CD16A Antibodies and Methods of Use

Abstract

The present disclosure provides antibodies or antigen-binding fragments that specifically bind to human CD16A. The antibodies and antigen-binding fragments are selective for CD16A over CD16B. The antibodies can activate NK cells upon binding to CD16A. In embodiments, the present disclosure is directed to anti-CD16A antibodies or antigen-binding fragments thereof that specifically bind to human CD16A. In embodiments, the anti-CD16A antibodies or antigen-binding fragments thereof are more selective for human CD16A over human CD16B. In embodiments, the anti-CD16A antibodies or antigen-binding fragments have cross-binding affinity for both human CD16A and cynomolgus CD16A.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to PCT Application No. PCT / CN2023 / 079509, entitled "CD16A Antibodies and Methods of Use," filed March 3, 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, which is provided as a file entitled "01368-0055-00PCT_SL.xml," created on February 27, 2024, and having a size of 119,384 bytes. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] Disclosed herein are antibodies that specifically bind to human CD16A with greater selectivity than human CD16B. [Background technology]

[0004] Natural killer (NK) cells are important innate immune lymphoid cells that mediate antiviral and antitumor responses. Tumor-targeting monoclonal antibodies (mAbs) can exert antibody-dependent cell-mediated cytotoxicity (ADCC) by binding to CD16A, a low-affinity Fc receptor on the surface of NK cells.

[0005] CD16A (FCGRIIIA, FCGR3A) is a type I membrane protein with two Ig-like domains that has low affinity for IgG. CD16A is widely found on myeloid cells such as macrophages, dendritic cells, mast cells, and eosinophils, as well as on NK cells and T cells, but not on neutrophils. Upon binding by antigen-IgG complexes, CD16A associates with the adaptor protein FcR gamma chain (FcγRI) to mediate signal transduction through its intracellular immunoreceptor tyrosine-based activation motifs (ITAMs), activating NK cells for cytolysis.

[0006] CD16A gene polymorphisms exist at two sites: 48 (L / R / H) and 158 (F / V). The presence of a valine at site 158 of CD16A (V / V or V / F) has been shown to enhance NK cell binding to IgG1 or IgG3 and generate higher levels of NK cell-mediated ADCC compared with CD16A(F / F158). Several clinical reports have shown that patients with CD16A(V / V158) had improved progression-free survival compared with patients with CD16A(F / F158) when treated with various monoclonal antibodies. These studies highlighted the importance of CD16A polymorphisms for NK cell effector function and the potential for controlling their cytotoxic activity by manipulating NK cell engagers.

[0007] Another CD16 isoform, CD16B (FCGR3B, FCGRIIIB), is present in humans but not in mice, rats, rabbits, llamas, or cynomolgus monkeys. CD16B is highly homologous to CD16A and can bind IgG with a low affinity range. CD16B is a GPI-anchored protein selectively expressed on neutrophils and eosinophils. It is generally believed to function as a decoy receptor, capable of binding IgG complexes without inducing activation. While it is unclear whether CD16B involvement on neutrophils will be clinically relevant, it is desirable to at least retain its role as a drug sink for neutrophils.

[0008] Although tumor-targeting therapeutic antibodies are successful in cancer treatment, they are not without limitations, such as off-target binding to other Fc receptors and competition with serum IgG for CD16A binding. Alternative approaches to enhancing NK cell-based ADCC function include antibodies with enhanced Fc binding activity through Fc engineering or glycoengineering, and bispecific or trispecific NK cell engagers (NKCEs). These NKCEs are designed so that one arm specifically recruits NK cells and the other arm binds to tumor cells. Compared to monospecific antibodies, NKCEs are more flexible with variable binding affinity, valency, and targeting. Therefore, they may offer unique opportunities for cancer therapy. Indeed, several NKCE molecules have entered clinical or preclinical stages. An anti-human CD16A-specific single-chain Fv (scFv) was first identified from a naive phage library and then affinity-matured. These NKCEs, which bind to NK cells via CD16A when paired with the disclosed tumor-associated antigens (TAA), e.g., CD30, EGFR, BCMA, or CD123, have already shown superior efficacy relative to antibodies in in vitro and in vivo assays.

[0009] There is a need for anti-CD16A antibodies that can specifically bind to human CD16A with minimal or no binding to human CD16B, and that are useful as NKCEs that pair with TAAs to treat TAA-positive tumors. Summary of the Invention

[0010] The present disclosure is directed to anti-CD16A antibodies and antigen-binding fragments thereof that specifically bind to CD16A (including CD16A 158F and CD16A 158V). The antibodies and antigen-binding fragments are selective for CD16A over CD16B. The antibodies can activate NK cells upon binding to CD16A.

[0011] In embodiments, the present disclosure is directed to anti-CD16A antibodies or antigen-binding fragments thereof that specifically bind to human CD16A.

[0012] In embodiments, the anti-CD16A antibody or antigen-binding fragment thereof is more selective for human CD16A over human CD16B.

[0013] In embodiments, the anti-CD16A antibody or antigen-binding fragment has cross-binding affinity for both human CD16A and cynomolgus CD16A.

[0014] In embodiments, the anti-CD16A antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a F(ab')2 fragment, a heavy-chain antibody (HcAb), or a VHH.

[0015] In embodiments, the anti-CD16A antibody or antigen-binding fragment thereof is a VHH.

[0016] In embodiments, the anti-CD16A antibody or antigen-binding fragment thereof comprises at least one heavy chain CDR selected from the group consisting of: a) the heavy chain CDR1 sequence of SEQ ID NO: 109; b) a heavy chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 110 and 114, and c) The heavy chain CDR3 sequence of SEQ ID NO: 111.

[0017] In embodiments, the anti-CD16A antibody or antigen-binding fragment thereof comprises each of the following heavy chain CDRs: a) the heavy chain CDR1 sequence of SEQ ID NO: 109; b) a heavy chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 110 and 114, and c) Heavy chain CDR3 sequence of SEQ ID NO: 111.

