Anti-CD16A antibodies and uses thereof

Abstract

Antibodies with high selectivity and specificity to human CD16A protein rather than CD16B protein are identified, and the antibodies can be used for preparing multifunctional antibodies with specificity to tumor-associated antigens. Such multifunctional antibodies can selectively activate CD16A in a target dependent manner, thereby exhibiting effective activation of NK cells and macrophages and lasting ADCC / ADCP function for the treatment of tumors, including cold tumors.

Description

Background Art

[0001] Immunotherapy is a revolutionary therapeutic strategy in the field of cancer treatment, which has brought promising clinical benefits by harnessing the body's innate immune system to fight tumor cells. Currently, a variety of immunotherapy strategies are under development, including monoclonal antibodies (mAbs) targeting tumor-associated antigens (TAAs), as well as immune checkpoint inhibitors, bispecific and multispecific antibodies, modified cytokines, tumor vaccines, adaptive cell therapies, and messenger RNA (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 main mechanisms of these drugs is that they mediate antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) through natural killer cells (NK cells) and macrophages, thereby achieving tumor cell clearance.

[0002] As our body's first line of defense, NK cells (a key component of the innate immune system) can rapidly and directly eliminate cancer cells, invading microorganisms, virus-infected cells, and transplanted cells in vitro through their activation-induced cytotoxic activity and immunomodulatory functions. Whether or not an NK cell is activated is determined by the balance between inhibitory and activating receptors on the NK cell surface. Among the numerous activating and inhibitory receptors expressed on the NK cell surface, CD16A (FcγRIIIA), a member of the Fc region of the gamma immunoglobulin IgG (FcγR) receptor family, is the only receptor that mediates ADCC, one of the primary 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 and induces cross-linking-based activation of downstream signaling, triggering the degranulation of cytotoxic molecules, including granzyme B and perforin, from the NK cell. These cytotoxic molecules mediate direct lysis of the target cell. At the same time, activated NK cells release pro-inflammatory cytokines such as IFN-γ and TNF-α, which can recruit adaptive immune cells to participate in the clearance of target cells.

[0003] Human CD16 exists in two isoforms, CD16A and CD16B (FcγRIIIB), both of which share 96% sequence identity in their immunoglobulin binding regions. Due to allelic polymorphism, CD16A has two allelic forms: CD16A-158F, which is the dominant allele in about 60% of humans and has low affinity for IgG; and CD16A-158V (or CD16A-176V based on the precursor sequence of SEQ ID NO: 55), which has high affinity for IgG. Clinical data reveal that high-affinity CD16A-158V predicts a favorable response to therapeutic IgG antibodies. In addition to being expressed on NK cells, CD16A is also present on the surface of monocytes and macrophages, mediating ADCP of phagocytes against target cells. CD16B, a GPI-anchored receptor, exhibits distinct functions due to its selective expression on neutrophils and eosinophils. CD16B reportedly acts as a decoy receptor, trapping a certain amount of IgG via the Fc fragment without triggering cell activation, leading to devastating impairment of ADCC function. Human CD16B has three allelic forms: CD16B-NA1 (R36, N65, D82, V106), CD16B-NA2 (S36, S65, N82, I106), and CD16B-SH (D78, N65), which differ in amino acids at five positions. CD32B (FcγRIIB), the only inhibitory receptor among FcγRs, is primarily expressed on B cells, macrophages, dendritic cells, neutrophils, and basophils. It plays a regulatory role in controlling the threshold and extent of cell activation through Fc engagement.

[0004] Because CD16A is crucial for NK cell- and macrophage-mediated ADCC and ADCP, the development of CD16A-targeted drugs is a promising strategy for activating NK cells and macrophages. An ideal strategy would selectively activate both CD16A-158F and CD16A-158V allelic forms, without bias in their interaction with the decoy receptor CD16B and the inhibitory receptor CD32B. However, due to the high amino acid sequence similarity between CD16A and CD16B, current strategies for protein engineering of Fc fragments have failed to prevent CD16B binding, although some engineered Fc fragments have partially achieved enhanced binding to both CD16A alleles and reduced binding to the inhibitory receptor CD32B. For example, Tafasitamab (MorphoSys), an anti-CD19 mAb with an engineered S239D / I332E (DE) mutation in the Fc region that contributes to enhanced ADCC and ADCP function, was approved by the EMA in 2020 for second-line therapy in combination with lenalidomide for DLBCL. Obinutuzumab (Roche), a second-generation anti-CD20 mAb with complete afucosylation in the Fc region, strongly binds to both CD16A alleles and enhances ADCC effects. However, these Fc engineerings also increase binding affinity for CD16B, which may limit their clinical benefits.

[0005] An alternative strategy is to develop anti-CD16A specific antibodies for the construction of bispecific or multispecific fragments to selectively and effectively 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 effective activation of NK cells and macrophages and persistent ADCC / ADCP functions without binding to CD16B and CD32B. For example, the anti-CD16A antibody 4-LS-21 developed by Affimed Therapeutics and its affinity matured version (i.e., P2C47) show similar high affinity for both CD16A-158V and CD16A-158F, and do not bind to CD16B and CD32B. Based on the CD16A antibody 4-LS21, two clinical assets, AFM13 (CD30-CD16A) without an Fc fragment and AFM24 (EGFR-CD16A) with an Fc disablement, are currently being clinically evaluated for the treatment of relapsed peripheral T-cell lymphoma, Hodgkin lymphoma (HL), and solid tumors expressing EGFR. Recently, it was reported that AFM13 had a clinical response with an overall response rate (ORR) of 100% in the recommended Phase II dose (RP2D) group when combined with NK cell therapy in CD30-positive lymphoma. Based on this previous knowledge, it is necessary to fully unleash the potential of this unique strategy in expanding the therapeutic field by developing effective CD16A-specific antibodies and exploring the appropriate composition and format of CD16A-specific bispecific or multispecific fragments.

[0006] The incidence of hepatocellular carcinoma (HCC) is increasing, particularly in East Asia. Since its discovery last century, when it was found to be highly expressed in HCC tissue, glypican 3 (GPC3) has attracted considerable attention. As a heparan sulfate (HS) glycoprotein, GPC3 interacts with growth factors, extracellular matrix proteins, and adhesion molecules to regulate cell proliferation, differentiation, adhesion, and migration. Because GPC3 expression is very limited in normal tissues, it is considered a specific TAA in HCC, and strategies targeting GPC3 as a therapeutic target would provide new approaches for the treatment of liver diseases. However, a randomized phase II trial (NCT01507168) evaluating GC33, a GPC3 monoclonal antibody with an ADCC-activating wild-type human IgG1 Fc region, failed to demonstrate clinical benefit in patients with advanced HCC who had failed prior systemic therapy. Biomarker analysis revealed that high CD16A expression on peripheral immune cells was associated with prolonged progression-free and overall survival. These results suggest that more effective strategies are needed to induce GPC3-targeted CD16A activation and the resulting ADCC and ADCP functions to contribute to better clinical outcomes. Summary of the Invention

[0007] In various embodiments, the present disclosure provides antibodies and antigen-binding fragments that are specific for human CD16A protein. Experimental tests have shown that these newly identified antibodies can potently and specifically bind to human CD16A protein without interacting with CD16B variants. Some of these antibodies also cross-react with cynomolgus monkey CD16 protein, thereby contributing to preclinical studies. In addition, in vitro and in vivo studies have shown 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 afucosylated Fc), multispecific fragments based on anti-CD16A show a stronger ADCC effect. More interestingly, when Fc function is intact, anti-tumor killing is further enhanced, indicating that there is a synergistic effect between anti-CD16A and Fc-mediated effector functions.

[0008] Therefore, one embodiment of the present disclosure provides an antibody or antigen-binding fragment thereof having binding specificity to human CD16A protein, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, the light chain variable region comprising VL CDR1, VL CDR2 and VLCDR3, wherein: the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the following amino acid sequences: (a) SEQ ID NO: 22, 23, 24, 37, 38 and 39; (b) SEQ ID NO: 13, 14, 15, 28, 29 and 30; (c) SEQ ID NO: 16, 17, 18, 31, 32 and 33; (d) SEQ ID NO: 19, 20, 21, 34, 35 and 36; or (e) SEQ ID NO: ID NO:25, 26, 27, 40, 41 and 42.

[0009] In one embodiment, a multispecific antibody is provided, comprising: an anti-CD16A antibody or antigen-binding fragment having binding specificity to human CD16A protein; and a second antibody or antigen-binding fragment having binding specificity to 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 act synergistically in exerting effector functions. 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] Also provided are methods and uses for treating diseases such as cancer. 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 DESCRIPTION OF THE DRAWINGS

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

[0013] Figure 2A 、 Figure 2B and Figure 2CThe binding ability of anti-CD16A antibodies to human CD16B-NA1-his, human CD16B-NA2-his, and human CD16B-SH-his in ELISA assays is shown.

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

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

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

[0017] Figure 6 Shown are cross-species binding abilities of anti-CD16A antibodies to cynomolgus monkey CD16 expressed on CHO-K1 cells measured by flow cytometry.

[0018] Figure 7A -D shows 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 measured by flow cytometry.

