does not mediate ADCC, ADCP and / or CDC, and the antibody or fragment thereof is used in combination with at least one immune checkpoint inhibitor (ICI).

[0518] The present invention also relates to the use of at least one antibody or antigen-binding fragment thereof of the present invention for detecting CD160-TM in a sample, preferably a biological sample, in vitro or in vivo. The present invention also relates to the use of at least one antibody or antigen-binding fragment thereof of the present invention for detecting and / or quantifying CD160-TM in a sample, preferably a biological sample, in vitro or in vivo.

[0519] The present invention also relates to an antibody or antigen binding fragment thereof according to the present invention for use in vivo in detecting a tumor expressing CD160-TM in a subject, preferably wherein the antibody or fragment thereof is coupled to a detectable label. Examples of cancers in which cancer cells express CD160-TM are provided above. Examples of detectable labels are provided above.

[0520] The present invention also relates to a method for detecting CD160-TM in a sample, preferably a biological sample, in vitro, comprising the step of contacting the sample with at least one antibody or antigen-binding fragment thereof as described above.

[0521] Examples of assays in which the antibodies or antigen-binding fragments thereof according to the present 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.

[0522] The present invention also relates to a method for detecting CD160-TM in a sample, comprising the step of contacting the sample with an antibody or antigen binding fragment thereof according to the present invention, and the step of detecting an anti-CD160-TM antibody or fragment thereof bound to CD160-TM, thereby indicating the presence of CD160-TM in the sample. In one embodiment, the method is an in vitro method.

[0523] In one embodiment, the sample is a biological sample. Examples of biological samples include, but are not limited to, body fluids, preferably blood, more preferably serum, plasma, synovial fluid, bronchoalveolar lavage fluid, sputum, lymph, ascites, urine, amniotic fluid, peritoneal fluid, cerebrospinal fluid, pleural fluid, pericardial fluid and alveolar macrophages, tissue lysates and extracts prepared from diseased tissues.

[0524] In one embodiment, the sample is a 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.

[0525] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is coupled to a detectable label and can be directly detected. Examples of detectable labels are provided above.

[0526] In one embodiment, the antibody or its antigen-binding fragment according to the present invention is unlabeled (and referred to as the first / primary antibody), and is used in combination with a second antibody or other molecules capable of binding to anti-CD160-TM antibodies, and is labeled. The example of a detectable label that can be combined with the second antibody or other molecules includes but is not limited to the detectable label provided above.

[0527] Compared with the anti-CD160-TM antibodies of the prior art, the antibodies or antigen-binding fragments according to the present invention may have one or more of the following advantages:

[0528] - In some embodiments, the antibodies or antigen-binding fragments according to the present invention do not require a second antibody to induce ADCC, CDC and / or ADCP;

[0529] - In some embodiments, the antibody or antigen-binding fragment according to the present invention exhibits increased ADCC, CDC and / or ADCP compared to the CD160-TM antibodies of the prior art;

[0530] - In some embodiments, the antibody or antigen-binding fragment according to the present invention exhibits increased NK cell activation capacity or induces increased NK cell-induced lysis compared to the CD160-TM antibody of the prior art;

[0531] - in some embodiments, the antibodies or antigen-binding fragments according to the invention are suitable for therapeutic use, e.g. in humans;

[0532] - In some embodiments, the antibody or antigen-binding fragment thereof according to the present invention exhibits increased affinity for CD160-TM compared to CD160-TM antibodies of the prior art, particularly exhibits increased affinity for CD160-TM in an ELISA assay.

[0533] - In some embodiments, the antibodies or antigen-binding fragments according to the present invention can detect non-native forms of CD160-TM, for example, on fixed samples or tissues; and can therefore be used in immunohistochemistry or immunofluorescence applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0534] Figure 1 is a combination of two graphs showing NK92 cell-induced K562 target cell lysis, represented by the percentage of 7AAD-positive cells, in the presence of control murine immunoglobulin G isotype 2a (muIgG2a) or anti-CD160-TM antibodies of the present invention (22B12 and 21C8 antibodies) at an effector cell (E) to target cell (T) ratio of 8 / 1. Figure 1A The percentage of 7AAD-positive cells in K562 cells in the presence of control mouse IgG2a antibody or 21C8 antibody at an E / T cell ratio of 8 / 1. n = 4 / group, *p < 0.05 vs muIgG 2a, paired t-test. Figure 1B The percentage of 7AAD-positive cells in K562 cells in the presence of control mouse IgG2a antibody or 22B12 antibody at an E / T cell ratio of 8 / 1. n = 4 / group, *p < 0.05 vsmuIgG2a, paired t-test.

[0535] Figure 2 Is a histogram showing the percentage of NK cells expressing CD107a (NK cell degranulation marker) in the presence of anti-CD160-TM antibodies (22B12 and 21C8) of the present invention and Raji cells at different E / T ratios (2.5 / 1, 5 / 1, 10 / 1, 20 / 1). The results are expressed as mean ± SEM (Ab-control isotype) (n = 3 donors).