[0018] In embodiments, the anti-CD16A antibody or antigen-binding fragment comprises a heavy chain variable region comprising: a) HCDR1 of SEQ ID NO: 109, (b) HCDR2 of SEQ ID NO: 110, and (c) HCDR3 of SEQ ID NO: 111, or (b) HCDR1 of SEQ ID NO: 109, (b) HCDR2 of SEQ ID NO: 114, and (c) HCDR3 of SEQ ID NO: 111.

[0019] In embodiments, the anti-CD16A antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising at least one amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 112, 115, 117, or 119.

[0020] In embodiments, the anti-CD16A antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising at least one amino acid sequence selected from SEQ ID NOs: 112, 115, 117, and 119, having a deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in at least one of SEQ ID NOs: 112, 115, 117, and 119.

[0021] In embodiments, the anti-CD16A antibody or antigen-binding fragment thereof comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 112, 115, 117, and 119.

[0022] The present disclosure is also directed to a multispecific antibody or antigen-binding fragment thereof comprising at least one first antigen-binding domain that specifically binds to human CD16A, wherein the first antigen-binding domain comprises an antibody-binding fragment that specifically binds to human CD16A.

[0023] In embodiments, in the multispecific antibody or antigen-binding fragment, the first antigen-binding domain is more selective for human CD16A than for human CD16B.

[0024] In embodiments, in the multispecific antibody or antigen-binding fragment, the first antigen-binding domain has cross-binding affinity for both human CD16A and cynomolgus CD16A.

[0025] In embodiments, in the multispecific antibody or antigen-binding fragment, the first antigen-binding domain is a VHH.

[0026] In embodiments, the multispecific antibody or antigen-binding fragment is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single chain antibody (scFv), a Fab fragment, a F(ab')2 fragment, a heavy chain antibody (HcAb), and a VHH.

[0027] In embodiments, the multispecific antibody or antigen-binding fragment comprises at least one heavy chain CDR selected from the group consisting of: a) the heavy chain CDR1 sequence of SEQ ID NO: 109; b) a heavy chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 110 and 114, and c) Heavy chain CDR3 sequence of SEQ ID NO: 111.

[0028] In embodiments, the multispecific antibody or antigen-binding fragment comprises each of the following heavy chain CDRs: a) the heavy chain CDR1 sequence of SEQ ID NO: 109; b) a heavy chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 110 and 114, and c) Heavy chain CDR3 sequence of SEQ ID NO: 111.

[0029] In embodiments, the multispecific antibody or antigen-binding fragment comprises a heavy chain variable region comprising: a) HCDR1 of SEQ ID NO: 109, (b) HCDR2 of SEQ ID NO: 110, and (c) HCDR3 of SEQ ID NO: 111, or (b) HCDR1 of SEQ ID NO: 109, (b) HCDR2 of SEQ ID NO: 114, and (c) HCDR3 of SEQ ID NO: 111.

[0030] In embodiments, the multispecific antibody or antigen-binding fragment comprises a heavy chain variable region (VH) comprising at least one amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 112, 115, 117, or 119.

[0031] In embodiments, the multispecific antibody or antigen-binding fragment comprises a heavy chain variable region (VH) comprising at least one amino acid sequence selected from SEQ ID NO: 112, 115, 117, or 119, having a deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in at least one of SEQ ID NO: 112, 115, 117, or 119.

[0032] In embodiments, the multispecific antibody or antigen-binding fragment comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 112, 115, 117, and 119.

[0033] The present disclosure is also directed to VHHs that specifically bind to human CD16A.

[0034] In embodiments, the VHH is more selective for human CD16A over human CD16B.

[0035] In embodiments, the VHH has cross-binding affinity for both human CD16A and cynomolgus CD16A.

[0036] In embodiments, the VHH is a humanized or human engineered antibody.

[0037] In embodiments, the VHH comprises at least one heavy chain CDR selected from the group consisting of: a) the heavy chain CDR1 sequence of SEQ ID NO: 109; b) a heavy chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 110 and 114, and c) Heavy chain CDR3 sequence of SEQ ID NO: 111.

[0038] In embodiments, the VHH comprises each of the following heavy chain CDRs: a) the heavy chain CDR1 sequence of SEQ ID NO: 109; b) a heavy chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 110 and 114, and c) Heavy chain CDR3 sequence of SEQ ID NO: 111.

[0039] In embodiments, the VHH comprises a heavy chain variable region comprising: a) HCDR1 of SEQ ID NO: 109, (b) HCDR2 of SEQ ID NO: 110, and (c) HCDR3 of SEQ ID NO: 111, or (b) HCDR1 of SEQ ID NO: 109, (b) HCDR2 of SEQ ID NO: 114, and (c) HCDR3 of SEQ ID NO: 111.

[0040] In embodiments, the VHH comprises a heavy chain variable region (VH) comprising at least one amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 112, 115, 117, or 119.

[0041] In embodiments, the VHH comprises a heavy chain variable region (VH) comprising at least one amino acid sequence selected from SEQ ID NO: 112, 115, 117, or 119, having an insertion, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in at least one of SEQ ID NO: 112, 115, 117, or 119.

[0042] In embodiments, the VHH comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 112, 115, 117, and 119.

[0043] The present disclosure also provides constructs comprising the antibodies or antigen-binding fragments disclosed herein.

[0044] In embodiments, the construct comprises one or more VHHs disclosed herein.

[0045] In embodiments, the construct is a multispecific antibody, a heavy chain antibody, a bivalent VHH, a double paratopic VHH, a bispecific VHH, a VHH-scFv, a VHH-cytokine, a VHH-drug, a VHH-nanoparticle, a VHH-virus, or a VHH-imaging probe.

[0046] In embodiments, the present disclosure provides anti-CD16A antibodies, antigen-binding fragments thereof, or constructs thereof that specifically bind to and have high affinity for human CD16A, exhibit excellent overall biophysical properties, and / or exhibit excellent pharmacokinetics. The excellent biophysical properties may be Tm and / or Tag.