[0019] FIG8 shows the binding ability of a series of humanized anti-CD16A antibodies with removed post-translational modification (PTM) sites to human CD16A-158V, CD16A-158F, CD16B-NA1 and cynomolgus monkey CD16 expressed on CHO-K1 cells, measured by flow cytometry.

[0020] Figure 9 Format 1 of a CD16A bispecific fragment is shown, containing a bivalent Fab format of the antigen-binding fragment and a bivalent scFv format of the anti-CD16A fragment at the N-terminus and C-terminus of a human IgG1 Fc fragment, respectively, with or without FcγR binding.

[0021] Figure 10A -D shows the expression of various GPC3-CD16A bispecific antibodies and anti-GPC3 mAbs against GPC3+HepG2 cells ( Figure 10A ) and GPC3-SK-HEP-1 cells ( Figure 10B) on human GPC3, human CD16A-158V ( Figure 10C ) and CD16A-158F( Figure 10D )’s combining ability.

[0022] Figure 11A -D shows 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 12A -C shows primary NK cell-mediated cytotoxicity induced by various chimeric and humanized GPC3-CD16 ABsAbs against GPC3-expressing and GPC3-negative tumor cells.

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

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

[0026] Figure 15A -C shows 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 16A -D shows primary NK cell-mediated cytotoxicity, degranulation, and cytokine production induced by CCR8-CD16A BsAb against CCR8-expressing tumor cells.

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

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

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

[0031] Figure 20 In vivo comparison of 194A1G9-42F5H2 BsAb and 194A1G9 mAb is shown

[0032] Figure 21 Shown is an in vivo comparison of the SA214G2-45H6E8 BsAb and the SA214G2 mAb. DETAILED DESCRIPTION definition

[0033] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "an antibody" should be understood to refer to one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0034] As used herein, the term "polypeptide" is intended to encompass both singular "polypeptides" and plural "polypeptides", and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, and does not refer to the specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to one or more chains of two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" can be used instead of any of these terms, or can be used interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting groups / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. The polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It can be produced in any way, including by chemical synthesis.

[0035] As used herein, the term "isolated" with respect to cells, nucleic acids such as DNA or RNA refers to molecules separated from other DNA or RNA present in the natural source of the macromolecule, respectively. As used herein, the term "isolated" also refers to nucleic acids or peptides that are substantially free of cellular material, viral material or culture medium when produced by recombinant DNA technology, or nucleic acids or peptides that are substantially free of chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that do not exist naturally as fragments and would not be found under natural conditions. The term "isolated" is also used herein to refer to cells or polypeptides separated 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 polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody as well as any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide containing a molecule that contains 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, a heavy or light chain complementary determining region (CDR) or ligand binding portion thereof, 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] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, an antibody fragment can bind to the same antigen 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 functions as an antibody by binding to a specific antigen to form a complex.

[0038] "Single-chain variable fragment" or "scFv" refers to an immunoglobulin heavy chain (V H ) and light chain (V L ) variable region. In some aspects, these regions are connected by a short connecting peptide of about 10 to about 25 amino acids in length. The connecting peptide can be rich in glycine to provide flexibility, as well as serine or threonine to improve solubility, and can connect V H The N-terminus and V L The C-terminus of the constant region is removed and a linker is introduced, but the protein still retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019.

[0039] The term antibody encompasses a wide range of polypeptide classes that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as γ, μ, α, δ, or ε (gamma, mu, alpha, delta, epsilon), including several subclasses (e.g., γ1-γ4). It is the properties of this chain that determine the "class" of the antibody, IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and known to confer functional specificity. In light of this disclosure, those skilled in the art can readily identify modified versions of these classes and isotypes, and accordingly, these modified versions are encompassed within the scope of this disclosure. While all immunoglobulin classes are clearly encompassed within the scope of this disclosure, the following discussion will generally be directed to immunoglobulin molecules of the IgG class. With respect to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of approximately 23,000 daltons and two identical heavy chain polypeptides of 53,000-70,000 daltons. The four chains are typically connected by disulfide bonds in a "Y" configuration, with the light chains wrapping around the heavy chains at the mouth of the "Y" and extending into the variable region.

[0040] The antibodies, antigen-binding polypeptides, variants or derivatives thereof disclosed herein 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, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising VK or VH domains, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecule disclosed herein can be 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 molecules.

[0041] Light chains are classified as either kappa or lambda (κ or lambda). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, the light and heavy chains are covalently bound to each other. When immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the "tail" portions of the two heavy chains are bound to each other by covalent disulfide bonds or non-covalent bonds. Within the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the base of each chain.

[0042] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains (VK and VH) of both the light and heavy chains determine antigen recognition and specificity. Conversely, the constant domains (CK and CH1, CH2, or CH3) of the light and heavy chains confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement fixation, etc. By convention, the farther the constant region domain is from the antibody's antigen-binding site or amino-terminus, the higher its number. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CK domains actually comprise the carboxyl termini of the heavy and light chains, respectively.

[0043] As mentioned above, variable region enables antibodies to selectively recognize and specifically bind epitopes on antigens. That is, the subset of the VK domain and VH domain or complementary determining region (CDR) of the antibody combines to form the variable region that defines the three-dimensional antigen binding site. This four-level antibody structure forms an antigen binding site located at the end of each arm of the Y configuration. More particularly, 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 VH and VK chain. In some cases, for example, certain immunoglobulin molecules (complete immunoglobulin molecules) derived from camelid species or based on camelid immunoglobulin engineering may consist only of heavy chains, without light chains. See, for example, Hamers-Casterman et al., Nature [nature] 363:446-448 (1993).

[0044] In naturally occurring antibodies, the six "complementarity determining regions," or "CDRs," in each antigen-binding domain are short, non-contiguous sequences of amino acids that are precisely positioned to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in these antigen-binding domains, termed "framework" regions, exhibit low inter-molecular variability. The framework regions primarily adopt a β-sheet conformation, with the CDRs forming loops that connect and, in some cases, form part of the β-sheet structure. Thus, the framework regions serve as a scaffold that positions the CDRs in the correct orientation through interchain, non-covalent interactions. The antigen-binding domain formed by these positioned CDRs defines a surface that is complementary to an epitope on an immunoreactive antigen. This complementary surface promotes non-covalent binding of the antibody to its cognate epitope. For any given heavy or light chain variable region, one of ordinary skill in the art can readily identify the amino acids comprising the CDRs and framework regions, respectively, since they have been 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. MoI. Biol., 196:901-917 (1987)).

[0045] In the case where there are two or more definitions for a term used and / or accepted in the art, unless clearly stated to the contrary, the definition of the term as used herein is intended to include all such meanings. A specific example is to use the term "complementarity determining region" ("CDR") to describe the non-continuous antigen binding sites found in the variable region of both heavy chain polypeptides and light chain polypeptides. This specific region has been described in the following documents: Kabat et al., US Department of Health and Human Services [U.S. Department of Health and Human Services], "Sequences of Proteins of Immunological Interest [immunology related protein sequences]" (1983) and Chothia et al., J.MoI.Biol [Molecular Biology Journal] .196:901-917 (1987), which are incorporated herein by reference in their entirety. According to the two CDR definitions of Kabat and Chothia, when compared to each other, overlap or subsets of amino acid residues are included. However, the application of any definition for referring to the CDR of an antibody or its variants is intended to be within the scope of the term as defined and used herein. For comparison, the corresponding amino acid residues encompassing these CDRs as defined in each of the references cited above are shown in the table below. The exact number of residues encompassing a particular CDR will vary depending on the sequence and size of the CDR. One skilled in the art can generally determine which residues comprise a particular CDR based on the variable region amino acid sequence of an antibody.

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

[0047] In addition to the above table, the Kabat numbering system describes these 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 at approximately the 15th residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the 33rd amino acid residue after the end of CDR-H2; includes 3-25 amino acids; and ends at the sequence WGXG, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., after a cysteine ​​residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the 16th residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins at about the thirty-third residue after the end of CDR-L2 (ie, after the cysteine ​​residue); comprises about 7-11 residues and ends at the sequence F or WGXG, where X is any amino acid.

[0048] The antibodies disclosed herein can be from any animal source, including birds and mammals. Preferably, these antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies. In another embodiment, the variable region may be derived from a cartilaginous fish (e.g., from a shark).

[0049] As used herein, the term "heavy chain constant region" includes amino acid sequences derived from immunoglobulin heavy chains. The polypeptide comprising the heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., upper, middle and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, the antigen-binding polypeptide used 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 and 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, the polypeptide of the present disclosure comprises a polypeptide chain comprising a CH3 domain. Further, the antibody used in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As described above, it will be understood by those of ordinary skill in the art that the heavy chain constant regions can be modified such that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0050] The heavy chain constant regions of the antibodies disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide may comprise a polypeptide derived from IgG l In another example, the heavy chain constant region may comprise a portion derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. l In another example, the heavy chain portion may comprise a portion derived from an IgG3 molecule. l molecule and is derived in part from the chimeric hinge of the IgG4 molecule.

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

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

[0053] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventive measures, wherein the purpose is to prevent or slow down (mitigate) a physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization (i.e., no worsening) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and alleviation (whether partial or complete), whether detectable or undetectable. "Treatment" can also refer to extending survival compared to the expected survival when not receiving treatment. Those in need of treatment include those already suffering from a disease or disorder and those who are prone to a disease or disorder or those in which the disease or disorder is to be prevented.

