CD16A antibody and its applications

Abstract

The present invention relates to single-domain antibodies that specifically bind to CD16A, heavy-chain antibodies containing the single-domain antibody, bispecific antibodies containing the single-domain antibody, immune conjugates containing the single-domain antibody, and pharmaceutical compositions containing the same, and their use in the treatment, prevention, or diagnosis of cancer, acute or chronic infections, or age-related diseases.

Description

[Technical Field]

[0001] This disclosure relates to single-domain antibodies, heavy-chain antibodies, bispecific antibodies, immunoconjugates, pharmaceutical compositions, and uses thereof against CD16A, which are used for the treatment, prevention, or diagnosis of cancer, acute or chronic infections, or age-related diseases. [Background technology]

[0002] Natural killer (NK) cells express the cell adhesion marker CD56 and lack the T cell receptor CD3 (CD56 + CD3 - NK cells are defined by the following: Based on the cell surface density of CD56, NK cells are classified into two functionally distinct subsets, namely CD56bright and CD56dim. CD56bright NK cells, which make up about 10% of circulating NK cells, are generally thought to have high proliferative capacity, high cytokine production capacity after stimulation with IL-12 and IL-18, and low cytotoxic effector activity at rest. In contrast, CD56dim NK cells exhibit potent cytotoxic activity even without stimulation and produce cytokines after target cell stimulation via antibody-dependent cell-mediated cytotoxicity (ADCC).

[0003] CD16(FCγRIII) binds to the Fc portion of IgG antibodies. One type, CD16A, is a transmembrane protein that co-localizes with CD3ζ and Fc-εRI-γ on NK cells. Ligand binding leads to potent signaling that induces ADCC-mediated cytokine production and cytotoxic effector activity. Another type, CD16B, is present in neutrophils and is not involved in tumor cell killing. Although their extracellular domains have high homology, CD16B is distinguished from CD16A by glycosylphosphatidylinositol binding. The majority of CD56bright NK cells in peripheral blood express little to no CD16A. In contrast, the majority of CD56dim cells express CD16A at high and uniform levels. CD16A expression decreases after mitogen stimulation or co-culture with malignant targets, and this effect is presumed to be mediated by metalloproteases. This process may be important for the rapid regulation of CD16A surface density and the resulting regulation of NK cell activation state and effector function.

[0004] The ADCC function of NK cells is attracting considerable attention in antibody-mediated immunotherapy. Bispecific antibodies, which have two targeting sites—one for recruiting the ADCC receptor CD16A and another for recognizing the antigen—are gaining widespread interest in both in vitro and in vivo settings due to their high efficacy in involving NK cells in target cell killing.

[0005] Currently, several formats of these bispecific antibodies are in the stages of basic research and clinical research, including Bispecific Killer Cell Engager (BiKE), bispecific diabody (BidAb), and tetravalent bispecific tandem diabody (TandAb). In recent years, the more promising format, TandAb, has been developed by Affimed Therapeutics AG (Heidelberg, Germany). TandAb is a tetravalent bispecific tandem diabody composed of a tail-to-head type homodimer in which two tandem scFvs are linked by three (GGS)3 linkers. One of the TandAbs, CD16 / CD30 AFM13, has completed a phase II clinical trial (clinical trial number NCT02321592) for patients with relapsed or refractory Hodgkin lymphoma. However, since TandAb contains more subdomains than BidAb, highly controlled structure formation is required for all of them to pair correctly, and there may be more production challenges. Furthermore, since TandAb has a tetravalent structure (bivalent for CD16A and bivalent for CD30), there is a risk of non-specifically activating CD16A and inducing side effects under conditions where CD30 is absent.

Summary of the Invention

Problems to be Solved by the Invention

[0006] The present invention aims to solve the problems of the background art.

Means for Solving the Problems

[0007] In one aspect, a single-domain antibody that specifically binds to CD16A is provided. The single-domain antibody contains an amino acid sequence shown in SEQ ID NO:1 having substitutions at one or any combination of positions selected from S54, G55, and S56 according to the Kabat numbering method.

[0008] In one embodiment, the substitution is selected from the group consisting of (i) S54N, S54D or S54T, (ii) G55V, (iii) S56Q, S56T, S56D or S56E, and any combination thereof.

[0009] In one embodiment, the substitution is selected from the group consisting of (i) S54N, (ii) S54D, (iii) S54T, (iv) G55V, (v) S56Q, (vi) S56T, (vii) S56D, (viii) S56E, (ix) S54N and S56Q, and (x) S54T and S56Q. In one aspect, a single-domain antibody that specifically binds to CD16A is provided. The single-domain antibody comprises the following components.

[0010] (a) According to the IMGT definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 41 or a conservative variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 42 or a conservative variant thereof, and CDR2 selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 and their respective conservative variants.

[0011] (b) According to the Kabat definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 53 or a conservative variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 54 or a conservative variant thereof, and CDR2 selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64 and their respective conservative variants.

[0012] (c) CDR1 having the amino acid sequence shown in SEQ ID NO:65 or a conservative variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:66 or a conservative variant thereof, and CDR2 selected from the group consisting of the amino acid sequences shown in SEQ ID NO:67, 68, 69, 70, 71, 72, 73, 74, 75 or 76 and their respective conservative variants, in accordance with the Chothia definition scheme.

[0013] (d) CDR1 having the amino acid sequence shown in SEQ ID NO:77 or a conservative variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:78 or a conservative variant thereof, and CDR2 selected from the group consisting of the amino acid sequences shown in SEQ ID NO:79, 80, 81, 82, 83, 84, 85, 86, 87 or 88 and their respective conservative variants, in accordance with the Contact definition scheme. However, the single-domain antibody shall not have the amino acid sequence shown in SEQ ID NO:1.

[0014] In one embodiment, the single-domain antibody includes an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, or 40, or amino acid sequences having at least 80% identity with each of these amino acid sequences. In one embodiment, the single-domain antibody is a humanized antibody, a human antibody, a chimeric antibody, or a camelized antibody. In other embodiments, the disclosure provides a single-domain antibody and a heavy-chain antibody comprising an Fc portion linked to the single-domain antibody. In other embodiments, the disclosure provides bispecific antibodies, including single-domain antibodies.

[0015] In one embodiment, the bispecific antibody comprises a single-domain antibody that specifically binds to CD16A and a second binding domain that specifically binds to a second target selected from tumor-associated antigens or tumor-specific antigens.

[0016] In one embodiment, the second target is HER2, 5T4, PSMA, BCMA, FGFR, CD20, CD33, CD19, CD22, CD123, CD30, GPC-3, CEA, EGFR1, EGFR2, EGFR3, TGF-β, ROR1, PD-L1, Claudin18.2, EpCAM, GD2, MSLN, EGFR, MUC1, MUC2, EGFRVIII, CD38, Trop-2, c-ME The following are selected from the group consisting of T, Nectin-4, CD79b, CCK4, GPA33, HLA-A2, CLEC12A, p-cadherin, TDO2, MART-1, Pmel17, MAGE-1, AFP, CA125, TRP-1, TRP-2, NY-ESO, PSA, CDK4, BCA225, CA125, MG7-Ag, NY-CO-1, RCAS1, SDCCAG16, TAAL6, and TAG72.

[0017] In one embodiment, the single-domain antibody is linked to a full-length IgG antibody, and may be linked via a peptide linker if necessary. In one embodiment, the peptide linker consists of a glycine residue and a serine residue. In one embodiment, the single-domain antibody is linked to the C-terminus of the heavy chain, the C-terminus of the light chain, or the N-terminus of the heavy chain of the full-length IgG antibody. In one embodiment, the full-length IgG antibody is an anti-HER2 antibody or an anti-5T4 antibody. In one embodiment, the anti-HER2 antibody includes the following components. (a) A heavy chain having the amino acid sequence shown in SEQ ID NO:12 and a light chain having the amino acid sequence shown in SEQ ID NO:13. (b) A heavy chain having the amino acid sequence shown in SEQ ID NO:37 and a light chain having the amino acid sequence shown in SEQ ID NO:38. (c) A heavy chain having the amino acid sequence shown in SEQ ID NO:25 and a light chain having the amino acid sequence shown in SEQ ID NO:13. (d) A heavy chain having the amino acid sequence shown in SEQ ID NO:26 and a light chain having the amino acid sequence shown in SEQ ID NO:13. In one embodiment, the bispecific antibody comprises the following components. (a) A heavy chain having the amino acid sequence shown in SEQ ID NO:14 and a light chain having the amino acid sequence shown in SEQ ID NO:13. (b) A heavy chain having the amino acid sequence shown in SEQ ID NO:15 and a light chain having the amino acid sequence shown in SEQ ID NO:13. (c) A heavy chain having the amino acid sequence shown in SEQ ID NO:17 and a light chain having the amino acid sequence shown in SEQ ID NO:13. (d) A heavy chain having the amino acid sequence shown in SEQ ID NO:12 and a light chain having the amino acid sequence shown in SEQ ID NO:16. (e) A heavy chain having the amino acid sequence shown in SEQ ID NO:18 and a light chain having the amino acid sequence shown in SEQ ID NO:13. (f) A heavy chain having the amino acid sequence shown in SEQ ID NO:19 and a light chain having the amino acid sequence shown in SEQ ID NO:13. (g) A heavy chain having the amino acid sequence shown in SEQ ID NO:20 and a light chain having the amino acid sequence shown in SEQ ID NO:13. (h) A heavy chain having the amino acid sequence shown in SEQ ID NO:21 and a light chain having the amino acid sequence shown in SEQ ID NO:13. (i) A heavy chain having the amino acid sequence shown in SEQ ID NO:22 and a light chain having the amino acid sequence shown in SEQ ID NO:13. (j) A heavy chain having the amino acid sequence shown in SEQ ID NO:23 and a light chain having the amino acid sequence shown in SEQ ID NO:13. (k) A heavy chain having the amino acid sequence shown in SEQ ID NO:24 and a light chain having the amino acid sequence shown in SEQ ID NO:13. (l) A heavy chain having the amino acid sequence shown in SEQ ID NO:27 and a light chain having the amino acid sequence shown in SEQ ID NO:13. (m) A heavy chain having the amino acid sequence shown in SEQ ID NO:28 and a light chain having the amino acid sequence shown in SEQ ID NO:13.

[0018] In one embodiment, the anti-5T4 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:29 and a light chain having the amino acid sequence shown in SEQ ID NO:30.

[0019] In one embodiment, the bispecific antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:31 and a light chain having the amino acid sequence shown in SEQ ID NO:30.

[0020] In other embodiments, the disclosure provides an immunoconjugate comprising one of a single-domain antibody, a heavy-chain antibody, or a bispecific antibody, further comprising an active group, a pharmaceutically acceptable salt thereof, or a solvate thereof. In one embodiment, the active group is selected from the group consisting of toxins, peptide tags, sotags, radionuclides, near-infrared fluorescent dyes, and nanoparticles.

[0021] In other embodiments, the Disclosure provides a pharmaceutical composition comprising one of a single-domain antibody, a heavy-chain antibody, a bispecific antibody, or an immunoconjugate, and a pharmaceutically acceptable carrier.

[0022] In yet another embodiment, the disclosure provides a nucleic acid molecule encoding one of a single-domain antibody, a heavy-chain antibody, a bispecific antibody, or an immunoconjugate, an expression vector containing the nucleic acid molecule, and a non-human host cell containing the expression vector.

[0023] In another embodiment, the disclosure provides for the use of any one of a single-domain antibody, a heavy-chain antibody, a bispecific antibody, an immunoconjugate, or a composition for the manufacture of pharmaceuticals for the treatment of cancer.

[0024] In further embodiments, the disclosure provides for the use of any one of a single-domain antibody, a heavy-chain antibody, a bispecific antibody, an immunoconjugate, or a composition for the treatment of cancer, acute or chronic infection, or age-related diseases.

[0025] In yet another embodiment, the Disclosure provides a method for treating cancer, an acute or chronic infection or an age-related disease, comprising administering a therapeutically effective amount of one of a single-domain antibody, a heavy-chain antibody, a bispecific antibody, an immunoconjugate, or a composition to a subject in need of treatment.

[0026] In yet another embodiment, the disclosure provides a polypeptide comprising a single-domain antibody or a heavy-chain antibody and one or more amino acid residues covalently linked to the N-terminus, C-terminus, or any position between them of the single-domain antibody or the heavy-chain antibody. These and other aspects of the present disclosure, as well as their advantages, will become apparent from the detailed description of the present invention that follows. [Brief explanation of the drawing]

[0027] [Figure 1] The binding of selected VH to CD16A 158V and CD16A 158F is shown by ELISA. BM156-01 was used as a control. [Figure 2] The results of measuring the binding affinity of selected VH to CD16A 158V and CD16A 158F using the BLI method are shown. [Figure 3]Different bispecific antibody formats are schematically shown. (A) Preferred anti-CD16 VH, (B) Trastuzumab IgG1 (SEQ ID NO: 12 and SEQ ID NO: 13), (C) Homodimerated construct with CD16 VH fused to the C-terminus of the heavy chain (SEQ ID NO: 14 and SEQ ID NO: 13), (D) Construct with VH linked to the C-terminus of the heavy chain via a G4S linker (SEQ ID NO: 15 and SEQ ID NO: 13), (E) Homodimerated construct with CD16 VH fused to the C-terminus of the light chain (SEQ ID NO: 12 and SEQ ID NO: 16), (F) Homodimerated construct with CD16 VH fused to the N-terminus of the heavy chain via a (G4S)3 linker (SEQ ID NO: 17 and SEQ ID NO: 13). [Figure 4] The ELISA binding of the bispecific antibodies to human CD16A 158F (left figure) and CD16A 158V (right figure) is shown. [Figure 5] The results of measuring the binding of different bispecific antibodies to CD16A 158V and CD16A 158F using the BLI method are shown. [Figure 6] The double binding of BMP01-16 to HER2 and CD16A is shown by ELISA. [Figure 7] This shows ELISA binding of BMP01-16 to human CD16A-158V and CD16B. BM130-92 (margituximab) and BM130-93 (trastuzumab) were used as controls. [Figure 8-1] The binding of different bispecific antibodies to the HER2 receptor is demonstrated on the surface of SK-BR-3, JIMT-1, and MDA-MB-231 cells. [Figure 8-2] The binding of different bispecific antibodies to the HER2 receptor is demonstrated on the surface of SK-BR-3, JIMT-1, and MDA-MB-231 cells. [Figure 8-3] The binding of different bispecific antibodies to the HER2 receptor is demonstrated on the surface of SK-BR-3, JIMT-1, and MDA-MB-231 cells. [Figure 9]This study demonstrates that BMP01 exhibits higher ADCC activity than trastuzumab in an ADCC reporter assay. [Figure 10] This study demonstrates that LALA and N297A mutations do not cause loss of ADCC activity in BMP01-12 in ADCC reporter assays. [Figure 11] This study demonstrates that BMP02-10 exhibits higher ADCC activity than m603 in an ADCC reporter assay. [Figure 12] This study demonstrates that a bispecific antibody against BMP01 enhances ADCC-induced cytotoxicity compared to trastuzumab. [Figure 13] This study demonstrates that the BMP02-10 bispecific antibody enhances ADCC-induced cytotoxicity compared to m603. [Figure 14] This study demonstrates that a bispecific antibody against BMP01 enhances IFN-γ production compared to trastuzumab. [Figure 15] This study demonstrates that BMP01-16 exhibits stronger antitumor activity than BM130-93 under low-dose conditions in a JIMT-1 / PBMC co-cultured tumor model. The top panel shows the average tumor growth across all groups, while the bottom panel shows individual mouse data for PBMC vs. PBMC + BM130-93 and PBMC vs. PBMC + BMP01-16. [Figure 16] This study demonstrates that BMP01-16 suppresses the growth of HCC1954 tumors in the huHSC-NCG-hIL15 model. The top panel shows the average tumor growth across all groups, while the bottom panel shows individual mouse data for Ctrl vs. BM130-93 and Ctrl vs. BMP01-16. [Modes for carrying out the invention]

[0028] [Definition] Unless otherwise specified in this specification, the following terms and phrases shall have the meanings set forth below.