[0047] In embodiments, the anti-CD16A antibody, antigen-binding fragment thereof, or construct has at least one of the following characteristics: (1) specific binding and high affinity to human CD16A; (2) Excellent pharmacokinetics (3) good overall biophysical properties, e.g., Tm or Tag, or (4) It is a humanized antibody with low immunogenicity risk in humans.

[0048] In embodiments, the present disclosure provides anti-CD16A antibodies, antigen-binding fragments thereof, or constructs thereof that are humanized antibodies that maintain specific binding to and high affinity for human CD16A, exhibit excellent overall biophysical properties, and have a low risk of immunogenicity in humans.

[0049] The present disclosure is also directed to pharmaceutical compositions comprising any of the anti-CD16A antibodies, antigen-binding fragments, or constructs disclosed herein and a pharmaceutically acceptable carrier.

[0050] The present disclosure also provides isolated nucleic acids encoding the anti-CD16A antibodies or antigen-binding fragments disclosed herein.

[0051] The present disclosure also provides vectors comprising the nucleic acids disclosed herein.

[0052] The present disclosure also provides a host cell comprising a nucleic acid or vector disclosed herein.

[0053] The present disclosure also provides a process for producing an anti-CD16A antibody or antigen-binding fragment thereof, comprising culturing a host cell disclosed herein and recovering the antibody or antigen-binding fragment from the culture. [Brief explanation of the drawings]

[0054] [Figure 1] 1 is a bar graph showing the results of ELISA analysis of a representative top clone, BG523P, against LS21. [Figure 2A] 1 is a line graph showing FACS analysis of BG523P, a representative top clone, relative to LS21 in NK92mi / CD16A(V158) cells. [Figure 2B] 1 is a line graph showing FACS analysis of BG523P, a representative top clone, relative to LS21 in NK92mi / CD16B(NA1) cells. [Figure 2C] 1 is a line graph showing FACS analysis of BG523P, a representative top clone, relative to LS21 in NK92mi / CD16B(NA2) cells. [Figure 3A] 1 is a line graph showing FACS binding comparison of BG523P and BG524P with CD16A 158F overexpressing cells (NK92mi / CD16A 158F cell line). [Figure 3B] 1 is a line graph showing FACS binding comparison of BG523P, BG525P, and BG526P with CD16A 158F overexpressing cells (NK92mi / CD16A 158F cell line). [Figure 4] 1 is a bar graph showing FACS binding signals of BG523P, BG525P, and BG526P at 300 nM on CD16B-overexpressing cells (NK92mi / CD16B NA1 and NK92mi / CD16B NA2 cell lines). [Figure 5A]1 is a line graph showing FACS-based human IgG competition for NK92mi / CD16A 158F binding to BG523P in the presence or absence of 10 mg / mL recombinant CB6 human IgG1. The effect of IgG competition on BG523P binding to NK92mi / CD16A 158F is shown. [Figure 5B] 1 is a line graph showing FACS-based human IgG competition for NK92mi / CD16A 158F binding to humanized VHH (BG525P) in the presence or absence of 10 mg / mL recombinant CB6 human IgG1. The effect of IgG competition on BG525P binding to NK92mi / CD16A 158F is shown. [Figure 5C] 1 is a line graph showing FACS-based human IgG competition for NK92mi / CD16A 158F binding to humanized VHH (BG526P) in the presence or absence of 10 mg / mL recombinant CB6 human IgG1. The effect of IgG competition on BG526P binding to NK92mi / CD16A 158F is shown. DETAILED DESCRIPTION OF THE INVENTION

[0055] definition Unless specifically defined below or elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0056] As used herein, including in the appended claims, singular terms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0057] The term "or" is used to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0058] Unless otherwise specified or clear from the context, the term "about" as used herein refers to a value or composition that falls within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, according to practice in the art. "About" can also mean a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% more or less than the stated value. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, these terms can mean values ​​up to an order of magnitude or up to 5 times greater. When a particular value or composition is provided in this disclosure, unless otherwise specified, the meaning of "about" should be assumed to be within the acceptable error range of that particular value or composition.

[0059] The term "CD16A" refers to a type I membrane protein with two Ig-like domains with low affinity for IgG, also known as FCGRIIIA and FCGR3A. The amino acid sequence of human CD16A (P08637) can be found in the Uniprot database at Uniprot P08637.

[0060] As used herein, the terms "administration" and "administering," when applied to an animal, human, subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent with the cell, as well as contact of a reagent with a fluid when the fluid is in contact with the cell.

[0061] The term "subject" or "patient" as used herein includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, primate), and most preferably a human (e.g., a patient having or at risk of having a disorder described herein).

[0062] In one aspect, "treating" any disease or disorder refers to ameliorating the disease or disorder (i.e., delaying, preventing, or reducing the onset of the disease or at least one of its clinical symptoms). In another aspect, "treat," "treating," or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another aspect, "treat," "treating," or "treatment" refers to modulating the disease or disorder either physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both.

[0063] The term "affinity" as used herein refers to the strength of the interaction between an antibody and an antigen. Within the antigen, the variable region of the antibody interacts with the antigen through non-covalent forces at multiple sites. Generally, the more interactions, the stronger the affinity.

[0064] The term "antibody," as used herein, refers to a polypeptide of the immunoglobulin family that can bind to a corresponding antigen in a reversible and specific manner other than by covalent bonds. For example, naturally occurring IgG antibodies are tetramers containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL or Vκ) 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 regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four framework regions (FRs), arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin 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.

[0065] The locations of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997); Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003).

[0066] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies. Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0067] The term "chimeric antibody" refers to a molecule composed of domains derived from different species, i.e., in which the variable domains of an antibody derived from one host species (e.g., mouse, rabbit, llama, etc.) are fused with the constant domains of an antibody derived from a different species (e.g., human).