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

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

[0056] Through trial and error, the inventors were able to identify new antibodies that can potently and specifically bind to the human CD16A protein. As shown in the experimental examples, all tested examples showed strong affinity for 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 with cynomolgus monkey CD16. Equally important, these new antibodies showed negligible binding to CD16B.

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

[0058] These newly developed antibodies have also been used to generate bispecific antibodies with a secondary specificity for the tumor-associated antigens (TAAs) GPC3 or CCR8. These bispecific antibodies are active in triggering CD16A signaling only when the coupled antigen-binding moiety also recognizes the corresponding antigen on the target cell. Therefore, these newly developed anti-CD16A antibodies are suitable for clinical use in preventing or treating various diseases.

[0059] Therefore, according to one embodiment of the present disclosure, an antibody or antigen-binding fragment thereof having binding specificity to human CD16A protein is provided, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, the light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3, wherein: the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 are the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the antibodies shown in Table 1, Table 6A and Table 7A, respectively. Representative CDR sequences (Kabat numbering) of these antibodies are shown in Tables 1A-B and 7B, 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.

[0060] In one embodiment, the antibody or antigen-binding fragment thereof of the present disclosure comprises the CDR region of antibody 45H6E8, which has a VH sequence of SEQ ID NO: 7 and a 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 NO: 22, 23, 24, 37, 38 and 39, respectively. In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise these CDR sequences and have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity with any of the antibodies 45H6E8 or its humanized counterparts. In one embodiment, VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 7. In one embodiment, VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity with 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 exemplary 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)-de-risked. An example is SEQ ID NO: 96 for VH CDR2. Exemplary VH sequences of humanized, PTM-de-risked 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] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on CD16A as 45H6E8 are also provided. Therefore, in some embodiments, antibodies and antigen-binding fragments thereof that compete with 45H6E8 for binding to CD16A are also provided.

[0068] In one embodiment, the antibody or antigen-binding fragment thereof of the present disclosure comprises the CDR region of antibody 23H7E4, which has a VH sequence of SEQ ID NO: 1 and a 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 NO: 13, 14, 15, 28, 29 and 30, respectively. In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise these CDR sequences and have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to antibody 23H7E4. 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, VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2.

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

[0071] In one embodiment, the antibody or antigen-binding fragment thereof of the present disclosure comprises the CDR region of antibody 37B3G11, which has a VH sequence of SEQ ID NO: 3 and a 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, the antibodies or antigen-binding fragments thereof of the present disclosure comprise these CDR sequences and have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to antibody 37B3G11. 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, VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4.

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

[0074] In one embodiment, the antibody or antigen-binding fragment thereof of the present disclosure comprises the CDR region of antibody 42F5H2, which has a VH sequence of SEQ ID NO: 5 and a 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 NO: 19, 20, 21, 34, 35 and 36, respectively. In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise these CDR sequences and have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to antibody 42F5H2. 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, VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6.

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

[0077] In one embodiment, the antibody or antigen-binding fragment thereof of the present disclosure comprises the CDR region of antibody 91A11D11, which has a VH sequence of SEQ ID NO: 9 and a 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, the antibodies or antigen-binding fragments thereof of the present disclosure comprise these CDR sequences and have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to antibody 91A11D11. 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, VH has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10.

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

[0080] In some embodiments, the antibodies of the present disclosure are full-length IgG antibodies, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibodies have an Fc fragment without effector function (e.g., unable to bind Fcγ receptors or complement proteins). Modifications to wild-type Fc fragments that are known to cause the Fc fragment to lose such binding activity are known. An 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, as well as to complement component C1q. Additional examples are L234F / L235E / P331S (FES), L234F / L235Q / K322Q (FQQ), L234A / G237A, L234A / L235A / G237A, L234A / L235A / G237A, L234A / L235A / G237A / P238S / H268A / A330S / P330S, L234A / L235E, G236R / L328R, and L234A / L235A / K322A. For IgG2 Fc, an example 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, one portion of which binds CD16A and another portion of which targets a specific antigen on target tumor cells, can exhibit effective activation of NK cells and macrophages and sustained ADCC / ADCP function.

[0083] Therefore, one embodiment provides a multispecific antibody comprising an anti-CD16A antibody or antigen-binding fragment having binding specificity to human CD16A protein; and a second antibody or antigen-binding fragment having binding specificity to 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 can be a human IgG1, IgG2, IgG3, or IgG4 Fc fragment, or a modified subtype thereof.

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

[0086] Example mutations that reduce or eliminate effector function of Fc 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 mutations that enhance the effector function of the antibody. In some embodiments, the Fc fragment does not contain post-translational modifications that 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 bind to CD64 and do not bind to CD16B or CD32B, which can inhibit or reduce ADCC activity. Enhanced effector function, such as those with mutations such as Fc-DLE (S239D / A330L / I332E) or Fc-DE (S239D / I332E), can induce or increase binding to CD16B and / or CD32B, thereby reducing ADCC efficacy.

[0089] The multispecific antibody can take any form, wherein the anti-CD16A and / or anti-TAA portion can comprise one, two, three or four antigen-binding fragments. In some embodiments, the anti-CD16A portion and the anti-TAA portion can be located on the same side of the Fc fragment (e.g., N-terminus).

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

[0091] When the anti-TAA portion is located at the N-terminus of the Fc fragment, it can form a conventional full-size IgG antibody with the Fc fragment, while the anti-CD16A portion (which can be 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, such as P2C47, or the currently identified antibodies or fragments, including 45H6E8, 23H7E4, 37B3G11, 42F5H2, and 91A11D11.

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

[0094] Example anti-CD16A antibodies or fragments comprise the CDR sequences of the antibodies shown in Table 1, Table 6A, and Table 7A. Representative CDR sequences (Kabat numbering) for these antibodies are shown in Tables 1A-B and 7B, such as those in the following: 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. Biological variants thereof, including optimized and humanized counterparts, are further described in the above sections and 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 example anti-GPC3 antibody comprising the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 57 has been prepared and tested in the Experimental Examples. It will be appreciated that other anti-GPC3 antibodies and variants may also be used herein.

[0097] Exemplary 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] Exemplary 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] Exemplary multispecific antibodies have been prepared and tested herein, such as the multispecific antibodies having the sequences provided in Table 9C. In some embodiments, the multispecific antibodies comprise 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 antibodies comprise 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 example anti-CCR8 antibody comprising the VH of SEQ ID NO: 117 and the VL of SEQ ID NO: 118 has been prepared and tested in the Experimental Examples. It will be appreciated that other anti-CCR8 antibodies and variants may also be used herein.

[0102] Exemplary 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 example anti-CCR8 antibody comprising the VH of SEQ ID NO: 77 and the VL of SEQ ID NO: 78 has been prepared and tested in the Experimental Examples. It will be appreciated that other anti-CCR8 antibodies and variants may also be used herein.

[0104] Exemplary multispecific antibodies have been prepared and tested herein, such as the multispecific antibodies having the sequences provided in Table 12. In some embodiments, the multispecific antibodies comprise 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 antibodies comprise 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 antibodies comprise 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 amino acid sequences or one or more moieties that are not typically associated with antibodies. Exemplary modifications are described in more detail below. For example, the antibodies of the present disclosure may comprise a flexible linker sequence or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0106] The antibodies (including multispecific or multifunctional antibodies) or fragments thereof disclosed herein comprise modified derivatives, i.e., by covalently attaching any type of molecule to the antibody such that the covalent attachment does not hinder the antibody from binding to the epitope. For example, but not limited to, the antibody can be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting groups / blocking groups, proteolytic cleavage, connection to cellular ligands or other proteins, and the like. Any of the many chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the antibody may contain one or more non-classical amino acids.

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

[0108] The antibodies may be conjugated or fused to therapeutic agents, which may include detectable labels (such as radiolabels), immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents (which may be drugs or toxins), ultrasound enhancing agents, non-radioactive labels, combinations thereof, and other such agents known in the art.

[0109] Antibodies can be detectably labeled by coupling them to a chemiluminescent compound. The presence of the chemiluminescent-labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence generated during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

[0110] Antibodies can also be made using fluorescent emitting metals such as 152Eu or other lanthanide metals are detectably labeled. These metals can be attached to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).Techniques for conjugating various moieties to antibodies are well known, see, e.g., 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 And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", 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 uses

[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 disorders such as cancer.

[0112] Thus, in some embodiments, methods of treating cancer in patients in need thereof are provided. In one embodiment, the method requires administering to the patient an effective amount of an antibody of the present disclosure. In some embodiments, at least one of the cancer cells (e.g., stromal cells) in the patient expresses, overexpresses, or is induced to express a tumor antigen (e.g., a TAA, such as GPC3) recognized by the multispecific antibody. For example, gene expression can be induced by administering a tumor vaccine or radiotherapy.

[0113] Tumors that may be suitable for treatment include bladder cancer, non-small cell lung cancer, kidney 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. Therefore, the antibodies disclosed in the present invention can be used to treat any one or more of these cancers.

