Anti-CD16A antibodies and uses thereof

Abstract

Antibodies have been identified that have highly selective specificity for human CD16A protein but not for CD16B protein, and are useful for preparing multifunctional antibodies that also have specificity for tumor-associated antigens. Such multifunctional antibodies can selectively activate CD16A in a target-dependent manner, and therefore exhibit efficient activation of NK cells and macrophages and durable ADCC / ADCP functions for treating tumors, including cold tumors.

Description

[Background technology]

[0001] background Immunotherapy is a revolutionary therapeutic strategy in the field of cancer treatment that has demonstrated promising clinical benefits by harnessing the body's innate immune system to combat tumor cells. Currently, several immunotherapy strategies are under development, including monoclonal antibodies (mAbs) and immune checkpoint inhibitors targeting tumor-associated antigens (TAAs), bispecific and multispecific antibodies, engineered cytokines, tumor vaccines, adaptive cell therapy, and mRNA drugs. Among monoclonal antibody drugs, for example, rituximab, trastuzumab, and cetuximab, which target CD20, HER2, and EGFR, respectively, have been approved for the treatment of non-Hodgkin's lymphoma (NHL), HER2-positive breast cancer, and colorectal / head and neck cancer. One of the primary mechanisms of action of these drugs is to achieve tumor cell clearance by mediating antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by natural killer cells (NK cells) and macrophages.

[0002] NK cells, a vital component of the innate immune system and the body's first line of defense, can rapidly and directly eliminate cancer cells, invading microorganisms, virus-infected cells, and exografted cells through cytotoxic activity and immunoregulatory functions induced by NK cell activation. The balance between inhibitory and activating receptors on the NK cell surface determines whether NK cells are activated. Among the numerous activating and inhibitory receptors expressed on the surface of NK cells, CD16A (FcγRIIIA), a member of the Fc region receptor (FcγR) family of gamma immunoglobulin IgG, is the only receptor that mediates ADCC, one of the major cytotoxic mechanisms used by NK cells to eliminate tumor cells. Mechanistically, when a specific antibody recognizes and binds to an antigen expressed on a target cell, the antibody's Fc fragment binds to CD16A on the NK cell, inducing downstream signaling through cross-linking, ultimately triggering the degranulation of NK cell cytotoxic molecules, including granzyme B and perforin, thereby mediating direct lysis of the target cell. On the other hand, activated NK cells can release proinflammatory cytokines, such as IFN-γ and TNF-α, to recruit adaptive immune cells to participate in the elimination of target cells.

[0003] Human CD16 exists in two isoforms, CD16A and CD16B (FcγRIIIB), which share 96% sequence identity in their immunoglobulin-binding regions. Due to allelic polymorphism, CD16A has two allelic forms: CD16A-158F, which has low affinity for IgG and is the dominant allele in approximately 60% of humans, and CD16A-158V (or CD16A-176V, based on the precursor sequence of SEQ ID NO: 55), which has high affinity for IgG. Clinical data demonstrate that the high-affinity CD16A-158V predicts a favorable response to therapeutic IgG antibodies. In addition to its expression on NK cells, CD16A is also present on the surface of monocytes and macrophages, where it mediates phagocytic ADCP of target cells. CD16B, a GPI-anchored receptor, exhibits distinct functions due to its selective expression on neutrophils and eosinophils. CD16B has been reported to function as a decoy receptor, capturing IgG by the Fc fragment without inducing cell activation, resulting in devastating damage to ADCC function. There are three allelic forms of human CD16B, including CD16B-NA1 (R36, N65, D82, V106), CD16B-NA2 (S36, S65, N82, I106), and CD16B-SH (D78, N65), which differ by five amino acids. CD32B (FcγRIIB), the only inhibitory receptor among the FcγRs, is primarily expressed on B cells, macrophages, dendritic cells, neutrophils, and basophils. This receptor plays a regulatory role in controlling the threshold and degree of cell activation by Fc binding.

[0004] Because CD16A is essential for ADCC and ADCP mediated by NK cells and macrophages, developing drugs targeting CD16A is a promising strategy for activating NK cells and macrophages. An ideal strategy would selectively activate both the CD16A-158F and CD16A-158V allelic forms without interacting with the decoy receptor CD16B and the inhibitory receptor CD32B. However, current strategies for protein engineering of Fc fragments have partially achieved enhanced binding to both CD16A alleles and attenuated binding to the inhibitory receptor CD32B, but have failed to suppress CD16B binding due to the high amino acid sequence similarity between CD16A and CD16B. For example, tafasitamab (MorphoSys), an anti-CD19 mAb with engineered S239D / I332E(DE) mutations in its Fc region that contribute to enhanced ADCC and ADCP functions, was approved by the EMA in 2020 for second-line treatment of DLBCL in combination with lenalidomide. Obinutuzumab (Roche), a second-generation anti-CD20 mAb with a fully defucosylated Fc region, strongly binds to both CD16A alleles, enhancing ADCC efficacy. Nevertheless, these Fc modifications also increased binding affinity for CD16B, limiting their clinical benefit.

[0005] An alternative strategy is to develop anti-CD16A-specific antibodies for the construction of bispecific or multispecific fragments to selectively and efficiently activate CD16A in a target-dependent manner. These types of molecules, in which one arm binds to CD16A and the other arm targets a specific antigen on the target cell, can exhibit conditional and efficient activation of NK cells and macrophages and persistent ADCC / ADCP functions without binding to CD16B or CD32B. For example, the anti-CD16A antibody 4-LS-21, developed by Affimed Therapeutics, and its affinity-matured version, named P2C47, showed similar high affinity for both CD16A-158V and CD16A-158F, but did not bind to CD16B or CD32B. Based on the CD16A antibody 4-LS21, two clinical assets, Fc fragment-less AFM13 (CD30-CD16A) and Fc-inactivated AFM24 (EGFR-CD16A), are currently under clinical evaluation for the treatment of relapsed peripheral T-cell lymphoma, Hodgkin's lymphoma (HL), and EGFR-expressing solid tumors. Recently, AFM13 was reported to demonstrate clinical responses with a 100% overall response rate (ORR) in the recommended phase II dose (RP2D) group when combined with NK cell therapy in CD30-positive lymphoma. Based on these preliminary findings, the potential of this unique strategy in a wide range of therapeutic areas must be fully explored by developing efficient CD16A-specific antibodies and exploring the appropriate components and formats of CD16A-specific bispecific or multispecific fragments.

[0006] The incidence of hepatocellular carcinoma (HCC) is on the rise, especially in East Asia. Since the discovery of glypican 3 (GPC3) in the last century, which shows high expression levels in liver cancer tissue, the application of GPC3 has attracted widespread attention. As a type of heparan sulfate (HS) glycoprotein, GPC3 can interact with growth factors, extracellular matrix proteins, and adhesion molecules to regulate cell proliferation, differentiation, adhesion, and migration. Due to its very limited expression in normal tissues, GPC3 is considered to be a specific TAA in HCC, and strategies exploring GPC3 as a therapeutic target may provide a new approach to treating liver disease. However, a randomized phase II trial (NCT01507168) evaluating the GPC3 monoclonal antibody GC33, which possesses a wild-type human IgG1 Fc region capable of ADCC, failed to demonstrate clinical benefit in patients with advanced HCC who had failed previous systemic therapy. Biomarker analysis demonstrated that high levels of CD16A on peripheral immune cells were associated with prolonged progression-free survival and overall survival. These results indicated that more efficient strategies for inducing GPC3-targeting CD16A activation and the resulting ADCC and ADCP functions are necessary to contribute to better clinical outcomes. Summary of the Invention [Means for solving the problem]

[0007] Abstract In various embodiments, the present disclosure provides antibodies and antigen-binding fragments specific for human CD16A protein. Experimental studies have shown that these newly identified antibodies can strongly and specifically bind to human CD16A protein without interacting with CD16B variants. Some of these antibodies also cross-react with cynomolgus monkey CD16 protein, thus facilitating preclinical studies. Furthermore, in vitro and in vivo studies have demonstrated that bispecific fragments constructed from anti-CD16A antibodies activate NK cells and mediate ADCC against target cells expressing certain antigens. Compared with other ADCC enhancement strategies, such as monoclonal antibodies with engineered Fc, including DE, DLE (S239D / A330L / I332E), or defucosylated Fc, anti-CD16A-based multispecific fragments exhibited stronger ADCC effects. More interestingly, anti-tumor killing was further enhanced when Fc function was intact. This suggests that anti-CD16A and Fc-mediated effector functions are synergistic.

[0008] Accordingly, one embodiment of the present disclosure provides an antibody or antigen-binding fragment thereof having binding specificity for human CD16A protein, comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of: (a) SEQ ID NOs: 22, 23, 24, 37, 38, and 39; (b) SEQ ID NOs: 13, 14, 15, 28, 29, and 30; (c) SEQ ID NOs: 16, 17, 18, 31, 32, and 33; (d) SEQ ID NOs: 19, 20, 21, 34, 35, and 36; or (e) SEQ ID NOs: 25, 26, 27, 40, 41, and 42, respectively.

[0009] Also provided in one embodiment is a multispecific antibody comprising an anti-CD16A antibody or antigen-binding fragment thereof that has binding specificity for human CD16A protein; and a second antibody or antigen-binding fragment thereof that has binding specificity for a tumor-associated antigen (TAA).

[0010] In some embodiments, the multispecific antibody further comprises an Fc fragment. In some embodiments, the anti-CD16A antibody or antigen-binding fragment and the Fc fragment have a synergistic effect when exerting effector function. In some embodiments, the Fc fragment can bind to CD64 but cannot bind to CD16B and / or CD32B. In some embodiments, the Fc fragment is a wild-type human IgG Fc fragment.

[0011] Methods and uses for treating diseases such as cancer are also provided. In some embodiments, the cancer is prostate cancer, pancreatic cancer, leukemia, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, liver cancer, esophageal cancer, cervical cancer, thyroid cancer, lung cancer, bladder cancer, kidney cancer, uterine cancer, or melanoma. [Brief explanation of the drawings]

[0012] [Figure 1] 1A-B show the binding ability of anti-CD16A antibodies to human CD16A-158V-his (A) and human CD16A-158F-his (B) in an ELISA assay.

[0013] [Figure 2] 2A, 2B, and 2C show the binding ability of anti-CD16A antibodies to human CD16B-NA1-his, human CD16B-NA2-his, and human CD16B-SH-his in an ELISA assay.

[0014] [Figure 3] FIG. 3 shows the cross-species binding ability of anti-CD16A antibodies to cynomolgus monkey CD16-his in an ELISA assay.

[0015] [Figure 4] 4A and 4B show the binding ability of anti-CD16A antibodies to human CD16A-158V and CD16A-158F expressed on CHO-K1 cells, as measured by flow cytometry.

[0016] [Figure 5] 5A and 5B show the binding ability of anti-CD16A antibodies to human CD16B-NA1 and human CD32B expressed on CHO-K1 cells, as measured by flow cytometry.

[0017] [Figure 6] FIG. 6 shows the cross-species binding ability of anti-CD16A antibodies to cynomolgus monkey CD16 expressed on CHO-K1 cells, as measured by flow cytometry.

[0018] [Figure 7] 7A-D show the binding ability of a series of humanized anti-CD16A antibodies to human CD16A-158V, CD16A-158F, CD16B-NA1, and cynomolgus monkey CD16 expressed on CHO-K1 cells, as measured by flow cytometry.

[0019] [Figure 8] Figure 8 shows the binding ability of a series of humanized anti-CD16A antibodies with deleted post-translational modification (PTM) sites to human CD16A-158V, CD16A-158F, CD16B-NA1, and cynomolgus monkey CD16 expressed on CHO-K1 cells, as measured by flow cytometry.

[0020] [Figure 9] Figure 9 shows a CD16A bispecific fragment of Format 1, which contains an antigen-binding fragment in the form of a bivalent Fab and an anti-CD16A fragment in the form of a bivalent scFv, separately at the N- and C-termini of a human IgG1 Fc fragment, with or without FcγR binding.

[0021] [Figure 10]Figures 10A-D show the binding ability of various GPC3-CD16A bispecific antibodies and anti-GPC3 mAbs to human GPC3 on GPC3+HepG2 cells (Figure 10A) and GPC3-SK-HEP-1 cells (Figure 10B), and to human CD16A-158V (Figure 10C) and CD16A-158F (Figure 10D) expressed on CHO-K1 cells.