[0029] As used herein, the term "complete IgG antibody" generally refers to a tetramer in which at least two heavy chains (H chains) and two light chains (L chains) are interconnected by disulfide bonds. Each heavy chain (HC) consists of a variable heavy chain region (abbreviated herein as VH) and a constant heavy chain region, the constant heavy chain region consisting of three domains: CH1, CH2, and CH3. Each light chain (LC) consists of a variable light chain region (abbreviated herein as VL) and a constant light chain region, the constant light chain region consisting of one CL domain. The VH and VL regions are further divided into highly variable regions (complementarity-determining regions, referred to as CDRs) and more conserved regions (framework regions, referred to as FRs). Each VH and VL consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen, while the constant region of the antibody mediates binding to host tissues or host factors (e.g., various cells of the immune system, such as effector cells, and the first component C1q of the classical complement system).

[0030] The term "full-length IgG antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, and chimeric antibodies. The antibody may be any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0031] A "heavy-chain antibody" refers to an antibody lacking a light chain, and in the case of cameroid-derived heavy-chain antibodies, the CH1 domain is also deleted. As a result, cameroid heavy-chain antibodies bind to the corresponding antigen via the variable heavy-chain domain (VHH) of the heavy-chain antibody. In this specification, the terms VHH, VH, nanobody, and single-domain antibody are used synonymously. Therefore, anti-CD16A VH means anti-CD16A VHH, anti-CD16A nanobody, or anti-CD16A single-domain antibody. Throughout this specification, CD16A may be referred to as CD16. VHH antibodies are the isolated VHH domains of heavy-chain-only antibodies produced by cameroid species such as alpacas, llamas, and camels. These proteins consist of a framework region and three highly variable loops (CDR1, CDR2, and CDR3). Studies have shown that the CDR3 loop enables broad binding specificity for VHH antibodies. The CDR3 loop is 3-4 residues longer than conventional antibodies, allowing it to protrude and bind to "hidden" epitope cavities. Furthermore, the CDR3 loop exhibits greater amino acid sequence diversity in VHH antibodies than conventional antibodies, with approximately 7% higher sequence diversity per residue.

[0032] The term "Fc portion" refers to a polypeptide that includes the C-terminal portion of an immunoglobulin H chain and retains at least one function of the IgG Fc region, particularly the function of binding to FcRn. The effector function of the antibody is determined by the sequence of the Fc region. The Fc portion can contain a CH2 domain, a CH3 domain, or a CH2-CH3 polypeptide chain. A CH2-CH3 polypeptide chain dimerizes with another CH2-CH3 polypeptide chain by covalent linkage at a hinge region located on the NC-terminal side of the CH2 domain. Therefore, in some embodiments, the Fc portion contains a dimer consisting of two CH2-CH3 polypeptide chains and a hinge region. Preferably, the Fc portion contains a constant domain of the Ig class (e.g., IgA, IgD, IgE, IgM, preferably IgG1, IgG2, IgG3, or IgG4, particularly IgG1). An example of an Fc portion is one having the amino acid sequence shown in SEQ ID NO:89.

[0033] The "hinge" domain may originate from the same or a different IgG class as the Fc portion, or it may be an artificially designed non-natural hinge domain. An example of an IgG hinge region is the one with the amino acid sequence shown in SEQ ID NO:90. This also includes variants of wild-type hinge regions, such as truncated forms.

[0034] A "linker" refers to an amino acid sequence containing a linker peptide that connects two adjacent variable domains, linking the C-terminus of one domain to the N-terminus of the other domain (or vice versa) to form an antigen-binding site. Regarding amino acid composition, a peptide linker sequence is selected that does not hinder Fv (i.e., VH / VL) formation, antigen-binding / recognition site formation, and multimerization (e.g., dimerization) of multispecific antigen-binding proteins. For example, linkers containing glycine and serine residues generally exhibit protease resistance. In some embodiments, (G2S) X Linker peptides are used, for example, x=1~20, i.e., (G2S), (G2S)2, (G2S)3, (G2S)4, (G2S)5, (G2S)6, (G2S)7 or (G2S)8, etc. Also, (G3S) X Linker (x=1~15) and (G4S) X Linkers (x=1-10, preferably 1-6) may also be used. The amino acid sequence of the linker may be optimized, for example, by phage display, to improve antigen-binding site formation and polypeptide production yield.

[0035] As used herein, the terms “target” or “target antigen” refer to an antigen expressed by or associated with cells that NK cells should direct to induce or elicit cytotoxic activity, i.e., target cells, or virus-infected cells. Examples of target antigens include tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs or TAAs may be expressed on the surface of target cells or may exist as MHC-restricted peptides presented by MHC complexes. Examples of tumor antigens include HER2, 5T4, PSMA, BCMA, FGFR, CD20, CD33, CD19, CD22, CD123, CD30, GPC-3, CEA, EGFR1, EGFR2, EGFR3, TGF-β, ROR1, PD-L1, Claudin18.2, EpCAM, GD2, MSLN, EGFR, MUC1, MUC2, EGFRVIII, CD38, Trop-2, c-MET, and Nectin. Examples include, but are not limited to, -4, CD79b, CCK4, GPA33, HLA-A2, CLEC12A, p-cadherin, TDO2, MART-1, Pmel17, MAGE-1, AFP, CA125, TRP-1, TRP-2, NY-ESO, PSA, CDK4, BCA225, CA125, MG7-Ag, NY-CO-1, RCAS1, SDCCAG16, TAAL6, and TAG72.

[0036] The term "binding domain" as used in connection with the present invention characterizes a domain that specifically binds to, interacts with, or recognizes a target site on a specific target epitope or target molecule (antigen), namely CD16 and the target cell surface antigen, respectively. The structure and function of the first binding domain (e.g., CD16 recognition), and preferably the structure and / or function of the second binding domain (target cell surface antigen recognition), are preferably based on the structure and / or function of an antibody, e.g., a full-length or full-length immunoglobulin molecule, or derived from the variable heavy chain (VH) and / or variable light chain (VL) domains or fragments thereof of the antibody. The first binding domain is preferably characterized by having three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region). The second binding domain also preferably possesses the minimum structural requirements of the antibody that enable target binding.

[0037] In this invention, the terms "specifically binds to," "specifically recognizes," "is specifically directed to," or "specifically reacts with" mean that the binding domain interacts with, or specifically interacts with, a specific target site on a particular epitope or target molecule (antigen), such as CD16A and a target cell surface antigen (e.g., HER2).

[0038] As used in the present invention, the terms "does not essentially / substantially bind" or "is not capable of binding" mean that the binding domain of the present invention does not bind to any protein or antigen other than CD16A and / or the target cell surface antigen; that is, it exhibits reactivity to any protein or antigen other than CD16A and / or the target cell surface antigen of no more than about 30%, preferably no more than about 20%, more preferably no more than about 10%, and particularly preferably no more than about 9%, 8%, 7%, 6%, or 5%, and is evaluated on a scale in which binding to CD16A and / or the target cell surface antigen is set to 100%.

[0039] As used herein, the term "complementarity determining domains" is used synonymously with "complementarity determining regions (CDRs)" and generally refers to the highly variable regions of the VL and VH. CDRs are the target protein binding sites of the antibody chain and are responsible for the specificity to that target protein. Human VL and VH each contain three CDRs (CDR1-3, numbered sequentially from the N-terminus), which constitute approximately 15-20% of the entire variable domain. CDRs are named by region and order; for example, "VH CDR1" or "HCDR1" both refer to the first CDR of the heavy chain variable region. CDRs are structurally complementary to the epitope of the target protein and are therefore directly involved in binding specificity. The residual portion of VL or VH, i.e., the framework region, has few amino acid sequence mutations (Kuby, Immunology, 4th ed., Chapter 4, WH Freeman & Co., New York, 2000), and the CDR of an antibody can be defined by various methods, including Kabat's definition method based on sequence mutations (Kabat et al., Protein Sequence in Immunology, 5th edition, NIH, Bethesda, 1991), Chothia's definition method based on the structural loop position (A1-Lazikani et al., J.Mol.Biol.273:927-948, 1997), and the IMGT definition method based on the concept and scientific rules of IMGT-ONTOLOGY. In some embodiments of this application, the IMGT rules are used to define the CDR of an antibody. Furthermore, the definition methods of Martin and PyIgClassify, as well as the combined definition method of Kabat, Chothia, IMGT, Martin, and PyIgClassify, are also included in this application (Mark L. Chiu et al., Antibodies 8(4), 55, 2019). Table A shows a comparison of CDR numbering between different definition schemes (based on Chothia numbering). [Table 1]

[0040] Here, Laa-Lbb or Haa-Hbb may refer to the amino acid sequence from amino acid number NO.aa to NO.bb of the light chain or heavy chain, respectively, when viewed from the N-terminus. For example, L24-L34 refers to the sequence from amino acid number 24 to 34 of the light chain.

[0041] Both the light and heavy chains are divided into regions based on structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it is understood that the variable regions of the light chain (VL) and heavy chain (VH) determine antigen recognition and specificity. In contrast, the constant regions of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, placental transfer, Fc receptor binding, and complement binding. By convention, the numbering of constant region domains increases as they move away from the antigen-binding site or the N-terminus of the antibody. The N-terminal side is the variable region, and the C-terminal side is the constant region. The CH3 and CL domains constitute the carboxyl-terminal domains of the heavy and light chains, respectively.

[0042] As used herein, the term "humanized antibody" generally refers to an antibody that contains heavy chain and light chain variable region sequences derived from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences has been modified to resemble the variable sequences of a human germline. For example, "humanized antibody" means an antibody or its variant, derivative, analogue, or fragment that can immunospecifically bind to a relevant antigen and contains a framework region (FR) substantially containing the amino acid sequence of a human antibody and a complementation-determining region (CDR) substantially containing the amino acid sequence of a non-human antibody. "Substantially" in the CDR means that the amino acid sequence of the CDR has at least 80%, e.g., at least 85%, 90%, 95%, 98%, or 99% identity with the CDR sequence of a non-human antibody. Humanized antibodies substantially contain at least one, usually two, variable domains (Fab, Fab', F(ab')2, Fab, Fv), where all or nearly all CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or nearly all framework regions consist of a frame region having the consensus sequence of a human immunoglobulin. In some embodiments, the humanized antibody may further contain at least a portion of the constant region of an immunoglobulin (Fc), typically the constant region of a human immunoglobulin.

[0043] As used herein, the term "human antibody" generally refers to an antibody having a variable region and a constant region based on an immunoglobulin sequence derived from human germline cells. Human antibodies are widely known in the prior art (e.g., van Dijk, MA and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374), and can also be obtained by transgenic animals (e.g., mice) that can produce a complete or selective human antibody assembly under conditions that do not produce endogenous immunoglobulins after immunization (Jakobovits, A. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A. et al., Nature 362 (1993) 255-258; Brueggemann, M. et al., Year Immunol. 7 (1993) 33-40). Furthermore, human antibodies can also be produced using phage display libraries (Hoogenboom, HR and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD et al., J. Mol. Biol. 222 (1991) 581-597). The term "human antibody" includes antibodies with modified constant regions.

[0044] As used herein, the term "chimeric antibody" in its broadest sense refers to an antibody designed to contain one or more regions derived from one antibody and one or more regions derived from one or more other antibodies. In particular, a chimeric antibody contains the VH and VL domains of an antibody derived from a non-human animal in combination with the CH and CL domains of another antibody, especially a human antibody. Non-human animals that can be used include camels, mice, rats, hamsters, rabbits, and others.

[0045] As used herein, the term "bispecific antibody" refers to an antibody that has two types of specificity for one or more antigens, or for different epitopes of the same antigen. For example, a bispecific antibody may have a first specificity for a first antigen (e.g., CD16A) and a second specificity for a second antigen different from the first antigen (e.g., a tumor-associated antigen or tumor-specific antigen). Alternatively, a bispecific antibody may have specificity for a first epitope of one antigen and specificity for a different second epitope of the same antigen (e.g., CD16A).

[0046] As used herein, the terms “purified” and “isolated” refer to polypeptides or nucleic acid sequences, meaning that the molecule is substantially free of other biomacromolecules of the same kind. “Purified” specifically means that the molecule contains at least 75%, 85%, 95%, or 98% by mass of biomacromolecules of the same kind. “Isolated” means that, when an isolated nucleic acid molecule codes for a particular polypeptide, the nucleic acid molecule is substantially free of other nucleic acid molecules that do not code for the target polypeptide, although it may contain additional bases or groups as long as the essential properties of the composition are not impaired. The present invention may include, for example, isolated antigen-binding proteins, isolated antibodies or their antigen-binding fragments, isolated polypeptides, isolated nucleic acid molecules, and the like.

[0047] An "isolated" nucleic acid molecule encoding a specific polypeptide means a nucleic acid molecule that substantially does not contain other nucleic acid molecules that do not encode the target polypeptide, provided that it does not contain additional bases or groups that do not impair the basic properties of its composition. The present invention may include, for example, isolated antigen-binding proteins, isolated antibodies or their antigen-binding fragments, isolated polypeptides, isolated nucleic acid molecules, and the like.

[0048] As used herein, the term "affinity" generally refers to the equilibrium binding between the antibody as a whole and the antigen. Affinity is, for example, the half-maximal effective concentration (EC2).50 Affinity can be expressed by the binding and dissociation rates measured by surface plasmon resonance (SPL) or by the equilibrium dissociation constant (KD). Affinity can be expressed by measuring the binding and dissociation rates by surface plasmon resonance (SPL) or by immunochemical assays (ELISA, FACS) in EC2. 50 It can be experimentally evaluated using various known methods, such as measurement.

[0049] The term “conservatively modified variant” applies to both amino acid sequences and nucleic acid sequences. For a given nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that codes for the same or substantially identical amino acid sequence, or, if it does not code for an amino acid sequence, a substantially identical nucleic acid sequence. Due to the degeneracy of the genetic code, there are numerous functionally identical nucleic acids that code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for alanine. Therefore, at any position where alanine is specified, it can be substituted for any of these codons without altering the coded polypeptide. Such nucleic acid variations are called “silent variations” and are a type of conservative variation. In this specification, all nucleic acid sequences that code for polypeptides also encompass all silent variations of those nucleic acids. Those skilled in the art will understand that each codon in a nucleic acid (except typically AUG, which codes for methionine, and TGG, which codes for tryptophan) can be modified to produce a functionally identical molecule. Therefore, each silent variation of a polypeptide-coding nucleic acid is inherently present in each described sequence.