[0068] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules within the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a large number of different antibodies with different amino acid sequences within their variable domains, particularly their CDRs, which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those skilled in the art. See, for example, Kohler et al., Nature 1975 256:495-497; U.S. Patent No. 4,376,110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory 1988; and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The antibodies disclosed herein can be of any immunoglobulin class, such as IgG, IgM, IgD, IgE, IgA, and any subclass thereof, e.g., IgG1, IgG2, IgG3, IgG4. Hybridomas producing monoclonal antibodies can be cultivated in vitro or in vivo. High-titer monoclonal antibodies can be obtained by in vivo production, where cells from individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb / c mice, to produce ascites fluid containing high concentrations of the desired antibody. Monoclonal antibodies of the IgM or IgG isotype can be purified from such ascites fluid or from the culture supernatant using column chromatography techniques well known to those skilled in the art.

[0069] Generally, the basic structural unit of an antibody comprises a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are usually classified as α, δ, ε, γ, or μ, and the antibody isotype is defined as IgA, IgD, IgE, IgG, and IgM, respectively.

[0070] Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including an additional "D" region of about 10 amino acids.

[0071] The variable regions of each light / heavy chain (VL / VH) pair form the antibody binding site. Thus, an intact antibody generally has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites generally have the same primary sequence.

[0072] Typically, both heavy and light chain variable domains contain three hypervariable regions, also called "complementarity-determining regions (CDRs)," which are located between relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. Generally, from the N-terminus to the C-terminus, both light and heavy chain variable domains contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (FR3), CDR-3 (CDR3), and FR-4 (FR4). The locations of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997) ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) (see "IMGT" numbering scheme)).The definition of antigen-binding sites is also described in Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). For example, in Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the Kabat and Chothia CDR definitions, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.In IMGT, the CDR amino acid residues of the VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues of the VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (Kabat numbering). In IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGapAlign.

[0073] The term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. A hypervariable region comprises amino acid residues from a "CDR" (e.g., LCDR1, LCDR2, and LCDR3 in the light-chain variable domain and HCDR1, HCDR2, and HCDR3 in the heavy-chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., which define antibody CDR regions by sequence. See also Chothia and Lesk (1987) J. Mol. Biol. 196:901-917, which define antibody CDR regions by structure. The term "framework" or "FR" residues refers to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0074] Unless otherwise specified, "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to the antigen bound by the full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules such as single-chain Fv (ScFv), nanobodies (or VHH antibodies), multispecific antibodies formed from antibody fragments, and bicyclic peptides (Hurov, K. et al., 2021. Journal for ImmunoTherapy of Cancer, 9(11)).

[0075] As used herein, an antibody or antigen-binding antibody fragment "specifically binds" to an antigen (e.g., a protein) means that the antibody exhibits preferential binding to its target relative to other proteins, although this specificity does not require absolute binding specificity. A "specific" or "selective" binding reaction determines the presence of an antigen in a heterogeneous population of proteins and other biologics, for example, in a biological sample, blood, serum, plasma, or tissue sample. Thus, under certain designated immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least twice as much as background levels and does not specifically bind in significant amounts to other antigens present in the sample. In one embodiment, under designated immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least 10 times as much as background levels of binding and does not specifically bind in significant amounts to other antigens present in the sample.

[0076] As used herein, an "antigen-binding domain" comprises at least six CDRs (or, in the context of a single-domain antibody, three CDRs) and specifically binds to an epitope. The "antigen-binding domain" of a multispecific antibody (e.g., a bispecific antibody) comprises a first antigen-binding domain that specifically binds to a first epitope and a second antigen-binding domain that also comprises at least three CDRs that specifically bind to a second epitope. Multispecific antibodies can be bispecific, trispecific, tetraspecific, etc., with an antigen-binding domain directed to each specific epitope. Multispecific antibodies can be multivalent (e.g., a bispecific tetravalent antibody) comprising multiple antigen-binding domains, for example, two, three, four, or more antigen-binding domains that specifically bind to a first epitope and two, three, four, or more antigen-binding domains that specifically bind to a second epitope. The "antigen-binding domain" of a single-chain antibody, such as a heavy-chain antibody, or a VHH, comprises an antigen-binding domain that specifically binds to an epitope without pairing with an additional variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH / VHH or VL domain.

[0077] As used herein, the terms "VHH domain," "VHH antibody," "VHH antibody fragment," and "VHH" (also known as nanobody) refer to the antibody binding domain originally described as a heavy-chain-only antibody produced by camelids (naturally occurring antibodies lacking light chains; C. Hamers-Casterman et al., Nature, volume 363, pages 446-448 (1993)), which is distinct from the heavy-chain variable domain (VH domain) of conventional tetrameric antibodies. VHH antibodies retain the immunoglobulin fold of conventional four-chain antibodies, with only three hypervariable loops, CDR1, CDR2, and CDR3, binding to their targets. Many VHHs bind to their targets with affinities similar to those of conventional full-sized antibodies and may have other superior properties.

[0078] The term "human antibody" herein refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse glycosylation if produced in a mouse, a mouse cell, or a mouse cell-derived hybridoma. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse immunoglobulin protein sequences or only rat immunoglobulin protein sequences, respectively.

[0079] The terms "humanized" or "humanized antibody" refer to forms of antibodies comprising sequences derived from non-human (e.g., murine) and human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. A humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region (Fc). Where necessary to distinguish humanized antibodies from rodent parent antibodies, the name of the antibody clone is prefixed with "hum," "hu," "Hu," or "h." Humanized forms of rodent antibodies generally contain the same CDR sequences of the parent rodent antibody, but can contain certain amino acid substitutions to increase affinity, increase the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0080] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. Corresponding human germline sequence can also refer to a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other evaluated variable region amino acid sequences. The corresponding human germline sequence can be framework regions only, complementarity determining regions only, framework and complementarity determining regions, variable regions, or other combinations of sequences or subsequences. Sequence identity can be determined using methods described herein, such as aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid or amino acid sequence of the reference variable region. Furthermore, if the antibody contains a constant region, the constant region also is derived from such a human sequence, e.g., a human germline sequence, or a mutated version of a human germline sequence, or an antibody containing a consensus framework sequence derived from human framework sequence analysis, e.g., as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000.