[0022] [Figure 11] Figures 11A-D show CD16A downstream signaling in Jurkat-NFAT cells induced by various chimeric and humanized GPC3-CD16A BsAbs against GPC3+HepG2, Huh-7, and PLC-PRF-5 cells or GPC3-SK-HEP-1 cells.

[0023] [Figure 12] Figures 12A-C show primary NK cell-mediated cytotoxicity against GPC3-expressing and GPC3-negative tumor cells induced by various chimeric and humanized GPC3-CD16A BsAbs.

[0024] [Figure 13] 13A-C show primary NK cell degranulation induced by GPC3-CD16A BsAb against GPC3-expressing and GPC3-negative tumor cells.

[0025] [Figure 14] Figures 14A-F show primary NK cell-mediated cytokine production against GPC3-expressing and GPC3-negative tumor cells induced by GPC3-CD16A BsAb.

[0026] [Figure 15] 15A-C show the binding ability of various CCR8-CD16A BsAbs and anti-hCCR8 mAbs to human CD16A-158V, CD16A-158F, and CD32B expressed on CHO-K1 cells.

[0027] [Figure 16] Figures 16A-D show primary NK cell-mediated cytotoxicity, degranulation and cytokine production against CCR8-expressing tumor cells induced by CCR8-CD16A BsAb.

[0028] [Figure 17] FIG. 17 shows primary NK cell-mediated cytotoxicity against CCR8-expressing tumor cells induced by various chimeric and humanized CCR8-CD16A BsAbs.

[0029] [Figure 18] Figures 18A-B show the binding ability of the in-house anti-mCCR8 clone 194A1G9 to human and mouse CCR8 expressed on cells.

[0030] [Figure 19] FIG. 19 shows ADCC signaling of 194A1G9-42F5H2 BsAb and 194A1G9 mAb.

[0031] [Figure 20-1] Figure 20 shows an in vivo comparison of 194A1G9-42F5H2 BsAb and 194A1G9 mAb. [Figure 20-2] Same as above.

[0032] [Figure 21] Figure 21 shows an in vivo comparison of SA214G2-45H6E8 BsAb and SA214G2 mAb. DETAILED DESCRIPTION OF THE INVENTION

[0033] Detailed Description definition It should be noted that the term "a" or "an" entity refers to one or more of that entity, e.g., "an antibody" is understood to represent one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0034] As used herein, the term "polypeptide" is intended to encompass the singular as well as the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modifications with non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. Polypeptides may be produced in any manner, including by chemical synthesis.

[0035] The term "isolated," as used herein with respect to cells, nucleic acids, e.g., DNA or RNA, refers to molecules separated from other DNA or RNA, respectively, present in the natural source of the polymer. The term "isolated," as used herein, also refers to nucleic acids or peptides that, when produced by recombinant DNA techniques, are substantially free of cellular material, viral material, and culture medium, or, when chemically synthesized, are substantially free of chemical precursors and other chemicals. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that do not naturally occur as fragments and are not found in their natural state. The term "isolated" is also used herein to refer to cells or polypeptides that are isolated from other cellular proteins or tissues. Isolated polypeptides are intended to encompass both purified and recombinant polypeptides.

[0036] As used herein, "antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide containing molecule comprising at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, the complementarity-determining regions (CDRs) of a heavy or light chain or its ligand-binding portion, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0037] The term "antibody fragment" or "antigen-binding fragment," as used herein, refers to a portion of an antibody, e.g., F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0038] A "single-chain variable fragment" or "scFv" is a fragment of an immunoglobulin heavy chain (V H ) and the variable region of the light chain (V L In some embodiments, the domains are joined by a short linker peptide of 10 to about 25 amino acids. The linker can contain many glycines for flexibility, as well as many serine or threonines for solubility, and the V H N-terminus of V L The scFv molecule can be linked either to the C-terminus of the IgG or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. scFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0039] The term antibody encompasses a wide variety of biochemically distinguishable polypeptide classes. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses within these (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, such as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to those skilled in the art in light of the present disclosure and, therefore, are within the scope of the present disclosure. All immunoglobulin classes are expressly within the scope of the present disclosure, and the following discussion will generally refer to the IgG class of immunoglobulin molecules. For IgG, a standard immunoglobulin molecule contains two identical light polypeptide chains of approximately 23,000 daltons molecular weight and two identical heavy polypeptide chains of 53,000-70,000 molecular weight. The four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains, starting at the mouth of the "Y" and continuing through the variable region.

[0040] Antibodies, antigen-binding polypeptides thereof, variants, or derivatives of the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments comprising the VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the LIGHT antibodies disclosed herein). Immunoglobulin or antibody molecules of the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule.

[0041] Light chains are classified as either kappa or lambda (K or λ). Each heavy chain class may be associated with either kappa or lambda light chains. Generally, light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by either a hybridoma, a B cell, or a genetically engineered host cell. For heavy chains, the amino acid sequence runs from the N-terminus at the forked ends of the Y-shape to the C-terminus at the base of each chain.

[0042] Both light and heavy chains are divided into structurally and functionally homologous regions. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light (VK) and heavy (VH) chains determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CK) and the heavy chain (CH1, CH2, or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of constant region domains increases as they become more distal from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region, with the CH3 and CK domains actually comprising the carboxy termini of the heavy and light chains, respectively.

[0043] As described above, the variable region enables an antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VK domain and VH domain, or a subset of complementarity-determining regions (CDRs), of an antibody combine to form the variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each of the VH and VK chains. In some cases, for example, certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, the complete immunoglobulin molecule may consist of only a heavy chain, without any light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

[0044] In naturally occurring antibodies, the six "complementarity-determining regions" or "CDRs" present in each antigen-binding domain are short, noncontiguous sequences of amino acids specifically arranged to form the antigen-binding domain when the antibody assumes its three-dimensional shape in an aqueous environment. The remaining amino acids within the antigen-binding domain, called "framework" regions, show little intermolecular variability. The framework regions primarily adopt a β-sheet conformation, while the CDRs form loops that connect, and in some cases become part of, the β-sheet structure. Thus, the framework regions act as a scaffold that allows for the correct orientation of the CDRs through interchain noncovalent interactions. The antigen-binding domain formed by the arranged CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes noncovalent binding of the antibody to its cognate epitope. Those skilled in the art can easily identify the amino acids that make up the CDRs and framework regions, respectively, for any given heavy or light chain variable region. because they are precisely defined (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0045] When there are two or more definitions for a term used and / or accepted in the art, the definition of that term used herein is intended to encompass all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe the discontinuous antigen-binding site found within the variable regions of both heavy and light chain polypeptides. This particular region is described by Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The CDR definitions by Kabat and Chothia include overlapping or subsets of amino acid residues when compared with each other. However, application of either definition to refer to the CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues that encompass the CDRs defined by each of the above-cited references are shown for comparison in the table below. The exact number of residues that encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the amino acid sequence of the variable region of an antibody. [Table A]

[0046] Kabat et al. also define a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain without reliance on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0047] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine ​​residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins 15 residues after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins approximately 33 amino acid residues after the end of CDR-H2, includes 3-25 amino acids, and ends with the sequence WGXG, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., after the cysteine ​​residue), includes approximately 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins approximately 16 residues after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins approximately 33 residues after the end of CDR-L2 (i.e., after the cysteine ​​residue), contains approximately 7 to 11 residues, and ends with the sequence F or WGXG, where X is any amino acid.

[0048] The antibodies disclosed herein can be of any animal origin, including birds and mammals.Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.In another embodiment, the variable region can be of chondrichthyan origin (e.g., from sharks).

[0049] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the present disclosure may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain, a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present disclosure comprises a polypeptide chain comprising a CH3 domain. Furthermore, an antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of the CH2 domain). As indicated above, it will be understood by those skilled in the art that the heavy chain constant regions can be modified so that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0050] The heavy chain constant region of the antibody disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide can include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region can include a hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another example, the heavy chain portion can include a chimeric hinge that is partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0051] As used herein, the term "light chain constant region" includes amino acid sequences derived from an antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.

[0052] "Specifically binds" or "having specificity for" generally means that an antibody binds to an epitope via its antigen-binding domain and that binding requires a degree of complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it binds to that epitope via its antigen-binding domain more readily than it would to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind epitope "C" with higher specificity than it has for related epitope "D."

[0053] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures in which the goal is to prevent or slow (reduce) an undesirable physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilized (i.e., not worsening) disease, delayed or slowed disease progression, improvement or reduction in disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already suffering from a condition or disorder, as well as those susceptible to the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0054] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, domestic animals, livestock, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, etc.

[0055] As used herein, phrases such as "patient in need of treatment" or "subject in need of treatment" include subjects, e.g., mammalian subjects, who would benefit from the administration of an antibody or composition of the present disclosure for use, e.g., in detection, diagnostic procedures, and / or treatment. Anti-CD16A antibody

[0056] Through trial and error, the inventors were able to identify new antibodies that can bind strongly and specifically to human CD16A protein. As shown in the experimental examples, all of the tested antibodies showed strong affinity to both the CD16A 158V polymorphic form (high affinity) and the more common 158F form (low affinity). Interestingly, one of the newly identified antibodies, 45H6E8, also showed cross-reactivity to cynomolgus monkey CD16. It is also important to note that these new antibodies showed negligible binding to CD16B.

[0057] Yet another important finding is that these new anti-CD16A antibodies showed minimal or no binding to CD32B, the only inhibitory FcγR. Therefore, reduced interaction with FcγRs contributes to the antitumor activity of these antibodies.

[0058] These newly developed antibodies were also used to create bispecific antibodies with a second specificity for the tumor-associated antigens (TAAs) GPC3 or CCR8. These bispecific antibodies only had the activity of inducing CD16A signaling, even though the coupled antigen-binding moiety also recognized the corresponding antigen on target cells. Therefore, these newly developed anti-CD16A antibodies are suitable for clinical use in the prevention or treatment of various diseases.

[0059] Thus, according to one embodiment of the present disclosure, there is provided an antibody or antigen-binding fragment thereof having binding specificity for human CD16A protein, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are those of an antibody shown in Tables 1, 6A, and 7A, respectively. Representative CDR sequences (Kabat numbering) of these antibodies, for example, in SEQ ID NOs: 22, 96, 24, 37, 38 and 39; SEQ ID NOs: 22, 23, 24, 37, 38 and 39; SEQ ID NOs: 13, 14, 15, 28, 29 and 30; SEQ ID NOs: 16, 17, 18, 31, 32 and 33; SEQ ID NOs: 19, 20, 21, 34, 35 and 36; or SEQ ID NOs: 25, 26, 27, 40, 41 and 42, are shown in Tables 1A-B and 7B.

[0060] In one embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises the CDR regions of antibody 45H6E8, which has the VH sequence of SEQ ID NO:7 and the VL sequence of SEQ ID NO:8.

[0061] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 have the sequences of SEQ ID NOs: 22, 23, 24, 37, 38, and 39, respectively. In some embodiments, an antibody or antigen-binding fragment of the disclosure comprises these CDR sequences and has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to antibody 45H6E8 or any of its humanized counterparts. In one embodiment, VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:8.

[0062] In some embodiments, the 45H6E8 antibody is humanized. Exemplary VH sequences of humanized 45H6E8 antibodies include SEQ ID NOs: 83-88. An example VL sequence is SEQ ID NO: 89. In some embodiments, one or more of the CDRs of the 45H6E8 antibody are post-translationally modified (PTM)-derisked. An example is SEQ ID NO: 96 for VH CDR2. Exemplary VH sequences of humanized, PTM-derisked 45H6E8 antibodies include SEQ ID NOs: 91, 93, and 95.

[0063] In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 83. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 89. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 84. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 89.

[0064] In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 85. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 89. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 86. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 89.

[0065] In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 86. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 89. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 87. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 89. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 88. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:89.

[0066] In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 91. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 89. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 93. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 89. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 95. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:89.

[0067] Thus, in some embodiments, antibodies and antigen-binding fragments are provided that bind to the same epitope on CD16A as 45H6E8, hi some embodiments, antibodies and antigen-binding fragments are provided that compete with 45H6E8 for binding to CD16A.

