Anti-CD16A antibodies and uses thereof

Abstract

Disclosed are a single-domain antibody specifically binding to CD16A, a heavy-chain antibody comprising the single-domain antibody, a bispecific antibody comprising the single-domain antibody, an immunoconjugate comprising the single-domain antibody, and a conjugate comprising the single-domain antibody. And a pharmaceutical composition and its use for the treatment, prevention or diagnosis of cancer, acute or chronic infection or aging-related diseases.

Description

Technical Field

[0001] This disclosure relates to anti-CD16A single-domain antibodies, heavy chain antibodies, bispecific antibodies, immune conjugates, pharmaceutical compositions, and their use in the treatment, prevention, or diagnosis of cancer, acute or chronic infections, or age-related diseases. Background Technology

[0002] Natural killer (NK) cells are characterized by the expression of the cell adhesion marker CD56 and the T cell receptor CD3 (CD56). + CD3 - The absence of CD56 is used to define NK cells. Based on the cell surface density of CD56, NK cells can be divided into two functionally distinct subsets: CD56-overexpressing and CD56-overexpressing. bright ) and CD56 low expression (CD56) dim CD56 accounts for approximately 10% of circulating NK cells. bright NK cells are generally considered more proliferative, exhibiting higher cytokine production capacity after stimulation with IL-12 and IL-18, and possessing poor cytotoxic effector activity in the resting state. However, CD56... dim NK cells exhibit potent cytotoxicity without stimulation, mediate antibody-dependent cytotoxicity (ADCC), and produce cytokines upon stimulation with target cells.

[0003] CD16 (FcγRIII) binds to the Fc portion of IgG antibodies; one type, CD16A, is a transmembrane protein co-localized with CD3ζ and Fc-εRI-γ on NK cells. Upon binding, it induces a potent signaling pathway leading to cytokine production and cytotoxic effector activity via ADCC. The second type, CD16B, is found on neutrophils and is not involved in tumor cell killing. Although the extracellular domains are highly homologous, the glycosylphosphatidylinositol bond distinguishes CD16B from CD16A. Most CD56 in peripheral blood... bright NK cells express almost no CD16A. In contrast, most CD56 cells express CD16A. dim Cells uniformly expressed high levels of CD16A. Downregulation of CD16A occurred after mitogen stimulation and co-culture with malignant targets, a process hypothetically mediated by metalloproteinases. This process may be important for rapidly regulating CD16A surface density, thereby modulating NK cell activation and effector function.

[0004] In antibody-mediated immunotherapy, the ADCC function of NK cells is highly sought after. Bispecific antibodies with two targeting modules (one for recruiting the ADCC receptor CD16A and the other for recognizing antigens) have attracted widespread attention due to their high efficiency in binding NK cells to kill target cells in vitro and in vivo.

[0005] Currently, several forms of these bispecific antibodies are under basic and clinical research, such as bispecific killer cell connectors (BiKE), bispecific biantibodies (BidAb), and tetravalent bispecific TandAb. Recently, Affimed Therapeutics AG (Heidelberg, Germany) developed a more promising form, TandAb. TandAb is a tetravalent bispecific tandem biantibody composed of head-to-tail homodimers of two tandem scFvs linked by three (GGS)3 linkers. One TandAb, CD16 / CD30 AFM13, has completed a phase II clinical trial in patients with relapsed or refractory Hodgkin lymphoma (Clinical Trial No. NCT02321592). However, TandAb may encounter more manufacturing challenges than BidAb because it contains more subdomains, all of which must be precisely paired to ensure the correct TandAb. Furthermore, due to its tetravalent nature (CD16A is bivalent, CD30 is bivalent), TandAb carries the risk of nonspecific activation of CD16A and inducing side effects in the absence of CD30. Summary of the Invention

[0006] In one aspect, a single-domain antibody that specifically binds to CD16A is provided, wherein the single-domain antibody has the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence comprising substitutions at positions selected from: S54, G55, S56 and any combination thereof according to the Kabat numbering system.

[0007] In some implementations, the substitution is selected from (i) S54N, S54D or S54T, (ii) G55V, (iii) S56Q, S56T, S56D or S56E, and any combination thereof.

[0008] In some implementations, the substitutions are 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.

[0009] In one aspect, a single-domain antibody that specifically binds to CD16A is provided, wherein the single-domain antibody comprises: (a) CDR1 having the amino acid sequence shown in SEQ ID NO: 41 or a conserved variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 42 or a conserved variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or a conserved variant thereof; (b) According to the Kabat definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 53 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 54 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or a conservatively modified variant thereof; (c) According to the Chothia definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 65 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 66 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or a conservatively modified variant thereof; or (d) According to the Contact definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 77 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 78 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or a conservatively modified variant thereof; The condition is that the single-domain antibody does not have the amino acid sequence shown in SEQ ID NO: 1.

[0010] In some embodiments, the single-domain antibody comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, 40 and an amino acid sequence having at least 80% identity with each of them.

[0011] In some embodiments, the single-domain antibody is a humanized antibody, a human antibody, a chimeric antibody, or a camel-derived antibody.

[0012] Another aspect of this disclosure provides a heavy chain antibody comprising a single-domain antibody as disclosed herein and an Fc portion linked to said single-domain antibody.

[0013] Another aspect of this disclosure provides a bispecific antibody comprising a single-domain antibody as disclosed herein.

[0014] In some embodiments, the bispecific antibody comprises the 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 and tumor-specific antigens.

[0015] In some embodiments, the second target is selected from: HER2, 5T4, PSMA, BCMA, FGFR, CD20, CD33, CD19, CD22, CD123, CD30, GPC-3, CEA, EGFR1, EGFR2, EGFR3, TGF-β, ROR1, PD-L1, tight junction protein 18.2, 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, Pmel 17, MAGE-1, AFP, CA125, TRP-1, TRP-2, NY-ESO, PSA, CDK4, BCA225, CA 125, MG7-Ag, NY-CO-1, RCAS 1, SDCCAG16, TAAL6, and TAG72.

[0016] In some embodiments, the single-domain antibody is optionally linked to a complete IgG antibody via a peptide linker. In some embodiments, the peptide linker is composed of glycine and serine residues. In some embodiments, 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 complete IgG antibody.

[0017] In some embodiments, the complete IgG antibody is an anti-HER2 antibody or an anti-5T4 antibody. In some embodiments, the anti-HER2 antibody comprises: (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; or (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.

[0018] In some embodiments, the bispecific antibody comprises: (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; or (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.

[0019] In some embodiments, 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.

[0020] In some embodiments, 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.

[0021] Another aspect of this disclosure provides an immunoconjugate, a pharmaceutically acceptable salt thereof, or a solvation thereof, comprising a single-domain antibody, a heavy-chain antibody, or a bispecific antibody as disclosed herein; and an active moiety.

[0022] In some embodiments, the active portion is selected from toxins, peptide tags, sortag, radionuclides, near-infrared fluorescent dyes, and nanoparticles.

[0023] Another aspect of this disclosure provides a pharmaceutical composition comprising a single-domain antibody, a heavy-chain antibody, a bispecific antibody, or an immunoconjugate as disclosed herein; and a pharmaceutically acceptable carrier.

[0024] Other aspects of this disclosure provide a nucleic acid molecule encoding a single-domain antibody, a heavy-chain antibody, a bispecific antibody, or an immunoconjugate as disclosed herein; an expression vector comprising the nucleic acid molecule disclosed herein; and a non-human host cell comprising the expression vector disclosed herein.

[0025] Another aspect of this disclosure provides the use of single-domain antibodies, heavy-chain antibodies, bispecific antibodies, immunoconjugates, or compositions disclosed herein in the preparation of medicaments for treating cancer.

[0026] Another aspect of this disclosure provides the use of single-domain antibodies, heavy-chain antibodies, bispecific antibodies, immunoconjugates, or compositions disclosed herein in the treatment of cancer, acute or chronic infections, or age-related diseases.

[0027] Another aspect of this disclosure provides a method for treating cancer, acute or chronic infection, or age-related disease, the method comprising administering to a subject in need a therapeutically effective amount of a single-domain antibody, a heavy-chain antibody, a bispecific antibody, an immunoconjugate, or a composition as disclosed herein.

[0028] Another aspect of this disclosure provides a polypeptide comprising a single-domain antibody or a heavy-chain antibody as disclosed herein; and one or more amino acid residues covalently linked to the N-terminus, C-terminus, or any position therebetween of the single-domain antibody or the heavy-chain antibody as disclosed herein.

[0029] These and other aspects and advantages of this disclosure will become apparent from the following detailed description of the invention. Attached Figure Description

[0030] Figure 1 The binding of VH selected on the ELISA with CD16A 158V and CD16A 158F. BM156-01 was used as a control.

[0031] Figure 2 The binding affinity of the selected VH to CD16A 158V and CD16A 158F was determined by biomembrane interferometry (BLI).

[0032] Figure 3 Figure 1 shows different bispecific forms of anti-CD16 VH. (A) Preferred anti-CD16 VH; (B) Trastuzumab IgG1 (Seq ID NO: 12 and Seq ID NO: 13); (C) Homodimeric constructs in which CD16 VH is fused to the C-terminus of the heavy chain (Seq ID NO: 14 and Seq ID NO: 13); (D) Multiple G4S linkers are available to attach VH to the C-terminus of the heavy chain (Seq ID NO: 15 and Seq ID NO: 13); (E) Homodimeric constructs in which CD16 VH is fused to the C-terminus of the light chain (Seq ID NO: 12 and Seq ID NO: 16); (F) Homodimeric constructs in which CD16 VH is fused to the N-terminus of the heavy chain using a (G4S)3 linker (Seq ID NO: 17 and Seq ID NO: 13).

[0033] Figure 4 The binding of bispecific antibodies to human CD16A 158F (left) and CD16A 158V (right) on ELISA.

[0034] Figure 5The binding of different bispecific antibodies to CD16A 158V and CD16A 158F was measured by BLI.

[0035] Figure 6 : BMP01-16 binds to both HER2 and CD16A on ELISA.

[0036] Figure 7 BMP01-16 binds to human CD16A-158V and CD16B on ELISA. BM130-92 (magutuximab) and BM130-93 (trastuzumab) were used as controls.

[0037] Figure 8 Different bispecific antibodies bind to the HER2 receptor expressed on the surface of SK-BR-3, JIMT-1, and MDA-MB-231 cells.

[0038] Figure 9 BMP01 showed higher ADCC potency than trastuzumab, as measured by ADCC reporter gene assay.

[0039] Figure 10 In ADCC reporter gene assays, the LALA and N297A mutations do not eliminate BMP01-12 ADCC activity.

[0040] Figure 11 According to ADCC reporter gene assay, BMP02-10 showed higher ADCC potency than m603.

[0041] Figure 12 BMP01 bispecific antibody enhances ADCC cell killing compared to trastuzumab.

[0042] Figure 13 BMP02-10 bispecific antibody enhances ADCC cell killing compared to m603.

[0043] Figure 14 BMP01 bispecific antibody enhances IFN-γ production compared to trastuzumab.

[0044] Figure 15 In the JIMT-1 / PBMC co-occurrence tumor model, BMP01-16 showed stronger antitumor activity than BMP130-93 at low doses. The average tumor growth for all groups is plotted at the top, and the tumor growth for individual mice with PBMC and PBMC+BM130-93, and PBMC and PBMC+BMP01-16 is plotted at the bottom.

[0045] Figure 16BMP01-16 inhibited HCC1954 tumor growth in the huHSC-NCG-hIL15 model. The mean tumor growth of all groups is plotted at the top, and the tumor growth of individual mice in control and BM130-93 and control and BMP01-16 are plotted at the bottom. Detailed Implementation

[0046] definition Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings.

[0047] As used herein, the term "complete IgG antibody" generally refers to a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain (HC) consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain (LC) consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and scattered with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0048] The term "complete IgG antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, and chimeric antibodies. Antibodies can be any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0049] "Heavy chain antibodies" lack a light chain, and in the case of heavy chain antibodies in camelids, the CH1 domain is also missing. Therefore, camelid heavy chain antibodies associate with their homologous antigens via a single domain, a variable heavy chain domain of the heavy chain antibody, or a VHH. The terms VHH, VH, nanobody, or single-domain antibody are used interchangeably herein. Thus, anti-CD16A VH is anti-CD16A VHH, or anti-CD16A nanobody, or anti-CD16A single-domain antibody. Throughout this disclosure, CD16A is sometimes referred to as CD16. VHH antibodies are isolated VHH domains of heavy chain-only antibodies produced by camelid 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 it is the CDR3 ring that enables VHH antibodies to have such broad binding specificity; the CDR3 ring is often 3-4 residues longer than that of conventional antibodies, allowing them to use these protrusions to extend into the “hidden” epitope cavity; the CDR3 ring also has more variation in the amino acid sequence of VHH antibodies than that of conventional antibodies, resulting in a sequence diversity of about 7% per residue.