[0536] Figure 3 It is a histogram showing NK cell degranulation when ADCC is applied to CD160 negative target cells (Raji cells) or CD160 positive target cells (Raji cells expressing CD160-TM) in the presence of 21C8 antibody. The results are expressed as mean ± SEM (Ab-control isotype) (n = 3), *p <0.05 vs Raji, unpaired t-test.

[0537] Figure 4 is a graph showing the induction of ADCC (surface monitored by green area) on spheroids of IJG1731 breast cancer cell line expressing GFP in response to the addition of anti-CD160-TM antibody of the present invention (22B12 antibody) or isotype control antibody (murine IgG2a).

[0538] Figure 5It is a combination of three histograms showing the induction of ADCP 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 the anti-CD160-TM antibody of the present invention (22B12 antibody) or an isotype control antibody (mouse IgG2a antibody), expressed as the percentage of phagocytosis.

[0539] Figure 6 It is a combination of immunofluorescence images of anti-CD160-TM antibody 22B12 or isotype control antibody (murine IgG2a antibody) in three TNBC cell lines: IJG1731, MDA-MB-231 and BT20, and one luminal A cancer cell line: MCF7.

[0540] Figure 7 It is a combination of two histograms showing the percentage of tumor cells expressing CD160-TM in breast tumor tissues of different cancers: HER2-positive cancer, TNBC, luminal A type and luminal B type (A) and the staining intensity with the anti-CD160-TM antibody of the present invention (22B12 antibody) (B).

[0541] Figure 8 is a combination of a panel (A) and a graph (B) showing the effect of administration of an anti-CD160-TM antibody of the present invention (21C8 antibody) on tumor volume in a breast tumor mouse model compared to control murine IgG2a (muIgG2a). Figure 8 A represents a flow chart of the protocol used, in which SCID mice were injected intramammarily with BT-20 cells and treated on days 10, 13, 17 and 19 with control murine IgG2a (muIgG2a) or anti-CD160-TM 21C8 antibody. Figure 8 B shows tumor volumes of mice treated with muIgG2a or anti-CD160-TM21C8 antibody monitored on days 10-20. n=6 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs isotype control, two-way ANOVA. DETAILED DESCRIPTION

[0542] The present invention is further illustrated by the following examples.

[0543] Materials and methods

[0544] NK cell activation

[0545] NK92 cells (effector cells) were pre-incubated with mouse IgG2a control isotype, CD160-TM 21C8 antibody or CD160-TM22B12 antibody (10 μg / ml) and then incubated with K562 cells (target cells) at an E / T cell ratio of 8 / 1 at 37°C for 4 hours and 30 minutes.

[0546] The cells were labeled by staining with CD56-PE and 7AAD apoptosis dyes. The cells were analyzed by flow cytometry and the results are expressed as % of 7AAD+ cells in K562; mean ± SEM (n = 4 experiments).

[0547] CD107a analysis

[0548] PBMCs were used as a source of NK cells. To evaluate the gain of degranulation induced by engagement of CD160-TM on NK cells, PBMCs were mixed with Raji cells at an E / T ratio of 2.5 / 1, 5 / 1, 10 / 1, or 20 / 1 in the presence of 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 incubation at 37°C for 24 hours, the cells were labeled with a mixture of anti-CD3, anti-CD19, anti-CD56, and anti-CD107a, and CD107a was evaluated by flow cytometry in the presence of CD3 gated cells. - CD56 + Expression on NK cells. Similar experiments were performed using Raji cells forced to express CD160-TM (Raji-CD160-TM) to evaluate the degranulation of NK cells when displaying ADCC.

[0549] Figure 2 The results are expressed as the gain in degranulation induced by CD160-TM antibodies (21C8 or 22B12) compared to that induced by the IgG2a control isotype, depending on the E / T ratio (% Ab - % control).

[0550] Immunofluorescence in breast cancer cell lines

[0551] After adhesion on the slide, breast cancer cell lines were fixed in PBS / 4% paraformaldehyde. After a blocking step in PBS / 3% goat serum / 3% BSA, anti-CD160-TM antibody 22B12 or control isotype (muIgG2a) was added. After washing, AlexaFluor 594-coupled anti-mouse Ig antibody (Invitrogen) was added. Slides were fixed with DAPI Fluoromount solution (Southern Biotech).

[0552] Antibody-dependent cellular cytotoxicity (ADCC)

[0553] The TNBC 1JG1731 cell line was stably transfected with GFP. To form spheroids, cells were plated in ultra-low adhesion 96-well plates supplemented with 2.5% Matrigel culture medium. After 96 hours, PBMC was added as a source of NK cells, as well as a mixture of IL2 and IL15 (to promote activation) and anti-CD160-TM antibody 22B12 or control isotype (muIgG2a). Live cell imaging was performed to monitor the green fluorescence associated with spheroids (one image was collected every 6 hours). The results were expressed as the mean ± SD of the green fluorescence surface repeated three times.