[0068] In one embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises the CDR regions of antibody 23H7E4, which has the VH sequence of SEQ ID NO:1 and the VL sequence of SEQ ID NO:2.

[0069] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 have the sequences of SEQ ID NOs: 13, 14, 15, 28, 29, and 30, respectively. In some embodiments, an antibody or antigen-binding fragment of the disclosure comprises these CDR sequences and has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to antibody 23H7E4 or any of its humanized counterparts. In one embodiment, VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:2.

[0070] Thus, in some embodiments, antibodies and antigen-binding fragments are provided that bind to the same epitope on CD16A as 23H7E4, hi some embodiments, antibodies and antigen-binding fragments are provided that compete with 23H7E4 for binding to CD16A.

[0071] In one embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises the CDR regions of antibody 37B3G11, which has the VH sequence of SEQ ID NO:3 and the VL sequence of SEQ ID NO:4.

[0072] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 have the sequences of SEQ ID NOs: 16, 17, 18, 31, 32, and 33, respectively. In some embodiments, an antibody or antigen-binding fragment of the disclosure comprises these CDR sequences and has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to antibody 37B3G11 or any of its humanized counterparts. In one embodiment, VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:3. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:4.

[0073] Thus, in some embodiments, antibodies and antigen-binding fragments are provided that bind to the same epitope on CD16A as 37B3G11. In some embodiments, antibodies and antigen-binding fragments are provided that compete with 37B3G11 for binding to CD16A.

[0074] In one embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises the CDR regions of antibody 42F5H2, which has the VH sequence of SEQ ID NO:5 and the VL sequence of SEQ ID NO:6.

[0075] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 have the sequences of SEQ ID NOs: 19, 20, 21, 34, 35, and 36, respectively. In some embodiments, an antibody or antigen-binding fragment of the disclosure comprises these CDR sequences and has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to antibody 42F5H2 or any of its humanized counterparts. In one embodiment, VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:5. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:6.

[0076] Thus, in some embodiments, antibodies and antigen-binding fragments are provided that bind to the same epitope on CD16A as 42F5H2, hi some embodiments, antibodies and antigen-binding fragments are provided that compete with 42F5H2 for binding to CD16A.

[0077] In one embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises the CDR regions of antibody 91A11D11, having the VH sequence of SEQ ID NO:9 and the VL sequence of SEQ ID NO:10.

[0078] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 have the sequences of SEQ ID NOs: 25, 26, 27, 40, 41, and 42, respectively. In some embodiments, an antibody or antigen-binding fragment of the disclosure comprises these CDR sequences and has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to antibody 91A11D11 or any of its humanized counterparts. In one embodiment, VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9. In one embodiment, the VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:10.

[0079] Thus, in some embodiments, antibodies and antigen-binding fragments are provided that bind to the same epitope on CD16A as 91A11D11, hi some embodiments, antibodies and antigen-binding fragments are provided that compete with 91A11D11 for binding to CD16A.

[0080] In some embodiments, the antibody of the present disclosure is a full-size IgG antibody, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody has an Fc fragment without effector function (e.g., unable to bind to Fcγ receptors or complement proteins). Modifications of wild-type Fc fragments that can deprive the Fc fragment of binding activity, etc., are known. One example is the IgG1 LALAPG mutant (L234A / L235A / P329G, EU numbering; SEQ ID NO: 76).

[0081] Another example is an IgG1 Fc with L234A / L235A (LALA), which reduces binding to the IgG Fc receptors FcγRI, FcγRII, and FcγRIII and to complement component C1q. Still further examples are L234F / L235E / P331S (FES), L234F / L235Q / K322Q (FQQ), L234A / G237A, L234A / L235A / G237A, L234A / L235A / G237A / P238S / H268A / A330S / P330S, L234A / L235E, G236R / L328R, and L234A / L235A / K322A. An example for IgG2 Fc is A330S / P331S (also EU numbering). multifunctional molecules

[0082] Another embodiment of the present disclosure provides multispecific / multifunctional antibodies that can selectively activate CD16A in a target-dependent manner. Such multifunctional molecules, with one moiety binding to CD16A and another moiety targeting a specific antigen on target tumor cells, can exhibit efficient activation of NK cells and macrophages and durable ADCC / ADCP functions.

[0083] Thus, one embodiment provides a multispecific antibody comprising an anti-CD16A antibody or antigen-binding fragment having binding specificity for human CD16A protein; and a second antibody or antigen-binding fragment having binding specificity for a tumor-associated antigen (TAA), which may also be referred to as an anti-TAA antibody or antigen-binding fragment.

[0084] In some embodiments, the multispecific antibody comprises an Fc fragment, which may be an Fc fragment of human IgG1, IgG2, IgG3, or IgG4 or an engineered isoform thereof.

[0085] In one embodiment, the Fc fragment has reduced or no effector function, which can be achieved by Fc mutations that reduce or abolish binding to Fcγ receptors or complement proteins.

[0086] Examples of mutations that reduce or eliminate Fc effector function include, but are not limited to, IgG1 L234A / L235A (LALA), L234A / L235A / P329G (LALAPG), L234F / L235E / P331S (FES), L234F / L235Q / K322Q (FQQ), L234A / G237A, L234A / L235A / G237A, 234A / L235A / G237A / P238S / H268A / A330S / P330S, L234A / L235E, G236R / L328R, and L234A / L235A / K322A, and IgG2 A330S / P331S (EU numbering). In a preferred embodiment, the Fc fragment is IgG1 LALAPG (SEQ ID NO: 76).

[0087] In another embodiment, the Fc fragment is a wild-type Fc, such as a wild-type IgG1 Fc, a wild-type IgG2 Fc, a wild-type IgG3 Fc, or a wild-type IgG4 Fc. In a specific embodiment, the Fc is a wild-type IgG1 Fc. In certain embodiments, the Fc fragment does not contain a mutation that can enhance the effector function of the antibody. In some embodiments, the Fc fragment does not contain a post-translational modification that can enhance the effector function of the antibody.

[0088] As demonstrated in the experimental examples, the natural effector functions of these wild-type Fc fragments can synergize with anti-CD16A antibodies / fragments. Such Fc fragments can bind to CD64 and do not bind to CD16B or CD32B, which can inhibit or reduce ADCC activity. Enhancement of effector function, for example, by mutations such as Fc-DLE (S239D / A330L / I332E) or Fc-DE (S239D / I332E), can reduce ADCC efficacy by causing or increasing binding to CD16B and / or CD32B.

[0089] Multispecific antibodies can take any format in which the anti-CD16A and / or anti-TAA portions can comprise one, two, three, or four antigen-binding fragments. In some embodiments, the anti-CD16A and anti-TAA portions can be on the same side (e.g., N-terminal) of the Fc fragment.

[0090] In some embodiments, the anti-CD16A and anti-TAA moieties may be on opposite sides of the Fc fragment. In one embodiment, the anti-CD16A moiety is on the N-terminal side of the Fc, and the anti-TAA moiety is on the C-terminal side of the Fc. In another embodiment, the anti-CD16A moiety is on the C-terminal side of the Fc, and the anti-TAA moiety is on the N-terminal side of the Fc.

[0091] The anti-TAA portion, when at the N-terminus of the Fc fragment, can form a conventional full-size IgG antibody with the Fc fragment, while the anti-CD16A portion, which can exist in the form of two separate single-chain fragments (scFv), is fused to the C-terminus of the Fc fragment.

[0092] The anti-CD16A antibody or fragment can be any known anti-CD16A antibody or fragment, for example, P2C47, or one presently identified, including 45H6E8, 23H7E4, 37B3G11, 42F5H2, and 91A11D11.

[0093] In some embodiments, the anti-CD16A antibody or fragment, eg, 45H6E8, preferably has minimal or no affinity for the human CD16B protein.

[0094] Exemplary anti-CD16A antibodies or fragments include the CDR sequences of the antibodies shown in Tables 1, 6A, and 7A. Representative CDR sequences (Kabat numbering) of these antibodies, such as those in SEQ ID NOs: 22, 96, 24, 37, 38, and 39; SEQ ID NOs: 22, 23, 24, 37, 38, and 39; SEQ ID NOs: 13, 14, 15, 28, 29, and 30; SEQ ID NOs: 16, 17, 18, 31, 32, and 33; SEQ ID NOs: 19, 20, 21, 34, 35, and 36; or SEQ ID NOs: 25, 26, 27, 40, 41, and 42, are shown in Tables 1A-B and 7B. Biological variants thereof, including optimized and humanized counterparts, are further described in the sections above and are incorporated herein.

[0095] In some embodiments, the TAA is selected from the group consisting of GPC3, claudin 18.2, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and tenascin. In some embodiments, the TAA is GPC3.

[0096] An exemplary anti-GPC3 antibody has been prepared and tested in the experimental examples, including VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57. It should be understood that other anti-GPC3 antibodies and variants may also be used herein.

[0097] Examples of multispecific antibodies, such as those having the sequences provided in Table 9A, have been prepared and tested herein. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 71 and a light chain having the amino acid sequence of SEQ ID NO: 61. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 65 and a light chain having the amino acid sequence of SEQ ID NO: 61. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 67 and a light chain having the amino acid sequence of SEQ ID NO: 61. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 69 and a light chain having the amino acid sequence of SEQ ID NO: 61. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 73 and a light chain having the amino acid sequence of SEQ ID NO: 61.

[0098] In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 75 and a light chain having the amino acid sequence of SEQ ID NO: 61.

[0099] Examples of multispecific antibodies, such as those having the sequences provided in Table 9B, have been prepared and tested herein. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 97 and a light chain having the amino acid sequence of SEQ ID NO: 98. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 100 and a light chain having the amino acid sequence of SEQ ID NO: 101. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 103 and a light chain having the amino acid sequence of SEQ ID NO: 104. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 106 and a light chain having the amino acid sequence of SEQ ID NO: 107. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 109 and a light chain having the amino acid sequence of SEQ ID NO: 110.

[0100] Examples of multispecific antibodies have been prepared and tested herein, such as those having the sequences provided in Table 9C. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 112 and a light chain having the amino acid sequence of SEQ ID NO: 113. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 115 and a light chain having the amino acid sequence of SEQ ID NO: 116.

[0101] An exemplary anti-CCR8 antibody has been prepared and tested in the experimental examples, comprising a VH of SEQ ID NO: 117 and a VL of SEQ ID NO: 118. It should be understood that other anti-CCR8 antibodies and variants may also be used herein.

[0102] Examples of multispecific antibodies, such as those having the sequences provided in Table 10B, have been prepared and tested herein. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 119 and a light chain having the amino acid sequence of SEQ ID NO: 120. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 121 and a light chain having the amino acid sequence of SEQ ID NO: 122. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 123 and a light chain having the amino acid sequence of SEQ ID NO: 124. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 125 and a light chain having the amino acid sequence of SEQ ID NO: 126. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 127 and a light chain having the amino acid sequence of SEQ ID NO: 128. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 129 and a light chain having the amino acid sequence of SEQ ID NO: 130. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 131 and a light chain having the amino acid sequence of SEQ ID NO: 132. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 133 and a light chain having the amino acid sequence of SEQ ID NO: 134. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 135 and a light chain having the amino acid sequence of SEQ ID NO: 136.

[0103] An exemplary anti-CCR8 antibody has been prepared and tested in the experimental examples, comprising a VH of SEQ ID NO: 77 and a VL of SEQ ID NO: 78. It should be understood that other anti-CCR8 antibodies and variants may also be used herein.

[0104] Examples of multispecific antibodies have been prepared and tested herein, such as those having the sequences provided in Table 12. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 79 and a light chain having the amino acid sequence of SEQ ID NO: 80. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 81 and a light chain having the amino acid sequence of SEQ ID NO: 80. In some embodiments, the multispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 82 and a light chain having the amino acid sequence of SEQ ID NO: 80.

[0105] In certain embodiments, antibodies (including multispecific or multifunctional antibodies) or fragments thereof comprise an amino acid sequence or one or more moieties not normally associated with antibodies. Exemplary modifications are described in more detail below. For example, antibodies of the present disclosure may comprise a flexible linker sequence or may be modified to attach a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0106] The antibodies (including multispecific or multifunctional antibodies) or fragments thereof of the present disclosure include derivatives that have been modified, i.e., modified by the covalent attachment of any type of molecule to the antibody such that the covalent attachment does not interfere with the antibody's binding to the epitope. By way of example, but not limitation, antibodies can be modified by, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like. Any of a wide variety of chemical modifications may be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, antibodies may contain one or more non-classical amino acids.