[0050] The term "Fc moiety" refers to a polypeptide that contains the C-terminal portion of the immunoglobulin H chain and retains at least one function of the Fc region of the IgG region, particularly the function of binding to FcRn. The antibody effector function is determined by the sequence in the Fc region. The Fc moiety may contain a CH2 domain, a CH3 domain, or a CH2-CH3 polypeptide chain. The CH2-CH3 polypeptide chain assembles with another CH2-CH3 polypeptide chain to form a dimer of two CH2-CH3 polypeptides combined with each other, wherein dimerization is facilitated by a covalent link between the NC-terminus of the hinge region and the CH2 domain. Thus, in some embodiments, the Fc moiety comprises a dimer of two CH2-CH3 polypeptide chains and the hinge region. Preferably, the Fc moiety contains an Ig class constant domain, such as IgA, IgD, IgE, IgM, preferably IgG1, IgG2, IgG4, particularly the IgG1 constant domain. Examples of Fc moieties have an amino acid sequence as shown in SEQ ID NO: 89.

[0051] The "hinge" domain can be an IgG class that is the same as or different from the Fc portion, or an engineered, non-naturally occurring hinge domain. Examples of IgG hinge regions have the amino acid sequence shown in SEQ ID NO: 90. Variants of wild-type hinge regions, such as shortened hinge regions, are also included.

[0052] A "connector" refers to an amino acid sequence containing a linker peptide that connects two juxtaposed variable domains forming an antigen-binding moiety, wherein the C-terminus of one domain is connected to the N-terminus of the other juxtaposed domain, and vice versa. Regarding amino acid composition, peptide linker sequences are selected that do not interfere with the formation of the Fv (i.e., VH / VL) antigen-binding and recognition sites, and do not interfere with the polymerization (e.g., dimerization) of polypeptides of multispecific antigen-binding proteins. For example, linkers containing glycine and serine residues typically provide protease resistance. In some embodiments, (G2S) is used. x Peptide linkers, where x = 1-20, such as (G2S), (G2S)2, (G2S)3, (G2S)4, (G2S)5, (G2S)6, (G2S)7 or (G2S)8, or using (G3S). x Peptide linkers, where x = 1-15 or (G4S) is used. x Peptide linkers, wherein x = 1-10, preferably 1-6, for example. The amino acid sequence of the linker can be optimized, for example, by phage display methods, to improve the formation of antigen-binding sites and the yield of peptides.

[0053] The term "target" or "target antigen" refers to an antigen expressed by or associated with a type of cell (i.e., a target cell or a virus-infected cell) to which NK cells should be directed to induce or trigger NK cell cytotoxicity. Examples of target antigens can be tumor-specific antigens (TSA) or tumor-associated antigens (TAA). TSA or TAA can be expressed on the surface of target cells or displayed as MHC-restricted peptides by the MHC complex. Examples of tumor antigens include, but are not limited to, HER2, 5T4, PSMA, BCMA, FGFR, CD20, CD33, CD19, CD22, CD123, CD30, GPC-3, CEA, EGFR1, EGFR2, EGFR3, TGF-β, ROR1, PD-L1, tight junction protein 18.2, 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, and RCAS. 1. SDCCAG16, TAAL6 and TAG72.

[0054] In conjunction with this invention, the term "binding domain" characterizes a domain that specifically binds to / interacts with / recognizes a given target epitope or target side on a target molecule (antigen) (e.g., CD16 and target cell surface antigen). The structure and function of the first binding domain (recognizing, for example, CD16), and preferably also the structure and / or function of the second binding domain (recognizing target cell surface antigen), are based on the structure and / or function of the antibody, such as the structure and / or function of a full-length or intact immunoglobulin molecule, and / or variable heavy chain (VH) and / or variable light chain (VL) domains derived from the antibody or fragments thereof. Preferably, the first binding domain is characterized by the presence of 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 preferably also includes the minimum structural requirements of the antibody that allow target binding.

[0055] According to the present invention, the terms “(specific) binding”, “(specific) recognition”, “(specific) targeting” or “(specific) reaction with” mean that the binding domain interacts or specifically interacts with a given epitope or a given target side on a target molecule (antigen) (e.g., CD16A) and a target cell surface antigen (e.g., HER2), respectively.

[0056] The terms “non-essential / non-significantly binding” or “cannot bind” mean that the binding domain of the present invention does not bind to proteins or antigens other than CD16A and / or target cell surface antigens, that is, it does not show more than about 30%, preferably no more than about 20%, more preferably no more than about 10%, particularly preferably no more than about 9%, about 8%, about 7%, about 6% or about 5% reactivity with proteins or antigens other than CD16A and / or target cell surface antigens, thereby setting the binding to CD16A and / or target cell surface antigens to about 100%.

[0057] As used herein, the term “complementarity-determining domain” is used interchangeably with the term “complementarity-determining region” (“CDR”) and generally refers to the hypervariable region of VL and VH. A CDR is a target protein binding site of an antibody chain that is specific to that target protein. There are three CDRs (CDR1-3, numbered sequentially from the N-terminus) in each VL or VH, constituting approximately 15-20% of the variable domain. CDRs can be referred to by their 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 responsible for binding specificity. The remaining segments of VL or VH, the so-called framework regions, exhibit less variation in their amino acid sequence (Kubie Immunology, 4th ed., Chapter 4, W.H. Freeman, New York, 2000). In this art, the CDR of an antibody can be defined by various methods / schemes, such as the Kabat definition scheme based on sequence variability (see Kabat et al., Protein Sequence in Immunology, 5th Edition, NIH, Bethesda, MD (1991)), the Chothia definition scheme based on the location of the structural loop region (see Al-Lazikani et al., Journal of Molecular Biology, 273: 927-48, 1997), and the IMGT definition scheme based on the concepts of IMGT ontology and the IMGT scientific chart rules. In some embodiments, this application uses IMGT rules to define the CDR of an antibody. The definition rules of Martin, PyIgClassify and the combined definition rules of Kabat, Chothia, IMGT, Martin, and PyIgClassify are also included in this application (see Mark L. Chiu et al., Antibody, 8(4), 55, 2019).

[0058] Table A: Comparison of CDR numbers (Chothia numbers) under different definition schemes

[0059] In this context, Laa-Lbb or Haa-Hbb can refer to the amino acid sequence from position aa to position bb of the light chain or heavy chain, respectively, starting from the N-terminus. For example, L24-L34 refers to the amino acid sequence from position 24 to position 34 of the light chain.

[0060] Both the light and heavy chains are divided into structurally and functionally homologous regions. The terms "constant" and "variable" are used functionally. In this respect, it should be understood that the variable domains of the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental transfer, Fc receptor binding, complement binding, etc. By convention, the numbering of constant domains increases as they move further away from the antibody's antigen-binding site or N-terminus. The N-terminus is the variable region, and the C-terminus is the constant region; the CH3 and CL domains actually contain the carboxyl-terminal domains of the heavy and light chains, respectively.

[0061] As used herein, the term "humanized antibody" generally refers to an antibody comprising a heavy chain variable region and a light chain variable region derived from a non-human species (e.g., mouse), but wherein at least a portion of the VH and / or VL sequences has been modified to resemble a human-derived variable sequence. For example, the term "humanized antibody" is an antibody or a variant, derivative, analog, or fragment thereof capable of binding to an associated antigen with immune specificity and comprising a frame region (FR) comprising substantially the amino acid sequence of a human antibody and a complementarity-determining region (CDR) comprising substantially the amino acid sequence of a non-human antibody. In the context of CDR, the term "substantially" means that the amino acid sequence of the CDR has at least 80%, such as at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, identity with the amino acid sequence of the CDR of a non-human antibody. Humanized antibodies generally include at least one, typically two, variable domains (Fab, Fab', F(ab')2, Fab, Fv), wherein all or substantially all CDR regions correspond to CDR regions of non-human immunoglobulins, and all or substantially all frame regions are frame regions having a common sequence of human immunoglobulins. In some embodiments, humanized antibodies may also include at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of human immunoglobulins.

[0062] As used herein, the term “human antibody” generally refers to an antibody having both variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the art (e.g., Van Dyck (MA) and JG Van der Winkel, New Insights in Chemical Biology, 5(2001)368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that can produce a complete or selected set of human antibodies in the absence of endogenous immunoglobulins produced after immunization (e.g., A. Jakobowitz et al., Proceedings of the National Academy of Sciences, 90(1993)2551-2555; A. Jakobowitz et al., Nature, 362(1993)255-258; M. Brügmann et al., Year Immunol., 7(1993)33-40). Human antibodies can also be generated in phage display libraries (e.g., HR. Hugenbohm and G. Winter, Journal of Molecular Biology, 227(1992)381-388; JD. Max et al., Journal of Molecular Biology, 222(1991)581-597). The term “human antibody” can also include antibodies modified in constant regions.

[0063] As used herein, the term "chimeric antibody" generally refers to an engineered antibody that, in its broadest sense, contains one or more regions derived from one antibody and one or more regions derived from one or more other antibodies. Specifically, a chimeric antibody comprises the VH and VL domains of an antibody derived from a non-human animal, and the CH and CL domains of another antibody (particularly a human antibody). Any animal can be used as a non-human animal, such as a camel, mouse, rat, hamster, rabbit, etc.

[0064] As used herein, the term "bispecific antibody" refers to an antibody that has two specificities against one or more antigens or against different epitopes of the same antigen. For example, a bispecific antibody may have a first specificity against a first antigen (e.g., CD16A) and a second specificity against a second antigen different from the first antigen (e.g., a tumor-associated antigen or a tumor-specific antigen). For example, a bispecific antibody may have a first specificity against a first epitope of an antigen and a second specificity against a second, different epitope of the same antigen (e.g., CD16A).

[0065] When referring to polypeptide or nucleotide sequences, "purified" and "isolated" mean that the molecule in question is present in the absence of other biomolecules of the same type. As used herein, the term "purified" specifically refers to the presence of at least 75 wt%, 85 wt%, 95 wt%, or 98 wt% of the same type of biomolecule. An "isolated" nucleic acid molecule encoding a specific polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, it may include some additional bases or portions that do not adversely affect the essential characteristics of the composition. The present invention may contain, for example, isolated antigen-binding proteins, isolated antibodies or antigen-binding fragments thereof, isolated polypeptides, or one or more isolated nucleic acid molecules.

[0066] As used herein, the term "affinity" is typically defined by the balanced association between the whole antibody and the antigen. Affinity can be expressed, for example, as the half-maximal effective concentration (EC50). 50 Affinity is expressed as either the equilibrium dissociation constant (KD). Affinity can be experimentally assessed using various known methods, such as measuring association and dissociation rates using surface plasmon resonance or measuring EC in immunochemical assays (ELISA, FACS). 50 .

[0067] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to a specific nucleic acid sequence, a conservatively modified variant refers to those nucleic acids that encode the same or substantially identical amino acid sequence, or, when the nucleic acid does not encode an amino acid sequence, substantially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position of alanine specified by a codon, the codon can be changed to any of the corresponding codons without altering the encoded polypeptide. Such nucleic acid variations are "silent variants," which are variations of conserved modification. Each nucleic acid sequence encoding a polypeptide described herein also describes every possible silent variant of the nucleic acid. Those skilled in the art will recognize that every codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, each silent variant of a nucleic acid encoding a polypeptide is implicit in each of the sequences described.

[0068] For polypeptide sequences, “conserved variants” include single substitutions, deletions, or additions in the polypeptide sequence that result in the substitution of an amino acid with a chemically similar amino acid. Tables providing conserved substitutions of functionally similar amino acids are well known in the art. Such conserved variants, in addition to the above, do not exclude polymorphic variants, interspecific homologs, and alleles. The following eight groups contain amino acids that are conserved in 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); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)). In some respects, the term "conservative sequence modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing an amino acid sequence.

[0069] As used herein, in the context of two or more nucleic acid or polypeptide sequences, the term "percent identical" or "percent identity" refers to the degree to which two or more sequences or subsequences are the same. Two sequences are "identical" if they have the same amino acid or nucleotide sequence in the regions being compared. When compared and aligned in a comparison window or specified region to obtain maximum correspondence, if the two sequences have a specified percentage of the same amino acid residues or nucleotides (i.e., 60% identity in the specified region or, when not specified, throughout the entire sequence, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity) as measured by manual alignment and visual inspection, the two sequences are "substantially identical". Optionally, identity is present in regions of at least about 30 nucleotides (or 10 amino acids) in length, or more preferably in regions of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length. Two examples of algorithms suitable for determining sequence identity and sequence similarity percentage are the BLAST and BLAST 2.0 algorithms, described by Altschul, Nucleic Acid Research, 25: 3389-3402, 1977; and Altschul et al., Journal of Molecular Biology, 215: 403-410, 1990.

[0070] Besides the aforementioned percentage of sequence identity, another indication that two nucleic acid sequences or peptides are substantially identical is the presence of immune cross-reactivity between the peptide encoded by the first nucleic acid and antibodies generated against the peptide encoded by the second nucleic acid. Therefore, the peptide is often substantially identical to the second peptide; for example, the two peptides differ only through conserved substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions. Yet another indication that two nucleic acid sequences are substantially identical is that the sequences can be amplified using the same primers.

[0071] As used herein, the term "nucleic acid molecule" is used interchangeably with the term "polynucleotide" and generally refers to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and their polymers. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, or non-natural, have similar binding properties to a reference nucleic acid, and are metabolized in a similar manner to a reference nucleotide. Examples of such analogs include, but are not limited to, phosphate thioesters, aminophosphate esters, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0072] As used herein, the term "peptide" is used interchangeably with the term "protein" and refers to a polymer of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular peptide sequence also implicitly encompasses variants with conserved modifications.