[0554] Antibody-dependent cellular phagocytosis (ADCP)

[0555] Breast cancer cell lines were loaded with CFSE and then incubated with CD33+THP-1 monocyte cell line at various E / T ratios at 37°C for 2 hours and 30 minutes in the presence of anti-CD160-TM or control Ab (1 or 10 mg / mL). ADCP was evaluated by flow cytometric analysis of CFSE staining transfer to THP-1 cells (detection of CD33+CFSE+ cells).

[0556] In vivo effects of CD160-TM depletion

[0557] 5 × 10 6 CD160-TM positive triple negative breast cancer cell line BT-20. When the tumor reaches about 100mm 3 At 14:10 (day 10), mice were randomly divided into 2 groups of 6 mice each, and antibody treatment was started on the second day. 21C8 or isotype-matched negative control (muIgG2a) (100 μg) was administered intraperitoneally twice a week. Tumor size was measured on each antibody injection day (days 10, 13, 17, and 19) and at sacrifice (day 20).

[0558] result

[0559] Functionality of CD160-TM Antibody

[0560] The ability of the anti-CD160-TM antibodies of the present invention to increase the natural cytotoxicity of NK cells was first evaluated. Figure 1A -B shows that compared with the control mouse antibody, the antibodies 21C8 and 22B12 of the present invention are both able to target CD160-TM and thus promote increased NK cell killing activity against target cells.

[0561] In addition, the expression of CD107a (NK cell degranulation marker) was evaluated in the presence of the CD160-TM antibodies of the present invention (21C8 and 22B12 antibodies). Figure 2 and 3 As shown, when used as an agonist antibody on effector cells ( Figure 2 ) or as a depleting antibody against target cells expressing CD160-TM ( Figure 3 ), the anti-CD160-TM antibody of the present invention can positively regulate NK cell degranulation.

[0562] The ability of the anti-CD160-TM antibody (22B12 antibody) of the present invention to induce ADCC was further evaluated on spheroids from the IJG1731 cell line expressing GFP. Figure 4 As shown, addition of 22B12 antibody induced a decrease in fluorescence associated with spheroids, indicating that 22B12 antibody induced ADCC, whereas control isotype antibody failed to promote the disappearance of GFP-positive cells.

[0563] The ability of the anti-CD160-TM antibody (22B12 antibody) of the present invention to induce ADCP was also evaluated on three breast cancer cell lines. Figure 5 As shown in A-5C, the presence of 22B12 antibody increased phagocytosis of breast cancer cell lines compared to a control isotype antibody.

[0564] In conclusion, these results clearly demonstrate that the anti-CD160-TM antibodies of the present invention can increase the cytotoxic activity of NK cells and promote ADCC and ADCP, confirming the fact that these antibodies can be used for human therapy.

[0565] Detection of CD160-TM in cancer

[0566] The CD160-TM antibodies of the present invention were then evaluated for their ability to detect cancer cells expressing CD160-TM.

[0567] Figure 6 It was shown 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.

[0568] like Figure 7 As shown in A and 7B, the anti-CD160-TM antibody of the present invention (22B12 antibody) was used to detect tumor cells expressing CD160-TM in breast cancer tissues (including tissues from luminal A breast cancer, luminal B breast cancer, HER2-positive breast cancer and TNBC).

[0569] Taken together, 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 the presence of those CD160-TM positive tumor cells.

[0570] In vivo effects of CD160-TM depletion

[0571] The ability of the anti-CD160-TM antibodies of the invention to treat cancer was evaluated in vivo in a breast cancer mouse model. Figure 8 AB shows that administration of the anti-CD160-TM antibody of the present invention (ie, 21C8 antibody) reduced tumor growth compared to a murine IgG2a control isotype.

[0572] These data clearly support the use of the anti-CD160-TM antibodies of the present invention for treating cancer, such as breast cancer.

Claims

1. An isolated anti-human CD160-TM antibody or an antigen-binding fragment thereof, wherein the light chain variable region (VL) of the antibody or antigen-binding fragment comprises the following complementarity determining region 3 (CDR3): X1QSX2SYPX3T (SEQ ID NO: 15), wherein X1 is G or L, X2 is Y or Q, and X3 is Y or F.

2. The isolated anti-human CD160-TM antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region (VH) comprises the following three 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 following three CDRs: -CDR1:QGINIW(SEQ ID NO:4), -CDR2: KAS, -CDR3: LQSQSYPFT (SEQ ID NO: 5).

3. The isolated anti-human CD160-TM antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region (VH) comprises the following three 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 following three CDRs: -CDR1:ENVGIY(SEQ ID NO:9), -CDR2: GSS, -CDR3: GQSYSYPYT (SEQ ID NO: 10).

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

12.

5. The 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 and a light chain, the heavy chain variable region comprising a sequence having at least 70% identity with SEQ ID NO: 13, and the light chain comprising a sequence having at least 70% identity with SEQ ID NO:

14.

6. The 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 wherein the antibody or antigen-binding fragment thereof is monoclonal.

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

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

11. A pharmaceutical composition comprising the isolated anti-human CD160-TM antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the fusion protein according to claim 9, and at least one pharmaceutically acceptable excipient.

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

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

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

15. The isolated anti-human CD160-TM antibody or antigen-binding fragment thereof of 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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