[0107] In some embodiments, the antibody may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

[0108] The antibody may be conjugated or fused to a therapeutic agent which may include a detectable label, for example, a radioactive label, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent which may be a drug or a toxin, an ultrasound enhancing agent; a non-radioactive label, combinations thereof, and other such agents known in the art.

[0109] An antibody can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide can then be determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0110] Fluorescent metals, e.g. 152Antibodies can also be detectably labeled using Eu, or others of the lanthanide series. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).Techniques for conjugating various moieties to antibodies are well known and are described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future See "Prospect of the Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy," in Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al. (eds.), Academic Press, pp. 303-16 (1985), and Thorpe et al., "The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates," Immunol. Rev. (52:119-58 (1982)). Treatment and Use

[0111] As described herein, the antibodies (including multispecific or multifunctional antibodies) or fragments thereof of the present disclosure can be used in certain treatment and diagnostic methods for diseases or conditions, such as cancer.

[0112] Thus, in some embodiments, a method for treating cancer in a patient in need thereof is provided. In one embodiment, the method involves administering to the patient an effective amount of an antibody of the present disclosure. In some embodiments, at least one cancer cell (e.g., stromal cell) in the patient expresses, overexpresses, or is induced to express a tumor antigen (e.g., a TAA, e.g., GPC3) recognized by the multispecific antibody. Induction of gene expression can be achieved, for example, by administering a tumor vaccine or radiation therapy.

[0113] Tumors that may be suitably treated include bladder cancer, non-small cell lung cancer, renal cancer, breast cancer, urethral cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, and small cell lung cancer. Thus, the antibodies of the present disclosure can be used to treat any one or more of such cancers.

[0114] In some embodiments, the tumor to be treated is particularly difficult to treat with conventional cancer immunotherapy, for example, with antibodies targeting immune checkpoints (ICPs). Such tumors are sometimes referred to as "cold tumors" or "non-immunogenic tumors." Thus, in some embodiments, the present disclosure provides methods and uses for treating cold tumors with the multispecific antibodies disclosed herein.

[0115] In some embodiments, non-immunogenic tumors are those that are not infiltrated by T cells, or that lack T cell infiltration, antigen-presenting cells (APCs), or T cell activation, or that lack T cell homing to the tumor bed. Prostate cancer, pancreatic cancer, and leukemia are all non-immunogenic. The majority of breast cancers (95%), the majority of colorectal cancers (95%), the majority of gastric cancers (87%), the majority of head and neck cancers (84%), the majority of liver cancers (83%), the majority of esophageal cancers (86%), the majority of cervical cancers (87%), and the majority of thyroid cancers (87%) are also non-immunogenic. In addition, 83% of lung cancers, 79% of bladder cancers, 77% of kidney cancers, 70% of uterine cancers, and 66% of melanomas are also non-immunogenic.

[0116] Non-immunogenic or cold tumors can also be identified by measuring the type, density, and location of immune cells within the tumor. For example, Galon and Bruni (Nature Reviews Drug Discovery volume 18, pages 197-218 (2019)) describe the Immunoscore, a standardized scoring system based on the quantification of two lymphocyte populations (CD3 and CD8) in, for example, excised tissue, for the guided stratification of hot and cold tumors. The Immunoscore ranges from Immunoscore 0 (I0, for low density, e.g., the absence of both cell types in both regions) to I4 (high immune cell density in both locations). By classifying cancers according to their immune infiltration, this scoring system provides an immune-based classification of tumors, including the definition of "hot" (highly invasive, Immunoscore I4) and "cold" (non-invasive, Immunoscore I0) tumors.

[0117] In some embodiments, the tumor is resistant to treatment with an immune checkpoint inhibitor, such as a PD-L1 inhibitor, a PD-1 inhibitor, a CTLA-4 inhibitor, or a combination thereof. In some embodiments, the cancer is prostate cancer, pancreatic cancer, or leukemia. In some embodiments, the cancer is breast cancer, colorectal cancer, gastric cancer, head and neck cancer, liver cancer, esophageal cancer, cervical cancer, or thyroid cancer. In some embodiments, the cancer is lung cancer, bladder cancer, kidney cancer, uterine cancer, or melanoma.

[0118] Additional diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed, and / or prognosed by the antibodies or variants, or derivatives thereof, of the present disclosure include, but are not limited to, malignancies and related disorders, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, and progression and / or metastasis of solid tumors, including sarcomas and carcinomas, such as fibrosarcoma. , myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, thyroid cancer, endometrial cancer, melanoma, prostate cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma These include, but are not limited to, myeloid leukemia, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0119] The specific dosage and treatment regimen for any particular patient will depend on various factors, including the specific antibody, its variant or derivative used, the patient's age, weight, overall health, sex and diet, as well as the number of administrations, excretion rate, drug combinations and the severity of the specific disease being treated.The judgment of such factors by medical caregivers is within the ordinary skill of the art.The amount will also depend on the individual patient to be treated, the administration route, the type of formulation, the characteristics of the compound used, the severity of the disease and the desired effect.The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0120] Methods of administration of antibodies or variants include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptides or compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Thus, pharmaceutical compositions containing antigen-binding polypeptides of the disclosure can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as powders, ointments, drops, or transdermal patches), bucally, or as an oral spray or nasal drops.

[0121] The term "parenteral," as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0122] Administration can be systemic or local.In addition, it may be desirable to introduce the antibody of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection, and intraventricular injection can be facilitated by an intraventricular catheter attached to a reservoir such as an Ommaya reservoir.Pulmonary administration can also be utilized, for example, by using an inhaler or nebulizer and a formulation containing an aerosolizing agent.

[0123] It may be desirable to administer an antibody polypeptide or composition of the disclosure locally to the area in need of treatment, which can be achieved, for example, and not by way of limitation, by local infusion during surgery, by topical application after surgery, for example in conjunction with wound dressings, by injection, by catheter, by suppository, or by a deposit, said deposit being of a porous, non-porous, or gelatinous material, including a membrane, for example, a sialastic membrane, or fiber. Preferably, when administering proteins, including antibodies, of the disclosure, care should be taken to use materials to which the protein does not absorb. Polynucleotides encoding antibodies and methods for preparing antibodies

[0124] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies of the present disclosure, their variants, or derivatives. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, its variant, or derivative, on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, its variant, or derivative, on the same polynucleotide molecule or on separate polynucleotide molecules.

[0125] Methods for producing antibodies are well known in the art and are described herein.In certain embodiments, both the variable region and the constant region of the antigen-binding polypeptide of the present disclosure are fully human.Fully human antibodies can be produced using techniques described in the art and as described herein.For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen administration, but whose endogenous gene locus has been disabled.Exemplary techniques that can be used to produce such antibodies are described in U.S. Patent Nos. 6,150,584, 6,458,592, and 6,420,140, ​​and these reference patent documents are incorporated herein by reference in their entirety.

[0126] In certain embodiments, the prepared antibodies will not provoke an adverse immune response in the animal to be treated, e.g., in a human. In one embodiment, the antigen-binding polypeptides of the present disclosure, their variants, or derivatives, are modified to reduce their immunogenicity using art-recognized techniques. For example, antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be generated. These types of antibodies are derived from non-human antibodies, typically murine or primate antibodies, that retain or substantially retain the antigen-binding properties of the parent antibody but are less immunogenic in humans. This can be achieved by various methods, including (a) grafting entire non-human variable domains onto human constant regions to generate chimeric antibodies; (b) grafting at least a portion of one or more non-human complementarity-determining regions (CDRs) onto human framework and constant regions, with or without retaining critical framework residues; or (c) grafting entire non-human variable domains but "cloaking" them with human-like sections by replacing surface residues.

[0127] Deimmunization can also be used to reduce the immunogenicity of antibodies. As used herein, the term "deimmunization" includes altering an antibody to modify its T cell epitopes (see, e.g., International Application Publication Nos. WO / 9852976A1 and WO / 0034317A2). For example, the variable heavy and variable light chain sequences from a starting antibody are analyzed, and a human T cell epitope "map" is created from each V region, showing the location of the epitope in relation to the complementarity-determining regions (CDRs) and other critical residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed to identify alternative amino acid substitutions that have a low risk of altering the activity of the final antibody. A variety of alternative variable heavy and variable light chain sequences containing combinations of amino acid substitutions are designed, and these sequences are then incorporated into various binding polypeptides. Typically, between 12 and 24 variant antibodies are generated and tested for binding and / or function. The complete heavy and light chain genes containing the modified variable and human constant regions are then cloned into expression vectors, and the subsequent plasmids are introduced into cell lines for the production of whole antibodies. The antibodies are then compared in appropriate biochemical and biological assays to identify the optimal variant.

[0128] The binding specificity of an antigen-binding polypeptide of the disclosure can be determined by in vitro assays, such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). composition

[0129] The present disclosure also provides pharmaceutical compositions, which comprise an effective amount of an antibody (including a multispecific antibody) and an acceptable carrier.

[0130] In specific embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia, for use in animals, and more particularly in humans. Moreover, a "pharmaceutically acceptable carrier" will generally be a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid of any type.

[0131] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates. Antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for adjusting osmotic tonicity, such as sodium chloride or dextrose, are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions can be formulated as suppositories using traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin, incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation should be suitable for the method of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0132] In some embodiments, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Optionally, the composition may also contain a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, these ingredients are supplied separately or mixed in unit dosage form, for example, as a dry, lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of active ingredient. When the composition is to be administered by injection, it can be dispensed using an infusion bottle containing pharmaceutical-grade sterile water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0133] The compounds of the present disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. [Example]

[0134] Example 1 Generation of mouse anti-human CD16A antibodies This example describes the generation of a mouse anti-human CD16A monoclonal antibody using hybridoma technology.

[0135] Immunogens: Two immunogens were used during the process of immunizing mice. The extracellular domain (ECD) of human CD16A-158V was fused to either a human IgG1 Fc region with a C-terminal N297A mutation or a human IgG1 Fc region with a C-terminal his-tag to generate hCD16A-158V-FcNA protein (Biointron) or hCD16A-158V-his protein (AcroBio, catalog number CD8-H52H4).

[0136] Mouse immunization scheme: To generate mouse monoclonal antibodies against human CD16A, BALB / c mice were selectively immunized intraperitoneally and subcutaneously with hCD16A-158V-FcNA or hCD16A-158V-his protein at biweekly intervals. Serum titers of immunized mice were monitored by ELISA against human CD16A-158V-his protein. After two to four rounds of immunization, mice with sufficient titers were boosted with hCD16A-158V-his protein and selected for fusion.

[0137] Cell fusion and hybridoma screening: Splenocytes from selected mice were fused with the mouse myeloma cell line Sp2 / 0 by electrofusion. These hybridoma cells were plated in 96-well flat-bottom microplates, and these cells secreted mouse antibodies into the supernatant. During primary screening, positive clones were screened in a high-throughput manner using protein-based binding to hCD16A-158V-his by ELISA or cell-based binding to hCD16A-158V overexpressed on CHO-K1 cells (CHO-K1-hCD16A-158V) using Mirrorball (SPT Labtech). Subsequent confirmatory screening was carried out to exclude clones that nonspecifically bound to hCD16B-SH-his protein (SinoBiological, catalog no. 11046-H08H2) by ELISA or to hCD16B-NA1 overexpressed on CHO-K1 cells (CHO-K1-hCD16B-NA1) by FACS, and to identify clones that bound to cynomolgus monkey CD16 overexpressed on CHO-K1 cells (CHO-K1-cynoCD16).

[0138] Subcloning, screening, and sequencing: Positive primary clones from each fusion were subcloned by limiting dilution to ensure that hybridoma subclones were derived from a single parent cell. Subclones were screened using the same criteria as for the primary clone screening described above. Subclones that had specific binding to hCD16A but not hCD16B were selected for subsequent sequencing.