[0050] Regarding polypeptide sequences, a "conservatively modified variant" refers to any individual substitution, deletion, or addition within a polypeptide sequence that consists of substitutions to chemically similar amino acids. Conservative substitution tables showing functionally similar amino acids are well-known in the field. These conservatively modified variants do not exclude polymorphic variants, interspecific homologs, and alleles. The following eight groups of amino acids are conservatively substituted for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) Phenylalanine (F), tyrosine (Y), tryptophan (W); 7) Serine (S), threonine (T); 8) Cysteine ​​(C), Methionine (M). (See Creighton, Proteins (1984)).

[0051] In some embodiments, "conservative sequence modifications" refer to amino acid modifications that do not substantially affect the binding properties of the amino acid sequence constituting the antibody.

[0052] As used herein, the terms “percent identical” or “percent identity” refer to the proportion of mutually identical subsequences or sequences between two or more nucleic acid or polypeptide sequences. Two sequences are considered “identical” if they have identical amino acid or nucleotide sequences in a comparison region. Two sequences are considered “substantially identical” if they have identical amino acid residues or nucleotides in a certain proportion (i.e., 60%, or arbitrarily 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%). This comparison is evaluated by one of the known sequence comparison algorithms or by manual alignment and visual inspection in a comparison window or specific region aligned to obtain maximum agreement. Optionally, identity is observed in a region of at least approximately 30 nucleotides (or 10 amino acids), more preferably 100 to 500 or 1000 nucleotides (or 20, 50, or 200 or more amino acids). Examples of suitable algorithms for determining the percentages of sequence identity and sequence similarity include BLAST and BLAST2.0, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1997) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively.

[0053] In addition to the sequence identity rate mentioned above, another indicator that two nucleic acid sequences or polypeptides are substantially identical is when the polypeptide encoded by the first nucleic acid immunologically cross-reacts with an antibody produced against the polypeptide encoded by the second nucleic acid. Therefore, for example, if two polypeptides differ only by conservative substitutions, they are usually considered substantially identical. Furthermore, if two nucleic acid molecules or their complementary chains hybridize with each other under strict conditions, it also indicates substantially identical status. Moreover, if both sequences can be amplified using the same primers, this is also an indicator of substantial identity.

[0054] As used herein, the term "nucleic acid molecule" is used synonymously with "polynucleotide" and generally refers to deoxyribonucleotides or ribonucleotides, or polymers thereof, in single-stranded or double-stranded form. This term encompasses nucleic acids containing known nucleotide analogs, modified skeletal residues, or linkers, which may be synthetic, natural, or unnatural, possessing similar binding properties to a reference nucleic acid and exhibiting similar metabolic modes. Examples of these analogs include thioates, amidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0055] As used herein, the term "polypeptide" is used synonymously with "protein" and refers to a polymer of amino acid residues. This term applies to amino acid polymers, both natural and unnatural, where one or more amino acid residues are artificial chemical mimetics of corresponding natural amino acids. Unless otherwise specified, a given polypeptide sequence also includes conservatively modified variants.

[0056] As used herein, the term "immunoconjugate" generally refers to a combination of an antibody or its antigen-binding fragment with another agent (payload, drug component, chemotherapeutic agent, toxin, immunotherapy agent, imaging probe, etc.). This binding may be covalent or non-covalent interaction, such as electrostatic interaction. Various linkers known in the art can be used to form immunoconjugates. Immunoconjugates may also be provided as expressible fusion proteins from polynucleotides encoding the immunoconjugate. As used herein, "fusion protein" refers to a protein created by linking two or more genes or gene fragments that originally encoded separate proteins (including peptides or polypeptides). Translation of the fusion gene produces a single protein with functional properties derived from each of the original proteins.

[0057] As used herein, the terms “toxin,” “cytotoxin,” or “cytotoxic agent” generally refer to any agent that has an adverse effect on cell growth and proliferation, and that reduces, inhibits, or destroys cells or malignant tumors.

[0058] As used herein, the terms "cancer" and "tumor" are interchangeable and refer to a group of diseases treatable in accordance with this disclosure that involve abnormal cell proliferation and have the potential to invade or metastasize to other parts of the body. Not all tumors are malignant, and benign tumors do not metastasize to other parts of the body. Signs or symptoms of onset include new lumps, abnormal bleeding, chronic cough, unexplained weight loss, and changes in bowel habits. There are currently more than 100 known types of human cancer. In this specification, "cancer" includes, but is not limited to, solid tumors and hematologic malignancies. A "hematologic malignancy" is a cancer that originates from blood-forming tissue, such as bone marrow or immune system cells, and is also called a blood cancer. Hematopoietic malignancies include leukemia (acute myeloid leukemia (AML), acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), mixed lineage acute leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia (CLL), hairy cell leukemia, large granular lymphocyte leukemia, etc.), myelodysplastic syndromes (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocythemia, primary myelofibrosis, and chronic myeloid leukemia), lymphoma, multiple myeloma, MGUS and similar diseases, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transforming follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies.

[0059] "Solid tumors" include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, synovial sarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal cancer, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cavity cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, and cysts. This includes adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, germ cell carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, malignant melanoma, neuroblastoma, and retinoblastoma.

[0060] As used herein, the terms “anti-tumor agent” or “antitumor drug” generally refer to any agent used to treat cell proliferation disorders such as cancer, and include, but are not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, and immunotherapeutic agents.

[0061] As used herein, the term "anti-tumor activity" refers to a reduction in the proliferation rate, viability, or metastatic potential of tumor cells. One way to demonstrate anti-tumor activity is by observing a decrease in tumor cell proliferation rate, maintenance of tumor size, or reduction in tumor size. Such activity can be evaluated using various in vitro or in vivo tumor models known in the industry for assessing anti-tumor activity, including xenograft models, allograft models, MMTV models, and others.

[0062] As used herein, the term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals other than primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, "patient" and "subject" are used interchangeably.

[0063] As used herein, the term "pharmaceutically acceptable" generally refers to a non-toxic substance that does not impair the efficacy of the biological activity of the active ingredient. Such formulations typically include salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Pharmacologically acceptable formulations also include solid or liquid fillers, diluents, or encapsulating materials suitable for administration to humans. In pharmaceutical applications, the salts used must be pharmaceutically acceptable; however, non-acceptable salts can also be used to prepare pharmaceutically acceptable salts and are not excluded from the scope of this invention. Examples of pharmacologically or pharmaceutically acceptable salts include salts prepared from acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, and succinic acid. Pharmacologically acceptable salts can also be prepared as alkali metal or alkaline earth metal salts such as sodium, potassium, and calcium. As used herein, the term "solvate" refers, as in conventional terms, to a complex of a solute (e.g., an active compound or its salt) and a solvent. Solvates typically do not significantly affect the physiological activity or toxicity of a compound and can function as pharmacological equivalents. When the solvent is water, the compound is called a hydrate (e.g., monohydrate, dihydrate, trihydrate, etc.).

[0064] As used herein, the terms “treat,” “treating,” or “treatment” mean improving a disease or disorder (i.e., delaying, halting, or mitigating its development, or reducing at least one clinical symptom). In other words, “treat,” etc., means alleviating or improving at least one physical parameter, including when it is not perceived by the patient. In yet another way, “treat,” etc., means regulating a disease or disorder by physical (e.g., symptom stabilization), physiological (e.g., physiological indicator stabilization), or both. In yet another way, these terms mean preventing or delaying the onset, progression, or worsening of a disease or disorder.

[0065] As used herein, the terms “therapeutically acceptable amount” or “therapeutically effective dose” refer to an amount sufficient to produce the desired outcome (i.e., reduction in tumor size, suppression of tumor growth, prevention of metastasis, or suppression or prevention of viral, bacterial, fungal, or parasitic infection). In some embodiments, a therapeutically acceptable amount does not cause adverse side effects. The therapeutically acceptable amount can be determined by starting with a low dose and gradually increasing the dose until the desired effect is achieved. “Prophylactically effective dose” and “therapeutically effective dose” refer to an amount that prevents the onset of the disease or reduces the severity of disease symptoms (including cancer-related symptoms), respectively.

[0066] In this specification, the terms “comprising,” “containing,” “having,” “include,” and “including” shall, unless otherwise specified, be interpreted as “including, but not limited to.” Furthermore, articles such as “a,” “an,” and “the,” and similar demonstrative pronouns, in the description of the present invention and especially in the appended claims, shall, unless otherwise specified, be interpreted as encompassing both singular and plural forms. Exemplary expressions such as “for example,” “eg,” and “such as” are used to illustrate one aspect or embodiment of the present invention and shall not limit the scope of the invention.

[0067] As used herein, the term "about," when applied to a measured value (such as quantity or duration), means to include a variation of ±20%, or in some cases ±10%, ±5%, ±1%, or ±0.1%, from the specified value. These variations are within a reasonable range for carrying out the methods of this disclosure.

[0068] As used herein, the term "vector" includes shuttle vectors and expression vectors. Typically, a vector is a plasmid construct containing an origin of replication (e.g., Col E1 origin of replication) and a selection marker (e.g., ampicillin resistance or tetracycline resistance) for replication and selection. An "expression vector" refers to a vector containing regulatory sequences or modulogen necessary for expressing the antibody or antibody fragment of this disclosure in bacterial or eukaryotic cells.

[0069] [Anti-CD16A single-domain antibody] In one embodiment, the present disclosure provides a single-domain antibody that specifically binds to CD16A. This single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:1, having a substitution at position S54, G55, S56, or any combination thereof, according to the Kabat numbering system.

[0070] In one embodiment, the anti-CD16A single-domain antibody comprises the amino acid sequence of SEQ ID NO: 1 having a substitution at position S54. In another embodiment, the anti-CD16A single-domain antibody comprises the amino acid sequence of SEQ ID NO: 1 having a substitution at position G55. In yet another embodiment, the anti-CD16A single-domain antibody comprises the amino acid sequence of SEQ ID NO: 1 having a substitution at position S56.

[0071] In one embodiment, the anti-CD16A single-domain antibody contains the amino acid sequence of SEQ ID NO: 1 having substitutions at positions S54 and G55. In another embodiment, it has substitutions at positions S54 and S56. In yet another embodiment, it has substitutions at positions G55 and S56. Furthermore, in yet another embodiment, it has substitutions at positions S54, G55, and S56.

[0072] In one embodiment, the anti-CD16A single-domain antibody has substitutions at positions S54, G55, and S56, and one or more substitutions at other positions. In such cases, the additional substitutions are preferably conservative substitutions.

[0073] In one embodiment, the serine residue at position 54 is replaced with an amino acid residue other than serine. For example, the serine residue at position 54 may be replaced with any of the following: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), threonine (T), tryptophan (W), tyrosine (Y), or valine (V). Preferably, the serine residue at position 54 is replaced with asparagine (N), aspartic acid (D), or threonine (T).

[0074] In one embodiment, the glycine residue at position 55 is replaced with an amino acid residue other than glycine. For example, the glycine residue at position 55 may be replaced with any of the following: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), or valine (V). Preferably, the glycine residue at position 55 is replaced with valine (V).

[0075] In one embodiment, the serine residue at position 56 is replaced with an amino acid residue other than serine. For example, the serine residue at position 56 may be replaced with any of the following: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), threonine (T), tryptophan (W), tyrosine (Y), or valine (V). Preferably, the serine residue at position 56 is replaced with glutamine (Q), threonine (T), aspartic acid (D), or glutamic acid (E).

[0076] In one embodiment, substitutions are selected from (i) S54N, S54D, or S54T; (ii) G55V; (iii) S56Q, S56T, S56D, or S56E; and any combination thereof.

[0077] In one embodiment, the single-domain antibody has an amino acid sequence shown in SEQ ID NO:1, having a single substitution at S54N according to the Kabat numbering system. In another embodiment, the single-domain antibody has an amino acid sequence shown in SEQ ID NO:1, having a single substitution at S54D according to the Kabat numbering system. In yet another embodiment, the single-domain antibody has an amino acid sequence shown in SEQ ID NO:1, having a single substitution at S54T according to the Kabat numbering system.

[0078] In one embodiment, the single-domain antibody has the amino acid sequence shown in SEQ ID NO:1, which has a single G55V substitution according to the Kabat numbering system.

[0079] In one embodiment, the single-domain antibody has an amino acid sequence shown in SEQ ID NO:1, having a single substitution of S56Q according to the Kabat numbering system. In another embodiment, the single-domain antibody has an amino acid sequence shown in SEQ ID NO:1, having a single substitution of S56T according to the Kabat numbering system. In yet another embodiment, the single-domain antibody has an amino acid sequence shown in SEQ ID NO:1, having a single substitution of S56D according to the Kabat numbering system. In yet another embodiment, the single-domain antibody has an amino acid sequence shown in SEQ ID NO:1, having a single substitution of S56E according to the Kabat numbering system.

[0080] In one embodiment, the substitution is selected from (i)S54N, (ii)S54D, (iii)S54T, (iv)G55V, (v)S56Q, (vi)S56T, (vii)S56D, (viii)S56E, (ix)S54N and S56Q, and (x)S54T and S56Q.

[0081] In one embodiment, the single-domain antibody has an amino acid sequence shown in SEQ ID NO:1, having double substitutions of S54N and S56Q according to the Kabat numbering system. In another embodiment, the single-domain antibody has an amino acid sequence shown in SEQ ID NO:1, having double substitutions of S54T and S56Q according to the Kabat numbering system.

[0082] In one embodiment, the single-domain antibody having substitutions at positions selected from S54, G55, and S56, or substitutions in any combination thereof, comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, and 40, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to each of them.

[0083] In one embodiment, the single-domain antibody having substitutions at positions selected from S54, G55, and S56, or substitutions in any combination thereof, comprises the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:3.

[0084] In one embodiment, the single-domain antibody having substitutions at positions selected from S54, G55, and S56, or any combination thereof, comprises the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:4.

[0085] In one embodiment, the single-domain antibody having substitutions at positions selected from S54, G55, and S56, or substitutions in any combination thereof, comprises the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:5.

[0086] In one embodiment, the single-domain antibody having substitutions at positions selected from S54, G55, and S56, or substitutions in any combination thereof, comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:6.

[0087] In one embodiment, the single-domain antibody having substitutions at positions selected from S54, G55, and S56, or substitutions in any combination thereof, comprises the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:7.

[0088] In one embodiment, the single-domain antibody having substitutions at positions selected from S54, G55, and S56, or substitutions in any combination thereof, comprises the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:8.

[0089] In one embodiment, the single-domain antibody having substitutions at positions selected from S54, G55, and S56, or substitutions in any combination thereof, comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:9.

[0090] In one embodiment, the single-domain antibody having substitutions at positions selected from S54, G55, and S56, or any combination thereof, comprises the amino acid sequence shown in SEQ ID NO:10, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:10.

[0091] In one embodiment, the single-domain antibody having substitutions at positions selected from S54, G55, and S56, or substitutions in any combination thereof, comprises the amino acid sequence shown in SEQ ID NO:39, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:39.

[0092] In one embodiment, the single-domain antibody having substitutions at positions selected from S54, G55, and S56, or substitutions in any combination thereof, comprises the amino acid sequence shown in SEQ ID NO:40, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:40. In another aspect of this disclosure, a single-domain antibody that specifically binds to CD16A is provided. This single-domain antibody includes the following:

[0093] (a) CDR1 having the amino acid sequence shown in SEQ ID NO:41 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:42 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or a conservative modified variant thereof, according to the IMGT definition scheme.