[0073] As used herein, the term "immunoconjugate" generally refers to the conjugation of an antibody or an antigen-binding fragment thereof to another reagent, such as a payload, pharmaceutical moiety, chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, etc. The conjugation can be covalent or non-covalent, for example, by electrostatic forces. Various linkers known in the art can be used to form immunoconjugates. Additionally, immunoconjugates can be provided in the form of fusion proteins, which can be expressed by polynucleotides encoding the immunoconjugate. As used herein, "fusion protein" refers to a protein produced by conjugating two or more genes or gene segments that originally encode different proteins, including peptides and polypeptides. Translation of the fusion gene produces a single protein having functional properties derived from each of the original proteins.

[0074] As used herein, the terms “toxin,” “cytotoxic agent,” or “cytotoxic agent” generally refer to any agent that is harmful to cell growth and proliferation and can reduce, inhibit, or destroy cells or malignant tumors.

[0075] As used herein, and as may be used interchangeably, “cancer” or “tumor” means a group of diseases that can be treated according to this disclosure and involve the growth of abnormal cells that have the potential to invade or spread to other parts of the body. Not all tumors are cancerous; benign tumors do not spread to other parts of the body. Possible signs and symptoms include: a new lump, unusual bleeding, prolonged cough, unexplained weight loss, and changes in bowel movements. There are more than 100 different known cancers affecting humans. As used herein, “cancer” includes, but is not limited to, solid tumors (such as tumors) and blood malignancies. “Blood malignancies,” also known as blood cancers, are cancers that originate in hematopoietic tissues (such as bone marrow or other cells of the immune system). Hematologic malignancies include, but are not limited to, leukemias (such as acute myeloid leukemia (ANIL), acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), acute mixed spectrum leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia (CLL), hairy cell leukemia, and large granular lymphocytic leukemia), myelodysplastic syndromes (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocythemia, primary myelofibrosis, and chronic myeloid leukemia), lymphomas, multiple myeloma, MGUS and similar disorders, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies. Solid tumors include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, laryngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and nipple carcinoma. Adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, retinoblastoma.

[0076] As used herein, the term “antitumor agent” or “antitumor drug” generally refers to any agent that can be used to treat cell-proliferating diseases such as cancer, including but not limited to cytotoxic agents, chemotherapy agents, radiotherapy and radiotherapy agents, targeted anticancer agents, and immunotherapy agents.

[0077] As used herein, the term "antitumor activity" refers to a reduction in the rate of tumor cell proliferation, viability, or metastatic activity. Possible ways to demonstrate antitumor activity include showing a decrease in tumor cell growth rate, tumor size arrest, or tumor size reduction. This activity can be assessed using recognized in vitro or in vivo tumor models, including but not limited to xenograft models, allogeneic transplant models, MMTV models, and other known models in the art for studying antitumor activity.

[0078] As used herein, the term “subject” includes both humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise stated, the terms “patient” or “subject” are used interchangeably herein.

[0079] As used herein, the term "pharmaceutically acceptable" generally refers to one or more non-toxic substances that do not interfere with the effectiveness of the bioactivity of the active ingredient. Such formulations may typically contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also typically contain compatible solid or liquid fillers, diluents, or encapsulating materials suitable for human administration. When used in pharmaceuticals, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used to prepare pharmaceutically acceptable salts and are not excluded from the scope of this invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, succinic acid, etc. Pharmacologically acceptable salts may also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium, or calcium salts. The term "solvent" is used in its conventional sense herein to refer to a complex of a solute (e.g., an active compound, a salt of an active compound) and a solvent. Solvents generally do not significantly alter the physiological activity or toxicity of a compound and can therefore function as pharmacological equivalents. If the solvent is water, the solvent compound may be conveniently referred to as a hydrate, such as a monohydrate, dihydrate, trihydrate, etc.

[0080] As used herein, the terms “treat,” “treating,” or “treatment” for any disease or condition generally refer in one respect to improving a disease or condition (i.e., slowing or stopping or reducing the development of a disease or at least one of its clinical symptoms). In another respect, “treat,” “treating,” or “treatment” refers to reducing or improving at least one bodily parameter, including those that the patient may not be able to discern. In yet another respect, “treat,” “treating,” or “treatment” refers to regulating a disease or condition physically (e.g., stabilizing discernible symptoms), physiologically (e.g., stabilizing bodily parameters), or both. In yet another respect, “treat,” “treating,” or “treatment” refers to preventing or delaying the onset, development, or progression of a disease or condition.

[0081] As used herein, the terms "therapeuticly acceptable amount" or "therapeutic effective dose" are interchangeable in meaning an amount sufficient to achieve the desired outcome (i.e., tumor size reduction, inhibition of tumor growth, prevention of metastasis, inhibition or prevention of viral, bacterial, fungal, or parasitic infections). In some embodiments, the therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically acceptable amount can be determined by initially administering a low dose and then gradually increasing that dose until the desired effect is achieved. The "preventive effective dose" and "therapeutic effective dose" of the molecules of this disclosure can respectively prevent the onset of disease symptoms (including cancer-related symptoms) or reduce the severity of disease symptoms (including cancer-related symptoms).

[0082] As used herein, unless otherwise stated, the terms “comprising,” “containing,” “having,” “including,” and “including” should be interpreted as “including, but not limited to.” Unless otherwise stated, in the context of describing the invention, and particularly in the context of the appended claims, the terms “an,” “a,” and “the,” and similar indicators should be interpreted to cover both the singular and plural. Unless otherwise required, the use of any and all example or exemplary language (“e.g.,” “such as,” “like”) is intended only to illustrate aspects or embodiments of the invention and should not be construed as limiting its scope.

[0083] As used herein, when referring to measurable values ​​such as quantity, duration, etc., the term “about” means to cover a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% compared to a specified value, because such variation is suitable for performing the disclosed methods.

[0084] The term "vector" includes shuttle vectors and expression vectors. Typically, a vector can be a plasmid construct that also includes an origin of replication (e.g., Col E1 origin of replication) and a selection marker (e.g., ampicillin or tetracycline resistance) for replication and selection of the plasmid in bacteria, respectively. "Expression vector" refers to a vector containing the control sequence or regulatory element necessary for expressing an antibody, including the antibody fragment of this disclosure, in bacteria or eukaryotic cells.

[0085] Anti-CD16A single-domain antibody

[0086] In one aspect, this disclosure provides an anti-CD16A single-domain antibody, wherein the single-domain antibody has the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence comprising substitutions at positions selected from: S54, G55, S56 and any combination thereof according to the Kabat numbering system.

[0087] In some embodiments, the anti-CD16A single-domain antibody has a substituted amino acid sequence as shown in SEQ ID NO: 1 at position S54. In some embodiments, the anti-CD16A single-domain antibody has a substituted amino acid sequence as shown in SEQ ID NO: 1 at position G55. In some embodiments, the anti-CD16A single-domain antibody has a substituted amino acid sequence as shown in SEQ ID NO: 1 at position S56.

[0088] In some embodiments, the anti-CD16A single-domain antibody has a substituted amino acid sequence as shown in SEQ ID NO: 1 at positions S54 and G55. In some embodiments, the anti-CD16A single-domain antibody has a substituted amino acid sequence as shown in SEQ ID NO: 1 at positions S54 and S56. In some embodiments, the anti-CD16A single-domain antibody has a substituted amino acid sequence as shown in SEQ ID NO: 1 at positions G55 and S56. In some embodiments, the anti-CD16A single-domain antibody has a substituted amino acid sequence as shown in SEQ ID NO: 1 at positions S54, G55, and S56.

[0089] In some embodiments, the anti-CD16A single-domain antibody has the amino acid sequence shown in SEQ ID NO: 1, which includes substitutions at positions S54, G55, and S56 and one or more additional substitutions at positions other than S54, G55, and S56. In these embodiments, the one or more additional substitutions are preferably conservative substitutions.

[0090] In some embodiments, the serine residue at position 54 is replaced by an amino acid residue other than serine. For example, the serine residue at position 54 is replaced by any one of 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 by asparagine (N), aspartic acid (D), or threonine (T).

[0091] In some embodiments, the glycine residue at position 55 is replaced by an amino acid residue other than glycine. For example, the glycine residue at position 55 is replaced by any one of 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 by valine (V).

[0092] In some embodiments, the serine residue at position 56 is replaced by an amino acid residue other than serine. For example, the serine residue at position 56 is replaced by any one of 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 by glutamine (Q), threonine (T), aspartic acid (D), or glutamic acid I.

[0093] In some implementations, the substitution is selected from (i) S54N, S54D or S54T; (ii) G55V; (iii) S56Q, S56T, S56D or S56E; and any combination thereof.

[0094] In some embodiments, the single-domain antibody has the amino acid sequence shown in SEQ ID NO: 1, having a single substitution of S54N according to the Kabat numbering system. In some embodiments, the single-domain antibody has the amino acid sequence shown in SEQ ID NO: 1, having a single substitution of S54D according to the Kabat numbering system. In some embodiments, the single-domain antibody has the amino acid sequence shown in SEQ ID NO: 1, having a single substitution of S54T according to the Kabat numbering system.

[0095] In some embodiments, the single-domain antibody has the amino acid sequence shown in SEQ ID NO: 1, which has a single substitution of G55V according to the Kabat numbering system.

[0096] In some embodiments, the single-domain antibody has the amino acid sequence shown in SEQ ID NO: 1, with a single substitution of S56Q according to the Kabat numbering system. In some embodiments, the single-domain antibody has the amino acid sequence shown in SEQ ID NO: 1, with a single substitution of S56T according to the Kabat numbering system. In some embodiments, the single-domain antibody has the amino acid sequence shown in SEQ ID NO: 1, with a single substitution of S56D according to the Kabat numbering system. In some embodiments, the single-domain antibody has the amino acid sequence shown in SEQ ID NO: 1, with a single substitution of S56E according to the Kabat numbering system.

[0097] In some implementations, the substitutions are 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.

[0098] In some embodiments, the single-domain antibody has the amino acid sequence shown in SEQ ID NO: 1, with two substitutions, S54N and S56Q, according to the Kabat numbering system. In some embodiments, the single-domain antibody has the amino acid sequence shown in SEQ ID NO: 1, with two substitutions, S54T and S56Q, according to the Kabat numbering system.

[0099] In some embodiments, the single-domain antibody having a substitution at a position selected from S54, G55, S56 and any combination thereof comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, 40, and 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 with each of them.

[0100] In some embodiments, the single-domain antibody having a substitution at a position selected from S54, G55, S56 and 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 with SEQ ID NO: 3.

[0101] In some embodiments, the single-domain antibody having a substitution at a position selected from S54, G55, S56 and 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 with SEQ ID NO: 4.

[0102] In some embodiments, the single-domain antibody having a substitution at a position selected from S54, G55, S56 and 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 with SEQ ID NO: 5.

[0103] In some embodiments, the single-domain antibody having a substitution at a position selected from S54, G55, S56 and 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 with SEQ ID NO: 6.

[0104] In some embodiments, the single-domain antibody having a substitution at a position selected from S54, G55, S56 and 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 with SEQ ID NO: 7.

[0105] In some embodiments, the single-domain antibody having a substitution at a position selected from S54, G55, S56 and 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 with SEQ ID NO: 8.

[0106] In some embodiments, the single-domain antibody having a substitution at a position selected from S54, G55, S56 and 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 with SEQ ID NO: 9.

[0107] In some embodiments, the single-domain antibody having a substitution at a position selected from S54, G55, S56 and 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 with SEQ ID NO: 10.

[0108] In some embodiments, the single-domain antibody having a substitution at a position selected from S54, G55, S56 and 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 with SEQ ID NO: 39.

[0109] In some embodiments, the single-domain antibody having a substitution at a position selected from S54, G55, S56 and 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 with SEQ ID NO: 40.

[0110] Another aspect of this disclosure provides a single-domain antibody that specifically binds to CD16A, wherein the single-domain antibody comprises: (a) CDR1 having the amino acid sequence shown in SEQ ID NO: 41 or a conserved variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 42 or a conserved variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or a conserved variant thereof; (b) According to the Kabat definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 53 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 54 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or a conservatively modified variant thereof; (c) According to the Chothia definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 65 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 66 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or a conservatively modified variant thereof; or (d) According to the Contact definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 77 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 78 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or a conservatively modified variant thereof; The condition is that the single-domain antibody does not have the amino acid sequence shown in SEQ ID NO: 1.

[0111] In some embodiments, a single-domain antibody that specifically binds to CD16A is provided, wherein, according to the IMGT definition scheme, the single-domain antibody comprises CDR1 having the amino acid sequence shown in SEQ ID NO: 41, CDR3 having the amino acid sequence shown in SEQ ID NO: 42, and CDR2 having the amino acid sequence selected from SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, and 52.

[0112] In some embodiments, a single-domain antibody that specifically binds to CD16A is provided, wherein, according to the Kabat definition scheme, the single-domain antibody comprises CDR1 having the amino acid sequence shown in SEQ ID NO: 53, CDR3 having the amino acid sequence shown in SEQ ID NO: 54, and CDR2 having the amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, and 64.