[0139] The resulting sequence for the Fab fragment of the murine antibody was fused to a human IgG1 Fc with L234A / L235A / P329G (LALAPG) mutations to generate a chimeric CD16A mAb. To verify the CD16A agonist efficacy of the Fab fragment of the CD16A mAb, the LALAPG mutation was introduced into the Fc region, completely abolishing Fc binding to all FcγRs and the resulting ADCC and ADCP effects. The DNA sequence of the chimeric antibody was cloned into the pcDNA3.4 plasmid and expressed in HEK-293F or CHO-K1 cells. The antibody was then purified from the culture supernatant using a protein A affinity chromatography column or beads. The purified chimeric antibody was subjected to a series of in vitro screening processes to determine affinity, binding capacity, specificity, species cross-reactivity, and agonist function.

[0140] Several hybridoma clones were selected for further analysis based on their performance in the screening assay, including 23H7E4, 37B3G11, 42F5H2, 45H6E8, and 91A11D11. The amino acid sequences of the variable regions of selected murine CD16A antibodies are provided in Table 1 below, and the CDR sequences are summarized in Tables 1A and 1B. The affinity-matured CD16A mAb P2C47 (sequence obtained from U.S. Pat. No. 11,001,633 B2) developed by Affimed was used as the benchmark antibody, with the sequences of the Fab fragments listed in Table 1. The sequences of the heavy and light chains of the antibodies are listed in Table 2. Table 1. Antibody variable region sequences (underlined CDRs) [Table 1-1] [Table 1-2] Table 1A. Heavy chain CDR sequences (Kabat numbering) [Table 1A] Table 1B. Light chain CDR sequences (Kabat numbering) [Table 1B] Table 2. CD16A chimeric Ab sequences [Table 2-1] [Table 2-2]

[0141] Example 2 Binding activity of chimeric anti-human CD16A monoclonal antibodies 2.1. ELISA binding to human CD16A To determine the binding ability of chimeric Abs to human CD16A protein, an ELISA-based binding assay was performed as follows. Briefly, hCD16A-158V-his and hCD16A-158F-his proteins (Acro Bio, catalog no. CDA-H5220) were diluted to 2 μg / mL in DPBS buffer and adsorbed to wells of a 96-well microplate overnight at 4°C. After blocking the wells with 2% bovine serum albumin (BSA) to prevent nonspecific binding, CD16A chimeric Abs, benchmark antibody P2C47, or an isotype control were titrated in 3-fold dilutions starting at 20 nM and added to the wells pre-adsorbed with hCD16A-his protein. The mixture was incubated for 1 hour at room temperature (RT). The primary antibody was recognized by a detection antibody against human IgG Fc conjugated with horseradish peroxidase (HRP) (Jackson Immuno, catalog number 109-035-008). Tetramethylbenzidine (TMB), a substrate for HRP, was added to the wells to visualize the binding signal. After sufficient color development, stop solution was added to the wells. The absorbance of the signal was detected at 450 nm using an Envision multilabel plate reader (PerkinElmer). Graphs and statistical analyses were generated by four-parameter nonlinear regression curve fitting using Graphpad Prism 9 software.

[0142] As shown in Figures 1A and 1B, all CD16A chimeric Abs efficiently bound to the high-affinity 158V and low-affinity 158F fragments of human CD16A protein. Furthermore, all antibodies, except for the 37B3G11 chimeric Ab, exhibit binding efficiencies comparable to that of the benchmark antibody P2C47. The binding EC values ​​of the tested CD16A chimeric Abs were 50 are listed in Table 3. 2.2. ELISA binding to human CD16B

[0143] Identifying the specificity of CD16A chimeric antibodies is extremely important because generating CD16A-specific antibodies is very difficult due to the very few amino acid differences between CD16A and its isoform CD16B.

[0144] To determine the binding ability of the chimeric Ab to human CD16B protein, ELISA binding assays were performed as described above. Three splice isoforms of CD16B, including human CD16B-NA1 ECD his-tagged protein (hCD16B-NA1-his, Acro Bio catalog no. CDB-H5227), human CD16B-NA2 ECD his-tagged protein (hCD16B-NA2-his, Acro Bio catalog no. CDB-H82Ea), and human CD16B-SH-his protein, were used as coating antigens at 2 μg / mL. With hCD16B in mind, the commercially available anti-CD16 antibody 3G8 (Stemcell, catalog no. 60041) was used as a positive control. As shown in Figures 2A, 2B, and 2C, all CD16A chimeric Abs displayed negligible binding to hCD16B-NA1, hCD16B-NA2, or hCD16B-SH proteins, similar to the benchmark antibodies, demonstrating the strict binding specificity of these chimeric CD16A antibodies, which recognize distinct epitopes on CD16A but not CD16B. 2.3 ELISA binding to cynomolgus monkey CD16 protein

[0145] To determine the cross-reactivity of chimeric Abs with cynomolgus monkey CD16, ELISA binding assays were performed as described above. Recombinant cyno CD16 ECD his-tagged protein (cynoCD16-his, Acro Bio catalog no. FC6-C52H9) was used as the coating antigen at 2 μg / mL. As shown in Figure 3, only 45H6E8 bound efficiently to cyno CD16 protein, while other chimeric Abs, including 23H7E4, 37B3G11, 42F5H2, and 91A11D11, showed only negligible binding to cyno CD16 protein. These data were likely due to the limited amino acid sequence that distinguishes human CD16A and cyno CD16 from all three isoforms of human CD16B. Nevertheless, 45H6E8 is a specific CD16A antibody that exhibited excellent cross-reactivity between human and cynomolgus monkey species and precise specificity for two human CD16A polymorphisms. Table 3. Binding activity of chimeric antibodies to antigenic proteins [Table 3] --: no join 2.4 Binding ability to human CD16A expressed on CHO-K1 cells

[0146] To evaluate the binding activity of chimeric mAbs to human CD16A expressed on cells, a cell-based binding assay was utilized as follows. Briefly, CHO-K1 cells stably expressing the hCD16A-158V or hCD16A-158F variants were constructed. The indicated CD16A chimeric Abs, benchmark antibody P2C47, or isotype controls were diluted in staining buffer (DPBS buffer containing 2% BSA) at 4-fold dilutions starting from a concentration of 100 nM. Antibody dilutions were adjusted to 1 × 10 5The cells were incubated with the indicated number of cells in a 96-well microplate for 1 hour at 4°C. Antibody binding to cell surface antigens was detected with a PE fluorescent dye-conjugated anti-human IgG Fc secondary antibody (ThermoFisher scientific, catalog number 12-4998-82) at a dilution of 1:1000. Cells were analyzed using a flow cytometer LSRFortessa Cell Analyzer (BD Biosciences). Data were analyzed using Flowjo 10.0 software. Graphs and statistical analyses were generated using Graphpad Prism 9 software by four-parameter nonlinear regression curve fitting.

[0147] As shown in Figures 4A and 4B, all CD16A chimeric antibodies efficiently bound to human CD16A-158V and human CD16A-158F expressed on CHO-K1 cells in a dose-dependent manner. Most of our CD16A chimeric antibodies, except for 23H7E4, showed comparable avidity compared to the benchmark antibody P2C47. The binding EC of the tested CD16A chimeric Abs 50 are listed in Table 4. 2.5. Binding Ability to Human CD16B and CD32B Expressed on CHO-K1 Cells

[0148] To exclude nonspecific binding of CD16A chimeric antibodies to human CD16B and CD32B expressed on cells, cell-based binding assays were performed as described above. First, CHO-K1 cells were stably transfected with one human CD16B splice isoform, hCD16B-NA1, and human CD32B. The cells were then incubated with the indicated concentrations of CD16A chimeric antibodies. Commercially available anti-CD16 antibody 3G8, designed with hCD16B in mind, and anti-CD32B antibody 6G11 (Bioinvent) were used as positive controls. As shown in Figures 5A and 5B, all CD16A chimeric antibodies showed little binding to hCD16B-NA1 and hCD32B expressed on cells. In summary, we confirmed that our CD16A chimeric antibody displays stringent binding specificity for human CD16A while avoiding nonspecific binding of human CD16B and all three isoforms of CD32B (Figures 2 and 5). 2.6. Binding Ability to Cynomolgus Monkey CD16 on CHO-K1 Cells

[0149] To confirm the cross-reactivity of the chimeric CD16A antibodies with cynomolgus monkey CD16, cell-binding assays were performed as described above. CHO-K1 cells stably expressing cynomolgus monkey CD16 were used in the following studies. As shown in Figure 6, consistent with their binding ability to cyno CD16 protein, only 45H6E8 efficiently bound to cyno CD16 expressed on CHO-K1 cells in a dose-dependent manner, with potency comparable to that of the benchmark antibody P2C47. In contrast, 23H7E4, 37B3G11, 42F5H2, and 91A11D11 showed only weak or negligible binding to cyno CD16. The binding EC of the tested CD16A chimeric Abs was 0.01. 50 are listed in Table 4. Table 4. Binding activity of chimeric antibodies to antigens expressed on cells [Table 4] --: no join 2.7. Affinity measurement

[0150] The binding affinity of chimeric antibodies to human CD16A was determined by Biacore™. Briefly, antibodies were captured using a Protein A chip. 50 nM of human CD16A-158V-his and CD16A-158F-his proteins were injected over the captured antibodies for 30 seconds at a flow rate of 30 μL / min. The antigen was allowed to dissociate for 500 seconds. Experiments were performed on a Biacore™ 8K. Data analysis was performed using Biacore™ 8K evaluation software. The results showed that all chimeric CD16A antibodies exhibited high and comparable affinity to both human CD16A-158V and CD16A-158F. Among them, 42F5H2 and 91A11D11 showed significantly superior binding affinity to both CD16A forms compared to the benchmark P2C47 (Tables 5A and 5B). 23H7E4, 37B3G11 and 45H6E8 showed binding affinities comparable to the benchmark P2C47 (Tables 5A and 5B). Table 5A. Chimeric antibody affinity ranking results [Table 5A] Table 5B. Chimeric antibody affinity ranking results [Table 5B]

[0151] Example 3 Humanization of CD16A chimeric antibody 3.1. Humanized design of CD16A chimeric antibody Based on the performance of the chimeric antibody, 45H6E8 was selected for further humanization. The variable regions were selected for humanization. Briefly, the amino acid sequences of the VH and VL were aligned with available databases of human Ig gene sequences to identify the best overall match for the human germline Ig gene sequence. The CDRs of the heavy and light chains of the 45H6E8 chimeric antibody were then grafted onto the candidate human germline sequences. A 3D model of the grafted antibody was generated using Molecular Operating Environment (MOE) to determine whether there were any critical human amino acids within the framework regions that would require backmutation to the corresponding mouse amino acids to maintain CDR conformation and function.

[0152] For the heavy chain of 45H6E8, the candidate human germline sequence was the IGHV1-24*01 gene. For the light chain of 45H6E8, the candidate human germline sequence was the IGKV1-39*02 gene. Within the heavy chain framework of the human germline sequence IGHV1-24*01, T30K, M48I, R67K, V68A, E72A, and A97T were backmutated. Within the light chain framework of the human germline sequence IGKV1-39*02, S60D and Y87F were backmutated.

[0153] Different combinations of backmutation sites were selected to generate the variable regions of humanized antibodies. The sequences of the heavy and light chain variable regions of humanized 45H6E8 are listed in Table 6A. The VH and VL pairings of each humanized antibody are listed in Table 6B. The variable regions of the humanized antibodies were then fused to the constant region of human IgG1 Fc with L234A / L235A / P329G (LALAPG) mutations for humanized antibody production and functional characterization. Table 6A. Variable region sequences of the 45H6E8 humanized antibody (underlined indicates CDRs; bold / italic indicates backmutations) [Table 6A] Table 6B. VH and VL pairing of 45H6E8 humanized antibody [Table 6B-1] [Table 6B-2] 3.2 Binding characteristics of CD16A humanized antibodies to CD16-expressing cells

[0154] The cell-binding activity of the CD16A humanized antibody to human CD16A-158V, CD16A-158F, CD16B-NA1, and cynomolgus monkey CD16 was confirmed in a cell-based binding assay using antigen-expressing CHO-K1 cells. The assay was performed according to the protocol described above. Binding of the antibody to the antigen on the cell surface was detected with Alexa Fluor® 647 AffiniPure™ F(ab')2 fragment goat anti-human IgG Fcγ fragment-specific secondary antibody (Jackson Immuno Research, catalog number 109-606-098) at a dilution of 1:2000.