[0081] The term "equilibrium dissociation constant" or "KD" or "M" refers to the dissociation rate constant (kd, time -1 ) to the association rate constant (ka, time -1 , M -l ) The equilibrium dissociation constant can be measured using any method known in the art. Antibodies of the present disclosure generally have an equilibrium dissociation constant of about 10 -7 Less than or equal to 10 -8 Less than m, e.g., about 10 -9 Less than M or 10 -10M, and in some embodiments, less than about 10 -11 Under M, 10 -12 Less than M or 10 -13 It is less than M.

[0082] The terms "cancer" or "tumor" as used herein have the broadest meaning understood in the art and refer to a physiological condition in mammals that is typically characterized by unregulated cell growth. In the context of this disclosure, cancer is not limited to any particular type or location.

[0083] In the context of the present disclosure, when referring to an amino acid sequence, the term "conservative substitution" refers to the substitution of an original amino acid with a new amino acid that does not substantially alter the chemical, physical, and / or functional properties of the antibody or fragment, e.g., its binding affinity to CD16A. Common conservative changes of amino acids are well known in the art.

[0084] As used herein, the term "knob-into-hole" technology refers to amino acids that direct pairing of two polypeptides either in vitro or in vivo by introducing a spatial protuberance (knob) in one polypeptide and a socket or cavity (hole) in the other polypeptide at the interface where they interact. For example, knob-into-hole technology can be used to direct pairing of two polypeptides, either in vitro or in vivo, ... L :C HIn some embodiments, knob-into-hole amino acids have been introduced into the VH / VL interface or the VH / VL interface (see, e.g., US2011 / 0287009, US2007 / 0178552, WO96 / 027011, WO98 / 050431, and Zhu et al., 1997, Protein Science, 6:781-788). In some embodiments, knob-into-hole amino acids ensure the correct pairing of two different heavy chains together during the production of multispecific antibodies. For example, multispecific antibodies with knob-into-hole amino acids in their Fc region may further comprise a single variable domain linked to each Fc region, or may further comprise a different heavy chain variable domain paired with a similar or different light chain variable domain. Knob-into-hole technology can also be used with VH or VL regions to ensure correct pairing.

[0085] An example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977, and Altschul et al., J. Mol. Biol. 215:403-410, 1990. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet a positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits serve as starting points for searches to find longer HSPs containing them. Word hits are extended outward along each end of each sequence as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted if the cumulative alignment score falls by an amount X from the maximum achieved value, if the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments, or if either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLAST program uses as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), an alignment (B) of 50, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two sequences of nucleotides or amino acids would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0086] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4:11-17, (1988), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight remainder table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970), which has been incorporated into the GAP program in the GCG software package, using either a BLOSUM62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0087] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, including synthetic, naturally occurring, and non-naturally occurring nucleic acids, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0088] The term "operably linked" in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences operably linked to a transcriptional sequence are physically contiguous to the transcriptional sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically contiguous to or located in close proximity to the coding sequence whose transcription they enhance.

[0089] In some aspects, the present disclosure provides compositions, e.g., pharmaceutically acceptable compositions, comprising an anti-CD16A antibody described herein formulated with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" includes all physiologically compatible solvents, dispersion media, isotonic and absorption delaying agents, and the like. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0090] The compositions disclosed herein can be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The appropriate form depends on the intended mode of administration and therapeutic application. One suitable mode of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0091] As used herein, the term "therapeutically effective amount" refers to the amount of an antibody that, when administered to a subject to treat a disease or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment for the disease, disorder, or condition. A "therapeutically effective amount" may vary depending on the antibody, the disease, disorder, and / or symptoms of the disease or disorder, the severity of the disease, disorder, and / or symptoms of the disease or disorder, the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in any given case will be apparent to one of skill in the art or can be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of the combined components.

[0092] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner. Such administration also includes co-administration in multiple or separate containers or formulations (e.g., capsules, powders, and liquids) for each active ingredient. The powders and / or liquids can be reconstituted or diluted to the desired dose before administration. Furthermore, "combination therapy" encompasses the use of each type of therapeutic agent in a sequential manner, either at about the same time or at different times. In either case, the treatment regimen provides the beneficial effects of the drug combination in treating the conditions or disorders described herein.

[0093] As used herein, the phrase "in combination with" means that the anti-CD16A antibody is administered to the subject simultaneously with, immediately before, or immediately after the administration of an additional therapeutic agent.

[0094] Detailed Description of the Invention The present disclosure is directed to anti-CD16A antibodies and antigen-binding fragments thereof, as well as multispecific antibodies or antigen-binding fragments thereof that recognize CD16A as one antigen and at least one tumor antigen as at least one second antigen. The antigen-binding fragments may be VHH antibody fragments. The disclosed antibodies and antigen-binding fragments have desirable pharmacokinetic properties, desirable biophysical properties, and other desirable attributes, such as selective binding to CD16A with negligible or no binding to CD16B. Pharmaceutical compositions comprising the antibodies or antigen-binding fragments are also disclosed.

[0095] Anti-CD16A antibody The present disclosure provides antibodies or antigen-binding fragments that specifically bind to CD16A. In embodiments, the anti-CD16A antibodies or antigen-binding fragments disclosed herein can activate human cells that express CD16A, including NK cells, and induce a signaling response through said binding. In embodiments, the antibodies or antigen-binding fragments are VHH antibodies. In embodiments, the antibodies or antigen-binding fragments cause antibody-dependent cellular cytotoxicity (ADCC). In embodiments, the antibodies or antigen-binding fragments cause CD16A-mediated cell killing through binding to CD16A-expressing cells, such as NK cells. The antibodies or antigen-binding fragments can be produced as described below.