[0114] In some embodiments, the tumors treated are those that are particularly challenging to treat with conventional immuno-oncology therapies, such as antibodies targeting immune checkpoints (ICPs). Sometimes, such tumors are referred to as "cold tumors" or "non-immunogenic tumors." Therefore, in some embodiments, the present disclosure provides methods and uses for treating cold tumors with the multispecific antibodies disclosed herein.

[0115] In certain embodiments, non-immunogenic tumors are tumors that are not infiltrated by T cells, or lack T cell filtration, antigen presenting cells (APC) or T cell activation, or lack T cells homing to the tumor bed. All prostate cancers, pancreatic cancers and leukemias are non-immunogenic. The vast majority of breast cancer (95%), colorectal cancer (95%), gastric cancer (87%), head and neck cancer (84%), liver cancer (83%), esophageal cancer (86%), cervical cancer (87%) and thyroid cancer (87%) are also non-immunogenic. In addition, 83% of lung cancer, 79% of bladder cancer, 77% of kidney cancer, 70% of uterine cancer and 66% of melanoma are also non-immunogenic.

[0116] Identification of non-immunogenic or cold tumors can also be performed by measuring the type, density, and location of immune cells within the tumor. For example, Galon and Bruni (Nature Reviews Drug Discovery, Vol. 18, pp. 197–218 (2019)) described a standardized scoring system, the Immunoscore, which is based on the quantification of two lymphocyte populations (CD3 and CD8) in, for example, resected tissue to guide the stratification of hot and cold tumors. The range of the Immunoscore is Immunoscore 0 (I0, representing low density, such as the absence of two cell types in two areas) to I4 (high density of immune cells in two locations). By classifying cancers according to their immune infiltration, the scoring system provides an immune-based tumor classification, including the definition of "hot" (highly infiltrated, Immunoscore I4) and "cold" (non-infiltrated, Immunoscore I0) tumors.

[0117] In some embodiments, the tumor is resistant to treatment with immune checkpoint inhibitors (such as PD-L1 inhibitors, PD-1 inhibitors, CTLA-4 inhibitors, or combinations 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 can be treated, prevented, diagnosed and / or predicted using the antibodies of the present disclosure, or variants or derivatives thereof, include, but are not limited to, progression and / or metastasis of malignancies and related disorders, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemias)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including, but not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma , angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, prostate 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, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, and retinoblastoma.

[0119] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody or variant thereof used, the patient's age, weight, overall health, sex, diet and time of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The judgment of the medical caregiver to such factors is within the ordinary skill of the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of preparation, 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] The method for administering antibody, variant includes but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral route.Antigen-binding polypeptide or composition can be applied by any convenient approach, for example, by infusion or push injection, by epithelial or mucocutaneous lining (for example, oral mucosa, rectum and intestinal mucosa etc.) absorption, and can be applied together with other bioactivators.Therefore, the pharmaceutical composition containing the antigen-binding polypeptide of the present disclosure can be applied orally, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, locally (as by powder, ointment, drops or transdermal patch), cheek or as mouth or nasal spray.

[0121] As used herein, the term "parenteral" refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intraarticular injection and infusion.

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

[0123] It is desirable to administer the antibody polypeptide or composition of the present disclosure locally to the area in need of treatment; this can be achieved, for example, but not limited to, by local infusion during surgery, topical application (e.g., in conjunction with a wound dressing after surgery), by injection, by catheter, by suppository, or by implant, wherein the implant is a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes or fibers. Preferably, when administering the proteins (including antibodies) of the present disclosure, care must be taken to use materials that the proteins are not absorbed. Polynucleotide encoding antibody and method for preparing antibody

[0124] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding antibodies, variants or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure can encode the entire heavy chain and light chain variable regions of antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on different polynucleotide molecules. In addition, the polynucleotides of the present disclosure can encode portions of the heavy chain and light chain variable regions of antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

[0125] Methods for preparing antibodies are well known in the art and described herein. In certain embodiments, the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies to 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 an antigenic challenge, but whose endogenous loci have failed. Exemplary techniques for preparing such antibodies are described in U.S. Patents 6,150,584, 6,458,592, and 6,420,140, ​​which are incorporated herein by reference in their entirety.

[0126] In certain embodiments, the prepared antibodies do not induce a harmful immune response in the animal to be treated (e.g., a human). In one embodiment, the antigen-binding polypeptides, variants or derivatives thereof disclosed herein are modified using techniques recognized in the art to reduce their immunogenicity. For example, antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies that retain or substantially retain the antigen-binding properties of the parent antibody, but are less immunogenic in humans, typically mouse or primate antibodies. This can be achieved by various methods, including (a) transplanting the entire non-human variable domain onto a human constant region to generate a chimeric antibody; (b) transplanting at least a portion of one or more non-human complementary determining regions (CDRs) into a human framework and constant region that retains or does not retain key framework residues; or (c) transplanting the entire non-human variable domain, but "dressing" it in the coat of a human-like segment by replacing surface residues.

[0127] Deimmunization can also be used to reduce the immunogenicity of antibody.As used herein, term " deimmunization " comprises changing antibody to modify T cell epitope (referring to, for example, International Application Publication No. WO / 9852976A1 and WO / 0034317A2).For example, analyze the variable heavy chain and variable light chain sequence from starting antibody, and produce the human T cell epitope " collection of maps " from each V district, which shows the position of epitope relative to other key residues in complementary determining region (CDR) and sequence.Analyze each T cell epitope from T cell epitope collection of maps, so as to identify the alternative amino acid replacement with lower risk of changing final antibody activity.Design a series of alternative variable heavy chain and variable light chain sequences, these sequences comprise the combination of amino acid replacement, and subsequently these sequences are incorporated into a series of binding polypeptides.Typically, generate variant antibody between 12 and 24 kinds and test its combination and / or function.Then the complete heavy chain and light chain gene comprising modified variable region and human constant region is cloned into expression vector, and subsequent plasmid is introduced into cell line to produce complete antibody. These antibodies are then compared in appropriate biochemical and biological assays, and the best variants are identified.

[0128] The binding specificity of the antigen-binding polypeptides 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 the Federal or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Further, a "pharmaceutically acceptable carrier" generally refers to any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0131] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle used together with the therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oil, including oils from petroleum, animal, plant or synthetic sources, for example peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and dextrose and glycerol aqueous solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffer, such as acetate, citrate or phosphate. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting tension such as sodium chloride or dextrose are also contemplated. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions can be formulated into suppositories along with conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grade 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 EW Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide (preferably in purified form) and an appropriate amount of carrier to provide the patient with an appropriate administration form. The formulation should be suitable for the mode of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple-dose vials made of glass or plastic.

[0132] In an embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to a human being according to conventional procedures. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine, to alleviate pain at the injection site. Generally speaking, the ingredients are provided in unit dosage form alone or mixed together, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or a pouch), and the amount of the active agent is indicated. When the composition is administered by infusion, an infusion bottle containing sterile pharmaceutical grade water or saline can be used for distribution. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided to mix the ingredients before administration.

[0133] The compounds of the present disclosure can be formulated in neutral form or salt form. Pharmaceutically acceptable salts include salts formed with anions, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and salts formed with cations, such as salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Examples Example 1. Generation of mouse anti-human CD16A antibodies

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

[0135] Immunogens: Two immunogens were used for mouse immunization: the extracellular domain (ECD) of human CD16A-158V was fused to a human IgG1 Fc region with an N297A mutation or a his tag at the C-terminus to generate hCD16A-158V-FcNA protein (Biointron) or hCD16A-158V-his protein (AcroBio, catalog number: CD8-H52H4).

[0136] Mouse Immunization Protocol: To generate mouse monoclonal antibodies against human CD16A, BALB / c mice were immunized biweekly with hCD16A-158V-FcNA or hCD16A-158V-his protein, alternating between intraperitoneal and subcutaneous administration. Serum titers from immunized mice were monitored by ELISA against human CD16A-158V-his protein. After 2-4 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 the supernatants secreted mouse antibodies. During the initial 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) by Mirrorball (SPT Labtech). The following confirmatory screen was performed to exclude clones that nonspecifically bound to hCD16B-SH-his protein (SinoBiological, catalog number 11046-H08H2) by ELISA or hCD16B-NA1 overexpressed on CHO-K1 cells (CHO-K1-hCD16B-NA1) by FACS, and to identify clones that bound to cynomolgus CD16 overexpressed on CHO-K1 cells (CHO-K1-cynoCD16).

[0138] Subclone Screening and Sequencing: Positive primary clones from each fusion were subcloned by limiting dilution to ensure that the hybridoma subclones were derived from a single parental cell. Subclone screening criteria were the same as those for primary clone screening described above. Subclones that showed specific binding potency to hCD16A and did not bind to hCD16B were selected for subsequent sequencing.

[0139] The sequence of the Fab fragment of the resulting mouse antibody was fused to a human IgG1 Fc with the L234A / L235A / P329G (LALAPG) mutations to generate a chimeric CD16A mAb. To validate the CD16A agonistic efficacy of the Fab fragment of the CD16A mAb, LALAPG mutations were introduced into the Fc region; these mutations completely abolished 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, after which the antibody was purified from the culture supernatant using a protein A affinity chromatography column or beads. The purified chimeric antibodies were subjected to a continuous in vitro screening process to determine affinity, binding capacity, specificity, species cross-reactivity, and agonistic function.