[0113] In some embodiments, a single-domain antibody that specifically binds to CD16A is provided, wherein, according to the Chothia definition scheme, the single-domain antibody comprises CDR1 having the amino acid sequence shown in SEQ ID NO: 65, CDR3 having the amino acid sequence shown in SEQ ID NO: 66, and CDR2 having the amino acid sequence selected from SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, and 76.

[0114] In some embodiments, a single-domain antibody that specifically binds to CD16A is provided, wherein, according to the Contact definition scheme, the single-domain antibody comprises CDR1 having the amino acid sequence shown in SEQ ID NO: 77, CDR3 having the amino acid sequence shown in SEQ ID NO: 78, and CDR2 having the amino acid sequence selected from SEQ ID NO: 79, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

[0115] In some embodiments, a single-domain antibody is provided comprising an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, and 40.

[0116] In some embodiments, a single-domain antibody is provided comprising 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 with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, or 40, and comprising, according to the IMGT definition scheme, CDR1 having an amino acid sequence as shown in SEQ ID NO: 41, CDR3 having an amino acid sequence as shown in SEQ ID NO: 42, and CDR2 having an amino acid sequence selected from SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, and 52.

[0117] In some embodiments, a single-domain antibody is provided comprising 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 with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, or 40, and, according to the Kabat definition scheme, comprising CDR1 having an amino acid sequence as shown in SEQ ID NO: 53, CDR3 having an amino acid sequence as shown in SEQ ID NO: 54, and CDR2 having an amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, and 64.

[0118] In some embodiments, a single-domain antibody is provided comprising 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 with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, or 40, and, according to the Chothia definition scheme, comprising CDR1 having an amino acid sequence as shown in SEQ ID NO: 65, CDR3 having an amino acid sequence as shown in SEQ ID NO: 66, and CDR2 having an amino acid sequence selected from SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, and 76.

[0119] In some embodiments, a single-domain antibody is provided comprising 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 with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, or 40, and, according to the Contact definition scheme, comprising CDR1 having an amino acid sequence as shown in SEQ ID NO: 77, CDR3 having an amino acid sequence as shown in SEQ ID NO: 78, and CDR2 having an amino acid sequence selected from SEQ ID NO: 79, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

[0120] 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 camel-derived antibody. Preferably, the single-domain antibody is a humanized antibody or a human antibody. More preferably, the single-domain antibody is a humanized antibody.

[0121] In some embodiments, the anti-CD16A single-domain antibody disclosed herein specifically binds to CD16A but not specifically to CD16B. In some embodiments, the anti-CD16A single-domain antibody disclosed herein specifically binds to human and monkey CD16A but not specifically to mouse or rat CD16A. The amino acid sequence of human CD16A is exemplarily shown in SEQ ID NO: 2 or 11. The amino acid sequence of human CD16B is exemplarily shown in SEQ ID NO: 36. The amino acid sequence of monkey CD16A is exemplarily shown in SEQ ID NO: 33. The amino acid sequence of mouse CD16A is exemplarily shown in SEQ ID NO: 34. The amino acid sequence of rat CD16A is exemplarily shown in SEQ ID NO: 35.

[0122] In some embodiments, the anti-CD16A single-domain antibody disclosed herein specifically binds to CD16A 158V and 158F. In some embodiments, the anti-CD16A single-domain antibody disclosed herein specifically binds to CD16A 158V and 158F with substantially the same affinity. In some embodiments, the anti-CD16A single-domain antibody disclosed herein binds to CD16A 158V and 158F with substantially the same affinity and, as determined by ELISA, with an EC50 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. In some embodiments, as determined by ELISA, the anti-CD16A single-domain antibody disclosed herein specifically binds to CD16A 158V with an EC50 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. In some embodiments, such as as determined by ELISA, the anti-CD16A single-domain antibody disclosed herein binds CD16A 158F specifically at EC50 in the range of about 1 to about 3 nM, about 1 to about 2 nM, or about 1 to about 1.2 nM.

[0123] In some embodiments, the anti-CD16A single-domain antibody disclosed herein exhibits improved binding affinity for CD16A, particularly CD16A 158V, compared to an anti-CD16A single-domain antibody having the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, as determined by BLI, the anti-CD16A single-domain antibody disclosed herein binds to CD16A 158V with EC50 specificity 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 some embodiments, as determined by BLI, the anti-CD16A single-domain antibody disclosed herein binds to CD16A 158F with EC50 specificity 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.

[0124] In some embodiments, a polypeptide is provided comprising an anti-CD16A single-domain antibody as described herein and one or more amino acid residues covalently linked to the N-terminus, C-terminus, or any position therebetween of the anti-CD16A single-domain antibody, and 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, and 40.

[0125] Anti-CD16A heavy chain antibody Another aspect of this disclosure provides a heavy chain antibody comprising a single-domain antibody disclosed herein and an Fc portion linked to said single-domain antibody.

[0126] In some embodiments, an anti-CD16A heavy chain antibody is provided, wherein the heavy chain antibody comprises a single-domain antibody, the single-domain antibody comprising: (a) CDR1 having the amino acid sequence shown in SEQ ID NO: 41 or a conserved variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 42 or a conserved variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or a conserved variant thereof; (b) According to the Kabat definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 53 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 54 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or a conservatively modified variant thereof; (c) According to the Chothia definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 65 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 66 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or a conservatively modified variant thereof; or (d) According to the Contact definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 77 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 78 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or a conservatively modified variant thereof; The condition is that the heavy chain antibody does not contain the amino acid sequence shown in SEQ ID NO: 1.

[0127] In some embodiments, an anti-CD16A heavy chain antibody is provided, wherein the heavy chain antibody comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39 and 40.

[0128] In some embodiments, an anti-CD16A heavy chain antibody is provided, wherein the heavy chain antibody comprises 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 with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, or 40, and, according to the IMGT definition scheme, comprises CDR1 having an amino acid sequence as shown in SEQ ID NO: 41, CDR3 having an amino acid sequence as shown in SEQ ID NO: 42, and CDR2 having an amino acid sequence selected from SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, and 52.

[0129] In some embodiments, an anti-CD16A heavy chain antibody is provided, wherein the heavy chain antibody comprises a single-domain antibody comprising an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39 and 40, and the single-domain antibody is linked to an Fc portion having the amino acid sequence shown in SEQ ID NO: 89.

[0130] In any embodiment of the anti-CD16A heavy chain antibody disclosed herein, the anti-CD16A heavy chain antibody may be a humanized antibody, a human antibody, a chimeric antibody, or a camel-derived antibody. Preferably, the anti-CD16A heavy chain antibody is a humanized antibody or a human antibody. More preferably, the anti-CD16A heavy chain antibody is a humanized antibody.

[0131] In some embodiments, a polypeptide is provided comprising an anti-CD16A heavy chain antibody as described herein and one or more amino acid residues covalently linked to the N-terminus, C-terminus, or any position therebetween of the anti-CD16A heavy chain antibody, and 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, and 40, and an Fc portion having the amino acid sequence shown in SEQ ID NO: 89.

[0132] Bispecific antibodies Another aspect of the invention provides a bispecific antibody comprising a single-domain antibody as disclosed herein.

[0133] In some embodiments, 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 and tumor-specific antigens.

[0134] In some embodiments, the second target is selected from: HER2, 5T4, PSMA, BCMA, FGFR, CD20, CD33, CD19, CD22, CD123, CD30, GPC-3, CEA, EGFR1, EGFR2, EGFR3, TGF-β, ROR1, PD-L1, tight junction protein 18.2, 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, Pmel 17, MAGE-1, AFP, CA125, TRP-1, TRP-2, NY-ESO, PSA, CDK4, BCA225, CA 125, MG7-Ag, NY-CO-1, RCAS 1, SDCCAG16, TAAL6, and TAG72. Preferably, the second target is HER2, 5T4, BCMA, CD20, CD19, CD30, tight junction protein 18.2, MUC1, Trop-2, c-MET, or Nectin-4. More preferably, the second target is HER2, 5T4, or CD30.

[0135] In some embodiments, 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 wherein the single-domain antibody comprises: (a) CDR1 having the amino acid sequence shown in SEQ ID NO: 41 or a conserved variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 42 or a conserved variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or a conserved variant thereof; (b) According to the Kabat definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 53 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 54 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or a conservatively modified variant thereof; (c) According to the Chothia definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 65 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 66 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or a conservatively modified variant thereof; or (d) According to the Contact definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 77 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 78 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or a conservatively modified variant thereof; The condition is that the single-domain antibody does not have the amino acid sequence shown in SEQ ID NO: 1.

[0136] In some embodiments, 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 wherein the single-domain antibody comprises: (a) CDR1 having the amino acid sequence shown in SEQ ID NO: 41 or a conserved variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 42 or a conserved variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or a conserved variant thereof; (b) According to the Kabat definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 53 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 54 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or a conservatively modified variant thereof; (c) According to the Chothia definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 65 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 66 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or a conservatively modified variant thereof; or (d) According to the Contact definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 77 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 78 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or a conservatively modified variant thereof; The condition is that the single-domain antibody does not have the amino acid sequence shown in SEQ ID NO: 1.

[0137] In some embodiments, 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 wherein the single-domain antibody comprises: (a) CDR1 having the amino acid sequence shown in SEQ ID NO: 41 or a conserved variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 42 or a conserved variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or a conserved variant thereof; (b) According to the Kabat definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 53 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 54 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or a conservatively modified variant thereof; (c) According to the Chothia definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 65 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 66 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or a conservatively modified variant thereof; or (d) According to the Contact definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 77 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 78 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or a conservatively modified variant thereof; The condition is that the single-domain antibody does not have the amino acid sequence shown in SEQ ID NO: 1.

[0138] In some embodiments, 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 wherein the single-domain antibody comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39 and 40.

[0139] In some embodiments, 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 wherein the single-domain antibody comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39 and 40.

[0140] In some embodiments, 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 wherein the single-domain antibody comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39 and 40.

[0141] In some embodiments, 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 and tumor-specific antigens. The first binding domain is a single-domain antibody that specifically binds to CD16A, and the second binding domain is a complete IgG antibody or its antigen-binding fragment, such as Fab, Fv, scFv, or F(ab')2.

[0142] In some embodiments, 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 and tumor-specific antigens. The first binding domain is a single-domain antibody that specifically binds to CD16A, and the second binding domain is a complete IgG antibody. 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 complete IgG antibody.

[0143] In some embodiments, 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 and tumor-specific antigens. The first binding domain is a single-domain antibody that specifically binds to CD16A, and the second binding domain is a complete IgG antibody. 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 complete IgG antibody via a peptide linker.

[0144] In some embodiments, 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 and tumor-specific antigens. The first binding domain is a single-domain antibody that specifically binds to CD16A, and the second binding domain is a complete IgG antibody. 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 complete IgG antibody via a peptide linker composed of glycine and serine residues. The peptide linker is as defined in the definition section.

[0145] In some embodiments, the bispecific antibody comprises a single-domain antibody that specifically binds to CD16A as disclosed herein and a fully anti-HER2 IgG antibody, and 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 fully anti-IgG antibody via a peptide linker composed of glycine and serine residues.

[0146] In some embodiments, the bispecific antibody comprises a single-domain antibody that specifically binds to CD16A as disclosed herein and a complete anti-5T4 IgG antibody, and 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 complete IgG antibody via a peptide linker composed of glycine and serine residues.

[0147] In some embodiments, the complete IgG antibody has a mutation in the Fc portion. In some embodiments, the complete IgG antibody has an L234A / L235A mutation (“LALA”). In some embodiments, the complete IgG antibody has an N297A mutation. In some embodiments, the complete IgG antibody has both the “LALA” and N297A mutations.

[0148] In some embodiments, the fully anti-HER2 IgG antibody comprises: (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; or (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.

[0149] In some embodiments, the bispecific antibody comprises: (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; or (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.

[0150] In a preferred embodiment, the bispecific antibody comprises: (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: 27 and a light chain having the amino acid sequence shown in SEQ ID NO: 13; or (f) The heavy chain having the amino acid sequence shown in SEQ ID NO: 28 and the light chain having the amino acid sequence shown in SEQ ID NO: 13.

[0151] In some embodiments, the fully 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.

[0152] In some embodiments, 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.

[0153] Immunoconjugates Another aspect of this disclosure relates to an immunoconjugate, a pharmaceutically acceptable salt thereof, or a solvation thereof, comprising a single-domain antibody according to the above-described "anti-CD16A single-domain antibody" portion, a heavy-chain antibody according to the above-described "anti-CD16A heavy-chain antibody" portion, or a bispecific antibody according to the above-described "bispecific antibody" portion; and an active portion conjugated to said single-domain antibody.

[0154] In some embodiments, the active portion is selected from toxins, peptide tags, sortag, radionuclides, near-infrared fluorescent dyes, and nanoparticles.