[0094] (b) CDR1 having the amino acid sequence shown in SEQ ID NO: 53 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 54 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or a conservative modified variant thereof, according to the Kabat definition scheme.

[0095] (c) CDR1 having the amino acid sequence shown in SEQ ID NO:65 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:66 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or their respective conservative modified variants, according to the Chothia definition.

[0096] (d) CDR1 having the amino acid sequence shown in SEQ ID NO:77 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:78 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or their respective conservative modified variants, according to the Contact definition method. However, this is conditional on the single-domain antibody not having the amino acid sequence shown in SEQ ID NO:1.

[0097] In one embodiment, a single-domain antibody that specifically binds to CD16A is provided. This single-domain antibody has an amino acid sequence selected from SEQ ID NO: 41 as CDR1, SEQ ID NO: 42 as CDR3, and SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 as CDR2, according to the IMGT definition scheme.

[0098] In one embodiment, a single-domain antibody that specifically binds to CD16A is provided. This single-domain antibody has an amino acid sequence selected from SEQ ID NO: 53 as CDR1, SEQ ID NO: 54 as CDR3, and SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64 as CDR2, according to the Kabat definition scheme.

[0099] In one embodiment, a single-domain antibody that specifically binds to CD16A is provided. This single-domain antibody has an amino acid sequence selected from SEQ ID NO: 65 as CDR1, SEQ ID NO: 66 as CDR3, and SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 as CDR2, according to the Chothia definition scheme.

[0100] In one embodiment, a single-domain antibody that specifically binds to CD16A is provided. This single-domain antibody has an amino acid sequence selected from SEQ ID NO:77 as CDR1, SEQ ID NO:78 as CDR3, and SEQ ID NO:79, 80, 81, 82, 83, 84, 85, 86, 87, or 88 as CDR2, according to the Contact definition scheme.

[0101] In one embodiment, a single-domain antibody is provided that contains an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, or 40.

[0102] In one embodiment, a single-domain antibody is provided that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 39, or 40, and has an amino acid sequence selected from SEQ ID NO:41 as CDR1, SEQ ID NO:42 as CDR3, and SEQ ID NO:43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 as CDR2, according to the IMGT definition scheme.

[0103] In one embodiment, a single-domain antibody is provided that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 39, or 40, and has an amino acid sequence selected from SEQ ID NO:53 as CDR1, SEQ ID NO:54 as CDR3, and SEQ ID NO:55, 56, 57, 58, 59, 60, 61, 62, 63, or 64 as CDR2, according to the Kabat definition scheme.

[0104] In one embodiment, a single-domain antibody is provided that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 39, or 40, and has an amino acid sequence selected from SEQ ID NO:65 as CDR1, SEQ ID NO:66 as CDR3, and SEQ ID NO:67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 as CDR2, according to the Chothia definition scheme.

[0105] In one embodiment, a single-domain antibody is provided that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 39, or 40, and has an amino acid sequence selected from SEQ ID NO:77 as CDR1, SEQ ID NO:78 as CDR3, and SEQ ID NO:79, 80, 81, 82, 83, 84, 85, 86, 87, or 88 as CDR2, according to the Contact definition scheme.

[0106] In any embodiment of the anti-CD16A single-domain antibody disclosed herein, the single-domain antibody may be a humanized antibody, a human antibody, a chimeric antibody, or a camelated antibody. Preferably, the single-domain antibody is a humanized antibody or a human antibody. More preferably, the single-domain antibody is a humanized antibody.

[0107] In some embodiments, the anti-CD16A single-domain antibody disclosed herein specifically binds to CD16A but not to CD16B. In some embodiments, the anti-CD16A single-domain antibody disclosed herein specifically binds to human and monkey CD16A but not to mouse or rat CD16A. The amino acid sequence of human CD16A is shown exemplary in SEQ ID NO: 2 or 11. The amino acid sequence of human CD16B is shown exemplary in SEQ ID NO: 36. The amino acid sequence of monkey CD16A is shown exemplary in SEQ ID NO: 33. The amino acid sequence of mouse CD16A is shown exemplary in SEQ ID NO: 34. The amino acid sequence of rat CD16A is shown exemplary in SEQ ID NO: 35.

[0108] In certain embodiments, the anti-CD16A single domain antibodies disclosed herein specifically bind to CD16A 158V and 158F. In certain embodiments, the antibody specifically binds to CD16A 158V and 158F with substantially equivalent affinity. In certain embodiments, the antibody specifically binds to CD16A 158V and 158F with substantially equivalent affinity and has an EC 50 in the range of about 1 to about 7 nM, about 1 to about 4 nM, about 1 to about 2 nM, or about 1 to about 1.5 nM, measured by ELISA. In certain embodiments, the antibody specifically binds to CD16A 158V and has an EC 50 in the range of about 1 to about 4 nM, about 1 to about 3 nM, about 1 to about 2 nM, or about 1 to about 1.5 nM, measured by ELISA. In certain embodiments, the antibody specifically binds to CD16A 158F and has an EC 50 in the range of about 1 to about 3 nM, about 1 to about 2 nM, or about 1 to about 1.2 nM.

[0109] In certain embodiments, the anti-CD16A single domain antibodies disclosed herein have improved binding affinity, particularly for CD16A 158V, compared to an anti-CD16A single domain antibody having the amino acid sequence set forth in SEQ ID NO:1. In certain embodiments, the antibody specifically binds to CD16A 158V, measured by BLI, and has an EC 50 in the range of about 2 to about 16 nM, about 2 to about 10 nM, about 2 to about 8 nM, about 2 to about 5 nM, or about 2 to about 3 nM. In certain embodiments, the antibody specifically binds to CD16A 158F, measured by BLI, and has an EC 50 in the range of about 1 to about 6 nM, about 1 to about 5 nM, about 1 to about 4 nM, about 1 to about 3 nM, or about 1 to about 2 nM.

[0110] In one embodiment, a polypeptide is provided comprising an anti-CD16A single-domain antibody and one or more amino acid residues covalently linked to the N-terminus, C-terminus, or any position between them of the antibody. Preferably, the anti-CD16A single-domain antibody comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, or 40.

[0111] [Anti-CD16A heavy chain antibody] In another aspect of this disclosure, a heavy chain antibody comprising a single-domain antibody disclosed herein and an Fc region linked to the single-domain antibody is provided. In one embodiment, an anti-CD16A heavy chain antibody is provided. This heavy chain antibody comprises the following single-domain antibody.

[0112] (a) CDR1 having the amino acid sequence shown in SEQ ID NO:41 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:42 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or a conservative modified variant thereof, according to the IMGT definition scheme.

[0113] (b) CDR1 having the amino acid sequence shown in SEQ ID NO: 53 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 54 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or a conservative modified variant thereof, according to the Kabat definition scheme.

[0114] (c) CDR1 having the amino acid sequence shown in SEQ ID NO:65 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:66 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or their respective conservative modified variants, according to the Chothia definition.

[0115] (d) CDR1 having the amino acid sequence shown in SEQ ID NO:77 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:78 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or their respective conservative modified variants, according to the Contact definition method. However, this is conditional on the heavy chain antibody not containing the amino acid sequence shown in SEQ ID NO:1.

[0116] In one embodiment, an anti-CD16A heavy chain antibody is provided, which comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, or 40.

[0117] In one embodiment, an anti-CD16A heavy chain antibody is provided, which has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 39, or 40, and has an amino acid sequence selected from SEQ ID NO:41 as CDR1, SEQ ID NO:42 as CDR3, and SEQ ID NO:43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 as CDR2, according to the IMGT definition scheme.

[0118] In one embodiment, an anti-CD16A heavy chain antibody is provided, which comprises a single-domain antibody having an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, or 40, wherein the single-domain antibody is ligated to an Fc region having the amino acid sequence shown in SEQ ID NO: 89.

[0119] In any embodiment of the anti-CD16A heavy chain antibody disclosed herein, the antibody may be a humanized antibody, a human antibody, a chimeric antibody, or a carmelized antibody. Preferably, it is a humanized antibody or a human antibody, and more preferably a humanized antibody.

[0120] In one embodiment, a polypeptide is provided comprising an anti-CD16A heavy chain antibody and one or more amino acid residues covalently linked to the N-terminus, C-terminus, or any position between them of the antibody. Preferably, the anti-CD16A heavy chain antibody comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, or 40, and further comprises an Fc region having the amino acid sequence shown in SEQ ID NO: 89.

[0121] [Bispecific antibody] In another aspect of the present invention, bispecific antibodies comprising the single-domain antibody disclosed herein are provided.

[0122] In one embodiment, the bispecific antibody comprises a single-domain antibody that specifically binds to CD16A as disclosed herein, and a second binding domain that specifically binds to a second target selected from tumor-associated antigens or tumor-specific antigens.

[0123] In one embodiment, the second target is HER2, 5T4, PSMA, BCMA, FGFR, CD20, CD33, CD19, CD22, CD123, CD30, GPC-3, CEA, EGFR1, EGFR2, EGFR3, TGF-β, ROR1, PD-L1, Claudin18.2, EpCAM, GD2, MSLN, EGFR, MUC1, MUC2, EGFRVIII, CD38, Trop-2, c -MET, Nectin-4, CD79b, CCK4, GPA33, HLA-A2, CLEC12A, p-cadherin, TDO2, MART-1, Pmel17, MAGE-1, AFP, CA125, TRP-1, TRP-2, NY-ESO, PSA, CDK4, BCA225, CA125, MG7-Ag, NY-CO-1, RCAS1, SDCCAG16, TAAL6, and TAG72 are selected. Preferably, the second target is HER2, 5T4, BCMA, CD20, CD19, CD30, Claudin18.2, MUC1, Trop-2, c-MET, or Nectin-4, more preferably HER2, 5T4, or CD30.

[0124] In one embodiment, the bispecific antibody comprises a first binding domain that specifically binds to CD16A and a second binding domain that specifically binds to HER2, wherein the first binding domain is a single-domain antibody that specifically binds to CD16A, and the single-domain antibody comprises the following:

[0125] (a) CDR1 having the amino acid sequence shown in SEQ ID NO:41 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:42 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or a conservative modified variant thereof, according to the IMGT definition scheme.

[0126] (b) CDR1 having the amino acid sequence shown in SEQ ID NO: 53 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 54 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or a conservative modified variant thereof, according to the Kabat definition scheme.

[0127] (c) CDR1 having the amino acid sequence shown in SEQ ID NO:65 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:66 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or their respective conservative modified variants, according to the Chothia definition.

[0128] (d) CDR1 having the amino acid sequence shown in SEQ ID NO:77 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:78 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or their respective conservative modified variants, according to the Contact definition method. However, this is conditional on the single-domain antibody not having the amino acid sequence shown in SEQ ID NO:1.

[0129] In one embodiment, the bispecific antibody comprises a first binding domain that specifically binds to CD16A and a second binding domain that specifically binds to 5T4, wherein the first binding domain is a single-domain antibody that specifically binds to CD16A, and the single-domain antibody comprises the following:

[0130] (a) CDR1 having the amino acid sequence shown in SEQ ID NO:41 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:42 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or a conservative modified variant thereof, according to the IMGT definition method.

[0131] (b) CDR1 having the amino acid sequence shown in SEQ ID NO: 53 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 54 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or a conservative modified variant thereof, according to the Kabat definition scheme.

[0132] (c) CDR1 having the amino acid sequence shown in SEQ ID NO:65 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:66 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or their respective conservative modified variants, according to the Chothia definition.

[0133] (d) CDR1 having the amino acid sequence shown in SEQ ID NO:77 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:78 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or their respective conservative modified variants, according to the Contact definition method. However, this is conditional on the single-domain antibody not having the amino acid sequence shown in SEQ ID NO:1.

[0134] In one embodiment, the bispecific antibody comprises a first binding domain that specifically binds to CD16A and a second binding domain that specifically binds to CD30, wherein the first binding domain is a single-domain antibody that specifically binds to CD16A, and the single-domain antibody comprises the following:

[0135] (a) CDR1 having the amino acid sequence shown in SEQ ID NO:41 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:42 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or a conservative modified variant thereof, according to the IMGT definition method.

[0136] (b) CDR1 having the amino acid sequence shown in SEQ ID NO: 53 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 54 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or a conservative modified variant thereof, according to the Kabat definition scheme.

[0137] (c) CDR1 having the amino acid sequence shown in SEQ ID NO:65 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:66 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or their respective conservative modified variants, according to the Chothia definition.

[0138] (d) CDR1 having the amino acid sequence shown in SEQ ID NO:77 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:78 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or their respective conservative modified variants, according to the Contact definition method. However, this is conditional on the single-domain antibody not having the amino acid sequence shown in SEQ ID NO:1.

[0139] In one embodiment, the bispecific antibody comprises a first binding domain that specifically binds to CD16A and a second binding domain that specifically binds to HER2, wherein the first binding domain is a single-domain antibody that specifically binds to CD16A, and the single-domain antibody comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, 40.

[0140] In one embodiment, the bispecific antibody comprises a first binding domain that specifically binds to CD16A and a second binding domain that specifically binds to 5T4, wherein the first binding domain is a single-domain antibody that specifically binds to CD16A, and the single-domain antibody comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, 40.

[0141] In one embodiment, the bispecific antibody comprises a first binding domain that specifically binds to CD16A and a second binding domain that specifically binds to CD30, wherein the first binding domain is a single-domain antibody that specifically binds to CD16A, and the single-domain antibody comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, 40.

[0142] In one embodiment, the bispecific antibody comprises a first binding domain that specifically binds to CD16A and a second binding domain that specifically binds to a second target selected from tumor-associated antigens or tumor-specific antigens, wherein the first binding domain is a single-domain antibody that specifically binds to CD16A, and the second binding domain is a full-length IgG antibody or its antigen-binding fragment (Fab, Fv, scFv, or F(ab')2).

[0143] In one embodiment, the bispecific antibody comprises a first binding domain that specifically binds to CD16A and a second binding domain that specifically binds to a second target selected from tumor-associated antigens or tumor-specific antigens, wherein the first binding domain is a single-domain antibody that specifically binds to CD16A, and the second binding domain is a full-length IgG antibody, and the single-domain antibody is ligated to the heavy chain C-terminus, light chain C-terminus, or heavy chain N-terminus of the full-length IgG antibody.

[0144] In one embodiment, the bispecific antibody comprises a first binding domain that specifically binds to CD16A and a second binding domain that specifically binds to a second target selected from tumor-associated antigens or tumor-specific antigens, wherein the first binding domain is a single-domain antibody that specifically binds to CD16A, and the second binding domain is a full-length IgG antibody, and the single-domain antibody is linked to the heavy chain C-terminus, light chain C-terminus, or heavy chain N-terminus of the full-length IgG antibody via a peptide linker.

[0145] In one embodiment, the bispecific antibody comprises a single-domain antibody that specifically binds to CD16A as disclosed herein and a full-length anti-HER2 IgG antibody, wherein the single-domain antibody is linked to the heavy chain C-terminus, light chain C-terminus, or heavy chain N-terminus of the full-length IgG antibody via a peptide linker consisting of glycine and serine residues. The peptide linker is as defined in the "Definitions" section.