[0140] Based on performance in the screening assay, several hybridoma clones, including 23H7E4, 37B3G11, 42F5H2, 45H6E8, and 91A11D11, were selected for further analysis. The amino acid sequences of the variable regions of the selected mouse 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 derived from US11001633 B2), which has been developed by Affimed, was used as a benchmark antibody with the sequences of the Fab fragments listed in Table 1. Table 2 lists the heavy and light chain sequences of the antibodies. Table 1. Antibody variable region sequences (underlined CDRs) Table 1A. Heavy chain CDR sequences (Kabat numbering) Table 1B. Light chain CDR sequences (Kabat numbering) Table 2. CD16A chimeric Ab sequences Example 2. Binding activity of chimeric anti-human CD16A monoclonal antibodies 2.1. ELISA binding to human CD16A

[0141] To determine the binding ability of the chimeric Ab to the human CD16A protein, an ELISA-based binding assay was performed as follows. Briefly, hCD16A-158V-his and hCD16A-158F-his proteins (AcroBio, catalog number CDA-H5220) were diluted to 2 μg / mL in DPBS buffer and adsorbed to the wells of a 96-well microplate at 4°C overnight. After blocking the wells with 2% bovine serum albumin (BSA) to prevent nonspecific binding, CD16A chimeric Ab, benchmark antibody P2C47, or isotype control were titrated at a 3-fold dilution rate starting from 20 nM and added to the wells pre-adsorbed with hCD16A-his protein. The mixture was incubated at room temperature (RT) for 1 hour. The primary antibody was recognized by a detection antibody (Jackson Immuno, catalog number 109-035-008) conjugated to human IgG Fc and horseradish peroxidase (HRP). Tetramethylbenzidine (TMB), a substrate for HRP, was added to the wells to visualize the binding signal. After sufficient color development, a stop solution was added to the wells. The absorbance of the signal was detected at 450 nm using an Envision multi-label plate reader (Perkin Elemer). Graphpad Prism 9 software was used to generate charts and statistical analysis using a four-parameter nonlinear regression curve fit.

[0142] like Figure 1A and Figure 1B As shown, all CD16A chimeric Abs effectively bound to both the high affinity 158V and low affinity 158F of the human CD16A protein. In addition, except for the 37B3G11 chimeric Ab, all antibodies showed comparable binding efficiency to the benchmark antibody P2C47. Table 3 lists the binding EC values ​​of the tested CD16A chimeric Abs. 50 . 2.2. ELISA binding to human CD16B

[0143] Identifying the specificity of CD16A chimeric antibodies is crucial because only very few amino acids distinguish CD16A from its isoform CD16B, making the generation of CD16A-specific antibodies extremely challenging.

[0144] In order to determine the binding ability of chimeric Ab to human CD16B protein, ELISA binding assay was performed as described above. Three splicing isoforms of CD16B, including human CD16B-NA1 ECD his tag protein (hCD16B-NA1-his, Akro Bio, catalog number CDB-H5227), human CD16B-NA2 ECD his tag protein (hCD16B-NA2-his, Akro Bio, catalog number CDB-H82Ea) and human CD16B-SH-his protein, were used as coating antigens at 2 μg / mL. The commercial anti-CD16 antibody 3G8 (Stemcell, catalog number 60041) with recognition of hCD16B was used as a positive control. Figure 2A 、 Figure 2B and Figure 2C As shown, all CD16A chimeric Abs showed negligible binding to hCD16B-NA1, hCD16B-NA2 protein or hCD16B-SH protein, similar to the benchmark antibodies, indicating the strict binding specificity of these chimeric CD16A antibodies recognizing different epitopes on CD16A but not CD16B. 2.3 ELISA binding to cynomolgus monkey CD16 protein

[0145] To determine the cross-reactivity of the chimeric Ab with cynomolgus monkey CD16, an ELISA binding assay was performed as described above. Recombinant cynomolgus monkey CD16 ECD his-tagged protein (cynoCD16-his, Acro Bio, catalog number FC6-C52H9) was used as the coating antigen at 2 μg / mL. Figure 3 As shown, only 45H6E8 efficiently bound to the cynomolgus CD16 protein, while other chimeric Abs, including 23H7E4, 37B3G11, 42F5H2, and 91A11D11, exhibited negligible binding to the cynomolgus CD16 protein. These data are likely due to the extremely limited number of amino acids that can distinguish human CD16A and cynomolgus CD16 from all three isoforms of human CD16B. However, 45H6E8 is a specific CD16A antibody that exhibits excellent cross-reactivity between human and cynomolgus species and precise specificity for both human CD16A polymorphisms. Table 3. Binding activity of chimeric antibodies to antigen proteins --: No combination 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, the following cell-based binding assay was used. Briefly, CHO-K1 cells stably expressing hCD16A-158V or hCD16A-158F variants were constructed. The designated CD16A chimeric Abs, benchmark antibody P2C47, or isotype control were diluted fourfold starting from a concentration of 100 nM in staining buffer (DPBS buffer containing 2% BSA). The antibody dilutions were mixed with 1×10 5 The designated cells were incubated in a 96-well microplate at 4°C for one hour. Binding of the antibody to the antigen on the cell surface was detected using a PE-fluorophore-conjugated anti-human IgG Fc secondary antibody (Thermo Fisher Scientific, catalog number 12-4998-82) at a dilution of 1:1000. Cells were analyzed by flow cytometry using an LSR Fortessa cell analyzer (BD Biosciences). Data were analyzed using FlowJo 10.0 software. Graphs and statistical analysis were generated using Graphpad Prism 9 software using a four-parameter nonlinear regression curve fit.

[0147] like Figure 4A and Figure 4B As shown, all CD16A chimeric antibodies effectively bound to human CD16A-158V and CD16A-158F expressed on CHO-K1 cells in a dose-dependent manner. When compared to the benchmark antibody P2C47, most of our CD16A chimeric antibodies (except 23H7E4) showed comparable binding potency. Table 4 lists the binding EC values ​​of the tested CD16A chimeric Abs. 50 . 2.5. Binding Ability to Human CD16B and CD32B Expressed on CHO-K1 Cells

[0148] In order to exclude the non-specific binding of CD16A chimeric antibodies to human CD16B and CD32B expressed on cells, a cell-based binding assay was performed as described above. First, CHO-K1 cells were stably transferred with a human CD16B splicing isoform hCD16B-NA1 and human CD32B. Then, the cells were incubated with the CD16A chimeric antibodies at the specified concentrations. The commercial anti-CD16 antibody 3G8, which recognizes hCD16B, and the anti-CD32B antibody 6G11 (Bioinvent) were used as positive controls. Figure 5A and Figure 5BAs shown, all CD16A chimeric antibodies showed minimal binding to hCD16B-NA1 and hCD32B expressed on cells. In summary, we have demonstrated that our CD16A chimeric antibodies exhibit strict binding specificity to human CD16A while avoiding nonspecific binding to all three isoforms of human CD16B and 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 antibody with cynomolgus monkey CD16, a cell binding assay was performed as described above. CHO-K1 cells stably expressing cynomolgus monkey CD16 were used in the following assays. Figure 6 As shown, consistent with the binding ability to cynomolgus monkey CD16 protein, only 45H6E8 effectively bound to cynomolgus monkey CD16 expressed on CHO-K1 cells in a dose-dependent manner, with a potency comparable to that of the benchmark antibody P2C47. In contrast, 23H7E4, 37B3G11, 42F5H2, and 91A11D11 showed only weak or negligible binding to cynomolgus monkey CD16. Table 4 lists the binding EC values ​​of the tested CD16A chimeric Abs. 50 . Table 4. Binding activity of chimeric antibodies to antigens expressed on cells --: No combination 2.7. Affinity measurement

[0150] Using Biacore TM The binding affinity of the chimeric antibodies to human CD16A was determined. Briefly, the antibodies were captured using a protein A chip. 50 nM human CD16A-158V-his and CD16A-158F-his proteins were injected over the captured antibodies at a flow rate of 30 μL / min for 30 s. The antigen was allowed to dissociate for 500 s. TM Experiments were performed on 8K. Using Biacore TM Data analysis was performed using 8K evaluation software. The results showed that all chimeric CD16A antibodies exhibited high and comparable affinities for both human CD16A-158V and CD16A-158F. Among them, 42F5H2 and 91A11D11 exhibited significantly superior binding affinities for both CD16A forms when compared to the benchmark P2C47 (Tables 5A and 5B). 23H7E4, 37B3G11, and 45H6E8 exhibited comparable binding affinities to the benchmark P2C47 (Tables 5A and 5B). Table 5A. Affinity ranking results of chimeric antibodies Table 5B. Affinity ranking results of chimeric antibodies Example 3. Humanization of CD16A chimeric antibody 3.1. Humanized Design of CD16A Chimeric Antibody

[0151] 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 VH and VL were compared with the existing human Ig gene sequence database to determine the most matching human germline Ig gene sequence overall. The CDRs of the heavy and light chains of the 45H6E8 chimeric antibody were then transplanted into the candidate germline. A 3D model of the transplanted antibody was generated using the Molecular Operating Environment (MOE) to determine whether there were any key human amino acids in the framework regions, which are crucial for backmutation to the corresponding mouse amino acids to maintain CDR conformation and function.