[0155] In some embodiments, the toxin is selected from cytotoxic agents, cytokines, nucleic acids, nucleic acid-related molecules, radionuclides, chemokines, co-stimulatory molecules, immunosuppressive molecules, death ligands, apoptosis-inducing proteins, kinases, prodrug converting enzymes, RNases, agonistic antibodies or antibody fragments, antagonistic antibodies or antibody fragments, growth factors, hormones, coagulation factors, fibrinolytic proteins, peptides that mimic these, fragments thereof, fusion proteins, and derivatives thereof.

[0156] In some embodiments, the radionuclide comprises: 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 Optionally, the radionuclide can be conjugated to the antibody using a chelating agent.

[0157] In some embodiments, an immunoconjugate, a pharmaceutically acceptable salt thereof, or a solvation thereof is provided, comprising a single-domain antibody, said single-domain antibody comprising: (a) CDR1 having the amino acid sequence shown in SEQ ID NO: 41 or a conserved variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 42 or a conserved variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or a conserved variant thereof; (b) According to the Kabat definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 53 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 54 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or a conservatively modified variant thereof; (c) According to the Chothia definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 65 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 66 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or a conservatively modified variant thereof; or (d) According to the Contact definition scheme, CDR1 having the amino acid sequence shown in SEQ ID NO: 77 or a conservatively modified variant thereof, CDR3 having the amino acid sequence shown in SEQ ID NO: 78 or a conservatively modified variant thereof, and CDR2 having the amino acid sequence selected from SEQ ID NO: 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or a conservatively modified variant thereof; The condition is that the single-domain antibody does not have the amino acid sequence shown in SEQ ID NO: 1; and the active portion conjugated to the single-domain antibody, wherein the active portion is selected from toxins, peptide tags, sortag, radionuclides, near-infrared fluorescent dyes, and nanoparticles.

[0158] In some embodiments, an immunoconjugate, a pharmaceutically acceptable salt thereof, or a solvation thereof is provided, comprising a single-domain antibody containing an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, and 40; and an active moiety conjugated to said single-domain antibody, wherein said active moiety is selected from toxins, peptide tags, sortag, radionuclides, near-infrared fluorescent dyes, and nanoparticles.

[0159] In some embodiments, an immunoconjugate, a pharmaceutically acceptable salt thereof, or a solvate thereof is provided, comprising a single-domain antibody containing an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, and 40; and an active moiety conjugated to said single-domain antibody, wherein said active moiety is a toxin, a radionuclide, or a near-infrared fluorescent dye.

[0160] In some embodiments, an immunoconjugate, a pharmaceutically acceptable salt thereof, or a solvation thereof is provided, comprising a single-domain antibody containing an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, and 40; and a cytotoxic agent conjugated to said single-domain antibody.

[0161] In some embodiments, an immunoconjugate, a pharmaceutically acceptable salt thereof, or a solvation thereof is provided, comprising a single-domain antibody containing an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, and 40; and a radionuclide reagent conjugated to said single-domain antibody.

[0162] Pharmaceutical Composition In another aspect, this disclosure provides a pharmaceutical composition comprising a single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate (hereinafter referred to as "antibody or immunoconjugate") as disclosed herein, and a pharmaceutically acceptable carrier. The composition is suitable for veterinary or human administration.

[0163] The compositions of the present invention can be in any form that allows for administration to a patient. For example, the compositions can be in solid, liquid, or gaseous (aerosol) form. Typical routes of administration include, but are not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intratumoral, and intranasal administration. Parenteral administration includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques. In one aspect, the composition is administered parenterally. In another aspect, the composition is administered intravenously.

[0164] Pharmaceutical compositions can be formulated such that the active ingredient is bioavailable when the composition is administered to a patient. The composition can be in the form of one or more dose units, wherein, for example, a tablet can be a single dose unit, and a container for an immunoconjugate in aerosol form can hold multiple dose units.

[0165] The materials used to prepare the pharmaceutical composition may be non-toxic in the amounts used. It will be apparent to those skilled in the art that the optimal dosage of the active ingredient in the pharmaceutical composition will depend on a variety of factors. These factors include, but are not limited to, the type of animal (e.g., human), the specific form of the immunoconjugate, the route of administration, and the composition used.

[0166] Pharmaceutically acceptable carriers or excipients may be granular, such that the composition is, for example, in tablet or powder form. The carrier may be a liquid, such as an oral syrup or injectable liquid. Furthermore, the carrier may be gaseous or granular to provide an aerosol composition suitable for, for example, inhalation administration.

[0167] The composition may be in liquid form, such as an elixir, syrup, solution, emulsion, or suspension. The liquid may be used for oral administration or delivery by injection. When intended for oral administration, the composition may contain one or more of sweeteners, preservatives, dyes / colorants, and flavor enhancers. In compositions intended for injection administration, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents may also be included.

[0168] Liquid compositions, whether they are solutions, suspensions, or other similar forms, may also include one or more of the following: sterile diluents, such as water for injection, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride, fixed oils (e.g., synthetic mono- or diglycerides of glycerol that can be used as a solvent or suspension medium), polyethylene glycol, glycerol, 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; buffers such as acetates, citrates, or phosphates; and tonic agents such as sodium chloride or dextrose. Parenteral compositions may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass, plastic, or other materials. Physiological saline is an exemplary adjuvant. Injectable compositions are preferably sterile.

[0169] The amount of antibody or immunoconjugate effective in treating a specific condition or symptom will depend on the nature of the condition or symptom and can be determined using standard clinical techniques. Additionally, in vitro or in vivo assays may be optionally used to help determine the optimal dosage range. The precise dosage used in the composition will also depend on the route of administration and the severity of the disease or symptom, and should be determined based on the physician's judgment and the individual patient's situation.

[0170] The composition contains an effective amount of antibody or immunoconjugate such that a suitable dosage will be obtained. Typically, this amount is at least about 0.01% of the antibody or immunoconjugate by weight of the composition. When intended for oral administration, this amount can vary from about 0.1 wt% to about 80 wt% of the composition. In one aspect, the oral composition may contain about 4% to about 50% of the antibody or immunoconjugate by weight of the composition. In another aspect, the compositions of the present invention are prepared such that a parenteral dose unit contains about 0.01 wt% to about 2 wt% of the antibody or immunoconjugate.

[0171] For intravenous administration, the composition may contain about 0.01 to about 100 mg of antibody or immunoconjugate per kg of animal body weight. In one aspect, the composition may include about 1 to about 100 mg of antibody or immunoconjugate per kg of animal body weight. In another aspect, the amount administered will be in the range of about 0.1 to about 25 mg / kg of antibody or immunoconjugate.

[0172] Typically, the dose of antibody or immunoconjugate administered to a patient is typically from about 0.01 mg / kg to about 2000 mg / kg of animal body weight. In one aspect, the dose administered to a patient is from about 0.01 mg / kg to about 10 mg / kg of animal body weight; in another aspect, the dose administered to a patient is from about 0.1 mg / kg to about 250 mg / kg of animal body weight; in yet another aspect, the dose administered to a patient is from about 0.1 mg / kg to about 20 mg / kg of animal body weight; in yet another aspect, the dose administered is from about 0.1 mg / kg to about 10 mg / kg of animal body weight; and in yet another aspect, the dose administered is from about 1 mg / kg to about 10 mg / kg of animal body weight.

[0173] Antibodies, immunoconjugates, or compositions can be administered via any convenient route, such as by infusion or bolus, or by absorption through epithelial or mucosal skin linings (e.g., oral mucosa, rectal and intestinal mucosa). Administration can be systemic or local. Various delivery systems are known, such as those encapsulated in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer antibodies or immunoconjugates or compositions. In some embodiments, more than one antibody, immunoconjugate, or composition is administered to the patient.

[0174] In specific embodiments, it may be necessary to apply one or more antibodies, immunoconjugates, or compositions topically to the area requiring treatment. This can be achieved, for example, but not limited to, local infusion during surgery; local application, such as after surgery in combination with a wound dressing; by injection; by means of a catheter; by means of a suppository; or by means of an implant, which is a porous, non-porous, or gel-like material, including membranes, such as silicone rubber membranes or fibers. In one embodiment, administration can be performed by direct injection at the site (or primary site) of cancer, tumor, or tumor tissue or precancerous tissue. In another embodiment, administration can be performed by direct injection at the site (or primary site) of an autoimmune disease manifestation.

[0175] In some implementations, it may be necessary to introduce one or more antibodies, immunoconjugates, or compositions into the central nervous system via any suitable route, including intraventricular and intrathecal injection. Intraventricular injection can be facilitated, for example, by an intraventricular catheter attached to a reservoir such as the Ommaya reservoir.

[0176] It can also be administered to the lungs, for example by using an inhaler or nebulizer, or by formulation with a nebulizer, or by perfusion in a fluorocarbon or synthetic pulmonary surfactant.

[0177] In another embodiment, the antibody, immunoconjugate, or composition can be delivered in a controlled-release system, such as, but not limited to, using a pump or various polymer materials. In yet another embodiment, the controlled-release system can be placed near the target (e.g., the brain) of the antibody, immunoconjugate, or composition, thus requiring only a portion of the systemic dose.

[0178] The term "carrier" refers to a diluent, adjuvant, or carrier administered with an antibody or immunoconjugate. Such drug carriers can be liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Carriers can be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Additionally, adjuvants, stabilizers, thickeners, lubricants, and colorants can be used. In one embodiment, when administered to a patient, the antibody or immunoconjugate or composition and the pharmaceutically acceptable carrier are sterile. Water is an exemplary carrier when the antibody or immunoconjugate is administered intravenously. Saline solutions and aqueous solutions of glucose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable drug carriers also include carriers such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the compositions of the present invention may also contain small amounts of wetting agents, emulsifiers, or pH buffers.

[0179] In one embodiment, the immunoconjugate is formulated into a pharmaceutical composition suitable for intravenous administration to animals, particularly humans, according to conventional procedures. Typically, the carrier or excipient for intravenous administration is a sterile isotonic buffer solution. If necessary, the composition may also include a solubilizer. The composition for intravenous administration may optionally contain a local anesthetic, such as lidocaine, to relieve pain at the injection site. Typically, the components are provided individually or mixed together in unit dosage forms, for example, as a dried lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachet indicating the amount of active agent. When the immunoconjugate is administered by infusion, it can be dispensed, for example, using an infusion bottle containing sterile pharmaceutical-grade water or saline. In the case of administration of the antibody or immunoconjugate by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0180] The composition may comprise a variety of materials in physical form that alters the dosage unit of a solid or liquid. For example, the composition may include a material that forms a coating around the active ingredient. The material forming the coating is typically inert and may be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0181] The composition can consist of gaseous dosage units, for example, it can be in the form of an aerosol. The term aerosol is used to refer to a variety of systems, ranging from colloidal systems to systems consisting of pressurized packaging. Delivery can be carried out by liquefying or compressing gas or by a suitable pump system dispensing the active ingredient.

[0182] Whether in solid, liquid, or gaseous form, the compositions of the present invention may include pharmacological agents for the treatment of cancer.

[0183] Uses and therapies Antibodies and immunoconjugates can be used to inhibit the proliferation of tumor cells or cancer cells, induce apoptosis in tumor or cancer cells, or treat cancer in patients. Therefore, antibodies and immunoconjugates can be used to treat animal cancers in a variety of settings. Antibodies and immunoconjugates can be used to deliver drugs or drug units to tumor cells or cancer cells. Without being bound by theory, in one embodiment, the antibody portion of the antibody or immunoconjugate disclosed herein binds to or associates with CD16A expressed on or associated with the surface of NK cells. In the case of bispecific antibodies, the antibody binds to CD16A via one arm and to a second antigen expressed on tumor cells via the other arm, forming an “NK adaptor” to enhance the function of NK cells in killing tumor cells.

[0184] In another aspect, this disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate disclosed herein.

[0185] On the other hand, this disclosure provides a method for preventing cancer in a subject at risk of developing cancer, the method comprising administering to the subject a preventatively effective amount of the single-domain antibody, heavy-chain antibody, bispecific antibody, or immune conjugate disclosed herein.

[0186] In another aspect, this disclosure provides a method for, for example, in vitro diagnosis of cancer in a subject, the method comprising: isolating a sample from the subject; contacting the sample, for example in vitro, with a single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate disclosed herein; detecting a signal generated by the contact; comparing the signal with a threshold; and determining, based on the result of the comparison, whether the subject has the cancer.

[0187] The cancer can be a solid tumor or a blood cancer. A blood cancer, also known as a leukemia, is a cancer that originates in hematopoietic tissues, such as bone marrow or other cells of the immune system. Hematologic malignancies include, but are not limited to, leukemias (such as acute myeloid leukemia (ANIL), acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), acute mixed spectrum leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia (CLL), hairy cell leukemia, and large granular lymphocytic leukemia), myelodysplastic syndromes (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocythemia, primary myelofibrosis, and chronic myeloid leukemia), lymphomas, multiple myeloma, MGUS and similar disorders, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies. Solid tumors include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, laryngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and nipple carcinoma. Adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, retinoblastoma. Preferably, cancers treatable by this disclosure include breast cancer.