[0146] In one embodiment, the bispecific antibody comprises a single-domain antibody that specifically binds to CD16A and a full-length anti-HER2 IgG antibody, wherein the single-domain antibody is linked to the heavy chain C-terminus, light chain C-terminus, or heavy chain N-terminus of the full-length IgG antibody via a peptide linker consisting of glycine and serine residues.

[0147] In one embodiment, the bispecific antibody comprises a single-domain antibody that specifically binds to CD16A as disclosed herein and a full-length anti-5T4 IgG antibody, wherein the single-domain antibody is linked to the heavy chain C-terminus, light chain C-terminus, or heavy chain N-terminus of the full-length IgG antibody via a peptide linker consisting of glycine and serine residues.

[0148] In one embodiment, the full-length IgG antibody has a mutation in its Fc region. In one embodiment, the full-length IgG antibody has the L234A and L235A mutations ("LALA"). In one embodiment, the full-length IgG antibody has the N297A mutation. In one embodiment, the full-length IgG antibody has the "LALA" and N297A mutations.

[0149] In one embodiment, the full-length anti-HER2 IgG antibody comprises the following: (a) The heavy chain has the amino acid sequence shown in SEQ ID NO:12, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (b) The heavy chain has the amino acid sequence shown in SEQ ID NO:37 and the light chain has the amino acid sequence shown in SEQ ID NO:38. (c) The heavy chain has the amino acid sequence shown in SEQ ID NO:25, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (d) The heavy chain has the amino acid sequence shown in SEQ ID NO:26, and the light chain has the amino acid sequence shown in SEQ ID NO:13. In one embodiment, the bispecific antibody comprises the following: (a) The heavy chain has the amino acid sequence shown in SEQ ID NO:14, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (b) The heavy chain has the amino acid sequence shown in SEQ ID NO:15, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (c) The heavy chain has the amino acid sequence shown in SEQ ID NO:17, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (d) The heavy chain has the amino acid sequence shown in SEQ ID NO:12, and the light chain has the amino acid sequence shown in SEQ ID NO:16. (e) The heavy chain has the amino acid sequence shown in SEQ ID NO:18, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (f) The heavy chain has the amino acid sequence shown in SEQ ID NO:19, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (g) The heavy chain has the amino acid sequence shown in SEQ ID NO:20, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (h) The heavy chain has the amino acid sequence shown in SEQ ID NO:21, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (i) The heavy chain has the amino acid sequence shown in SEQ ID NO:22, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (j) The heavy chain has the amino acid sequence shown in SEQ ID NO:23, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (k) The heavy chain has the amino acid sequence shown in SEQ ID NO:24, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (l) The heavy chain has the amino acid sequence shown in SEQ ID NO:27, and the light chain has the amino acid sequence shown in SEQ ID NO:13. The (m) heavy chain has the amino acid sequence shown in SEQ ID NO:28, and the light chain has the amino acid sequence shown in SEQ ID NO:13.

[0150] In a preferred embodiment, the bispecific antibody comprises the following: (a) The heavy chain has the amino acid sequence shown in SEQ ID NO:14, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (b) The heavy chain has the amino acid sequence shown in SEQ ID NO:15, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (c) The heavy chain has the amino acid sequence shown in SEQ ID NO:17, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (d) The heavy chain has the amino acid sequence shown in SEQ ID NO:12, and the light chain has the amino acid sequence shown in SEQ ID NO:16. (e) The heavy chain has the amino acid sequence shown in SEQ ID NO:27, and the light chain has the amino acid sequence shown in SEQ ID NO:13. (f) The heavy chain has the amino acid sequence shown in SEQ ID NO:28, and the light chain has the amino acid sequence shown in SEQ ID NO:13.

[0151] In one embodiment, the full-length anti-5T4 antibody has a heavy chain having the amino acid sequence shown in SEQ ID NO:29 and a light chain having the amino acid sequence shown in SEQ ID NO:30.

[0152] In one embodiment, the bispecific antibody has a heavy chain having the amino acid sequence shown in SEQ ID NO:31 and a light chain having the amino acid sequence shown in SEQ ID NO:30.

[0153] [Immune Conjugates]

[0154] Other aspects of this disclosure relate to immunoconjugates, pharmaceutically acceptable salts or solvates thereof, comprising any single-domain antibody, heavy-chain antibody, or bispecific antibody described in the sections “Anti-CD16A Single-Domain Antibody”, “Anti-CD16A Heavy-Chain Antibody”, or “Bispecific Antibody”, and an active site conjugated to the single-domain antibody. In one embodiment, the active site is selected from the group consisting of toxins, peptide tags, sol tags, radionuclides, near-infrared fluorescent dyes, and nanoparticles.

[0155] In one embodiment, the toxin is selected from the group consisting of cytotoxic agents, cytokines, nucleic acids, nucleic acid-related molecules, radionuclides, chemokines, immunostimulatory or costimulatory molecules, immunosuppressive molecules, death ligands, apoptosis-inducing proteins, kinases, prodrug-converting enzymes, RNases, agonist antibodies or their antibody fragments, antagonist antibodies or their antibody fragments, growth factors, hormones, coagulation factors, fibrinolytic proteins, peptides that mimic these, and their fragments, fusion proteins, or derivatives.

[0156] In one embodiment, the radioactive nuclide is At 211 , I 131 , I 125 , I 123 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 , Pb 212 , Tc 99 , S 35 , F 19 , N 15 , C 14 , C 13 or H 3 It may contain and, if necessary, be conjugated to the antibody via a chelating agent. In one embodiment, an immunoconjugate, a pharmaceutically acceptable salt or solvate thereof is provided. The immunoconjugate comprises the following single-domain antibody:

[0157] (a) CDR1 having the amino acid sequence shown in SEQ ID NO:41 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:42 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or a conservative modified variant thereof, according to the IMGT definition scheme.

[0158] (b) CDR1 having the amino acid sequence shown in SEQ ID NO: 53 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 54 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or a conservative modified variant thereof, according to the Kabat definition scheme.

[0159] (c) CDR1 having the amino acid sequence shown in SEQ ID NO:65 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:66 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or their respective conservative modified variants, according to the Chothia definition.

[0160] (d) CDR1 having the amino acid sequence shown in SEQ ID NO:77 or a conservative modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO:78 or a conservative modified variant thereof, and CDR2 having an amino acid sequence selected from SEQ ID NO:79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or their respective conservative modified variants, according to the Contact definition method. However, this is conditional on the single-domain antibody not having the amino acid sequence shown in SEQ ID NO:1.

[0161] Furthermore, the antibody has an active site bound to the single-domain antibody, and the active site is selected from the group consisting of toxins, peptide tags, sol tags, radionuclides, near-infrared fluorescent dyes, and nanoparticles.

[0162] In one embodiment, an immunoconjugate, a pharmaceutically acceptable salt or solvate thereof is provided. The immunoconjugate comprises a single-domain antibody having an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, or 40, and an active site conjugated to the single-domain antibody, the active site being selected from the group consisting of toxins, peptide tags, sol tags, radionuclides, near-infrared fluorescent dyes, and nanoparticles.

[0163] In one embodiment, an immunoconjugate, a pharmaceutically acceptable salt or solvate thereof is provided. The immunoconjugate comprises a single-domain antibody having an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, and 40, and an active site conjugated to the single-domain antibody, wherein the active site is a toxin, a radionuclide, or a near-infrared fluorescent dye.

[0164] In one embodiment, an immunoconjugate, a pharmaceutically acceptable salt thereof, or a solvate thereof is provided. The immunoconjugate comprises a single-domain antibody having an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, and 40, and a cytotoxic agent conjugated to the single-domain antibody.

[0165] In one embodiment, an immunoconjugate, a pharmaceutically acceptable salt or solvate thereof is provided. The immunoconjugate comprises a single-domain antibody having an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, and 40, and a radionuclide conjugated to the single-domain antibody.

[0166] [Pharmaceutical composition] In another aspect of this disclosure, a pharmaceutical composition is provided comprising a single-domain antibody, a heavy-chain antibody, a bispecific antibody, or an immunoconjugate (collectively, "antibody or immunoconjugate") and a pharmaceutically acceptable carrier. The composition is suitable for administration to animals or humans.

[0167] The composition may be in any form that allows for administration to a patient. For example, the composition may be in the form of a solid, liquid, or gas (aerosol). Typical routes of administration include oral, transdermal, parenteral, sublingual, rectal, vaginal, ocular, intratumoral, and intranasal administration. Parenteral administration includes subcutaneous injection, intravenous administration, intramuscular administration, intrasternal injection, or infusion techniques. In one embodiment, the composition is administered parenterally. In yet another embodiment, the composition is administered intravenously.

[0168] Pharmaceutical compositions can be formulated so that the active ingredient becomes bioavailable after administration. Compositions can take the form of one or more dosage units. For example, a tablet is a single dosage unit, and a container containing an aerosol-form immunoconjugate may hold multiple dosage units.

[0169] The materials used in the preparation of pharmaceutical compositions may be nontoxic in the amounts used. As will be apparent to those skilled in the art, the optimal dose of the active ingredient in a pharmaceutical composition depends on various factors. These factors include, but are not limited to, the type of animal (e.g., human), the form of the immunoconjugate, the method of administration, and the composition used.

[0170] The pharmaceutically acceptable carrier or medium may be particulate, in which case the composition may be, for example, in tablet or powder form. The carrier may also be liquid, in which case the composition may be, for example, an oral syrup or an injectable liquid. The carrier may also be gaseous or particulate, providing, for example, an aerosol composition useful for inhalation administration.

[0171] The composition may be in liquid form, for example, as an elixir, syrup, solution, emulsion, or suspension. Liquids are useful for oral or injectable administration. When prepared for oral administration, the composition may contain one or more sweeteners, preservatives, colorants, and flavor enhancers. Compositions for injectable administration may contain one or more surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0172] The liquid composition may be in the form of a solution, suspension, or other, and may further contain one or more of the following: sterile diluents such as water for injection, physiological saline (preferably isotonic physiological saline), Ringer's solution, or isotonic sodium chloride solution; fixing oils such as synthetic monoglycerides or diglycerides that can act as a solvent or suspension medium; polyethylene glycol, glycerin, cyclodextrin, propylene glycol, or other solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetates, citrates, or phosphates; and isotonic agents such as sodium chloride or glucose. The parenteral composition may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass, plastic, or other materials. Physiological saline is a typical adjuvant, and the injectable composition is preferably sterile.

[0173] The amount of antibody or immunoconjugate effective in treating a particular disease or condition depends on the nature of the disease or condition and can be determined by standard clinical methods. Furthermore, in vitro or in vivo assays may be used to identify the optimal dosage range. The exact dose used should depend on the route of administration and the severity of the disease, and should be determined according to the physician's judgment and the individual patient's situation.

[0174] The composition contains an effective amount of antibody or immunoconjugate sufficient to obtain an appropriate dose. Typically, this amount is at least about 0.01% of the composition's weight of antibody or immunoconjugate. For oral administration, this percentage can vary in the range of about 0.1% to about 80%. In one embodiment, the oral composition may contain about 4% to about 50% of antibody or immunoconjugate relative to the composition's weight. In yet another embodiment, the composition for parenteral administration is prepared to contain about 0.01% to about 2% of antibody or immunoconjugate per dose unit relative to the composition's weight.

[0175] For intravenous administration, the composition may contain approximately 0.01 to 100 mg of antibody or immunoconjugate per kg of animal body weight. In one embodiment, the composition contains approximately 1 to 100 mg of antibody or immunoconjugate per kg of body weight. In another embodiment, the dosage ranges from approximately 0.1 to 25 mg per kg of body weight.

[0176] Generally, the dosage of antibodies or immune conjugates administered to patients is typically around 0.01 mg to 2000 mg per kg of body weight. In one embodiment, the dosage is in the range of approximately 0.01 mg to 10 mg per kg of body weight; in another embodiment, it is in the range of approximately 0.1 mg to 250 mg; in yet another embodiment, it is in the range of approximately 0.1 mg to 20 mg; in yet another embodiment, it is in the range of approximately 0.1 mg to 10 mg; and in yet another embodiment, it is in the range of approximately 1 mg to 10 mg.

[0177] Antibodies, immunoconjugates, or compositions can be administered by any convenient route, such as by injection or bolus injection, or by absorption through epithelial tissue or mucous membranes (e.g., oral mucosa, rectal mucosa, intestinal mucosa, etc.). Administration may be systemic or topical. Various known delivery systems, such as encapsulation in liposomes, microparticles, microcapsules, or capsules, can be used for the administration of antibodies, immunoconjugates, or compositions. In certain embodiments, multiple antibodies, immunoconjugates, or compositions may also be administered to a patient.

[0178] In certain embodiments, it may be desirable to administer one or more antibodies, immunoconjugates, or compositions topically to the site requiring treatment. This can be achieved, for example, by local injection during surgery, topical application in combination with wound dressings after surgery, injection, catheter, suppository, or implant (including porous, non-porous, or gelatinous materials, such as silastic membranes or fibers). In one embodiment, it can be injected directly into the site of cancer, tumor, or neonatal or prenatal tissue (or the site after its removal). In another embodiment, it can be injected directly into the symptomatic site of an autoimmune disease (or the site after its removal).

[0179] In certain embodiments, it may be desirable to introduce antibodies, immunoconjugates, or compositions into the central nervous system via any suitable route, such as intracerebroventricular injection or subarachnoid injection. Intracerebroventricular injection can be easily performed, for example, by an intracerebroventricular catheter connected to a reservoir such as an Omaya reservoir.

[0180] Lung administration is also available, for example, using an inhaler or nebulizer, using a formulation containing an aerosolizing agent, or by perfusion into a fluorocarbon or synthetic lung surfactant.

[0181] In yet another embodiment, the antibody, immunoconjugate, or composition may be administered using a controlled-release system such as a pump or various polymer materials. In yet another embodiment, the controlled-release system may be positioned near the target (e.g., the brain) of the antibody, immunoconjugate, or composition, so that only a small fraction of the systemic dose can exert its effect.

[0182] A "carrier" refers to a diluent, adjuvant, or carrier administered together with an antibody or immunoconjugate. Such pharmaceutical carriers can be liquids such as water or oil (e.g., petroleum-based, animal-based, vegetable-based, or synthetic oils, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). Edible rubber, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc., can also be used as carriers. Adjuvants, stabilizers, thickeners, lubricants, and colorants can also be used. In one embodiment, when administered to a patient, the antibody or immunoconjugate and the pharmaceutically acceptable carrier are sterile. When antibodies or immunoconjugates are administered intravenously, water is a typical carrier. Saline solution, aqueous glucose solution, and glycerol solution can also be used as liquid carriers, especially for injection. Suitable pharmaceutical carriers include starch, glucose, lactose, sucrose, gelatin, malt, rice flour, chalk, silica gel, sodium stearate, glycerin monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, and ethanol. Small amounts of surfactants and pH buffers may be added as needed.

[0183] In one embodiment, the immunoconjugate is formulated according to conventional methods as a pharmaceutical composition suitable for intravenous administration to animals (particularly humans). The carrier or medium for intravenous administration is usually a sterile isotonic aqueous buffer. A solubilizer may be included as needed. The intravenous administration composition may optionally contain a local anesthetic such as lidocaine to reduce pain at the injection site. Generally, the components are packaged separately or mixed, for example, as a dried frozen powder or a water-free concentrate, in unit dosing containers such as ampoules or sealed sachets, with the amount of the active ingredient indicated. When the immunoconjugate is administered by infusion, it is dispensed, for example, in an infusion vial containing sterile pharmaceutical-grade water or saline. When the antibody or immunoconjugate is administered by injection, an ampoule of sterile water for injection or saline may be provided and mixed before administration.