[0096] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to CD16A, wherein the antibodies or antibody fragments (e.g., antigen-binding fragments) comprise a VHH domain comprising the amino acid sequence of SEQ ID NO: 112, SEQ ID NO: 115, SEQ ID NO: 117, or SEQ ID NO: 119 (Table 2). The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to CD16A, wherein the antibodies or antigen-binding fragments comprise an HCDR (heavy chain complementarity determining region) comprising the amino acid sequence of any one of the HCDRs listed in Table 2. In one aspect, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to CD16A, wherein the antibodies comprise one, two, three, or more HCDRs (or alternatively consist of one, two, three, or more HCDRs) comprising the amino acid sequence of any one of the HCDRs listed in Table 2.

[0097] Other antibodies or antigen-binding fragments thereof of the present disclosure include amino acid alterations in the CDR regions that have at least 60%, 70%, 80%, 90%, 95%, or 99% identity to the CDR regions disclosed in Table 2. In some embodiments, this includes amino acid alterations in which no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acids are altered in the CDR regions when compared to the CDR regions shown in the sequences in Table 2.

[0098] Other antibodies of the present disclosure include those in which the amino acids or nucleic acids encoding the amino acids have alterations but are at least 60%, 70%, 80%, 90%, 95%, or 99% identical to the sequences disclosed in Table 2. In some embodiments, this includes alterations in the amino acid sequence in which no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acids are altered in the variable regions when compared to the variable regions set forth in the sequences disclosed in Table 2, while retaining substantially the same therapeutic activity.

[0099] The present disclosure also provides nucleic acid sequences encoding VH domain antibodies and full-length heavy chains of antibodies that specifically bind to CD16A, which nucleic acid sequences can be optimized for expression in mammalian cells.

[0100] Identification of epitopes and antibodies that bind to the same epitopes The present disclosure provides antibodies and antigen-binding fragments that bind to an epitope of human CD16A. In certain embodiments, the antibodies and antigen-binding fragments can bind to the same epitope of CD16A.

[0101] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as the anti-CD16A antibodies described in Table 2. Accordingly, additional antibodies and antigen-binding fragments thereof can be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with other antibodies in binding assays. The ability of a test antibody to inhibit the binding of an antibody and antigen-binding fragment thereof of the present disclosure to CD16A demonstrates that the test antibody can compete with that antibody or antigen-binding fragment thereof for binding to CD16A. Without being bound by any one theory, such antibodies may bind to the same or related (e.g., structurally similar or spatially proximal) epitope on CD16A as the competing antibody or antigen-binding fragment thereof. In certain embodiments, antibodies that bind to the same epitope on CD16A as the antibodies or antigen-binding fragments thereof of the present disclosure are human or humanized monoclonal antibodies. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0102] Fc region modification In some embodiments, the Fc region is modified by substituting at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be substituted with a different amino acid residue, resulting in an antibody with altered affinity for an effector ligand but retaining the antigen-binding ability of the parent antibody. The affinity-altered effector ligand can be, for example, an Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0103] In another embodiment, one or more amino acid residues can be substituted with one or more different amino acid residues such that the antibody has altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al.

[0104] In yet another embodiment, one or more amino acid residues are altered to modify the antibody's ability to fix complement. This approach is described, for example, in publication WO 94 / 29351 by Bodmer et al. In certain embodiments, one or more amino acids of an antibody or antigen-binding fragment thereof of the present disclosure are replaced with one or more allotypic amino acid residues for the IgG1 subclass and kappa isotype. Allotypic amino acid residues include, but are not limited to, the heavy chain constant regions of the IgG1, IgG2, and IgG3 subclasses and the light chain constant region of the kappa isotype, as described by Jefferis et al., MAbs.1:332-338 (2009).

[0105] In another embodiment, the Fc region is modified by modifying one or more amino acids to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors. This approach is described, for example, in publication WO 00 / 42072 by Presta. Furthermore, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0106] In yet another embodiment, the glycosylation of the multispecific antibody is modified. For example, an aglycosylated antibody (i.e., the antibody lacks or has reduced glycosylation) can be generated. Altering glycosylation can, for example, increase the affinity of the antibody for an "antigen." Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to remove one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. Such aglycosylation can increase the affinity of the antibody for the antigen. Such approaches are described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0107] Additionally or alternatively, antibodies can be generated with altered types of glycosylation (e.g., hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNAc structures). Such altered glycosylation patterns have been shown to enhance the ADCC ability of antibodies. Such glycosylation modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation pathway. Cells with altered glycosylation pathways have been described in the art and can be used as host cells for expressing recombinant antibodies, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line in which the FUT8 gene, encoding fucosyltransferase, has been functionally disrupted, such that antibodies expressed in such a cell line exhibit hypofucosylation. Publication WO 03 / 035835 by Presta describes a mutant CHO cell line, Lecl3 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). WO 99 / 54342 by Umana et al. describes cell lines engineered to express a glycoprotein-modifying glycosyltransferase (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibody (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0108] Anti-CD16A antibody production Anti-CD16A antibodies, antigen-binding fragments, and multispecific antibodies can be produced by any means known in the art, including, but not limited to, recombinant expression of antibody tetramers, chemical synthesis, and enzymatic digestion, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0109] The present disclosure further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding a heavy or light chain variable region or a segment comprising a complementarity determining region described herein. In some embodiments, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 113, SEQ ID NO: 116, SEQ ID NO: 118, or SEQ ID NO: 120.

[0110] The polynucleotides of the present disclosure can encode the variable region sequences of anti-CD16A antibodies. They can also encode both the variable and constant regions of the antibodies. Some of the polynucleotide sequences encode polypeptides containing both the heavy and light chain variable regions of one of the exemplified anti-CD16A antibodies.