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

[0153] Different combinations of back mutation sites were selected to generate the variable regions of humanized antibodies. The sequences of the variable regions of the heavy and light chains of humanized 45H6E8 are listed in Table 6A. The VH and VL pairings of individual humanized antibodies are listed in Table 6B. The variable regions of the humanized antibodies were then fused to the constant region of a human IgG1 Fc with the L234A / L235A / P329G (LALAPG) mutation for humanized antibody production and functional characterization. Table 6A. Sequences of the variable regions of the 45H6E8 humanized antibody (underlined CDRs; bold / italics indicate back mutations) Table 6B. VH and VL pairings of 45H6E8 humanized antibody 45H6E8 HU-VL1-3 VL HU-VH1-1 z13 HU-VH1-5 z17 HU-VH1-6 z19 HU-VH1-7 z20 HU-VH1-8 z21 VH Chimerism 3.2 Binding properties of humanized CD16A antibodies to cells expressing CD16

[0154] The cell binding activity of the humanized CD16A antibody to human CD16A-158V, CD16A-158F, CD16B-NA1, and cynomolgus monkey CD16 was confirmed in a cell-based binding assay using CHO-K1 cells expressing the antigen. The assay was performed according to the above protocol. 647AffiniPure TM Binding of the antibody to the antigen on the cell surface was detected by using a 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] like Figure 7A and Figure 7B All of the CD16A humanized antibodies, including 45H6E8-z13, 45H6E8-z17, 45H6E8-z19, 45H6E8-z20, and 45H6E8-z21, showed comparable cell-binding abilities to human CD16A-158V and CD16A-158F as their chimeric antibodies. Meanwhile, when compared to the chimeric 45H6E8 antibody, these CD16A humanized antibodies showed similar cell-binding abilities to cynomolgus monkey CD16, as Figure 7C In contrast, all of these CD16A humanized antibodies showed negligible binding to human CD16B-NA1 expressed on cells ( Figure 7D ). Example 4. Optimization of humanized CD16A antibody

[0156] After antibody humanization, CDR regions are often further optimized to remove potential post-translational modification (PTM) sites to improve the developability of humanized antibodies, including long-term stability, manufacturability, and homogeneity. 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 case, computational tools were used to predict PTM-susceptible sites to promote engineered antibodies with better physical and chemical properties. A careful examination of the CDR regions of the humanized 45H6E8 mAb identified an NG motif in the CDR2 of the VH region that may undergo deamidation at the asparagine (N) amino acid and impair the binding and function of the CD16A antibody. Therefore, an amino acid substitution of asparagine to glutamine (Q) was performed at this position to remove the potential PTM site. The sequences of the variable regions of the 45H6E8 humanized mAb with PTM removed are listed in Table 7A, and the mutated CDRs are summarized in Table 7B. Table 7A. Sequences of the variable regions of the CD16A humanized antibody (underlined CDRs; bold / italics indicate PTM mutations) Table 7B. Optimized heavy chain CDR2 of 45H6E8 Antibody H-CDR2 SEQ ID NO: 45H6E8-VH WIDPENGDTEYAPKFQG 23 45H6E8-p1 VH WIDPEQGDTEYAPKFQG 96 4.2 Binding properties of PTM-removed CD16A humanized antibodies to cells expressing human CD16

[0158] The cell binding activity of the PTM-depleted humanized CD16A 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 above protocol.

[0159] like Figure 8A and Figure 8B As shown, all PTM-depleted antibodies 45H6E8-z19p1, 45H6E8-z20p1, and 45H6E8-z21p1 retained the same binding potency to human CD16A-158V and CD16A-158F expressed on cells as their parental antibodies 45H6E8-z19, 45H6E8-z20, and 45H6E8-z21. Figure 8C As shown, the cell binding efficacy of these PTM-depleted CD16A humanized antibodies to cynomolgus monkey CD16 was maintained. Consistent with the parental humanized antibodies, all of these PTM-depleted mAbs 45H6E8-z19p1, 45H6E8-z20p1, and 45H6E8-z21p1 showed minimal binding to human CD16B-NA1 expressed on cells ( Figure 8D ). Example 5. Binding ability of CD16A bispecific fragments

[0160] The development strategy for targeting CD16A is to achieve conditional activation of CD16A through the other arm of the binding fragment in bispecific or multispecific fragments. Bispecific or multispecific fragments contain one arm that binds hCD16A and another arm that binds one or more specific antigens. In this context, we set out to evaluate whether crosslinking of CD16A with tumor-associated antigen (TAA)-specific fragments could induce TAA-dependent, conditional activation of CD16A and the resulting functional activation of effector cells expressing CD16A (such as NK cells and macrophages). 5.1 Generation of humanized monoclonal antibodies against human GPC3

[0161] As previously mentioned, GPC3 is a specific TAA for HCC due to its high expression levels in tumor cells and limited expression in normal tissues. We have 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, immune responses were assessed by serum ELISA against GPC3-his protein and FACS analysis of serum against a GPC3-overexpressing CHO-K1 cell line, with the parental CHO-K1 cell line serving as a negative control. The resulting mice were used for fusions. Positive hybridoma clones were selected by ELISA and FACS analysis. After subcloning, hybridoma clone 52H5D3B8 was selected for optimization and humanization. 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. Sequences of the variable regions of humanized GPC3 antibodies (CDRs are underlined) 5.2 Construction of CD16A bispecific fragments

[0162] We began to explore the possibility of conditional activation of CD16A by cross-linking bispecific fragments to activate cells expressing CD16A. We explored the possibility of conditional activation of CD16A by cross-linking bispecific fragments. Figure 9) to generate bispecific fragments. More specifically, Format 1 was used to construct a bispecific fragment comprising two anti-human CD16A scFv fragments fused to the C-terminus of the ADCC- and ADCP-inactivated Fc fragment of the GPC3-hIgG1LALAPG antibody (SEQ ID NOs: 59 and 61 for the heavy and light chains, respectively) with a (G4S)4 linker (SEQ ID NO: 62). In the scFv of the anti-CD16A fragment, the VH and VL were conjugated to 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 carrying 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, respectively, and SEQ ID NO: 61 for the light chain, respectively). GPC3-hIgG1LALAPG mAb carrying the LALAPG mutation in the Fc region was used as a negative control (SEQ ID NOs: 59 and 61 for the heavy and light chains, respectively). 5.3 CD16A Bispecific Fragments Binding to Human GPC3 Expressed on Cells

[0163] To evaluate the binding activity of the GPC3-CD16A bispecific fragment to GPC3, a cell-based binding assay was performed using HepG2 cells (a human hepatocellular carcinoma cell line with high expression levels of GPC3), and the SK-HEP-1 cell line lacking GPC3 served as a negative control. Figure 10A As shown, all tested GPC3-CD16A bispecific fragments showed dose-dependent and specific binding activity to GPC3-positive HepG2 cells, while no detectable binding was observed to GPC3-negative SK-HEP-1 cells ( Figure 10B More importantly, these bispecific fragments bind GPC3 with potency nearly comparable to that of their parental GPC3 mAb ( Figure 10A ). 5.4 CD16A Bispecific Fragments Binding to Human CD16A Expressed on Cells

[0164] The binding ability of the 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 showed effective binding to both high-affinity hCD16A-158V and low-affinity hCD16A-158F expressed on cells ( Figure 10C and Figure 10D When compared to the parental CD16A mAb, a slightly reduced binding capacity of the bispecific fragment to CD16A was observed, which may be caused by a change in the Fab-scFv format or steric hindrance induced by the C-terminal conjugation of the CD16A-scFv fragment. In contrast, GPC3-hIgG1DE showed relatively modest binding capacity to hCD16A-158V expressed on cells, although improved binding was observed compared to GPC3-hIgG1, which bound poorly to hCD16A-158V ( Figure 10C Neither GPC3-hIgG1DE nor GPC3-hIgG1 showed binding to low-affinity CD16A-158F ( Figure 10D ). Therefore, it is speculated that the long-lasting and strong binding efficacy of CD16A antibodies to both CD16A-158V and CD16A-158F may facilitate the sustained activation of NK cells and macrophages compared to monoclonal antibodies with ADCC- and ADCP-enhanced engineered Fc regions. Table 9A. Sequences of GPC3 mAbs and GPC3-CD16A bispecific fragments Table 9B. Sequences of GPC3-CD16A humanized bispecific fragments Table 9C. Sequences of GPC3-CD16A humanized bispecific fragments after PTM removal Example 6. CD16A agonistic signaling mediated by CD16A bispecific fragments

[0165] In order to evaluate the conditional activation of CD16A by the CD16A bispecific fragment, a CD16A signaling reporter gene assay was established. In brief, first, full-length human CD16A-158V was introduced into a previously constructed Jurkat-NFAT luciferase reporter cell line, in which the expression of the luciferase gene was under the control of a NFAT transcription factor responsive promoter. The resulting cell line allows us to conveniently evaluate the activation of CD16A signaling by examining downstream NFAT signaling. The Jurkat-hCD16A-158V-NFAT reporter cell line was then co-cultured with three human hepatoma cell lines (HepG2, Huh-7, and PLC / PRF / 5 with high, medium, and low expression levels of GPC3, respectively). SK-HEP-1, in the absence of GPC3 expression, was used as a negative control to exclude GPC3-independent activation of CD16A. Once CD16A is activated by the binding on GPC3-expressing cells mediated by the GPC3 targeting fragment, the NFAT signaling pathway is stimulated. Luciferase-based chemiluminescence can be detected by ONE-Glo TM Detection was performed using the luciferase assay system (Promega, catalog number E6110) and the Envision multilabel microplate reader (PerkinElmer). Graphs and statistical analysis were generated using Graphpad Prism 9 software using four-parameter nonlinear regression curve fitting.