[0155] As shown in Figures 7A and 7B, all CD16A humanized antibodies, including 45H6E8-z13, 45H6E8-z17, 45H6E8-z19, 45H6E8-z20, and 45H6E8-z21, exhibited cell-binding abilities to human CD16A-158V and CD16A-158F comparable to their chimeric counterparts. Meanwhile, these CD16A humanized antibodies displayed similar cell-binding abilities to cyno CD16 compared to their chimeric 45H6E8 counterparts, as shown in Figure 7C. In contrast, all CD16A humanized antibodies exhibited negligible binding to human CD16B-NA1 expressed on cells (Figure 7D).

[0156] Example 4 Optimization of a humanized CD16A antibody After antibody humanization, the CDR regions often need to be further optimized to remove potential post-translational modification (PTM) sites to increase the long-term stability, manufacturability, and homogeneity of the humanized antibody, which can impair the potency, efficacy, and safety of therapeutic antibodies. PTMs, such as deamidation, isomerization, glycosylation, and oxidation, can impair the potency, efficacy, and safety of therapeutic antibodies. 4.1 PTM removal design of humanized CD16A antibody

[0157] In this regard, computational tools were used to predict PTM-prone sites to facilitate the engineering of antibodies with better physical and chemical properties. By closely examining the CDR regions of the humanized 45H6E8 mAb, we identified one NG motif located within CDR2 of the VH region that could potentially undergo deamidation of the asparagine (N) amino acid, potentially impairing the binding and function of the CD16A antibody. Consequently, a glutamine (Q) to asparagine amino acid replacement was performed at this position to remove the potential PTM site. The sequences of the PTM-depleted variable regions of the 45H6E8 humanized mAb are listed in Table 7A, and the mutated CDRs are summarized in Table 7B. Table 7A. CD16A humanized antibody variable region sequences (underlined indicates CDRs; bold / italic indicates PTM mutations) [Table 7A] Table 7B. Optimized heavy chain CDR2 of 45H6E8 [Table 7B] 4.2 Binding characteristics of PTM-deleted CD16A humanized antibodies to human CD16-expressing cells

[0158] The cell-binding activity of the PTM-deleted humanized CD16A antibodies to human CD16A-158V, CD16A-158F, and CD16B-NA1, as well as cynomolgus monkey CD16, was confirmed in a cell-based binding assay using antigen-expressing CHO-K1 cells. The assay was performed according to the protocol described above.

[0159] As shown in Figures 8A and 8B, the PTM-depleted 45H6E8-z19p1, 45H6E8-z20p1, and 45H6E8-z21p1 antibodies all retained binding to human CD16A-158V and CD16A-158F expressed on cells, similar to their parent antibodies 45H6E8-z19, 45H6E8-z20, and 45H6E8-z21. Meanwhile, the cell-binding efficacy of these PTM-depleted CD16A humanized antibodies to cyno CD16 was maintained, as shown in Figure 8C. Consistent with their parent humanized antibodies, all of these PTM-depleted mAbs 45H6E8-z19p1, 45H6E8-z20p1, and 45H6E8-z21p1 showed little binding to human CD16B-NA1 expressed on cells (Figure 8D).

[0160] Example 5 Binding capacity of CD16A bispecific fragments The development strategy for targeting CD16A is to achieve conditional activation of CD16A by the other arm of the binding fragment in a bispecific or multispecific fragment. The bispecific or multispecific fragment contained one arm that binds to hCD16A and the other arm that binds to one or more specific antigens. In this regard, the inventors began to evaluate whether cross-linking of CD16A with tumor-associated antigen (TAA)-specific fragments could induce TAA-dependent conditional activation of CD16A on CD16A-expressing effector cells, such as NK cells and macrophages, and the resulting functional activation. 5.1 Generation of humanized monoclonal antibodies against human GPC3

[0161] As mentioned above, GPC3 is a specific TAA for HCC due to its high expression level in tumor cells and limited expression in normal tissues. We generated a potent humanized monoclonal antibody targeting human GPC3. Briefly, BALB / c and C57BL / 6 mice were immunized with human GPC3-his protein. After several rounds of immunization, the immune response was tested by serum ELISA against GPC3-his protein and by serum FACS against a CHO-K1 cell line overexpressing GPC3, with the CHO-K1 parent cell line serving as a negative control. The resulting mice were used for fusion. Positive hybridoma clones were selected by ELISA and FACS. After subcloning, hybridoma clone 52H5D3B8 was selected, optimized, and humanized. The amino acid sequences of the heavy and light chain variable regions of the humanized GPC3 antibody are listed in Table 8 below. Table 8. Variable region sequences of humanized GPC3 antibodies (underlined CDRs) [Table 8] 5.2 Construction of CD16A bispecific fragments

[0162] The present inventors began exploring the possibility of activating CD16A-expressing cells by achieving conditional activation of CD16A through cross-linking with bispecific fragments. Format 1 (Figure 9), containing two antigen-binding fragments and two CD16A scFv-binding fragments, was explored to generate bispecific fragments. More specifically, a bispecific fragment with Format 1 was constructed, containing two anti-human CD16A scFv fragments fused to the C-terminus of the ADCC- and ADCP-neutralizing Fc fragment of the GPC3-hIgG1LALAPG antibody (heavy and light chains, SEQ ID NOs: 59 and 61, respectively) via a (G4S)4 linker (SEQ ID NO: 62). The VH and VL in the scFv of the anti-CD16A fragments were conjugated via a (G4S)3 linker (SEQ ID NO: 63). The sequences of the heavy and light chains of the GPC3-CD16A bispecific fragment are listed in Tables 9A, 9B, and 9C. GPC3-hIgG1 with a wild-type Fc region and GPC3-hIgG1DE mAb with ADCC-enhancing S239D and I332E mutations in the Fc region were used as positive controls (SEQ ID NOs: 58 and 60 for the heavy chain, and SEQ ID NO: 61 for the light chain, respectively). GPC3-hIgG1LALAPG mAb with LALAPG mutations in the Fc region was used as a negative control (SEQ ID NOs: 59 and 61 for the heavy and light chain, respectively). 5.3 Binding of CD16A bispecific fragments to human GPC3 expressed on cells

[0163] To evaluate the binding activity of the GPC3-CD16A bispecific fragments to GPC3, we performed cell-based binding assays using HepG2 cells, a human hepatocellular carcinoma cell line with high GPC3 expression levels, and the SK-HEP-1 cell line, which lacks GPC3 and served as a negative control. As shown in Figure 10A, all tested GPC3-CD16A bispecific fragments displayed dose-dependent specific binding activity to GPC3-positive HepG2 cells, while no binding to GPC3-negative SK-HEP-1 cells was detectable (Figure 10B). More importantly, the binding avidity of these bispecific fragments to GPC3 was nearly equivalent to that of their parent GPC3 mAbs (Figure 10A). 5.4 Binding of CD16A bispecific fragments to human CD16A expressed on cells

[0164] The binding ability of GPC3-CD16A bispecific fragments to human CD16A-158V and CD16A-158F expressed on CHO-K1 cells was evaluated by cell-based binding assays. All tested GPC3-CD16A bispecific fragments displayed efficient binding to both the high-affinity hCD16A-158V and low-affinity hCD16A-158F expressed on cells (Figures 10C and 10D). A slightly reduced binding ability of the bispecific fragments to CD16A was observed compared to that of their parent CD16A mAb, which could be due to Fab-scFv format changes or steric hindrance induced by the C-terminal conjugation of the CD16A-scFv fragment. In contrast, GPC3-hIgG1DE displayed relatively weak binding to hCD16A-158V expressed on cells, but improved binding was observed when compared with GPC3-hIgG1, which poorly bound to hCD16A-158V (Figure 10C). Neither GPC3-hIgG1DE nor GPC3-hIgG1 showed binding to the low-affinity CD16A-158F (Figure 10D). Therefore, the persistent and focused binding of CD16A antibodies to both CD16A-158V and CD16A-158F may favor the prolonged activation of NK cells and macrophages compared with monoclonal antibodies with engineered Fc regions that have enhanced ADCC and ADCP. Table 9A. Sequences of GPC3 mAb and GPC3-CD16A bispecific fragments [Table 9A-1] [Table 9A-2] [Table 9A-3] [Table 9A-4] [Table 9A-5] Table 9B. Sequences of GPC3-CD16A humanized bispecific fragments [Table 9B-1] [Table 9B-2] [Table 9B-3] [Table 9B-4] Table 9C. Sequence of the GPC3-CD16A humanized bispecific fragment after PTM removal [Table 9C-1] [Table 9C-2]

[0165] Example 6 CD16A bispecific fragments mediated CD16A agonistic signaling To evaluate the conditional activation of CD16A by the CD16A bispecific fragment, a CD16A signaling reporter assay was constructed. Briefly, full-length human CD16A-158V was first introduced into a previously constructed Jurkat-NFAT luciferase reporter cell line, in which luciferase gene expression is under the control of an NFAT transcription factor-responsive promoter. The resulting cell line allows for convenient evaluation of CD16A signaling activation by examining downstream NFAT signaling. Therefore, this Jurkat-hCD16A-158V-NFAT reporter cell line was cocultured with three human hepatocellular carcinoma cell lines, including HepG2, Huh-7, and PLC / PRF / 5, which express high, medium, and low levels of GPC3, respectively. SK-HEP-1, which lacks GPC3 expression, was used as a negative control to rule out GPC3-independent activation of CD16A. The NFAT signaling pathway is stimulated when CD16A is activated by GPC3 targeting fragment-mediated binding on GPC3-expressing cells. Luciferase-based chemiluminescence can be detected using the ONE-Glo™ Luciferase Assay System (Promega, Cat. No. E6110) and the Envision multilabel plate reader (PerkinElmer). Graphs and statistical analysis were generated using Graphpad Prism 9 software with four-parameter nonlinear regression curve fitting.

[0166] The results showed that all tested GPC3-CD16A bispecific fragments efficiently activated CD16A signaling in the presence of GPC3-positive HepG2 (Figure 11A), Huh7 (Figure 11B), and PLC / PRF / 5 (Figure 11C) cell lines, but not in the presence of the GPC3-negative SK-HEP-1 cell line (Figure 11D), indicating efficient and specific GPC3-mediated conditional activation of CD16A signaling by GPC3-CD16A bispecific fragments even in the presence of moderate or low levels of GPC3.

[0167] Example 7 CD16A bispecific fragments activate NK cell function To assess whether conditional activation of CD16A signaling by the CD16A bispecific fragment could lead to NK cell activation and cytotoxic effects and demonstrate functional advantages over Fc-engineered mAbs upon target cell binding, several primary NK cell-based functional assays were performed, including NK cell cytotoxicity, degranulation, and effector cytokine production. 7.1 GPC3-CD16A bispecific fragment mediated NK cytotoxicity against target cells

[0168] To evaluate whether GPC3-mediated CD16A signaling activation induces the cytotoxic effect of NK cells against target cells, a human primary NK cell-mediated cytotoxicity assay was performed. Briefly, fresh human primary CD3-CD56+ NK cells were isolated from the buffy coats of healthy donors by negative selection with magnetic beads (Miltenyi, catalog no. 130-092-657). The purity of the isolated NK cells was typically greater than 90%, as monitored by FACS analysis. NK cells were then rested in complete culture medium at 37°C and 5% CO2 for 24 hours or freshly prepared as effector cells. Three human hepatocellular carcinoma cell lines, including HepG2, Huh-7, and PLC / PRF / 5, which have high, medium, and low GPC3 expression levels, respectively, were used as target cells. SK-HEP-1 cells were used as a negative control to rule out nonspecific killing by NK cells. NK cells were cultured at 1.0 × 10 4Effector-target cells were co-cultured with target cells at a 10:1 effector-to-target ratio in a 96-well microplate. Starting at 3 nM, serially diluted antibodies were added to the corresponding wells. After 4 h of incubation at 37°C, supernatant lactate dehydrogenase (LDH) released by injured cells was measured using an LDH Cytotoxicity Detection Kit (Roche, catalog no. 04744926001) and detected using an Envision multilabel plate reader (PerkinElmer). Calculation formula: % cytotoxicity = (LDH release of effector-target cell mix - spontaneous LDH release of effector cells - spontaneous LDH release of untreated target cells) / (maximum LDH release of target cells - spontaneous LDH release of untreated target cells) × 100. Graphs and statistical analyses were generated using Graphpad Prism 9 software by four-parameter nonlinear regression curve fitting.