[0111] The present disclosure also provides expression vectors and host cells for producing anti-CD16A antibodies. The choice of expression vector depends on the intended host cell in which the vector will be expressed. Typically, expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the anti-CD16A antibody chain or antigen-binding fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence except under the control of inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be grown under non-inducing conditions without biasing the population toward coding sequences whose expression products are better tolerated by the host cell. In addition to promoters, other regulatory elements may be required or desired for efficient expression of anti-CD16A antibodies or antigen-binding fragments. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. Furthermore, the efficiency of expression can be increased by incorporating enhancers appropriate for the cell system being used (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994, and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0112] Host cells for harboring and expressing anti-CD16A antibody vectors can be prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other suitable microbial hosts include bacilli such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors can also be made in these prokaryotic hosts, typically containing expression control sequences compatible with the host cell (e.g., an origin of replication). Additionally, various well-known promoters exist, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or promoter systems derived from phage lambda. The promoter typically controls expression, optionally via an operator sequence, and contains, for example, ribosome binding site sequences for initiating and completing transcription and translation. Other microorganisms, such as yeast, can also be used to express anti-CD16A polypeptides. Insect cells can also be used in conjunction with baculovirus vectors.

[0113] In other embodiments, mammalian host cells are used to express and produce the anti-CD16A polypeptides of the present disclosure. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines harboring exogenous expression vectors. These include any normal mortal, or normal or abnormal immortal, animal or human cells. For example, several suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell culture to express polypeptides is generally discussed, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells can include expression control sequences such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or tunable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP pol III promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0114] Anti-CD16A multispecific antibodies and constructs In one embodiment, the anti-CD16A antibody or antigen-binding fragment disclosed herein can be incorporated into an anti-CD16A×TAA multispecific antibody, where the TAA is an antibody or fragment thereof directed against any human tumor-associated antigen (TAA). For example, when the antibody comprises several antigen-binding domains, the antibody is a multispecific antibody molecule, where at least one antigen-binding domain sequence specifically binds to CD16A and a second antigen-binding domain sequence specifically binds to a TAA. In embodiments, the multispecific antibody comprises a third, fourth, or fifth antigen-binding domain. In embodiments, the multispecific antibody is a bispecific, trispecific, or tetraspecific antibody. In each embodiment, the multispecific antibody comprises at least one anti-CD16A antigen-binding domain and at least one anti-TAA antigen-binding domain. In embodiments, binding of the multispecific antibody or fragment thereof to CD16A via the anti-CD16A binding domain arm can result in the activation of NK cells and, through binding thereto, the killing of tumor cells expressing antigens directed against the other arm(s) of the multispecific antibody or fragment thereof. Thus, anti-CD16A multispecific antibodies can be used to treat a variety of cancers or other diseases, depending on the specificity of the other arm(s) of the multispecific antibody.

[0115] In one embodiment, a multispecific antibody is a bispecific antibody. As used herein, a bispecific antibody specifically binds to only two antigens. A bispecific antibody comprises a first antigen-binding domain that specifically binds to CD16A and a second antigen-binding domain that specifically binds to a TAA. This includes bispecific antibodies comprising a heavy chain variable domain that specifically binds to CD16A and a heavy chain variable domain and a light chain variable domain that specifically bind to a TAA. In another embodiment, a bispecific antibody comprises an antigen-binding fragment of an antibody that specifically binds to CD16A and an antigen-binding fragment that specifically binds to a TAA. When a bispecific antibody comprises an antigen-binding fragment, the antigen-binding fragment can be a Fab, F(ab')2, Fv, or single-chain Fv (scFv).

[0116] Previous work (Coloma and Morrison, Nature Biotech. 15:159-163 (1997)) described tetravalent bispecific antibodies engineered by fusing DNA encoding a single-chain anti-dansyl antibody Fv (scFv) to the C-terminus (CH3-scFv) or hinge (hinge-scFv) of an IgG3 anti-dansyl antibody. The present disclosure provides multivalent antibodies (e.g., tetravalent antibodies) having at least two antigen-binding domains, which can be readily produced by recombinant expression of nucleic acids encoding antibody polypeptide chains. The multivalent antibodies herein contain three to eight, preferably four, antigen-binding domains that specifically bind to at least two antigens.

[0117] The present disclosure provides a bispecific tetravalent antibody comprising VD1-CL-(X1)n-VD2-CH1-Fc or VD1-CH-(X1)n-VD2-CL-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, CH or CL is a constant heavy chain domain or constant light chain domain, respectively, and (X1)n is a linker of at least two amino acids.

[0118] In one embodiment, a bispecific tetravalent antibody can be a multimer of four polypeptide chains, each of two heavy chains comprising a first VH domain (VH1), a first CH1 domain, a second VH domain (VH2), and an Fc region comprising a second CH1, hinge, CH2, and CH3; and each of two light chains comprising a first VL domain (VL1), a first CL region, a second VL domain (VL2), and a second CL region. In another embodiment, a bispecific tetravalent antibody can comprise multiple antibody Fab fragments linked together to a single Fc domain. For example, Fab1 can be linked to Fab2 via a polypeptide linker, with Fab2 comprising one CH1 domain of the Fab, the hinge region, and then the CH2 and CH3 of the Fc domain. For example, an anti-TAA Fab can be linked via a linker from the CL domain of the anti-TAA Fab to the VH domain of the anti-CD16A Fab, and from the CH1 domain, hinge region, CH2 domain, and CH3 domain of the anti-CD16A Fab. In another example, an anti-CD16A Fab can be linked via a linker from the CL domain of the anti-CD16A Fab to the VH domain of the anti-TAA Fab, and from the CH1 domain, hinge region, CH2 domain, and CH3 domain of the anti-TAA Fab.

[0119] In one embodiment, the anti-CD16A antibody or antigen-binding fragment disclosed herein can be incorporated into a construct. The antibody or antigen-binding fragment in the construct can be a VHH. In some examples, the VHH is combined with a second antigen-binding fragment. In some examples, the VHH is combined with a payload such as a therapeutic agent or a tool such as a visualization agent. The construct can be a multispecific antibody, a heavy-chain antibody, a bivalent VHH, a dual paratopic VHH, a bispecific VHH, a VHH-scFv, a VHH-cytokine, a VHH-drug, a VHH-nanoparticle, a VHH-virus, or a VHH-imaging probe. The construct is a multispecific antibody. The multispecific antibody is a bispecific antibody.