[0184] The composition may include various materials for altering the physical form of the solid or liquid dosage unit. For example, it may include materials that form a coating shell covering the active ingredient. These coating materials are typically inert and are selected from sugar, shellac, and other enteric coatings. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0185] The compositions of the present invention may be in the form of gaseous dosing units, such as aerosols. The term "aerosol" is used as a concept that encompasses various systems, from colloidal systems to systems in pressurized packaging. Delivery can be carried out by liquefied gas or compressed gas, or by a suitable pump system for distributing the active ingredient. The compositions of the present invention may contain pharmacologically active substances used in cancer treatment, whether in solid, liquid, or gaseous form.

[0186] [Uses and Treatments] Antibodies and immunoconjugates are useful for inhibiting the proliferation of tumor or cancer cells, inducing apoptosis in tumor or cancer cells, or treating cancer in patients. These antibodies and immunoconjugates can be used in a variety of settings in the treatment of cancer in animals. Antibodies or immunoconjugates can also be used to deliver drugs or drug units to tumor or cancer cells. While not bound by theory, in one embodiment, the antibody portion of the antibody or immunoconjugate disclosed herein binds to or associates with CD16A expressed or associated on the surface of NK cells. In the case of bispecific antibodies, the antibody binds to CD16A with one arm and to another antigen expressed on tumor cells with the other arm, forming an "NK engager (NK cell activating antibody)" that enhances the tumor cell-killing function of NK cells.

[0187] In other embodiments of this disclosure, a method for treating cancer is provided, comprising administering a therapeutically effective dose of a single-domain antibody, a heavy-chain antibody, a bispecific antibody, or an immunoconjugate to a subject.

[0188] In other aspects of this disclosure, a method for cancer prevention is provided, comprising administering a prophylactic dose of a single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate to a subject at risk of developing cancer.

[0189] In other embodiments of the present disclosure, a cancer diagnostic method is provided, for example in vitro, which includes separating a sample from a subject, contacting the sample with a single-domain antibody, a heavy-chain antibody, a bispecific antibody, or an immunoconjugate, detecting a signal produced by the contact, comparing the signal to a threshold, and determining whether the subject has cancer based on the comparison result.

[0190] The aforementioned cancers may be solid tumors or hematological malignancies. "Hematological malignancies" refer to cancers originating from hematopoietic tissues such as bone marrow and immune system cells, and are also called blood cancers. Hematological malignancies include, but are not limited to, the following: Specifically, these include leukemia (acute myeloid leukemia (AML), acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), acute mixed lineage leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia (CLL), pilocytic cell leukemia, large granular lymphocytic leukemia, etc.), myelodysplastic syndromes (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocythemia, primary myelofibrosis, chronic myeloid leukemia), lymphoma, multiple myeloma, MGUS and similar diseases, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), mediastinal large cell B lymphoma, diffuse large cell B lymphoma, follicular lymphoma, transforming follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, T-cell lymphoma and other B-cell malignancies.

[0191] While not limited to "solid tumors," this category includes fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, synovial sarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cavity cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, and papilloma. Cancers include papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminomas, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astroglioma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, and retinoblastoma. Preferably, cancers that can be treated by this disclosure include breast cancer.

[0192] In one embodiment, a method for treating or preventing cancer is provided. The method comprises administering a therapeutically effective dose of the antibody or immunoconjugate disclosed herein and a chemotherapeutic agent to a patient in need. In one embodiment, the chemotherapeutic agent is a drug to which resistance has not been observed for the cancer treatment. In another embodiment, the chemotherapeutic agent is a drug to which resistance has been observed for the cancer treatment. The antibody or immunoconjugate may also be administered to a patient who has undergone surgical treatment as part of the cancer treatment. In one embodiment, the patient is a patient who is resistant to a HER2 antibody, such as trastuzumab or margetuzumab.

[0193] In certain embodiments, the antibody or immunoconjugate is administered concurrently with chemotherapeutic agents or radiotherapy. In other embodiments, chemotherapeutic agents or radiotherapy are administered before or after the administration of the antibody or immunoconjugate. In some embodiments, administration may occur at least 1 hour, 5 hours, 12 hours, 1 day, 1 week, 1 month, or longer (e.g., up to 3 months) before or after.

[0194] Chemotherapy agents can be administered in multiple sessions. Applicable chemotherapeutic agents include methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, topotecan, nitrogen mustard, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin derivatives, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. Regarding radiotherapy, any radiotherapy protocol can be used depending on the type of cancer being treated. For example, X-ray irradiation may be performed, and high-energy megavoltage radiation exceeding 1 MeV can be used particularly for deep tumors. Furthermore, electron beam and orthovoltage X-ray irradiation can be used to treat skin cancer. In addition, radioactive isotopes that emit gamma rays, such as radium, cobalt, and other elements, may be administered.

[0195] Furthermore, in cases where chemotherapy or radiotherapy is excessively toxic to the subject (e.g., causing unbearable side effects), this disclosure also provides alternative cancer treatment methods using antibodies or immunoconjugates. The treated animals may also receive other cancer treatments, such as surgery, radiotherapy, or chemotherapy, as needed.

[0196] Antibodies or immunoconjugates can be used in vitro or ex vivo. For example, they can be used in the treatment of certain cancers such as leukemia and lymphoma, in combination with autologous hematopoietic stem cell transplantation. This involves a multi-step process in which autologous hematopoietic stem cells are collected from animals, all cancer cells are removed, the remaining bone marrow cells are destroyed with high doses of antibodies or immunoconjugates (and high-dose radiotherapy if necessary), and then purified stem cells are reinfused. Supportive care is then provided to support bone marrow function recovery and the animal's recovery.

[0197] Similarly, this disclosure provides single-domain antibodies, heavy-chain antibodies, bispecific antibodies, or immunoconjugates for use in the treatment of cancer in subjects. Furthermore, this disclosure also provides the use of these antibodies or immunoconjugates in the manufacture of pharmaceuticals for the treatment of cancer in subjects.

[0198] This disclosure further provides methods for treating acute or chronic infections caused by viruses, fungi, or bacteria. The methods include administering a therapeutically effective amount of a single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate to a subject as needed. For example, infections that can be treated with the antibody or immunoconjugate include, but are not limited to, infections caused by adenoviruses, herpesviruses (HSV-I, HSV-II, CMV, or VZV), poxviruses (orthopoxviruses [smallpox virus, vaccinia virus], or molluscum contagiosum virus), picornaviruses (rhinovirus or enterovirus), orthomyxoviruses (influenza virus), paramyxoviruses (parainfluenza virus, mumps virus, measles virus, RSV), coronaviruses (such as SARS), papovaviruses (such as human papillomavirus), hepadnaviruses (hepatitis B virus), flaviviruses (such as hepatitis C virus, dengue virus), and retroviruses (such as HIV).

[0199] Bacterial infections also include infections caused by bacteria of the genera Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, and Bordetella. Furthermore, Chlamydia infections, fungal infections (candidiasis, aspergillosis, histoplasmosis, cryptococcal meningitis, etc.), and parasitic infections (malaria, Pneumocystis pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, trypanosomiasis infection, etc.) are also covered.

[0200] This disclosure further provides a method for treating age-related diseases. The method comprises administering a therapeutically effective amount of a single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate to a subject as needed. Age-related diseases include arteriosclerosis, cardiovascular disease, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, and Alzheimer's disease.

[0201] This disclosure also provides single-domain antibodies, heavy-chain antibodies, bispecific antibodies, or immunoconjugates for use in the treatment of acute or chronic infections in subjects. Similarly, this disclosure also provides the use of these antibodies or immunoconjugates in the manufacture of pharmaceuticals for the treatment of acute or chronic infections in subjects.

[0202] This disclosure also provides single-domain antibodies, heavy-chain antibodies, bispecific antibodies, or immunoconjugates for use in the treatment of age-related diseases in subjects. Similarly, this disclosure also provides the use of these antibodies or immunoconjugates in the manufacture of pharmaceuticals for the treatment of age-related diseases in subjects.

[0203] This disclosure further provides a method for preventing an acute or chronic infection in a subject at risk of developing such infection. The method comprises administering to the subject a prophylactic dose of a single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate disclosed herein.

[0204] This disclosure further provides a method for preventing age-related diseases in subjects at risk of developing such diseases. The method comprises administering to a subject a prophylactic dose of a single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate disclosed herein.

[0205] This disclosure further provides a method for diagnosing acute or chronic infections, for example, in vitro. The method includes separating a sample from a subject, contacting the sample with, for example, a single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate disclosed herein in vitro, detecting a signal generated by such contact, comparing the signal to a threshold, and determining, based on the comparison, whether the subject has an acute or chronic infection.

[0206] This disclosure further provides a method for diagnosing age-related diseases, for example, in vitro. The method includes separating a sample from a subject, contacting the sample with, for example, a single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate disclosed herein in vitro, detecting a signal generated by the contact, comparing the signal to a threshold, and determining, based on the comparison, whether the subject has an age-related disease.

[0207] This disclosure further provides a method for detecting CD16A-positive specimens in vitro. The method includes contacting a specimen in vitro with a single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate disclosed herein, detecting a signal generated by such contact, comparing the signal to a threshold, and determining whether the specimen is CD16A-positive based on the comparison result.

[0208] [Table 2] TIFF2026516120000004.tif248166TIFF2026516120000005.tif248166TIFF2026516120000006.t if248166TIFF2026516120000007.tif248166TIFF2026516120000008.tif248166TIFF20265161200 00009.tif248166TIFF2026516120000010.tif248166TIFF2026516120000011.tif248166TIFF202 6516120000012.tif248166TIFF2026516120000013.tif248166TIFF2026516120000014.tif248166

[0209] Note: Where this specification contains references to CDRs (e.g., references to SEQ ID NO: 88), the terms “IMGT,” “Kabat,” “Chothia,” or “Contact” refer to different CDR definition schemes as defined in the Definitions section. The following examples are included to illustrate preferred embodiments of the present invention and do not limit the scope of the invention.

[0210] [Example 1] CD16A VH affinity maturation This example demonstrates a method for affinity maturation by panning using a random mutant phage library. A random mutant library was constructed using the already identified CD16A VH BM156-01 (SEQ ID NO:1) as a template. NNB primers were designed for each amino acid in the annotated CDR (shown in Table 3). Antibody numbering and CDR annotation were performed according to the IMGT method. PCR was performed to construct a random mutant library for each amino acid within each CDR region.

[0211] [Table 3] TIFF2026516120000016.tif70166

[0212] Phage library panning was performed according to the following procedure: Biotin-labeled recombinant human CD16A protein (hCD16A, SEQ ID NO:2) was immobilized on streptavidin-coated magnetic beads and washed three times with PBS buffer containing 2% BSA. Then, the CD16A-immobilized beads were panned 10 times. 10 The individual phages were incubated at room temperature for 2 hours.

[0213] After incubation, the magnetic beads were washed with PBS buffer containing 0.05% Tween 20 (PBST) and resuspended in PBS. This was added to 2 mL of fresh TG1 E. coli culture and infected in a shaking incubator at 37°C and 100 rpm for 45 minutes. After infection, 10 μL of M13 helper phage was added to concentrate the library for re-amplification of the phage for the next round of panning.

[0214] The random mutant library was panned four times, using 5, 4, 2, and 1 μg of hCD16A antigen, respectively. The number of PBST washing steps was also increased stepwise with each round. After the completion of the fourth round of panning, single colonies were randomly selected from TG1 cells infected with eluted phages, and CD16A-specific conjugates were identified by monoclonal ELISA.

[0215] Specifically, hCD16A protein was coated onto 96-well ELISA plates and blocked overnight at 4°C with PBS buffer containing 2% BSA. Subsequently, 100 μL of TG1 culture medium was added to each well and incubated at 37°C for 30 minutes. After washing the plates twice with PBS containing 0.05% Tween 20, VH binding was detected using HRP-labeled anti-Flag antibody.

[0216] Clones exhibiting high binding affinity were selected, and the corresponding phagemids were isolated from TG1 cells and DNA sequencing was performed to identify the enriched VH sequences. The resulting enriched VH sequences were named CD16-VH-RM72, CD16-VH-RM73, CD16-VH-RM75, CD16-VH-RM76, CD16-VH-RM77, CD16-VH-RM80, CD16-VH-RM81, and CD16-VH-RM82. The amino acid sequences of these enriched VH sequences are shown in the sequence listing section.

[0217] [Example 2] Evaluation of VH protein production in E. coli and its binding characteristics to CD16 158F / V protein by ELISA and biolayer interferometry (BLI).

[0218] The VH proteins selected by phage panning were further validated using purified antibodies. This example describes the expression and purification of the CD16A VH protein in E. coli, as well as the evaluation of its binding characteristics to the hCD16A protein using ELISA and biolayer interferometry (BLI).

[0219] Selected VH plasmids were used to transform HB2151 competent cells. Single colonies cultured overnight in ampicillin plates were inoculated into SB medium, and after cooling the culture to 30°C, IPTG was added to induce protein expression. After culturing, the cells were collected, and VH was purified using a Ni-NTA column according to the manufacturer's instructions. The obtained purified antibody was used to evaluate its in vitro binding characteristics by ELISA.

[0220] Specifically, human CD16 158V and 158F proteins (SEQ ID NO:2 and SEQ ID NO:11) were coated onto ELISA plates and left to stand overnight at 4°C. After washing with PBS containing 0.05% Tween 20 (PBST), serially diluted VH was added and incubated at room temperature for 1 hour. Subsequently, the binding ability of VH to CD16A was detected using an anti-FLAG tag antibody.

[0221] In experiments conducted based on the above procedure, compared to BM156-01, it showed a lower bound EC. 50 Clones exhibiting values ​​(such as CD16-VH-RM72, CD16-VH-RM77, and CD16-VH-RM81) were obtained. The ELISA results are shown in Figure 1, and the EC of each VH is shown. 50 The values ​​are shown in Table 4.

[0222] [Table 4]

[0223] The binding properties of CD16A VH to the CD16A protein were also evaluated by BLI (biolayer interferometry). Biotinylated CD16A-158V or 158F proteins were immobilized on avidin-coated probes according to the manufacturer's (Octet) recommendations. CD16A VH was prepared in a 2-fold dilution series from 500 nM to 31.3 nM. The equilibrium dissociation constant (KD) for monovalent receptor binding was calculated by fitting a 1:1 Langmuir model to the obtained data.

[0224] Experiments were conducted using essentially the same procedure as described above, and it was confirmed that the binding affinity of CD16-VH-RM72, CD16-VH-RM75, CD16-VH-RM77, and CD16-VH-RM81 to CD16A-158F / V was improved compared to BM156-01. The results are shown in Figure 2 and Table 5.

[0225] [Table 5]

[0226] [Example 3] Design of bispecific antibodies with different formats and N297A / LALA mutations This embodiment demonstrates the construction and engineering of bispecific antibodies having different structural formats.