[0169] All tested GPC3-CD16A bispecific fragments induced NK cell-mediated cytotoxicity against the GPC3+ target cells HepG2 (Figure 12A) and Huh-7 (Figure 12B) in a GPC3-dependent and dose-dependent manner, whereas no cytotoxicity was observed against GPC3-negative SK-HEP-1 cells (Figure 12C). 7.2 GPC3-CD16A bispecific fragment stimulated NK cell degranulation

[0170] To investigate the ability of the GPC3-CD16A bispecific fragment to stimulate NK cell degranulation, the expression level of the degranulation marker CD107a was analyzed in NK cells. Briefly, human primary NK cells generated as described above were cultured at 1.0 × 10 cells, including HepG2, Huh7, and SK-HEP-1. 5 The antibodies were co-cultured with 1x10 target cells at a 1:1 E / T ratio in a 96-well microplate. 3-fold serial dilutions of antibodies starting at 10 nM were added to the corresponding wells. After 4 hours of incubation at 37°C, the mixed cells were harvested, washed, and stained at 1x10 in ice-cold staining buffer (2% BSA in DPBS). 6The cells were adjusted to a concentration of 1000 cells / mL. After pretreatment with Fc blocker, the cells were stained with PE-conjugated human CD107a antibody (2 μL per test, Biolegend, catalog number 328608) for 30 minutes at 4°C in the dark. The samples were then fixed with 4% paraformaldehyde and analyzed using a flow cytometer LSRFortessa Cell Analyzer (BD Biosciences). Data were analyzed using Flowjo 10.0 software. Graphs and statistical analyses were generated using Graphpad Prism 9 software by four-parameter nonlinear regression curve fitting.

[0171] The results showed that the GPC3-CD16A bispecific fragment induced NK cell-mediated degranulation in a GPC3-dependent and dose-dependent manner, as indicated by CD107 upregulation in the GPC3+ target cells HepG2 (Figure 13A) and Huh-7 (Figure 13B), whereas no NK cell degranulation was observed in the GPC3-negative SK-HEP-1 cells (Figure 13C). More importantly, the GPC3-CD16A bispecific fragment showed enhanced induction of NK cell degranulation compared to either the GPC3-IgG1DE or GPC3-hIgG1 mAb (Figures 13A and 13B). This indicates the superiority of the CD16A bispecific fragment over the Fc-engineered mAb in NK cell activation. 7.3 GPC3-CD16A bispecific fragment stimulated cytokine production in NK cells

[0172] To investigate the ability of the bispecific fragment GPC3-CD16A to stimulate NK cell cytokine production in the presence of target cells, intracellular IFN-γ and TNF-α in NK cells were analyzed by FACS staining. Briefly, 1.0 × 10 primary human NK cells were generated as effector cells, including HepG2, Huh7, and SK-HEP-1. 5NK cells were co-cultured with target cells (E / T ratio = 1:1) in a 96-well microplate. The protein transport inhibitor brefeldin A (BFA) was added to the co-culture system to prevent cytokine secretion into the supernatant during NK cell activation. Antibodies were serially diluted 3-fold, starting at 10 nM, and added to corresponding wells. After 4 hours of incubation at 37°C, the mixed cells were washed, fixed with 4% paraformaldehyde, and then permeabilized with 1x permeabilization buffer (Invitrogen, catalog no. 00-8333-56) for 20 minutes at room temperature. The cell pellet was resuspended in staining buffer containing diluted APC mouse anti-human IFN-γ antibody (Biolegend, catalog no. 506510) and BV421 mouse anti-human TNF-α antibody (BD bioscience, catalog no. 562783), followed by incubation for 30 minutes at 4°C in the dark. Samples were washed, resuspended, and analyzed using a flow cytometer LSRFortessa Cell Analyzer (BD Biosciences). Data were analyzed using Flowjo 10.0 software. Graphs and statistical analyses were generated using Graphpad Prism 9 software by four-parameter nonlinear regression curve fitting.

[0173] The results showed that the GPC3-CD16A bispecific fragment induced NK cell-mediated cytokine production in a GPC3-dependent and dose-dependent manner, as indicated by the upregulation of intracellular IFN-γ and TNF-α in the GPC3+ target cells HepG2 (Figures 14A and 14D) and Huh-7 (Figures 14B and 14E), whereas no NK cell cytokine production was observed in the GPC3-negative SK-HEP-1 cells (Figures 14C and 14F). More importantly, the GPC3-CD16A bispecific fragment showed enhanced induction of NK cell cytokine release compared to either the GPC3-IgG1DE or GPC3-hIgG1 mAb (Figures 14A-14D). This demonstrates the superiority of the CD16A bispecific fragment over Fc-engineered mAbs in NK cell activation.

[0174] Example 8 In vitro characterization of CCR8-CD16A bispecific antibodies To assess whether cross-linking of CD16A by other binding fragments could also induce conditional activation of CD16A downstream signaling, we selected CCR8, a chemokine receptor highly expressed on tumor-infiltrating Treg cells, as the other binding arm of the CD16A bispecific antibody (BsAb).

[0175] The CC motif chemokine receptor 8 (CCR8) represents another type of tumor-associated antigen. It has recently been identified that CCR8 is exclusively expressed on a subset of highly suppressive tumor-infiltrating regulatory T cells (Tregs) found in multiple cancers, including breast, colorectal, and lung cancers. Meanwhile, high abundance of CCR8+ Tregs is associated with poor prognosis. Therefore, CCR8 is a promising therapeutic target for enhancing antitumor immunity.

[0176] The discovery of the CCR8 antibodies tested in this example is described as follows: BALB / c, C57BL / 6, and SJL mice were immunized with full-length human CCR8 DNA and the CHO-K1 / HEK293 hCCR8 cell line. After the routine process of hybridoma technology, including several rounds of mouse immunization and fusion with immortalized myeloma cells followed by clonal characterization, hybridoma clones were selected for strong binding to human CCR8 (Table 10A). Table 10A. CCR8 antibody sequence (underlined CDRs) [Table 10A] 8.1 Construction of CCR8-CD16A bispecific antibodies

[0177] A CCR8-CD16A-hIgG1LALAPG bispecific antibody was constructed using Format 1 (Figure 9), which contains two anti-human CD16A scFv fragments fused to the C-terminus of the ADCC-neutralizing Fc fragment of the CCR8-hIgG1 antibody. To assess whether the wild-type Fc fragment synergizes with the CD16A-specific binding fragment and contributes to different binding and functional activities, we also generated a CCR8-CD16A-hIgG1 bispecific antibody with a wild-type Fc fragment. We generated anti-hCCR8 monoclonal antibodies with Fc-DLE (S239D / A330L / I332E), Fc-DE (S239D / I332E), Fc-defucosylation (in which N-glycan residues within the IgG Fc region lack the core fucose sugar unit), or wild-type Fc hIgG1, compared to the ADCC-enhanced monospecific antibody (eADCC CCR8 mAb). All CCR8-CD16A BsAbs and CCR8 mAbs used the same anti-CCR8 Fab sequence. The sequences of the CCR8-CD16A BsAbs and CCR8 mAbs are listed in Table 10B. Table 10B. Sequences of CCR8-CD16A BsAb and CCR8 mAb [Table 10B-1] [Table 10B-2] [Table 10B-3] [Table 10B-4] 8.2 Binding Ability of CCR8-CD16A Bispecific Antibodies to CD16A

[0178] The CCR8-45H6E8-hIgG1 BsAb, CCR8-45H6E8-hIgG1LALAPG BsAb, and eADCC CCR8 mAb were tested in a cell-based binding assay using CHO-K1 cell lines stably expressing different human Fcγ receptors: hCD16A-158V, hCD16A-158F, or hCD32B, respectively. The purpose of the assay was to evaluate the binding of the BsAbs to each of the hFcγRs.

[0179] As shown in Figures 15A and 15B, the CCR8-45H6E8-hIgG1 bsAb strongly bound to both human CD16A-158V and human CD16A-158F in a dose-dependent manner. More importantly, compared with various eADCC CCR8 mAbs, the CCR8-45H6E8-hIgG1 BsAb and CCR8-45H6E8-HIgG1LALAPG BsAb showed superior binding to both CD16A forms. Notably, the CCR8-45H6E8-hIgG1 BsAb displayed stronger binding ability to human CD16A than the CCR8-45H6E8-hIgG1LALAPG BsAb or CCR8-hIgG1 mAb. This phenomenon indicated that wild-type Fc could synergize with a CD16A-specific binding fragment to enhance binding to CD16A. Figure 15C shows that the CCR8-45H6E8 WT BsAb and CCR8-45H6E8 LALAPG BsAb have lower binding affinity to the inhibitory Fcγ receptor hCD32B compared with various CCR8 mAbs. Surprisingly, compared with CCR8-hIgG1 mAb, the CCR8-45H6E8-hIgG1 BsAb showed significantly reduced binding ability to CD32B. 8.3 CCR8-CD16A bispecific antibody stimulated NK cell function

[0180] To assess whether CCR8-CD16A BsAb could induce the cytotoxic effect of NK cells against target cells, NK cell degranulation, and NK cell cytokine production, a series of functional assays using human primary NK cells as effector cells and the human T lymphoblastoid cell line MT-4, which naturally expresses human CCR8, as target cells were set up as described in Example 7.

[0181] Both CCR8-CD16A bispecific antibodies, CCR8-45H6E8-hIgG1LALAPG BsAb and CCR8-45H6E8-hIgG1 BsAb, effectively enhanced NK cell function in a dose-dependent and CCR8-dependent manner (Figures 16A-16D). More importantly, these two CCR8-CD16A BsAbs demonstrated more potent in vitro cytotoxicity against target cells (Figure 16A), NK cell degranulation (Figure 16B), and NK cell cytokine production (Figures 16C and 16D) than the ADCC-enhancing CCR8-hIgG1DE mAb. These findings demonstrate the superiority of CD16A bispecific fragments over Fc-engineered mAbs in NK cell activation.

[0182] As shown in Figure 17, humanized CCR8-CD16A BsAbs, including CCR8-45H6E8-z17-hIgG1, CCR8-45H6E8-z19-hIgG1, and CCR8-45H6E8-z20-hIgG1, showed maintained NK cell-mediated killing efficacy compared to the chimeric CCR8-45H6E8-hIgG1 BsAb.

[0183] Example 9 In vitro potency of surrogate CCR8-CD16A BsAbs This example describes the in vitro efficacy of a CCR8-CD16A BsAb and compares it in parallel with an eADCC CCR8 mAb. 9.1 Generation of CCR8 surrogate antibodies

[0184] The surrogate anti-CCR8 antibody 194A1G9 was generated and selected as previously described in Example 8 for its strong binding to human CCR8 while maintaining cross-reactivity to mouse CCR8, facilitating preclinical efficacy evaluation in mouse models. The amino acid sequences of the heavy and light chain variable regions of the surrogate CCR8 antibody are listed below in Table 11. Table 11. CCR8 antibody sequence (underlined CDRs) [Table 11] 9.2 Binding Ability of CCR8 Surrogate Antibodies to CCR8

[0185] To confirm the binding activity of the surrogate anti-CCR8 antibody 194A1G9, this antibody was subjected to FACS analysis using HEK293 hCCR8 cell line, CHO-K1 mCCR8 cell line and their parental cell lines.

[0186] Briefly, HEK293 hCCR8 cell line, CHO-K1 mCCR8 cell line, and parental cell line were first incubated with serially diluted 194A1G9 for 30 minutes at 4°C. After washing with FACS buffer, PE goat anti-human IgG Fc secondary antibody (eBioscience™, Invitrogen) was added to each well and incubated for 30 minutes at 4°C. Samples were washed twice with FACS staining buffer. The mean fluorescence intensity (MFI) of PE was assessed using a MACSQuant Analyzer 16.