[0166] The results showed that all tested GPC3-CD16A bispecific fragments were Figure 11A )、Huh7( Figure 11B ) and PLC / PRF / 5( Figure 11C ) cell lines, but in the GPC3-negative SK-HEP-1 cell line ( Figure 11D ), indicating that even in the presence of moderate or low levels of GPC3, efficient and specific GPC3-mediated conditional activation of CD16A signaling by the GPC3-CD16A bispecific fragment can be achieved. Example 7. CD16A bispecific fragment activates NK cell function

[0167] To evaluate whether conditional activation of CD16A signaling by CD16A bispecific fragments could translate into NK cell activation and cytotoxicity and demonstrate functional superiority relative to Fc-engineered mAbs upon target cell engagement, 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 cell cytotoxicity against target cells

[0168] In order to evaluate whether GPC3-mediated CD16A signaling activation can induce NK cell cytotoxicity against target cells, a human primary NK cell-mediated cytotoxicity assay was established. Briefly, fresh human primary CD3-CD56+ NK cells were isolated from the buffy coat of healthy donors by negative selection with magnetic beads (Miltenyi, catalog number 130-092-657). The purity of the isolated NK cells monitored by FACS analysis was typically greater than 90%. The NK cells were then allowed to stand in complete medium at 37°C and 5% CO2 for 24 hours, or freshly prepared as effector cells. Three human hepatoma cell lines (HepG2, Huh-7, and PLC / PRF / 5 with high, medium, and low expression levels of GPC3, respectively) were used as target cells. SK-HEP-1 cells were used as negative controls to exclude nonspecific killing of NK cells. NK cells were incubated with 1.0×10 4 Target cells were co-cultured in 96-well microplates at an effector to target ratio of 10:1. The antibodies were serially diluted 4-fold starting from 3 nM and added to the corresponding wells. After incubation at 37°C for 4 hours, lactate dehydrogenase (LDH) released from the supernatant of damaged cells was measured by LDH cytotoxicity detection kit (Roche, catalog number 04744926001) and detected by Envision multi-label microplate reader (PerkinElmer). Calculation formula: % cytotoxicity = (LDH release of the mixture of effector cells and target cells - 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 analysis were generated using four-parameter nonlinear regression curve fitting in Graphpad Prism9 software.

[0169] All tested GPC3-CD16A bispecific fragments induced NK cell-mediated inhibition of GPC3+ target cells HepG2 ( Figure 12A ) and Huh-7( Figure 12B ) cells, while no cytotoxicity was observed against GPC3-negative SK-HEP-1 cells ( Figure 12C ). 7.2 Degranulation of NK cells stimulated by GPC3-CD16A bispecific fragment

[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, primary human NK cells generated as described above were incubated with 1.0 × 10 5 Target cells (including HepG2, Huh7, and SK-HEP-1) were co-incubated in a 96-well microplate at an E / T ratio of 1:1. Antibodies were serially diluted 3-fold starting from 10 nM and added to the corresponding wells. After incubation at 37°C for 4 hours, the mixed cells were harvested and washed to adjust the concentration to 1×10 in ice-cold staining buffer (2% BSA in DPBS). 6 The concentration of cells / mL was 400 μg / mL. After pretreatment with Fc blocker, the cells were stained with PE-conjugated human CD107a antibody (2 μL per test, Biolegend, catalog number 328608) at 4 ° C in the dark for 30 min. The samples were then fixed with 4% paraformaldehyde and analyzed by flow cytometry LSRFortessa cell analyzer (Bidec Biosciences). Data analysis was performed using Flowjo 10.0 software. Graphpad Prism 9 software was used to generate charts and statistical analysis using 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 shown by the inhibition of GPC3+ target cells HepG2 ( Figure 13A ) and Huh-7( Figure 13B ), while no degranulation of GPC3-negative SK-HEP-1 cells by NK cells was observed ( Figure 13C More importantly, the GPC3-CD16A bispecific fragment showed enhanced NK cell degranulation induction when compared to GPC3-IgG1DE or GPC3-hIgG1 mAb ( Figure 13A and Figure 13B ), indicating that CD16A bispecific fragments are superior to Fc-engineered mAbs in NK cell activation. 7.3GPC3-CD16A bispecific fragment-stimulated NK cell cytokine production

[0172] To investigate the ability of the bispecific fragment GPC3-CD16A to stimulate NK cells to produce cytokines in the presence of target cells, intercellular IFN-γ and TNF-α in NK cells were analyzed by FACS staining. Briefly, primary human NK cells generated as effector cells were co-cultured with 1.0 × 10 5Target cells (including HepG2, Huh7, and SK-HEP-1) (E / T ratio = 1:1) were co-cultured in 96-well microplates. The protein trafficking inhibitor Brefeldin A (BFA) was added to the co-culture system to prevent cytokine secretion into the supernatant during NK cell activation. The antibodies were serially diluted 3-fold starting from 10 nM and added to the corresponding wells. After incubation at 37°C for 4 hours, the mixed cells were washed and fixed with 4% paraformaldehyde, and then permeabilized with 1x permeabilization buffer (Invitrogen, catalog number 00-8333-56) at room temperature for 20 minutes. The cell pellet was resuspended in staining buffer containing diluted APC mouse anti-human IFN-γ antibody (Baijin Company, catalog number 506510) and BV421 mouse anti-human TNF-α antibody (Biobionics, catalog number 562783), and then incubated in the dark at 4°C for 30 minutes. Samples were washed, resuspended, and analyzed using a flow cytometer (LSFR Fortessa) (BD Biosciences). Data were analyzed using FlowJo 10.0 software. Graphs and statistical analysis were generated using Graphpad Prism 9 software using 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 shown by the inhibition of GPC3+ target cells HepG2 ( Figure 14A and Figure 14D ) and Huh-7( Figure 14B and Figure 14E ), as indicated by the upregulation of intercellular IFN-γ and TNF-α in GPC3-negative SK-HEP-1 cells, whereas no cytokine production by NK cells was observed on GPC3-negative SK-HEP-1 cells ( Figure 14C and Figure 14F More importantly, the GPC3-CD16A bispecific fragment showed enhanced induction of NK cell cytokine release when compared to GPC3-IgG1DE or GPC3-hIgG1 mAb ( Figures 14A-14D ), indicating that CD16A bispecific fragments are superior to Fc-engineered mAbs in NK cell activation. Example 8: In vitro properties of CCR8-CD16A bispecific antibodies

[0174] To evaluate whether cross-linking of CD16A by other binding fragments could also induce conditional activation of CD16A downstream signaling, the chemokine receptor CCR8, which is highly expressed on tumor-infiltrating Treg cells, was selected 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. Recently, CCR8 has been found to be expressed exclusively on a subset of highly suppressive tumor-infiltrating regulatory T cells (Tregs) found in a variety of cancers, including breast, colorectal, and lung cancers. Furthermore, a high abundance of CCR8+ Tregs is associated with a poor prognosis. Therefore, CCR8 is a promising therapeutic target for enhancing anti-tumor immunity.

[0176] The discovery of the CCR8 antibodies tested in this example is described below. BALB / c mice, C57BL / 6 mice, and SJL mice were immunized with full-length human CCR8 DNA and the CHO-K1 / HEK293hCCR8 cell line. After the conventional 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 their potent binding to human CCR8 (Table 10A). Table 10A. CCR8 antibody sequences (CDRs are underlined) 8.1 Construction of CCR8-CD16A Bispecific Antibody

[0177] Use form 1 ( Figure 9 ) constructed a CCR8-CD16A-hIgG1LALAPG bispecific antibody, which comprises two anti-human CD16AscFv fragments fused to the C-terminus of the ADCC-invalidated Fc fragment of the CCR8-hIgG1 antibody. To evaluate whether wild-type Fc would synergize with the CD16A-specific binding fragment to promote differentiated binding and functional activity, we also generated CCR8-CD16A-hIgG1 bispecific antibodies with wild-type Fc fragments. Compared to ADCC-enhanced monospecific antibodies (eADCC CCR8 mAbs), we generated anti-hCCR8 monoclonal antibodies with Fc-DLE (S239D / A330L / I332E), Fc-DE (S239D / I332E), Fc-afucosylation (in which the N-glycan residues in the IgG Fc region lack the core fucose sugar unit), or wild-type Fc hIgG1. All CCR8-CD16ABsAb and CCR8 mAb use the same anti-CCR8 Fab sequence. The sequences of CCR8-CD16ABsAb and CCR8 mAb are listed in Table 10B. Table 10B. Sequences of CCR8-CD16A BsAb and CCR8 mAb 8.2 Binding Ability of CCR8-CD16A Bispecific Antibody to CD16A

[0178] 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. The purpose of the assay was to evaluate the binding of the BsAb to each of the hFcγRs.