[0227] We selected BM130-93 (trastuzumab: SEQ ID NO: 12 and SEQ ID NO: 13), which has been successfully used in clinical treatment of HER2-positive breast cancer and gastric cancer, as the basic structure and constructed a bispecific antibody by fusing it with CD16A VH. CD16A VH can be fused to the heavy chain C-terminus of BM130-93 in Morrison body format via (G4S)3 linkers (SEQ ID NO: 14 and SEQ ID NO: 13) or (G4S)3 linkers (SEQ ID NO: 15 and SEQ ID NO: 13). CD16A VH can also be fused to the light chain C-terminus (SEQ ID NO: 12 and SEQ ID NO: 16) or positioned at the heavy chain N-terminus (SEQ ID NO: 17 and SEQ ID NO: 13). These different construction formats are shown in Figure 3.

[0228] The bispecific antibodies BMP01-16 to BMP01-23 shown in the examples of this specification were constructed by fusing CD16A VH to the heavy chain C-terminus of BM130-93 via a (G4S)3 linker, without modifying the light chain of BM130-93.

[0229] [Example 4] Production of bispecific antibody in CHO cells and evaluation of binding properties to CD16A (ELISA and BLI) This example shows the expression and purification of bispecific antibodies in CHO cells and the evaluation of binding to CD16A protein by ELISA and BLI.

[0230] Different heavy chains and corresponding light chains were transiently co-expressed in ExpiCHO cell lines to generate bispecific antibodies. The antibodies in the obtained culture supernatant were purified using Capturem TM Protein A Miniprep Columns (Takarabio, product number 635717) according to the manufacturer's instructions. The purified antibodies were evaluated for their binding and functional properties in vitro.

[0231] Human CD16 158V and 158F proteins (SEQ ID NO:2 and SEQ ID NO:11) were coated on ELISA plates at 4 °C overnight. After washing with PBS containing 0.05% Tween 20 (PBST), serially diluted bispecific antibody BMP01 was added and incubated at room temperature for 1 hour. An anti-human Fc tag antibody was used to detect the binding ability of the bispecific antibody to CD16A. From the ELISA results, it was confirmed that each of the bispecific antibodies BMP01-16 to BMP01-23 retained the binding ability to CD16A 158V and 158F. The detailed results are shown in Figure 4.

[0232] The binding affinity of the bispecific antibody to CD16A protein was evaluated by BLI (Biolayer Interferometry). Biotinylated CD16A-158V or 158F protein was immobilized on an avidin-coated probe according to the manufacturer's (Octet) recommendation. Bispecific antibodies and BM130-93 (trastuzumab) were prepared in a 2-fold dilution series from 500 nM to 31.3 nM. The equilibrium dissociation constant (KD) in single binding was calculated by fitting the obtained data to a 1:1 Langmuir model.

[0233] Experiments based on the above procedure confirmed that BMP01-16~19 and BMP01-21, 22 all bound to CD16A-158V with high affinity of less than nanomolar. Furthermore, compared to BM130-93 (trastuzumab), which did not show clear binding to the CD16A 158F mutant, BMP01-16~23 all showed strong binding, with affinities generally on the order of nanomolars. Detailed results are shown in Figure 5 and Table 6.

[0234] [Table 6]

[0235] [Example 5] Confirmation of the dual-target simultaneous binding ability of the bispecific antibody BMP01. This example demonstrates that the BMP01 antibody can simultaneously bind to two different targets using the sandwich ELISA method.

[0236] 50 μL of PBS solution containing human HER2 / ErbB2 protein (His-tagged, 1 μg / mL, SEQ ID NO: 32) or BSA (1 μg / mL) was added to a 96-well microplate, and the plate was incubated overnight at 4°C to prepare a HER2 antigen-coated ELISA plate. The following day, the coating solution was removed, and the plates were washed three times with 300 μL / well of PBS (PBST) containing 0.05% Tween 20. Subsequently, 300 μL / well of PBS solution containing 4% skim milk powder was added, and the plates were incubated at 37°C for 1 hour to block nonspecific binding.

[0237] The control and test samples were diluted to 15 μg / mL (initial concentration) with PBS, then serially diluted 2.5 times to create a total of 8 concentration points. After blocking, the plates were washed three times with PBST, and 50 μL / well of the diluted samples and control samples were added and incubated at 300 rpm and 37°C for 1.5 hours. After washing three more times with PBST, 50 μL / well of biotin-labeled 158V CD16A-His protein (1 μg / mL) was added and reacted at 300 rpm and 37°C for 1.5 hours. After washing three more times with PBST, 50 μL / well of detection antibody SA-HRP diluted 1:5000 was added and incubated at 300 rpm and 37°C for 1.5 hours. Finally, the plates were washed six times with PBST, 50 μL / well of the color development solution equilibrated to room temperature was added, and the plates were allowed to develop color in the dark for 15 minutes. Then, 25 μL / well of the reaction stop solution (1 M HCl) was added. The absorbance (OD value) at 450 nm was measured using a microplate reader. Using the obtained OD values, a 4-parameter fitting was performed with concentration on the x-axis and mean value on the y-axis, and the EC was calculated. 50 The result was calculated.

[0238] Based on the experimental results using the above procedure, only BMP01-16 and BMP01-20 can simultaneously bind to two types of targets, and each EC 50 The nucleotide levels were 0.2642 nM for BMP01-16 and 0.2947 nM for BMP01-20. On the other hand, no double bonds were observed in BM130-93 (trastuzumab) and the isotype control. Detailed results are shown in Figure 6.

[0239] [Example 6] Evaluation of cross-binding properties for CD16A of different origins.

[0240] The binding affinity of bispecific antibodies to human, monkey (SEQ ID NO:33), mouse (SEQ ID NO:34), and rat (SEQ ID NO:35) CD16 proteins was evaluated by BLI (Biolayer Interferometry). Biotinylated CD16A-158V or 158F proteins were immobilized on avidin-coated probes according to the manufacturer's (Octet) recommendations. BMP01-16 was prepared in a 2-fold dilution series starting from 200 nM, and trastuzumab was prepared in a 2-fold dilution series starting from 140 nM. The equilibrium dissociation constant (KD) for monovalent binding was calculated by fitting a 1:1 Langmuir model to the obtained data.

[0241] Experiments conducted using the same procedure as described above showed that BMP01-16 bound to human and monkey-derived CD16A with high affinity (0.11 nM and 3.31 nM, respectively). In contrast, it showed significantly lower affinity to mouse and rat-derived CD16 proteins (0.645 μM and 0.0958 μM, respectively). Trastuzumab, used as a control, showed weak binding to CD16 of all species. Detailed results are shown in Table 7.

[0242] [Table 7]

[0243] [Example 7] Evaluation of coupling compatibility with CD16B

[0244] Human CD16A and CD16B (SEQ ID NO: 36) proteins were coated onto ELISA plates overnight at 4°C. After washing with PBS (PBST) containing 0.05% Tween 20, serially diluted BMP01-16, BM130-92 (margituzumab: SEQ ID NO: 37 and SEQ ID NO: 38), and BM130-93 (trastuzumab) antibodies were added to the plates and incubated at room temperature for 1 hour. The binding ability of bispecific antibodies and control antibodies to CD16 was detected using anti-human Fc-tagged antibodies.

[0245] As a result of experiments conducted by basically the same procedure as described above, the EC values of BMP01-16 for CD16A-158V and CD16B were 1.255 nM and 1.296 μM, respectively. From this result, it was suggested that BMP01-16 has a binding affinity for CD16A-158V that is more than 1000 times stronger than that for CD16B. In contrast, BM130-92 (margetuximab) and BM130-93 (trastuzumab) bound to CD16A-158V at EC values of 45.09 nM and 110.9 nM, respectively, but almost no binding to CD16B was observed. Detailed results are shown in Figure 7 and Table 8. 50 values, respectively, bound to CD16A-158V, but almost no binding to CD16B was observed. Detailed results are shown in Figure 7 and Table 8. 50

[0246]

Table 8

[0247] [Example 8] Evaluation of the Cell Surface HER2 Binding Ability of Bispecific Antibodies In this example, the binding ability of the bispecific antibody BMP01 to cell surface HER2 was evaluated by FACS analysis.

[0248] 5×10 cells each of tumor cells with different HER2 expression levels, namely the high-expression strain SK-BR-3, the medium-expression strain JIMT-1, and the low-expression strain MDA-MB-231, were used. Each cell was incubated with Human TruStain FcX at room temperature for 10 minutes. 5 cells were used. Each cell was incubated with Human TruStain FcX at room temperature for 10 minutes. TM ​After blocking with Biolegend (Catalog No. 422302), antibody variants (100 nM, 10 nM, 1 nM) were added and the mixture was reacted at 4°C for 30 minutes. IgG1 was used as the isotype control. After the reaction, the cells were washed with PBS containing 3% BSA, and the amount of antibody bound to the cell surface was detected using APC-labeled AffiniPure F(ab')2 Fragment Goat Anti-Human IgG (Jackson ImmunoResearch Laboratories). The geometric mean of fluorescence intensity was measured using an Attune cell analyzer (Invitrogen), and the data was analyzed using Flowjo software (Tree Star Inc.).

[0249] Experiments based on the above procedure showed that BM130-93 and all BMP01 antibody variants bound to HER2 on the cell surface to a degree similar to that of HER2 receptors. A tendency was observed for the amount of bound BM130-93 and BMP01 antibodies to increase with higher levels of HER2 receptor expression on the cell surface.

[0250] [Example 9] ADCC Reporter Assay

[0251] In this example, the ADCC (antibody-dependent cell-mediated cytotoxicity) of the BMP01 antibody was evaluated using a luciferase reporter assay with Jurkat / NFAT-luc-CD16A transgenic cell line as the effector cell line and targeting HER2 or 5T4-expressing tumor cell lines.

[0252] On the day before the ADCC experiment, HER2-positive or 5T4-positive target cells were administered in 1 × 10⁶ units. 4 Cells were seeded at a density of cells / well (100 μL / well) in a 96-well plate and cultured overnight at 37°C under 5% CO2 conditions. On the day of the experiment, the test antibody was serially diluted 5-fold in RPMI-1640 medium containing 10% heat-inactivated FBS, and 50 μL was added to each well of target cells. BM130-93 (trastuzumab) or m603 (anti-5T4 antibody, SEQ ID NO: 29 and SEQ ID NO: 30) were used as positive controls. Next, 1 × 10⁶ cells were sampled.4 Jurkat-NFAT-Luc2-CD16A-158V or 158F effector cells (50 μL / well) were added to each well and cultured at 37°C under 5% CO2 conditions for 6 hours. After culturing, 100 μL of the Steady-Glo Luciferase Assay System reaction substrate was added to each well, the cells were lysed by pipetting, and then allowed to stand at room temperature for 5-30 minutes. The cells were transferred to a 96-well white plate for fluorescence intensity measurement, and the fluorescence intensity was measured using a microplate reader, taking care to avoid introducing air bubbles. The ADCC luciferase induction factor was calculated using the relative luminescence unit (RLU) values ​​of the test well and control well using the following formula: Induction ratio = (RLU test drug - RLU medium control) / (RLUNC - RLU medium control) GraphPad Prism software was used for statistical analysis and graph creation. Culture medium control: Wells to which only culture medium has been added. NC control: Wells in which only target cells and effector cells were co-cultured without the addition of the test antibody.

[0253] Experiments based on the same procedure as described above confirmed ADCC induction in all antibody-treated groups, depending on antibody concentration and target cells. BMP01 is EC 50 (See Table 9) In terms of both ADCC activity and maximum induction ratio, it showed superior ADCC activity compared to BM130-93 (trastuzumab). Detailed results are shown in Figure 9.

[0254] [Table 9]

[0255] Mutants BM130-90 and BM130-91, created by substituting the L234 and L235 residues or the N297 residue in the Fc region of trastuzumab with alanine, both completely lost ADCC activity. On the other hand, BM130-93 retained its activity. In contrast, the LALA mutant (BMP01-24) and N297A mutant (BMP01-25) of BMP01-16 still showed activity in the same assay, albeit with a slight decrease. These results suggest that CD16A VH significantly contributes to the excellent NK cell stimulating activity of the BMP01 antibody. Detailed results are shown in Figure 10.

[0256] In experiments conducted according to the above procedure, treatment with BMP02-10 (SEQ ID NO:30 and SEQ ID NO:31) enhanced ADCC activity to varying degrees depending on the cell type and 5T4 expression level. Compared to the control 5T4 antibody m603, BMP02-10 enhanced EC 50 It showed excellent ADCC activity in both the EC2 and maximum induction ratios. For example, in the JIMT-1 assay... 50 The values ​​were 2.8 nM for BMP02-10 and 95 nM for m603. Detailed results are shown in Figure 11 and Table 10.

[0257] [Table 10]

[0258] [Example 10] NK cell killing assay

[0259] In this example, PBMCs were used as effector cells and HER2 or 5T4-expressing tumor cell lines were used as target cells. The enhancement of NK cell-killing activity by the BMP01 antibody was evaluated using a lactate dehydrogenase (LDH) release assay.

[0260] The day before the NK cell killing experiment, 2 x 10 target cells 4Cells / 100 μL were seeded into a 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator. On the day of the experiment, the test antibody was serially diluted 5-fold in RPMI-1640 medium, and after removing the medium from the target cells, 50 μL of fresh medium and 50 μL of diluted antibody were added to each well. Subsequently, 5 × 10¹⁶ PBMC cells isolated from fresh blood were added. 5 Cells were added at a rate of 100 μL / well. Simultaneously, the following three control groups were established: (1) Spontaneous target cell release group: 50 μL of target cells + 150 μL of test medium (no test antibody); (2) Antibody-independent cytotoxicity group: 50 μL of target cells + 100 μL of PBMC cells + 50 μL of medium (no test antibody); (3) Maximum target cell release group: 50 μL of target cells + 120 μL of medium, with 30 μL / well of lysate added at the end of the experiment to measure maximum LDH release. The cells were further cultured for 4-6 hours under conditions of 37°C and 5% CO2.

[0261] After incubation, 50 μL of the supernatant from each well was transferred to a 96-well transparent flat-bottom plate, and 50 μL of CytoTox 96(R) reagent was added. The mixture was reacted at room temperature in the dark for 30 minutes. Subsequently, 50 μL of stop solution was added, and the absorbance was measured at 490 nm or 492 nm within 1 hour. The cell elimination rate was calculated using the following formula. GraphPad Prism software was used for statistical analysis and graph creation of the data. Cell killing rate (%) = (OD of test antibody group - OD of antibody-independent cell-mediated cytotoxicity group) / (OD of maximum target cell release group - OD of spontaneous target cell release group) × 100

[0262] Experiments based on the above procedure showed that BMP01 treatment induced cell killing to varying degrees depending on the cell type and HER2 expression level. In all cases except HT-29, BMP01-16 was EC 50 It showed superior enhancement of NK cell killing activity compared to BM130-93 (trastuzumab) in both the catalytic and maximal induction ratios. Consistent with the previous luciferase reporter assay, the LALA mutant (BMP01-24) and N297A mutant (BMP01-25) of BMP01-16 still retained NK cell killing activity, albeit with some reduction. Detailed results are shown in Figure 12.

[0263] Experiments based on the same procedure as described above showed that treatment with BMP02-10 enhanced NK cell killing against target cells to varying degrees, depending on cell type and 5T4 expression level. Compared to the control 5T4 antibody m603, BMP02-10 enhanced EC 50 It showed excellent cell-killing activity at both the maximum induction magnification and the cytotoxicity level. Detailed results are shown in Figure 13.