[0188] In some embodiments, a method for treating or preventing cancer is provided, comprising administering to a patient in need a therapeutically effective amount of the disclosed antibody or immunoconjugate and a chemotherapeutic agent. In one embodiment, the chemotherapeutic agent is one for which cancer has not been found to be refractory. In another embodiment, the chemotherapeutic agent is one for which cancer has been found to be refractory. The antibody or immunoconjugate may be administered to a patient who has also undergone surgery as cancer treatment. In some embodiments, the patient has been found to be refractory to HER2 antibodies such as trastuzumab or magnituximab.

[0189] In one specific embodiment, the antibody or immune conjugate is administered concurrently with chemotherapy or radiotherapy. In another specific embodiment, chemotherapy or radiotherapy is administered before or after the administration of the antibody or immune conjugate, in one aspect at least one hour, five hours, twelve hours, one day, one week, or one month before or after the administration of the antibody or immune conjugate, and in other aspects for several months (e.g., up to three months).

[0190] Chemotherapy agents can be administered in a series of treatments. Suitable chemotherapeutic agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, topotecan, nitrogen mustard, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, actinomycin D, procainamide, mitoxantrone, asparaginase, vincristine, vinorelbine, paclitaxel, and docetaxel. Regarding radiation, any radiation therapy regimen can be used depending on the type of cancer to be treated. For example, but not as a limitation, X-ray radiation can be administered; in particular, high-energy megavolts (radiation with energies greater than 1 MeV) can be used for deep tumors, and electron beam and positive voltage X-ray radiation can be used for skin cancer. Radioactive isotopes that emit gamma rays, such as radium, cobalt, and other radioactive isotopes of the elements, can also be used.

[0191] Additionally, methods for treating cancer with the antibodies or immune conjugates disclosed herein are provided as alternatives to chemotherapy or radiation therapy, where chemotherapy or radiation therapy has been demonstrated or may be demonstrated to be too toxic, for example, causing unacceptable or intolerable side effects in the treated subjects. The treated animals may optionally be treated with another cancer treatment such as surgery, radiation therapy, or chemotherapy, depending on which treatment is found to be acceptable or tolerable.

[0192] Antibodies or immunoconjugates can also be used in vitro or ex vivo, for example, to treat certain cancers, including but not limited to leukemia and lymphoma, where such treatments involve autologous stem cell transplantation. This can involve a multi-step process in which autologous hematopoietic stem cells are harvested from an animal and all cancer cells are removed. The remaining bone marrow cell population is then eradicated by administering high doses of antibodies or immunoconjugates, with or without high-dose radiation therapy, and the stem cell graft is infused back into the animal. Supportive care is then provided as bone marrow function recovers and the animal recovers.

[0193] Equivalently, this disclosure also provides the use of single-domain antibodies, heavy-chain antibodies, bispecific antibodies, or immunoconjugates as disclosed herein in the treatment of a subject with cancer. Equivalently, this disclosure also provides the use of single-domain antibodies, heavy-chain antibodies, bispecific antibodies, or immunoconjugates as disclosed herein in the preparation of a medicament for treating a subject with cancer.

[0194] This disclosure also provides a method for treating acute or chronic infections (e.g., infections caused by viruses, fungi, or bacteria), the method comprising administering to a subject in need a therapeutically effective amount of a single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate as disclosed herein. For example, an infection to be treated with a single-domain antibody, heavy-chain antibody, bispecific antibody, or immune conjugate as disclosed herein can be caused by adenovirus, herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), poxvirus (e.g., orthopoxvirus such as smallpox or cowpox, or molluscum contagiosum), microRNA virus (e.g., rhinovirus or enterovirus), orthomyxovirus (e.g., influenza virus), paramyxovirus (e.g., parainfluenza virus, mumps virus, measles virus, and respiratory syncytial virus (RSV)), coronavirus (e.g., SARS), papillomavirus (e.g., papillomavirus, such as those causing genital warts, common warts, or plantar warts), hepatotropic DNA virus (e.g., hepatitis B virus), flavivirus (e.g., hepatitis C virus or dengue virus), or retrovirus (e.g., lentivirus, such as HIV). Infections caused by bacteria such as Escherichia coli, Enterobacter spp., Salmonella spp., Staphylococcus spp., Shigella spp., Listeria spp., Aerobacter spp., Helicobacter spp., Klebsiella spp., Proteus spp., Pseudomonas spp., Streptococcus spp., Chlamydia spp., Mycoplasma spp., Pneumococcus spp., Neisseria spp., Clostridium spp., Bacillus spp., Corynebacterium spp., Mycobacterium spp., Campylobacter spp., Vibrio spp., Serratia spp., Providencia spp., Chromobacterium spp., Brucella spp., Yersinia spp., Haemophilus spp., or Bordetella spp.; or other infectious diseases, such as chlamydia, fungal diseases, including but not limited to candidiasis, aspergillosis, histoplasmosis, cryptococcal meningitis, or parasitic diseases, including but not limited to malaria, Pneumocystis pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosomiasis.

[0195] This disclosure also provides a method for treating age-related minor ailments or diseases, the method comprising administering to a subject in need a therapeutically effective amount of a single-domain antibody, heavy-chain antibody, bispecific antibody, or immune conjugate as disclosed herein. For example, the age-related disease is any one of atherosclerosis, cardiovascular disease, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, and Alzheimer's disease.

[0196] Similarly, this disclosure also provides for the use of single-domain antibodies, heavy-chain antibodies, bispecific antibodies, or immunoconjugates as disclosed herein in the treatment of acute or chronic infections in subjects. Similarly, this disclosure also provides for the use of single-domain antibodies, heavy-chain antibodies, bispecific antibodies, or immunoconjugates as disclosed herein in the preparation of medicaments for the treatment of acute or chronic infections in subjects.

[0197] Similarly, this disclosure also provides the use of single-domain antibodies, heavy-chain antibodies, bispecific antibodies, or immunoconjugates as disclosed herein in the treatment of age-related diseases in subjects. Similarly, this disclosure also provides the use of single-domain antibodies, heavy-chain antibodies, bispecific antibodies, or immunoconjugates as disclosed herein in the preparation of medicaments for the treatment of age-related diseases in subjects.

[0198] In another aspect, this disclosure provides a method for preventing acute or chronic infection in a subject at risk of acute or chronic infection, the method comprising administering to the subject a preventatively effective amount of the single-domain antibody, heavy chain antibody, bispecific antibody, or immune conjugate disclosed herein.

[0199] In another aspect, this disclosure provides a method for preventing age-related diseases in a subject at risk of such diseases, the method comprising administering to the subject a preventatively effective amount of the single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate disclosed herein.

[0200] In another aspect, this disclosure provides a method for, for example, in vitro diagnosis of an acute or chronic infection in a subject, the method comprising: isolating a sample from the subject; contacting the sample, for example in vitro, with a single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate disclosed herein; detecting a signal generated by the contact; comparing the signal with a threshold; and determining, based on the result of the comparison, whether the subject has the acute or chronic infection.

[0201] In another aspect, this disclosure provides a method for, for example, in vitro diagnosis of an aging-related disease in a subject, the method comprising: isolating a sample from the subject; contacting the sample, for example in vitro, with a single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate disclosed herein; detecting a signal generated by the contact; comparing the signal with a threshold; and determining, based on the result of the comparison, whether the subject suffers from the aging-related disease.

[0202] In another aspect, this disclosure provides a method for in vitro detection of CD16A-positive samples, the method comprising contacting the sample in vitro with a single-domain antibody, heavy-chain antibody, bispecific antibody, or immunoconjugate disclosed herein; detecting a signal generated by the contact; comparing the signal with a threshold; and determining whether the sample is CD16A-positive.

[0203] sequence

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215] Note: In the context of descriptions involving CDRs (e.g., SEQ ID NO: 88), the terms IMGT, Kabat, Chothia, or Contact refer to different CDR definition schemes as described in the definitions section.

[0216] Example The following examples are included to illustrate preferred embodiments of the invention and are not intended to limit the scope of the invention.

[0217] Example 1: CD16A VH affinity maturation This embodiment illustrates a method for affinity maturation using a randomly mutant phage library panning. The previously identified CD16A VH BM156-01 (SEQ ID NO: 1) was used as a template for constructing the random mutant library. NNB primers were designed for each amino acid in the annotated CDR (as shown in Table 1). Antibody numbering and CDR annotation were performed according to the IMGT protocol. PCR was performed to construct an arbitrary mutant library for each amino acid in the CDR region.

[0218] Table 1: List of mutagenic primers used for library construction

[0219]

[0220] Phage library panning was performed according to the following procedure. Biotin-labeled recombinant human CD16A protein (hCD16ASeq ID NO: 2) was immobilized on streptavidin-coated magnetic beads, and the beads were washed three times with PBS buffer containing 2% BSA. Then, the CD16A-coated beads were mixed with 10... 10 The bacteriophages were incubated at room temperature for 2 hours.

[0221] After washing with PBS buffer containing 0.05% Tween-20 (PBST), the magnetic beads were resuspended in PBS and added to 2 ml of fresh TG1 bacterial culture. The mixture was then incubated at 37°C and 100 rpm for 45 min for infection. After infection, 10 μL of M13 helper phage was added to re-amplify the phage for use as an enriched library in the next round of panning.

[0222] The random mutant library was panned four times, with 5, 4, 2, and 1 μg hCD16A antigen used in each round, and the number of PBST washing steps gradually increased with each round. At the end of the fourth round of panning, single colonies were randomly picked from the eluted phage-infected TG1 cells for monoclonal ELISA to identify CD16A-specific binders. In short, a 96-well ELISA plate was coated with hCD16A protein and blocked overnight at 4°C with PBS buffer containing 2% BSA. Then, 100 μL of TG1 culture was added to the plate and incubated at 37°C for 30 min. After washing the plate twice with PBS buffer containing 0.05% Tween 20, VH binding was detected using an HRP-conjugated anti-Flag antibody. The top conjugates were picked, the corresponding phage particles were isolated from TG1 cells, and DNA sequencing was performed to identify the enriched VH. The enriched VHs 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 the enriched VHs are listed in the sequence portion.

[0223] Example 2: Protein production of VH in Escherichia coli and characterization of its binding to CD16 158F / V protein by ELISA and biolayer interferometry. The VH selected from phage panning was further validated using purified antibodies. This example illustrates the protein production of CD16A VH in E. coli and the characterization of binding to the hCD16A protein by ELISA and biolayer interferometry (BLI).

[0224] The selected VH plasmid was transformed into HB2151 competent cells. Single colonies from overnight-grown ampicillin plates were inoculated into SB bacterial medium, and the bacterial cultures were cooled to 30°C before IPTG was added to induce protein expression. The bacterial cultures were harvested, and VH was purified using a Ni-NTA column according to the manufacturer's instructions. The purified antibody was characterized for in vitro binding by ELISA. Briefly, human CD16 158V and 158F proteins (SEQ ID NO 2 and SEQ ID NO 11) were coated onto ELISA plates overnight at 4°C. After washing with PBS containing 0.05% Tween 20 (PBST), serially diluted VH was added to the coated ELISA plates and incubated at room temperature for 1 hour. The binding ability of VH to CD16A was detected using an anti-FLAG-tagged antibody.

[0225] In the experiments conducted essentially as described above, compared to BM156-01, samples with lower binding EC were obtained from the panning process. 50 Several clones were used, such as CD16-VH-RM72, CD16-VH-RM77, and CD16-VH-RM81. ELISA results are as follows... Figure 1 As shown, EC 50 As shown in Table 2.

[0226] Table 2: EC5 binding of different VHs to CD16A 158V and CD16A 158F as determined by ELISA 50

[0227] Binding of CD16A VH to the CD16A protein was also characterized by BLI. Biotinylated CD16A-158V or 158F was immobilized on an avidin-coated probe, as recommended by the manufacturer (Aukite). CD16A VH was serially diluted 1:2 from 500 nM to 31.3 nM. The equilibrium dissociation constant (KD) for monovalent receptor binding was determined by fitting a 1:1 Langmuir model to the data.

[0228] In experiments conducted essentially as described above, 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. Results are as follows... Figure 2 As shown in Table 3.

[0229] Table 3: Binding affinity of different single-domain antibodies to CD16A 158V and CD16A 158V as determined by BLI.

[0230]

[0231] Example 3: Design of bispecific antibodies with different forms and N297A and LALA mutations This example demonstrates the construction and engineering of bispecific antibodies in different forms.

[0232] BM130-93 (trastuzumab SEQ ID NO 12 and SEQ ID NO 13) was selected as a building block to construct a bispecific antibody with the selected CD16A VH for its successful clinical treatment of HER2+ breast cancer and gastric cancer. CD16A VH can be fused to the C-terminus of the BM130-93 heavy chain using a (G4S)3 linker (SEQ ID NO 14 and SEQ ID NO 13) or a (G4S)4 linker (SEQ ID NO 15 and SEQ ID NO 13) to form a Morrison body. CD16A VH can also be fused to the C-terminus of the light chain (SEQ ID NO 12 and SEQ ID NO 16) or placed at the N-terminus of the heavy chain (SEQ ID NO 17 and SEQ ID NO 13). These different forms are as follows: Figure 3 As shown.

[0233] The bispecific antibodies BMP01-16 to BMP01-23 illustrated in the examples were constructed by fusing CD16A VH to the C-terminus of the BM130-93 heavy chain using the (G4S)3 linker, with the unmodified light chain of BM130-93.