[0187] As shown in Figures 18A and 18B, 194A1G9 specifically bound to human CCR8 and mCCR8 expressed on cells in a dose-dependent manner. 9.3 Construction of surrogate CCR8-CD16A bispecific antibodies

[0188] To understand the activity of CCR8-CD16A bispecific antibodies, we generated bispecific antibodies constructed in Format 1 ( FIG. 9 ), comprising the anti-CD16A scFv fragment 42F5H2 scFv (SEQ ID NO: 68) conjugated to the C-terminus of the mCCR8-hIgG1 or mCCR8-hIgG1LALA (194A1G9) antibody Fc via a (G4S)3 linker (SEQ ID NO: 63). The heavy and light chain sequences of the 194A1G9-42F5H2-hIgG1 BsAb and 194A1G9-42F5H2-hIgG1LALA BsAb are listed in Table 12. For control, eADCC mAb 194A1G9 hIgG1DE was compared in parallel with the heavy and light chain sequences listed in Table 12. Table 12. Sequences of CCR8-CD16A BsAb antibodies and CCR8 mAbs [Table 12-1] [Table 12-2] 9.4 Surrogate CCR8-CD16A bispecific antibody mediated activation of ADCC signaling

[0189] Previously, in Example 8, we observed enhanced cell binding of the CCR8-CD16A bsAb to the activating FcγR CD16A and reduced cell binding to the inhibitory FcγR CD32B. To assess whether this biased binding profile contributes to improved ADCC signaling, we performed an in vitro assay to compare the ADCC signaling of these antibodies. In this assay, Jurkat cells stably co-expressed hCD16A-158V, hCD32B, and a luciferase reporter driven by an NFAT response element (Jurkat-hCD16A-hCD32B-NFAT) as effector cells. Mouse CCR8-expressing CHO-K1 cells (CHO-K1-mCCR8) were used as target cells. The protocol was as previously described in Example 6.

[0190] The results suggested that both 194A1G9-42F5H2-hIgG1 and 194A1G9-42F5H2-hIgG1LALA BsAb could mediate stronger ADCC signaling compared to 194A1G9-hIgG1DE in the same experimental setting (Figure 19). Interestingly, 194A1G9-42F5H2-hIgG1 BsAb showed superior CD16A activation compared to 194A1G9-42F5H2-hIgG1LALA BsAb, indicating an additive effect from Fc-mediated CD16A signaling (Figure 19).

[0191] Example 10 Synergism between CD16A-bsAb-mediated and Fc-mediated effector functions in vivo To further confirm the function of the CD16A BsAb, we tested the in vivo antitumor efficacy of the molecule using syngeneic CD16A humanized C57BL / 6 mice in which the mouse FcγRIV gene was replaced with human CD16A 158V. MC38 cells resuspended in PBS were cultured at 5 × 10 in a volume of 0.1 ml. 5 Cells were administered subcutaneously (sc) to the right flank of mice at a concentration of 1000x1000x1000 cells. The mean tumor volume was approximately 72 mm. 3 Once tumor volume reached 1000 mg / kg, animals were randomly assigned to experimental groups with 7 animals per group according to tumor volume. Equimolar concentrations of surrogate anti-mCCR8 antibodies, including 194A1G9-hIgG1DE (6 mg / kg), 194A1G9-42F5H2-hIgG1 BsAb (8 mg / kg), and 194A1G9-42F5-hIgG1LALA BsAb (8 mg / kg), were administered intraperitoneally twice weekly. Body weight and tumor volume were measured twice weekly.

[0192] As shown in Figure 20, compared with the PBS control, the 194A1G9-42F5H2-hIgG1LALA BsAb exhibited tumor growth inhibition comparable to that of the ADCC-enhanced 194A1G9-hIgG1DE mAb, with inhibition rates of 31.5% and 36%, respectively. These data demonstrated that the 194A1G9-42F5H2-hIgG1LALA BsAb can efficiently induce CCR8-dependent CD16A activation and mediate effector cell-mediated depletion of CCR8+ Treg cells and tumor control. Remarkably, the 194A1G9-42F5H2-hIgG1 BsAb exhibited significantly improved tumor growth inhibition compared with the 194A1G9-42F5H2-hIgG1LALA BsAb and 194A1G9-hIgG1DE mAb, with an inhibition rate of 75.7% (Figure 20). This indicates a significant contribution of synergy between CD16A antibody-mediated effector function and Fc-mediated effector function.

[0193] In another independent study using the same mouse model, we further confirmed the antitumor effect of CD16A BsAb, which is mediated by the expression of a TAA such as CCR8 in this example. We used another mCCR8 clone, SA214G2 (Biolegend), and another CD16A clone, 45H6E8, to generate a CCR8-CD16A bispecific antibody with Format 1 (FIG. 9). Two Fc-engineered mAbs, SA214G2-hIgG1DE and SA214G2-hIgG1DLE, were compared in parallel.

[0194] As shown in Figure 21, compared with the PBS control, SA214G2-45H6E8-hIgG1LALAPG BsAb was able to efficiently inhibit tumor growth even at a very low treatment dose (1.33 mg / kg). This indicates efficient activation of effector cells and inhibition of tumor growth mediated solely by CCR8-dependent CD16A activation in the absence of Fc-mediated effector function. Notably, SA214G2-45H6E8-hIgG1 showed significantly improved tumor growth inhibition compared with other treatments, with more animals being tumor-free 21 days after the first dose (Figure 21). This further confirmed the synergistic interaction between CD16A antibody-mediated effector function and Fc-mediated effector function. * * *

[0195] The present disclosure is not limited in scope by the specific embodiments described; these embodiments are intended as single illustrations of individual aspects of the disclosure; any compositions or methods that are functionally equivalent are within the scope of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Accordingly, the present disclosure is intended to cover modifications and variations of the present disclosure, provided they come within the scope of the appended claims and their equivalents.

[0196] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. An antibody or antigen-binding fragment thereof having binding specificity for human CD16A protein, comprising a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, and a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are, respectively: (a) SEQ ID NOs: 22, 96, 24, 37, 38 and 39; (b) SEQ ID NOs: 22, 23, 24, 37, 38 and 39; (c) SEQ ID NOs: 13, 14, 15, 28, 29 and 30; (e) SEQ ID NOs: 16, 17, 18, 31, 32, and 33; (e) SEQ ID NOs: 19, 20, 21, 34, 35, and 36; or (f) SEQ ID NOs: 25, 26, 27, 40, 41, and 42 An antibody or antigen-binding fragment thereof comprising the amino acid sequence of:

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 22, 96, 24, 37, 38, and 39, or SEQ ID NOs: 22, 23, 24, 37, 38, and 39, respectively.

3. 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 22, 96, 24, 37, 38, and 39, respectively.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 91, 93 and 95, and the VL comprises the amino acid sequence of SEQ ID NO:

89.

5. 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 22, 23, 24, 37, 38, and 39, respectively.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 83 to 87, and the VL comprises the amino acid sequence of SEQ ID NO:

89.

7. The antibody or antigen-binding fragment thereof of claim 1 , wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 14, 15, 28, 29, and 30, respectively.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1 and the VL comprises the amino acid sequence of SEQ ID NO:

2.

9. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 16, 17, 18, 31, 32, and 33, respectively.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the VH comprises the amino acid sequence of SEQ ID NO: 3 and the VL comprises the amino acid sequence of SEQ ID NO:

4.

11. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 19, 20, 21, 34, 35, and 36, respectively.

12. The antibody or antigen-binding fragment thereof of claim 11, wherein the VH comprises the amino acid sequence of SEQ ID NO: 5 and the VL comprises the amino acid sequence of SEQ ID NO:

6.

13. 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 25, 26, 27, 40, 41, and 42, respectively.

14. The antibody or antigen-binding fragment thereof of claim 13, wherein the VH comprises the amino acid sequence of SEQ ID NO: 9 and the VL comprises the amino acid sequence of SEQ ID NO:

10.

15. The antibody or antigen-binding fragment thereof of claim 1 , further comprising an IgG Fc fragment.

16. The antibody or antigen-binding fragment thereof of claim 15, wherein the Fc fragment is incapable of binding to an Fcγ receptor or a complement protein.

17. The antibody or antigen-binding fragment thereof according to claim 16, wherein the Fc fragment preferably comprises the amino acid sequence of SEQ ID NO:

76.

18. an anti-CD16A antibody or antigen-binding fragment having binding specificity for human CD16A protein; a second antibody or antigen-binding fragment having binding specificity for a tumor-associated antigen (TAA); and Fc fragment A multispecific antibody comprising:

19. The multispecific antibody of claim 18 , wherein the anti-CD16A antibody or antigen-binding fragment and the Fc fragment have a synergistic effect when exerting effector functions.

20. 20. The multispecific antibody of claim 19, wherein the Fc fragment is capable of binding to CD64 and is incapable of binding to CD16B and / or CD32B.

21. 21. The multispecific antibody of claim 20, wherein the Fc fragment is a wild-type human IgG Fc fragment.

22. 22. The multispecific antibody of claim 21 , wherein the Fc fragment is a wild-type human IgG Fc fragment selected from IgG1 Fc, IgG2 Fc, IgG3 Fc and IgG4 Fc.

23. 23. The multispecific antibody of claim 22, wherein the Fc fragment is a wild-type human IgGl Fc fragment.

24. 24. The multispecific antibody of any one of claims 21 to 23, wherein the wild-type Fc fragment has not been modified to have an enhanced effector function.

25. 25. The multispecific antibody of claim 24, wherein the wild-type Fc fragment is not defucosylated.

26. 26. The multispecific antibody of any one of claims 18 to 25, wherein the anti-CD16A antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are both located N-terminal to the Fc fragment.

27. 26. The multispecific antibody of any one of claims 18 to 25, wherein the anti-CD16A antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are located on either side of the Fc fragment, and the anti-CD16A antibody or antigen-binding fragment thereof is preferably located on the C-terminal side of the Fc fragment.

28. 28. The multispecific antibody of any one of claims 18 to 27, wherein the anti-CD16A antibody or antigen-binding fragment thereof binds to at least 158Y of SEQ ID NO:

55.

29. 29. The multispecific antibody of any one of claims 18 to 28, wherein the anti-CD16A antibody or antigen-binding fragment thereof does not bind to CD16B.

30. 30. The multispecific antibody of any one of claims 18 to 29, wherein the anti-CD16A antibody or antigen-binding fragment thereof is an anti-CD16A antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

31. 31. The multispecific antibody of any one of claims 18 to 30, wherein the TAA is selected from the group consisting of CCR8, CD25, GPC3, Claudin 18.2, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and tenascin.

32. 32. The multispecific antibody of claim 31 , wherein the TAA is CCR8, CD25 or GPC3.

33. 32. The multispecific antibody of claim 31 , wherein the second antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 56, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

57.

34. 32. The multispecific antibody of claim 31 , wherein the second antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 117, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

118.

35. 35. The multispecific antibody of claim 34, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 129 and a light chain comprising the amino acid sequence of SEQ ID NO: 130, or comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 131 and a light chain comprising the amino acid sequence of SEQ ID NO:

132.

36. 32. The multispecific antibody of claim 31 , wherein the second antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 77, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

78.

37. 37. One or more polynucleotides encoding the antibody or antigen-binding fragment of any one of claims 1 to 36.

38. 38. A cell comprising one or more polynucleotides of claim 37.

39. A composition comprising an antibody described in any one of claims 1 to 36, one or more polynucleotides described in claim 37, or a cell described in claim 38.

40. 37. A method for treating cancer, comprising administering to a cancer patient an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 36.

41. 40. Use of the antibody or antigen-binding fragment of any one of claims 1 to 36 for the manufacture of a medicament for treating cancer.

42. 42. The method of claim 40 or the use of claim 41, wherein the cancer is a non-immunogenic tumor.

43. 43. The method or use of claim 42, wherein the non-immunogenic tumor is not infiltrated by T cells or is deficient in T cell infiltration or T cell activation.

44. 44. The method or use of claim 42 or 43, wherein the cancer is resistant to treatment with an immune checkpoint inhibitor.

45. 45. The method or use of claim 44, wherein the immune checkpoint inhibitor is a PD-L1 or PD-1 inhibitor.

46. 46. ​​The method or use of any one of claims 40 to 45, wherein the cancer is prostate cancer, pancreatic cancer, leukemia, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, liver cancer, esophageal cancer, cervical cancer, thyroid cancer, lung cancer, bladder cancer, kidney cancer, uterine cancer, or melanoma.

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