[0264] [Example 11] In vitro IFN-γ release assay

[0265] In this example, the innate immune cell stimulating activity of BMP01 was evaluated by an IFN-γ release assay using PBMCs as effector cells and HER2-expressing tumor cell lines as target cells.

[0266] The day before the IFN-γ release experiment, 2 x 10⁶ target cells 4 Cells / 100 μL were seeded into a 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator. On the day of the experiment, the test antibody was serially diluted 5-fold in RPMI-1640 medium. After removing the medium from the target cells, 50 μL of fresh medium and 50 μL of diluted antibody were added to each well. Subsequently, 5 × 10¹⁶ PBMC cells isolated from fresh blood were added. 5 The cells were added at a rate of 100 μL / well and incubated at 37°C under 5% CO2 conditions for 72 hours. After incubation, 50 μL of the supernatant was transferred to a 96-well clear flat-bottom plate, and the IFN-γ concentration was measured according to the recommended conditions of the Human IFN-γ Quantikine ELISA Kit (R&D Systems).

[0267] Experiments based on the above procedure revealed that BMP01-16 treatment induced varying degrees of IFN-γ depending on cell type and HER2 expression level. In all cases except HT-29, BMP01-16 induced higher IFN-γ production than BM130-93, suggesting stronger NK cell activation by the BMP01 bispecific antibody. In this assay, the LALA variant (BMP01-24) and N297A variant (BMP01-25) of BMP01-16 showed some enhancement compared to BMP01-16, although there were differences in their activity. Detailed results are shown in Figure 14.

[0268] [Example 12] In vivo efficacy evaluation of BMP01-16 in a JIMT-1 / PBMC mixed mouse model. In this example, the in vivo antitumor effect of the BMP01-16 antibody was evaluated using a JIMT-1 / PBMC co-mouse model.

[0269] On day 0 of the experiment, fresh human PBMC cells (5 × 10 6 (1 x 10) and human breast cancer cell line JIMT-1 (1 x 10) 6 The cells were pre-mixed at an effector-to-target ratio (E / T ratio) of 5:1 and subcutaneously transplanted into 6-8 week old female NOD / SCID mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.). After transplantation, the mice were randomly divided into 8 groups of 5 mice each. From day 0, BM130-93 or BMP01-16 were administered intraperitoneally at three different doses (BM130-93: 0.5, 1.5, 5 mg / kg; BMP01-16: 0.6, 1.8, 6 mg / kg). Administration was carried out twice a week for 4 weeks. In addition, two control groups were established: one transplanted with only JIMT-1 tumor cells and another administered via vehicle. Tumor volume was measured twice a week from the day of transplantation, and data analysis and graphing were performed using GraphPad Prism software.

[0270] Based on the experimental results using the procedure described above, significant tumor growth inhibition was observed in both the medium-dose and high-dose BM130-93 (trastuzumab) and BMP01-16 groups at the end of the study (day 32). In contrast, no tumor inhibition was observed in the low-dose group of BM130-93 (0.5 mg / kg), but significant tumor growth inhibition was observed in the low-dose group of BMP01-16 (0.6 mg / kg). This result indicates that BMP01-16 has higher antitumor activity in this model. Detailed results are shown in Figure 15.

[0271] [Example 13] In vivo efficacy evaluation of BMP01-16 in the huHSC-NCG-hIL15 / HCC1954 model

[0272] In this example, the in vivo antitumor effect of the BMP01-16 antibody was evaluated using a mouse subcutaneous transplantation model of the human breast cancer cell line HCC1954 / huHSC-NCG-hIL15.

[0273] NCG-IL15 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Human CD34 was injected into the tail vein of irradiated NCG-hIL15 mice. + Hematopoietic stem cells were transplanted to create huHSC-NCG-hIL15 human immune system reconstituted mice. After 4-6 weeks, hCD45, which accounted for more than 20% of viable cells in the peripheral blood, was observed. + Cells were detected. The human breast cancer cell line HCC1954 was purchased from Nanjing Kebai Biotechnology Co., Ltd. and cultured in RPMI-1640 medium containing 10% heat-inactivated FBS. When the required number of cells was reached, 0.2 mL of suspension (5.0 × 10⁶) was added. 6 We prepared individual HCC1954 cells + 50% Matrigel (PBS:Matrigel = 1:1) and transplanted them subcutaneously into the right forelimb of each mouse (30 mice in total).

[0274] Prior to group assignment, the level of immune reconstitution in mouse peripheral blood was analyzed by flow cytometry. The biomarkers used were hCD45, hCD3, hCD19, hCD4, hCD8, hCD56, and hCD16. The average tumor volume was approximately 100–120 mm². 3 Upon reaching a certain stage, 18 tumor-carrying mice were randomly divided into three groups (6 mice per group) based on tumor volume, body weight, and immune reconstitution level, with the coefficient of variation (CV) of tumor volume within each group being less than 1 / 3. The group division day was defined as PG-D0, and the test antibody was administered on that day. The doses were BM130-93 (trastuzumab) 0.5 mg / kg and BMP01-16 0.6 mg / kg, and both antibodies were administered intraperitoneally twice a week for 5 weeks. Body weight and tumor volume were measured twice a week, and the tumor growth inhibition rate (TGI) was calculated using the following formula. TGI (%) = (1 - T / C) × 100

[0275] Here, T and C represent the mean tumor volume (TV) at the end of the experiment for the test group and the control group. Data analysis was performed using GraphPad Prism software, and the results are shown as mean tumor volume ± SEM.

[0276] Based on the experimental results using the above procedure, the tumor volume was approximately 100-120 mm². 3 When administration was initiated at the point where the target tumor size (TGI) was reached, the BMP01-16 treatment group at 0.6 mg / kg showed significant suppression of tumor growth with a TGI of 38.9% at 38 days after group division. On the other hand, no tumor suppression effect was observed in the BM130-93 group at 0.5 mg / kg in this model. Detailed results are shown in Figure 16.

Claims

1. A single-domain antibody that specifically binds to CD16A, wherein the single-domain antibody contains the amino acid sequence shown in SEQ ID NO: 1, having substitutions at positions selected individually or in any combination from S54, G55, and S56 according to the Kabat numbering system.

2. A single-domain antibody according to claim 1, wherein the substitution is selected from the group consisting of i) S54N, S54D or S54T, ii) G55V, iii) S56Q, S56T, S56D or S56E, and any combination thereof.

3. A single-domain antibody according to claim 1, wherein the substitution is selected from i) S54N, ii) S54D, iii) S54T, iv) G55V, v) S56Q, vi) S56T, vii) S56D, viiii) S56E, ix) S54N and S56Q, or x) S54T and S56Q.

4. A single-domain antibody that specifically binds to CD16A, wherein the single-domain antibody comprises the following components. (a) A configuration in which, according to the IMGT definition scheme, CDR1 is the amino acid sequence shown in SEQ ID NO: 41 or a conservative variant thereof, CDR3 is the amino acid sequence shown in SEQ ID NO: 42 or a conservative variant thereof, and CDR2 is an amino acid sequence selected from the group SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51 and 52 or their respective conservative variants. (b) A configuration in which, according to the Kabat definition scheme, CDR1 is the amino acid sequence shown in SEQ ID NO: 53 or a conservative variant thereof, CDR3 is the amino acid sequence shown in SEQ ID NO: 54 or a conservative variant thereof, and CDR2 is an amino acid sequence selected from the group SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63 and 64 or their respective conservative variants. (c) A configuration in which, according to the Chothia definition scheme, CDR1 is the amino acid sequence shown in SEQ ID NO: 65 or a conservative variant thereof, CDR3 is the amino acid sequence shown in SEQ ID NO: 66 or a conservative variant thereof, and CDR2 is an amino acid sequence selected from the group SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75 and 76 or their respective conservative variants. (d) A configuration in which, according to the Contact definition scheme, CDR1 is the amino acid sequence shown in SEQ ID NO: 77 or a conservative variant thereof, CDR3 is the amino acid sequence shown in SEQ ID NO: 78 or a conservative variant thereof, and CDR2 is an amino acid sequence selected from the group SEQ ID NO: 79, 80, 81, 82, 83, 84, 85, 86, 87, and 88 or their respective conservative variants. However, the single-domain antibody does not have the amino acid sequence shown in SEQ ID NO:

1.

5. A single-domain antibody according to claim 4, wherein the single-domain antibody comprises an amino acid sequence selected from the group SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, and 40, or an amino acid sequence having at least 80% identity with each of them.

6. A single-domain antibody according to any one of claims 1 to 5, wherein the single-domain antibody is a humanized antibody, a human antibody, a chimeric antibody, or a camelized antibody.

7. A heavy chain antibody comprising a single-domain antibody according to any one of claims 1 to 6 and an Fc portion linked to the single-domain antibody.

8. A bispecific antibody comprising a single-domain antibody according to any one of claims 1 to 6.

9. A bispecific antibody according to claim 8, wherein the bispecific antibody comprises a single-domain antibody that specifically binds to CD16A and a second binding domain that specifically binds to a second target selected from tumor-associated antigens or tumor-specific antigens.

10. The bispecific antibody according to claim 9, wherein the second target is HER2, 5T4, PSMA, BCMA, FFFR, CD20, CD33, CD19, CD22, CD123, CD30, GPC-3, CEA, EGFR1, EGFR2, EGFR3, TGF-β, ROR1, PD-L1, Claudin18.2, EpCAM, GD2, MSLN, EGFR, MUC1, MUC2, EGFRVIII, CD38, Trop-2, c-M A bispecific antibody selected from the group consisting of ET, Nectin-4, CD79b, CCK4, GPA33, HLA-A2, CLEC12A, p-cadherin, TDO2, MART-1, Pmel17, MAGE-1, AFP, CA125, TRP-1, TRP-2, NY-ESO, PSA, CDK4, BCA225, CA125, MG7-Ag, NY-CO-1, RCAS1, SDCCAG16, TAAL6, and TAG72.

11. A bispecific antibody according to any one of claims 8 to 10, wherein the single-domain antibody is optionally linked to a full-length IgG antibody via a peptide linker.

12. A bispecific antibody according to claim 11, wherein the peptide linker is composed of a glycine residue and a serine residue.

13. A bispecific antibody according to claim 11 or claim 12, wherein the single-domain antibody is ligated to the C-terminus of the heavy chain, the C-terminus of the light chain, or the N-terminus of the heavy chain of the full-length IgG antibody.

14. A bispecific antibody according to claim 13, wherein the full-length IgG antibody is an anti-HER2 antibody or an anti-5T4 antibody.

15. A bispecific antibody according to claim 14, wherein the anti-HER2 antibody comprises the following components. (a) A heavy chain having the amino acid sequence shown in SEQ ID NO: 12 and a light chain having the amino acid sequence shown in SEQ ID NO:

13. (b) A heavy chain having the amino acid sequence shown in SEQ ID NO: 37 and a light chain having the amino acid sequence shown in SEQ ID NO:

38. (c) A heavy chain having the amino acid sequence shown in SEQ ID NO: 25 and a light chain having the amino acid sequence shown in SEQ ID NO:

13. (d) A heavy chain having the amino acid sequence shown in SEQ ID NO: 26 and a light chain having the amino acid sequence shown in SEQ ID NO:

13.

16. A bispecific antibody according to claim 14, wherein the anti-5T4 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 29 and a light chain having the amino acid sequence shown in SEQ ID NO:

30.

17. A bispecific antibody according to claim 14, wherein the bispecific antibody comprises the following components. (a) A heavy chain having the amino acid sequence shown in SEQ ID NO: 14 and a light chain having the amino acid sequence shown in SEQ ID NO:

13. (b) A heavy chain having the amino acid sequence shown in SEQ ID NO: 15 and a light chain having the amino acid sequence shown in SEQ ID NO:

13. (c) A heavy chain having the amino acid sequence shown in SEQ ID NO: 17 and a light chain having the amino acid sequence shown in SEQ ID NO:

13. (d) A heavy chain having the amino acid sequence shown in SEQ ID NO: 12 and a light chain having the amino acid sequence shown in SEQ ID NO:

16. (e) A heavy chain having the amino acid sequence shown in SEQ ID NO: 18 and a light chain having the amino acid sequence shown in SEQ ID NO:

13. (f) A heavy chain having the amino acid sequence shown in SEQ ID NO: 19 and a light chain having the amino acid sequence shown in SEQ ID NO:

13. (g) A heavy chain having the amino acid sequence shown in SEQ ID NO: 20 and a light chain having the amino acid sequence shown in SEQ ID NO:

13. (h) A heavy chain having the amino acid sequence shown in SEQ ID NO: 21 and a light chain having the amino acid sequence shown in SEQ ID NO:

13. (i) A heavy chain having the amino acid sequence shown in SEQ ID NO: 22 and a light chain having the amino acid sequence shown in SEQ ID NO:

13. (j) A heavy chain having the amino acid sequence shown in SEQ ID NO: 23 and a light chain having the amino acid sequence shown in SEQ ID NO:

13. (k) A heavy chain having the amino acid sequence shown in SEQ ID NO: 24 and a light chain having the amino acid sequence shown in SEQ ID NO:

13. (l) A heavy chain having the amino acid sequence shown in SEQ ID NO: 27 and a light chain having the amino acid sequence shown in SEQ ID NO:

13. (m) A heavy chain having the amino acid sequence shown in SEQ ID NO: 28 and a light chain having the amino acid sequence shown in SEQ ID NO:

13.

18. A bispecific antibody according to claim 14, wherein the bispecific antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 31 and a light chain having the amino acid sequence shown in SEQ ID NO:

30.

19. An immunoconjugate comprising a single-domain antibody according to any one of claims 1 to 6, a heavy-chain antibody according to claim 7, or a bispecific antibody according to any one of claims 8 to 18, further comprising an active site, a pharmaceutically acceptable salt thereof, or a solvate thereof.

20. An immunoconjugate according to claim 19, wherein the active site is selected from the group consisting of toxins, peptide tags, sotags, radionuclides, near-infrared fluorescent dyes, and nanoparticles.

21. A pharmaceutical composition comprising a single-domain antibody according to any one of claims 1 to 6, a heavy-chain antibody according to claim 7, a bispecific antibody according to any one of claims 8 to 18, or an immunoconjugate according to claim 19 or 20, further comprising a pharmaceutically acceptable carrier.

22. A single-domain antibody according to any one of claims 1 to 6, a heavy-chain antibody according to claim 7, a bispecific antibody according to any one of claims 8 to 18, or a nucleic acid molecule encoding an immunoconjugate according to claim 19 or claim 20.

23. An expression vector comprising the nucleic acid molecule described in claim 22.

24. A non-human host cell comprising the expression vector according to claim 23.

25. Use in the manufacture of a pharmaceutical product for the treatment, prevention or diagnosis of cancer, acute or chronic infectious disease or age-related disease, using a single-domain antibody according to any one of claims 1 to 6, a heavy-chain antibody according to claim 7, a bispecific antibody according to any one of claims 8 to 18, an immunoconjugate according to claim 19 or 20, or a pharmaceutical composition according to claim 21.

26. A polypeptide comprising a single-domain antibody according to any one of claims 1 to 6 or a heavy-chain antibody according to claim 7, further comprising one or more amino acid residues covalently bonded to the N-terminus, C-terminus, or any position between them of the single-domain antibody or the heavy-chain antibody.

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