[0234] Example 4: Characterization of CHO generation and bispecific antibody binding to CD16A by ELISA and BLI This example demonstrates the generation of CHO and the binding of bispecific antibodies to the CD16A protein via ELISA and BLI.

[0235] Different heavy chains were transiently co-expressed with their corresponding light chains in the ExpiCHO cell line to generate bispecific antibodies. Proteins from the ExpiCHO supernatant were purified using a Capturem™ Protein A miniaturization column (Takarabio, #635717) according to the manufacturer's instructions. The purified antibodies were then subjected to in vitro binding and functional characterization.

[0236] After washing with PBS containing 0.05% Tween 20 (PBST), human CD16 158V and 158F proteins (SEQ ID NO 2 and SEQ ID NO 11) were coated onto ELISA plates overnight at 4°C. Serially diluted bispecific antibody BMP01 was added to the coated ELISA plates and incubated at room temperature for 1 hour. The binding affinity of the bispecific antibody to CD16 was detected using an anti-human Fc-tagged antibody. ELISA results showed that the bispecific antibodies BMP01-16 to BMP01-23 maintained binding to CD16A 158V and 158F. Detailed results are as follows... Figure 4 As shown.

[0237] The binding affinity of the bispecific antibody to the CD16A protein was characterized using BLI. Biotinylated CD16A-158V or 158F was immobilized on an avidin-coated probe, as recommended by the manufacturer (Aukite). The bispecific antibody and BM130-93 (trastuzumab) were serially diluted 1:2 from 500 nM to 31.3 nM. The equilibrium dissociation constant (KD) for monovalent receptor binding was determined by fitting a 1:1 Langmuir model to the data.

[0238] In experiments conducted essentially as described above, bispecific antibodies BMP01-16 to 19, BMP01-21, and 22 all bound to CD16A-158V with less than nanomolar affinity. Compared to BM130-93 (trastuzumab), which showed no significant binding to the CD16A 158F variant, BMP01-16 to 23 all exhibited strong binding with approximately nanomolar affinity. Detailed results are as follows... Figure 5 As shown in Table 4.

[0239] Table 4: Binding affinity of different bispecific antibodies to CD16A 158V and CD16A 158F

[0240] Example 5: Dual binding of BMP01 to two targets simultaneously This example demonstrates that the BMP01 antibody can be used to detect the simultaneous binding of dual targets using a sandwich ELISA.

[0241] Add 50 μL of PBS solution containing human HER2 / ErbB2 protein (His tag, 1 μg / mL) (SEQ ID NO 32) or BSA (1 μg / mL) to a 96-well microplate and incubate the antigen solution overnight at 4°C to prepare a HER2 antigen-coated ELISA plate. The next day, discard the coating solution and wash the plate three times with 300 μL / well of PBST solution containing 0.05% Tween 20 (500 μl Tween 20 + 1000 ml PBS). Add 300 μL / well of PBS containing 4% skim milk powder and incubate at 37°C for 1 hour to block non-specific binding. Pre-dilute the control and test samples to 15 μg / mL (first-point concentration) with PBS, then serially dilute 2.5-fold for a total of 8 concentration points. After blocking, the plate was washed three times with PBST, and 50 μL / well of diluted control and test samples were added. The plate was incubated at 37°C and 300 rpm for 1.5 h. The plate was then washed three times with PBST, and 50 μL / well of 1 μg / mL biotin-labeled 158V CD16a-His was added. The plate was incubated at 37°C and 300 rpm for 1.5 h. The plate was washed three more times with PBST, and 50 μL / well of 1:5000 diluted SA-HRP detection antibody was added. The plate was incubated at 37°C and 300 rpm for 1.5 h. The plate was washed six times with PBST, and 50 μL / well of pre-equilibrated chromogenic solution to room temperature was added. The plate was incubated at room temperature in the dark for 15 min. 25 μL / well of 1M HCl stop solution was added. The OD value at 450 nm was measured using a microplate reader.

[0242] Four-parameter fitting was performed on the OD values ​​to calculate EC. 50 The concentration is used as the horizontal axis and the average value is used as the vertical axis.

[0243] In the experiments conducted essentially as described above, only BMP01-16 and BMP01-20 were able to bind to both targets simultaneously, with BMP01-16's EC50 being significantly higher. 50 The EC value of BMP01-20 is 0.2642 nM. 50 The value was 0.2947 nM, while BMP130-93 or the isotype control did not. Detailed results are as follows... Figure 6 As shown.

[0244] Example 6: Cross-binding with CD16A from different species The binding of the bispecific antibody to CD16 protein from different species (including human, monkey (SEQ ID NO 33), mouse (SEQ ID NO 34), and rat (SEQ ID NO 35)) was characterized by BLI. Biotinylated CD16A 158V or 158F was immobilized on avidin-coated probes as recommended by the manufacturer (Aukite). BMP01-16 was serially diluted 1:2 from 200 nM, while trastuzumab was serially diluted 1:2 from 140 nM. The equilibrium dissociation constant (KD) for monovalent receptor binding was determined by fitting a 1:1 Langmuir model to the data.

[0245] In experiments conducted essentially as described above, the bispecific antibody BMP01-16 bound to human and monkey CD16A with high affinities of 0.11 nM and 3.31 nM, respectively. BMP01-16 showed much weaker binding to mouse or rat CD16 protein, with affinities of 0.645 μM and 0.0958 μM, respectively. However, trastuzumab showed only weak binding to CD16 from all species. Detailed results are shown in Table 5.

[0246] Table 5: Binding affinity of BMP01-16 and trastuzumab to CD16 protein in humans, monkeys, mice, and rats.

[0247] Example 7: CD16B binding Human CD16A and CD16B (Seq ID NO 36) proteins were coated onto ELISA plates overnight at 4°C. After washing with PBS containing 0.05% Tween 20 (PBST), serially diluted BMP01-16 and BM130-92 (magutuximab, SEQ ID NO 37 and SEQ ID NO 38) and BM130-93 (trastuzumab) antibodies were added to the coated ELISA plates and incubated at room temperature for 1 hour. The binding ability of the bispecific and control antibodies to CD16 was detected using anti-human Fc-tagged antibody.

[0248] In experiments conducted essentially as described above, BMP01-16 bound to CD16A-158V and CD16B at concentrations of 1.255 nM and 1.296 μM, respectively. This result indicates that BMP01-16 binds much more strongly to CD16A-158V than to CD16B protein, EC... 50 >1000 times. In comparison, BM130-92 (magutuximab) and BMP130-93 (trastuzumab) bound to CD16A-158V, EC 50The molecular weights were 45.09 nM and 110.9 nM, respectively, while the binding to CD16B was negligible. Detailed results are as follows... Figure 7 As shown in Table 6.

[0249] Table 6: ECGs of BMP01-16, BM130-92, and BM130-93 binding to human CD16A-158V and CD16B on ELISA 50

[0250] Example 8: Evaluation of the binding of bispecific antibodies to HER2 on the cell surface This example demonstrates the binding of the BMP01 antibody to cell surface HER2 as assessed by FACS analysis.

[0251] 5×10 with different HER2 expression levels 5 Tumor cells, such as SK-BR-3 cells (high expression), JIMT-1 (intermediate expression), or MDA-MB-231 (low expression), were blocked with human Trustain FcX™ (Biolegend, 422302) at room temperature for 10 minutes, and then incubated with various concentrations of antibody variants (100 nM, 10 nM, 1 nM) at 4°C for 30 minutes. IgG1 was used as an isotype control. After incubation, cells were washed with 3% BSA in PBS. Cell-bound antibody levels were detected by APC-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG (Jackson Immunological Research Laboratory). Geometric mean fluorescence intensity was measured on an Attune Cell Analyzer (Engineer). Data were analyzed using FlowJo software (Tree Star Inc.).

[0252] In experiments conducted essentially as described above, results showed that BM130-93 and all BMP01 antibody variants bound to cell surface HER2 to similar degrees. Higher expression of the HER2 receptor on the cell surface resulted in the detection of more BM130-93 and BMP01 antibodies bound to cells.

[0253] Example 9: ADCC reporter gene assay This example demonstrates that the ADCC bioactivity of the BMP01 antibody was evaluated using the Jurkat / NFAT-luc-CD16A transgenic cell line as effector cells and tumor cell lines expressing HER2 or 5T4 as target cells via luciferase reporter gene assay.

[0254] The day before the ADCC experiment, HER2+ or 5T4+ target cells were cultured at a density of 1×10⁶ cells per well. 4The antibody was seeded at a density of 100 μl / well onto 96 plates and incubated overnight at 37°C in a 5% CO2 incubator. On the day of the experiment, the antibody to be tested was diluted 5-fold in RPMI-1640 medium containing 10% heat-inactivated FBS. 50 μL of the diluted antibody was added to each well containing target cells. BM130-93 (trastuzumab) or m603 (anti-5T4 antibody, SEQ ID NO 29 and SEQ ID NO 30) was used as a positive control. Then 1×10 4 Add 50 μl / well of Jurkat-NFAT-Luc2-CD16A-158V or Jurkat-NFAT-Luc2-CD16A-158F effector cells to each well and incubate for 6 hours at 37°C in a 5% CO2 incubator. At the end of incubation, add 100 μL of Steady-Glo luciferase assay substrate to each well, repeatedly pipette to lyse the cells, incubate at room temperature for 5–30 minutes, then transfer the lysate to a 96-well white plate to avoid bubble formation, and detect fluorescence values ​​using a microplate reader.

[0255] The ADCC luciferase induction fold was calculated by the fold change in the relative light units (RLU) values ​​of the test wells and control wells (reacted with simulated buffers for target cells and effector cells): fold change = (RLU test drug - RLU medium control) / (RLU NC - RLU medium control). Statistical analysis and plotting were performed using GraphPad Prism software.

[0256] Culture medium control: only culture medium was added. NC control: No target cells + effector cells in the test sample In experiments conducted essentially as described above, all antibody treatments induced ADCC in a dose- and target cell-dependent manner. Compared to BM130-93 (trastuzumab), BMP01 exhibited superior ADCC activity in terms of EC50 (Table 7) and maximum fold induction. Detailed results are as follows... Figure 9 As shown.

[0257] Table 7. EC50 luciferase activity of Jurkat T-CD16A-158F or 158V cells after antibody incubation. 50

[0258] Once L234 and L235 in the Fc region of trastuzumab or N297 are mutated to alanine to produce variants BM130-90 and BM130-91, both variants completely lose their ADCC activity, while BM130-93 retains its activity. However, the BMP01-16 LALA variant BMP01-24 and the N297A variant BMP01-25 remain active in the same assay, albeit at slightly lower levels. This result indicates that CD16A VH significantly contributes to the excellent NK-stimulating activity of the BMP01 antibody. Detailed results are as follows... Figure 10 As shown.

[0259] In experiments based on those described above, BMP02-10 (SEQ ID NO 30 and SEQ ID NO 31) treatment enhanced ADCC activity to varying degrees, depending on cell type and 5T4 expression level. Compared to the control 5T4 antibody m603, BMP02-10 significantly enhanced ADCC activity in EC2 cells. 50 They exhibit excellent ADCC activity in terms of maximum fold induction, for example, BMP02-10 and m603 EC. 50 The values ​​were 2.8 nM and 95 nM, respectively, as measured by JIMT-1. Detailed results are as follows... Figure 11 As shown in Table 8.

[0260] Table 8. EC50 values ​​of luciferase activity in Jurkat T-CD16A-158V cells after antibody incubation. 50

[0261] Example 10: NK cell killing assay In this embodiment, PBMCs were used as effector cells and tumor cell lines expressing HER2 or 5T4 were used as target cells. The enhanced NK cell killing activity of the BMP01 antibody was demonstrated by lactate dehydrogenase release assay.

[0262] The day before the NK lethality experiment, 2×10 4 100 μl of target cells were seeded into 96 plates and incubated overnight at 37°C in a 5% CO2 incubator. On the day of the experiment, the antibody to be tested was serially diluted 5-fold with RPMI-1640 medium. The target cell medium was discarded, 50 μl of fresh medium was added, and 50 μl of diluted antibody was added to each well containing target cells. Then, 5 × 10⁶ cells isolated from fresh blood were added. 5One PBMC cell was added to each well at 100 μl / well. Three control groups were set up: (1) Spontaneous release of target cells: 50 μL target cells and 150 μL test medium without the test molecule; (2) Antibody-independent cytotoxicity: 50 μL target cells, 100 μL PBMC cells, and 50 μL test medium without the test molecule; and (3) Maximum release of target cells: at the end of the experiment, 50 μL target cells and 120 μL culture medium were added, along with 30 μL / well lysis solution to determine maximum LDH release. The cells were further incubated at 37°C for 4–6 hours in a 5% CO2 incubator.

[0263] After incubation, transfer 50 μl / well of LDH-containing culture supernatant to a 96-well transparent plate, add 50 μl of CytoTox 96® reagent to the supernatant, and incubate at room temperature in the dark for 30 minutes. Then add 50 μl of stop solution and measure the absorbance at 490 nm or 492 nm within 1 hour.

[0264] The cell killing rate was calculated using the following formula. Statistical analysis and graphing of the data were performed using GraphPad Prism software.