[0179] like Figure 15A and Figure 15B As shown, CCR8-45H6E8-hIgG1 bsAb strongly bound to both human CD16A-158V and human CD16A-158F in a dose-dependent manner. More importantly, when compared with various eADCC CCR8 mAbs, CCR8-45H6E8-hIgG1 BsAb and CCR8-45H6E8-HIgG1LALAPG BsAb exhibited excellent binding potency to both CD16A forms. Notably, when compared with CCR8-45H6E8-hIgG1LALAPG BsAb or CCR8-hIgG1 mAb, CCR8-45H6E8-hIgG1 BsAb displayed stronger binding ability to human CD16A. This phenomenon suggests that the wild-type Fc may synergize with the CD16A-specific binding fragment to generate enhanced binding to CD16A. Figure 15C The results indicate that CCR8-45H6E8 WT BsAb and CCR8-45H6E8 LALAPG BsAb have lower binding affinity for the inhibitory Fcγ receptor hCD32B when compared to various CCR8 mAbs. Unexpectedly, CCR8-45H6E8-hIgG1 BsAb exhibited significantly reduced binding ability to CD32B when compared to CCR8-hIgG1 mAb. 8.3 Cellular Function of NK Cells Stimulated by CCR8-CD16A Bispecific Antibody

[0180] To evaluate whether CCR8-CD16ABsAb can induce NK cell cytotoxicity 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 established as described in Example 7.

[0181] CCR8-CD16A bispecific antibodies (both 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-CD16ABsAbs exhibited enhanced in vitro cytotoxicity against target cells when compared with the ADCC-enhanced CCR8-hIgG1DE mAb ( Figure 16A ), NK cell degranulation ( Figure 16B ) and NK cell cytokine production ( Figure 16C and Figure 16D ), indicating that CD16A bispecific fragments are superior to Fc-engineered mAbs in NK cell activation.

[0182] like Figure 17 As shown, humanized CCR8-CD16A BsAbs including CCR8-45H6E8-z17-hIgG1, CCR8-45H6E8-z19-hIgG1, and CCR8-45H6E8-z20-hIgG1 showed sustained NK cell-mediated killing efficacy compared with the chimeric CCR8-45H6E8-hIgG1 BsAb. Example 9: In vitro efficacy of surrogate CCR8-CD16A BsAb

[0183] This example describes a side-by-side comparison of the in vitro effects of a CCR8-CD16A BsAb and an eADCC CCR8 mAb. 9.1 Generation of CCR8 Surrogate Antibodies

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

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

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

[0187] like Figure 18A and Figure 18B As shown, 194A1G9 specifically bound to human CCR8 and mCCR8 expressed on cells in a dose-dependent manner. 9.3 Construction of Surrogate CCR8-CD16A Bispecific Antibody

[0188] To understand the activity of CCR8-CD16A bispecific antibodies, we generated Figure 9 ) bispecific antibodies constructed with the anti-CD16A scFv fragment 42F5H2 scFv (SEQ ID NO: 68) conjugated to the C-terminus of the Fc of mCCR8-hIgG1 or mCCR8-hIgG1LALA (194A1G9) antibody with a (G4S)3 linker (SEQ ID NO: 63). The heavy and light chain sequences of 194A1G9-42F5H2-hIgG1 BsAb and 194A1G9-42F5H2-hIgG1LALA BsAb are listed in Table 12. For comparison, the 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 BsAbs and CCR8 mAbs 9.4 Activation of ADCC Signaling Mediated by CCR8-CD16A Bispecific Antibody

[0189] Previously, we observed in Example 8 that CCR8-CD16A bsAb binds to the enhanced cells of activation FcγR CD16A and binds to the reduced cells of inhibitory FcγR CD32B. In order to evaluate whether this biased binding spectrum helps to improve ADCC signal transduction, we performed in vitro assays to compare the ADCC signal transduction of these antibodies. In this assay, Jurkat cells stably co-express hCD16A-158V and hCD32B as effector cells and luciferase reporter genes (Jurkat-hCD16A-hCD32B-NFAT) driven by NFAT response elements. The CHO-K1 cells (CHO-K1-mCCR8) expressing mouse CCR8 were used as target cells. The protocol was as previously described in Example 6.

[0190] The results showed that both 194A1G9-42F5H2-hIgG1 and 194A1G9-42F5H2-hIgG1LALA BsAbs could mediate stronger ADCC signaling compared with 194A1G9-hIgG1DE in the same experimental setting ( Figure 19 Interestingly, 194A1G9-42F5H2-hIgG1 BsAb showed superior CD16A activation when compared to 194A1G9-42F5H2-hIgG1LALA BsAb, suggesting an additive effect from Fc-mediated CD16A signaling ( Figure 19 ). Example 10: Synergy between CD16A-bsAb and Fc-mediated effector function in vivo

[0191] To further confirm the functionality of CD16A BsAb, we used syngeneic CD16A humanized C57BL / 6 mice, in which the murine FcγRIV gene was replaced by human CD16A 158V, to test the in vivo antitumor efficacy of the molecule. MC38 cells resuspended in PBS were cultured at 5 × 10 5 The concentration of cells was subcutaneously (sc) administered into the right flank of mice. When the average tumor volume reached about 72 mm 3 At 4 hr, animals were randomly assigned to experimental groups of 7 animals per group based on tumor volume. Equimolar concentrations of surrogate anti-mCCR8 antibodies, including 194A1G9-hIgG1DE (6 mg / kg), 194A1G9-42F5H2-hIgG1BsAb (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] like Figure 20As shown, compared with the PBS control, 194A1G9-42F5H2-hIgG1LALA BsAb showed comparable tumor growth inhibition to the ADCC-enhanced 194A1G9-hIgG1DE mAb, with inhibition rates of 31.5% and 36%, respectively. These data indicate that 194A1G9-42F5H2-hIgG1LALA BsAb can effectively exhibit CCR8-dependent CD16A activation and mediate effector cell-mediated CCR8+ Treg cell depletion and tumor control. Strikingly, when compared with 194A1G9-42F5H2-hIgG1LALA BsAb and 194A1G9-hIgG1DE mAb, 194A1G9-42F5H2-hIgG1 BsAb showed significantly improved tumor growth inhibition, with an inhibition rate of 75.7% ( Figure 20 ), which indicates a significant contribution of synergy between CD16A antibody-mediated effector functions and Fc-mediated effector functions.

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

[0194] like Figure 21 As shown, SA214G2-45H6E8-hIgG1LALAPG BsAb can effectively inhibit tumor growth compared with PBS control even at a very low therapeutic dose (1.33 mg / kg), indicating that in the absence of Fc-mediated effector function, the effector cells are effectively activated and the tumor growth inhibition is mediated solely by CCR8-dependent CD16A activation. 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 administration ( Figure 21 ), which further confirmed the synergistic effect between the effector function mediated by CD16A antibody and the effector function mediated by Fc. ***

[0195] The scope of the present disclosure is not limited by the specific embodiments described, which are intended to serve as single illustrations of various aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations may be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to cover modifications and variations of the present disclosure as long as they are 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. An antibody or antigen-binding fragment thereof having binding specificity to human CD16A protein, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2 and VHCDR3, the light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3, wherein: The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the following amino acid sequences: (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 NO: 19, 20, 21, 34, 35 and 36; or (f) SEQ ID NO: 25, 26, 27, 40, 41 and 42.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH CDR1, VH CDR2, VH CDR3, VLCDR1, 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. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH CDR1, VH CDR2, VH CDR3, VLCDR1, 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. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH CDR1, VH CDR2, VH CDR3, VLCDR1, 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 of claim 5, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-87, 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, VLCDR1, 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 of 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, VLCDR1, 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, VLCDR1, 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. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH CDR1, VH CDR2, VH CDR3, VLCDR1, 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 according to any one of claims 1 to 14, further comprising an IgG Fc fragment.

16. The antibody or antigen-binding fragment thereof of claim 15, wherein the Fc fragment is unable to bind to Fcγ receptors or complement proteins.

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. A multispecific antibody, comprising: an anti-CD16A antibody or antigen-binding fragment that has 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. The multispecific antibody of claim 18 , wherein the anti-CD16A antibody or antigen-binding fragment and the Fc fragment have a synergistic effect in exerting effector function.

20. The multispecific antibody of claim 19, wherein the Fc fragment is capable of binding to CD64 but is unable to bind to CD16B and / or CD32B. The multispecific antibody of claim 20 , wherein the Fc fragment is a wild-type human IgG Fc fragment. 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. The multispecific antibody of claim 22 , wherein the Fc fragment is a wild-type human IgG1 Fc fragment.

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

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 at the N-terminus of the Fc fragment.

27. 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 opposite sides of the Fc fragment, wherein the anti-CD16A antibody or antigen-binding fragment thereof is preferably located at the C-terminus of the Fc fragment.

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

55.

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

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

31. The multispecific antibody of any one of claims 18-30, 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. The multispecific antibody of claim 31, wherein the TAA is CCR8, CD25, or GPC3.

33. 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), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

57.

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

118.

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. 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), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

78.

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

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

39. A composition comprising the antibody of any one of claims 1-36, the one or more polynucleotides of claim 37, or the cell of claim 38.

40. 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 1-36.

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

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

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

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

46. ​​The method or use of any one of claims 40-45, 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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