[0265] Cell killing % = (sample antibody OD - antibody-independent cytotoxicity group OD) / (target cell maximum release group OD - target cell spontaneous release group OD) * 100 In experiments conducted essentially as described above, BMP01 treatment resulted in varying degrees of cytotoxicity induction, depending on cell type and HER2 expression levels. In all cases except HT-29, BMP01-16 showed significantly lower cytotoxicity in ECGs compared to BM130-93 (trastuzumab). 50 It exhibited superior enhanced NK cell killing activity in terms of maximum fold induction. Consistent with previous luciferase reporter gene assays, the BMP01-16 LALA variant BMP01-24 and the N297A variant BMP01-25 retained the stimulation of NK cell killing activity, although less than BMP01-16 in some cases. Detailed results are as follows... Figure 12 As shown.

[0266] In experiments based on those described above, BMP02-10 treatment enhanced NK cell killing of target cells to varying degrees, depending on cell type and 5T4 expression levels. Compared to the control 5T4 antibody m603, BMP02-10 significantly increased NK cell killing of target cells in EC2 cells. 50 It exhibited excellent cell-killing activity in terms of maximum fold induction. Detailed results are as follows: Figure 13 As shown.

[0267] Example 11: In vitro IFN-γ release assay This example demonstrates the innate immune cell stimulatory activity of BMP01, assessed by IFN-γ release assay, using PBMCs as effector cells and HER2-expressing tumor cell lines as target cells.

[0268] One day before the IFN-γ release experiment, 2×10 4 100 μl of target cells were seeded into 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. On the day of the experiment, the antibody to be tested was serially diluted 5-fold with RPMI-1640 medium. The target cell medium was discarded, 50 μl of fresh medium was added, and 50 μl of diluted antibody was added to each well containing target cells. Then, 5 × 10⁶ cells isolated from fresh blood were added. 5 One PBMC cell was added to each well at 100 μl / well and incubated for 72 hours at 37°C in a 5% CO2 incubator. After incubation, 50 μl / well of culture supernatant was transferred to a 96-well clear plate, and the concentration of IFN-γ was determined using the Human IFN-γ Quantikine ELISA kit under the manufacturer's (R&D Systems) recommended assay conditions.

[0269] In experiments conducted essentially as described above, BMP01-16 treatment resulted in varying degrees of IFN-γ induction, depending on cell type and HER2 expression levels. In all cases except HT-29, BMP01-16 induced more IFN-γ production than BMP130-93, indicating that the BMP01 bispecific antibody induced greater NK cell activation. In this assay, the BMP01-16 LALA variant BMP01-24 and N297A variant BMP01-25 showed an enhanced degree of mixing compared to BMP01-16. Detailed results are as follows... Figure 14 As shown.

[0270] Example 12: In vivo efficacy of BMP01-16 in a JIMT-1 / PBMC blend mouse model This example demonstrates the in vivo efficacy testing of the BMP01-16 antibody using a JIMT-1 / PBMC co-mixed mouse model.

[0271] On day 0, fresh human PBMC cells (5 × 10⁻⁶) were added. 6 (10 cells) and JIMT-1 human breast cancer cells (1×10) 6JIMT-1 cells were premixed at an E / T ratio of 5:1 and subcutaneously implanted into 6-8 week old female NOD / SCID mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.). The transplanted mice were randomly divided into 8 groups of 5 mice each. Starting from day 0, mice were treated intraperitoneally with BMP130-93 or BMP01-16 at three different dose levels (0.5 mg / kg, 1.5 mg / kg, 5 mg / kg of BMP130-93 and 0.6 mg / kg, 1.8 mg / kg, 6 mg / kg of BMP01-16) twice weekly for 4 weeks. Two control groups were also established: a JIMT-1 cancer cell group and a vector group. Tumor volume was recorded twice weekly from the day of tumor cell implantation. Data analysis and plotting were performed using GraphPad Prism software.

[0272] In the experiments conducted essentially as described above, by day 32 (the end of the study), moderate and high doses of BMP130-93 (trastuzumab) and BMP01-16 showed significant tumor growth inhibition. In contrast, a low dose of BM130-93 (trastuzumab) at 0.5 mg / kg had no tumor-suppressive effect, but a low dose of BMP01-16 at 0.6 mg / kg significantly inhibited tumor growth, indicating that BMP01-16 had better antitumor activity in this experimental setting. Detailed results are as follows... Figure 15 As shown.

[0273] Example 13: In vivo efficacy of BMP01-16 in the huHSC-NCG-hIL15 / HCC1954 model This example demonstrates the in vivo efficacy of the BMP01-16 antibody in a mouse subcutaneous transplantation model of human breast cancer HCC1954 / huHSC-NCG-hIL15.

[0274] NCG-IL15 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Human CD34+ hematopoietic stem cells were transplanted into the tail vein of irradiated NCG-hIL15 mice to construct huHSC-NCG-hIL15 human immune reconstitution mice. After 4-6 weeks, hCD45+ cells could be detected in peripheral blood, accounting for more than 20% of viable cells. The HCC1954 tumor cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. HCC1954 cells were cultured in RPMI-1640 medium containing 10% heat-inactivated FBS, and tumor cells were collected when the desired cell number was reached. For a total of 30 mice, 0.2 ml of 5.0 × 10⁶ cells was used. 6 HCC1954 cells + 50% Matrigel gel (PBS: Matrigel at a 1:1 volume ratio) were subcutaneously injected into the right forelimb of each mouse for tumor implantation.

[0275] Prior to grouping, the level of immune reconstitution in the peripheral blood of mice was analyzed by flow cytometry, using the following biomarkers: hCD45, hCD3, hCD19, hCD4, hCD8, hCD56, and hCD16. When the average tumor volume reached approximately 100-120 mm... 3 Eighteen tumor-bearing mice were randomly divided into three groups (n=6 per group) based on tumor volume, body weight, and immune reconstitution level, with a tumor volume CV ≤ 1 / 3 within each group. The day of grouping was defined as PG-D0, and the test antibodies were administered on the same day. The doses were 0.5 mg / kg BM130-93 (trastuzumab) and 0.6 mg / kg BMP01-16, respectively. For the next five weeks, both antibodies were administered intraperitoneally twice weekly. Body weight and tumor volume were measured twice weekly. Tumor growth inhibition (TGI) was calculated as follows: TGI% = (1 - T / C) × 100%, where T and C are the mean TV of the treatment and control groups at the end of the experiment. Data analysis was performed using GraphPadPrism software, and the results are shown as mean tumor volume ± SEM.

[0276] In the experiments conducted essentially as described above, when the tumor size reached approximately 100-120 mm... 3 Treatment began at [time]. 0.6 mg / kg BMP01-16 significantly inhibited tumor growth on day 38 post-grouping (the study termination point), with a TGI of 38.9%. Conversely, 0.5 mg / kg BM130-93 showed no tumor-suppressive effect in this model. Detailed results are as follows... Figure 16 As shown.

Claims

1. A single domain antibody that specifically binds to CD16A, wherein the single domain antibody has an amino acid sequence set forth in SEQ ID NO: 1 comprising a substitution at a position selected from the group consisting of S54, G55, S56, and any combination thereof, according to the Kabat numbering system.

2. The single domain antibody of 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. The single domain antibody of claim 1, wherein 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.

4. A single domain antibody that specifically binds to CD16A, wherein the single domain antibody comprises: (a) a CDR1 having an amino acid sequence set forth in SEQ ID NO: 41 or a conservatively modified variant thereof, a CDR3 having an amino acid sequence set forth in SEQ ID NO: 42 or a conservatively modified variant thereof, and a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, and a conservatively modified variant thereof, according to the International ImMunoGeneTion Society (IMGT) definition scheme; (b) a CDR1 having an amino acid sequence set forth in SEQ ID NO: 53 or a conservatively modified variant thereof, a CDR3 having an amino acid sequence set forth in SEQ ID NO: 54 or a conservatively modified variant thereof, and a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, and a conservatively modified variant thereof, according to the Kabat definition scheme; (c) a CDR1 having an amino acid sequence set forth in SEQ ID NO: 65 or a conservatively modified variant thereof, a CDR3 having an amino acid sequence set forth in SEQ ID NO: 66 or a conservatively modified variant thereof, and a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, and a conservatively modified variant thereof, according to the Chothia definition scheme; or (d) a CDR1 having an amino acid sequence set forth in SEQ ID NO: 77 or a conservatively modified variant thereof, a CDR3 having an amino acid sequence set forth in SEQ ID NO: 78 or a conservatively modified variant thereof, and a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, and a conservatively modified variant thereof, according to the Contact definition scheme. ​ ​ ​ ​ with the proviso that the single domain antibody does not have the amino acid sequence of SEQ ID NO:

1.

5. The single domain antibody of claim 4, wherein the single domain antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 39, 40 and an amino acid sequence that is at least 80% identical to each thereof.

6. The single domain antibody of 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 the single domain antibody of any one of claims 1 to 6 and an Fc portion linked to the single domain antibody.

8. A bispecific antibody comprising the single domain antibody of any one of claims 1 to 6.

9. The bispecific antibody of claim 8, wherein the bispecific antibody comprises the single domain antibody that specifically binds to CD16A and a second binding domain that specifically binds to a second target selected from the group consisting of a tumor associated antigen and a tumor specific antigen.

10. The bispecific antibody of claim 9, wherein the second target is selected from the group consisting of: HER2, 5T4, PSMA, BCMA, FGFR, CD20, CD33, CD19, CD22, CD123, CD30, GPC-3, CEA, EGFR1, EGFR2, EGFR3, TGF-beta, ROR1, PD-L1, Claudin 18.2, 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, Pmel 17, MAGE-1, AFP, CA125, TRP-1, TRP-2, NY-ESO, PSA, CDK4, BCA225, CA 125, MG7-Ag, NY-CO-1, RCAS 1, SDCCAG16, TAAL6, and TAG72.

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

12. The bispecific antibody of claim 11, wherein the peptide linker consists of glycine and serine residues.

13. The bispecific antibody of claim 11 or 12, wherein the single domain antibody is linked to the C-terminus of a heavy chain, the C-terminus of a light chain, or the N-terminus of a heavy chain of the full IgG antibody.

14. The bispecific antibody of claim 13, wherein the full IgG antibody is an anti-HER2 antibody or an anti-5T4 antibody.

15. The bispecific antibody of claim 14, wherein the anti-HER2 antibody comprises: (a) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 12 and a light chain having the amino acid sequence set forth in SEQ ID NO: 13; (b) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain having the amino acid sequence set forth in SEQ ID NO: 38; (c) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 25 and a light chain having the amino acid sequence set forth in SEQ ID NO: 13; or (d) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 26 and a light chain having the amino acid sequence set forth in SEQ ID NO:

13.

16. The bispecific antibody of claim 14, wherein the anti-5T4 antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 29 and a light chain having the amino acid sequence set forth in SEQ ID NO:

30.

17. The bispecific antibody of claim 14, wherein the bispecific antibody comprises: (a) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 14 and a light chain having the amino acid sequence set forth in SEQ ID NO: 13; (b) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain having the amino acid sequence set forth in SEQ ID NO: 13; (c) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 17 and a light chain having the amino acid sequence set forth in SEQ ID NO: 13; (d) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 12 and a light chain having the amino acid sequence set forth in SEQ ID NO: 16; (e) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain having the amino acid sequence set forth in SEQ ID NO: 13; (f) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 19 and a light chain having the amino acid sequence set forth in SEQ ID NO: 13; (g) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 20 and a light chain having the amino acid sequence set forth in SEQ ID NO: 13; (h) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 21 and a light chain having the amino acid sequence set forth in SEQ ID NO: 13; (i) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 22 and a light chain having the amino acid sequence set forth in SEQ ID NO: 13; (j) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 23 and a light chain having the amino acid sequence set forth in SEQ ID NO: 13; (k) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 24 and a light chain having the amino acid sequence set forth in SEQ ID NO: 13; (l) a heavy chain having the amino acid sequence of SEQ ID NO: 27 and a light chain having the amino acid sequence of SEQ ID NO: 13; or (m) a heavy chain having the amino acid sequence of SEQ ID NO: 28 and a light chain having the amino acid sequence of SEQ ID NO:

13.

18. The bispecific antibody of claim 14, wherein the bispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 31 and a light chain having the amino acid sequence of SEQ ID NO:

30.

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

20. The immunoconjugate of claim 19, wherein the active moiety is selected from the group consisting of a toxin, a peptide tag, a sortag, a radionuclide, a near-infrared fluorescent dye, and a nanoparticle.

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

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

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

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

25. Use of the single-domain antibody of any one of claims 1 to 6, the heavy-chain antibody of claim 7, the bispecific antibody of any one of claims 8 to 18, the immunoconjugate of claim 19 or 20, or the composition of claim 21, in the manufacture of a medicament for the treatment, prevention, or diagnosis of cancer, an acute or chronic infection, or a disease associated with aging.

26. A polypeptide comprising the single-domain antibody of any one of claims 1 to 6 or the heavy-chain antibody of claim 7; and one or more amino acid residues covalently linked to the N-terminus, C-terminus, or any position therebetween, of the single-domain antibody of any one of claims 1 to 6 or the heavy-chain antibody of claim 7.

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