[0120] Linker The domains and / or regions of the polypeptide chains of the bispecific tetravalent antibodies or constructs disclosed herein can be separated by linker regions of various lengths. In some embodiments, the antigen-binding domains are separated from each other, from the CL, CH1, hinge, CH2, CH3, or the entire Fc region by linker regions. For example, a polypeptide chain can comprise the sequence VL1-CL-(linker)VH2-CH1, VH-linker-VL. Such linker regions can comprise a random assortment of amino acids or a limited set of amino acids. Such linker regions can be flexible or rigid (see US2009 / 0155275).

[0121] Multispecific antibodies can be constructed by genetically fusing two single-chain Fv (scFv) or Fab fragments with or without a flexible linker (Mallender et al., J. Biol. Chem. 1994 269:199-206; Mack et al., Proc. Natl. Acad. Sci. USA 1995 92:7021-5; Zapata et al., Protein Eng. 1995 8:1057-62), via dimerization devices such as leucine zippers (Kostelny et al., J. Immunol. 1992 148:1547-53; de Kruifetal J. Biol. Chem. 1996 271:7630-4) and Ig C / CH1 domains (Muller et al., FEBS Lett. 422:259-64), or via diabodies (Holliger et al., (1993) Proc. Nat. Acad. Sci. USA. 1998 90:6444-8; Zhu et al., Bio / Technology (NY) 1996 14:192-6), Fab-scFv fusion (Schoonjans et al., J. Immunol. 2000 165:7050-7), and miniantibody formats (Pack et al., Biochemistry 1992.31:1579-84; Pack et al., Bio / Technology 1993 11:1271-7).

[0122] The multispecific antibodies and constructs disclosed herein may comprise a linker region of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or more amino acid residues between one or more of their antigen-binding domains, CL domain, CH1 domain, hinge region, CH2 domain, CH3 domain, or Fc region. In some embodiments, the linker region comprises the amino acids glycine and serine.The linkers are selected from the group consisting of the sequences GS (SEQ ID NO: 239), GGS (SEQ ID NO: 240), GSG (SEQ ID NO: 241), SGG (SEQ ID NO: 242), GGG (SEQ ID NO: 243), GGGS (SEQ ID NO: 244), SGGG (SEQ ID NO: 245), GGGGS (SEQ ID NO: 246), GGGGSGS (SEQ ID NO: 247), GGGGSGS (SEQ ID NO: 248), GGGGSGGS (SEQ ID NO: 249), GGGGSGGGGS (SEQ ID NO: 250), GGGSGGGGSGGGGS (SEQ ID NO: 251), A KTTPKLEEGEFSEAR (SEQ ID NO: 252), AKTTPKLEEGEFSEARV (SEQ ID NO: 253), AKTTPKLGG (SEQ ID NO: 254), SAKTTPKLGG (SEQ ID NO: 255), AKTTPKLEEGEFSEARV (SEQ ID NO: 256), SAKTTP (SEQ ID NO: 257), SAKTTPKLGG (SEQ ID NO: 258), RADAAP (SEQ ID NO: 259), RADAAPTVS (SEQ ID NO: 260), RADAAAAGGPGS (SEQ ID NO: 261), RAD AAAA(G4S)4 (SEQ ID NO: 262), SAKTTP (SEQ ID NO: 263), SAKTTPKLGG (SEQ ID NO: 264), SAKTTPKLEEGEFSEARV (SEQ ID NO: 265), ADAAP (SEQ ID NO: 266), ADAAPTVSIFPP (SEQ ID NO: 267), TVAAP (SEQ ID NO: 268), TVAAPSVFIFPP (SEQ ID NO: 269), QPKAAP (SEQ ID NO: 270), QPKAAPSVTLFPP (SEQ ID NO: 271), AKTTPP (SEQ ID NO: 272), A KTTPPSVTPLAP (SEQ ID NO: 273), AKTTAP (SEQ ID NO: 274), AKTTAPSVYPLAP (SEQ ID NO: 275), ASTKGP (SEQ ID NO: 276), ASTKGPSVFPLAP (SEQ ID NO: 277), GENKVEYAPALMALS (SEQ ID NO: 278), GPAKELTPLKEAKVS (SEQ ID NO: 279), and GHEAAAVMQVQYPAS (SEQ ID NO: 280), or any combination thereof (see WO2007 / 024715).

[0123] Dimerization-specific amino acids In one embodiment, the multivalent antibody or construct comprises at least one dimerization-specific amino acid change. Dimerization-specific amino acid changes can result in "knob-into-hole" interactions, increasing the likelihood of correctly combining the desired multivalent antibody. The dimerization-specific amino acid can be in the CH1 domain or the CL domain, or a combination thereof. Suitable dimerization-specific amino acids used to pair a CH1 domain with another CH1 domain (CH1-CH1) and a CL domain with another CL domain (CL-CL) can be found in at least WO2014082179, WO2015181805, and WO2017059551. The dimerization-specific amino acid can also be in the Fc domain or combined with a dimerization-specific amino acid in the CH1 or CL domain. In one embodiment, the present disclosure provides a bispecific antibody comprising at least one dimerization-specific amino acid pair.

[0124] Methods of detection and diagnosis The antibodies or antigen-binding fragments of the present disclosure are useful for a variety of applications, including, but not limited to, methods for detecting CD16A. In one embodiment, the antibodies or antigen-binding fragments are useful for detecting the presence of CD16A in a biological sample. As used herein, the term "detecting" includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues. In other embodiments, such tissues include normal and / or cancerous tissues that express CD16A at higher levels than other tissues.

[0125] In one aspect, the present disclosure provides a method for detecting the presence of CD16A in a biological sample. In certain aspects, the method includes contacting the biological sample with an anti-CD16A antibody or antigen-binding fragment thereof under conditions that allow the antibody to bind to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample may include, but is not limited to, urine, tissue, sputum, or blood.

[0126] Also included are methods for diagnosing disorders associated with CD16A expression. In certain embodiments, the methods include contacting the test cells with an anti-CD16A antibody or antigen-binding fragment, determining the expression level (either quantitatively or qualitatively) of CD16A expressed by the test cells by detecting binding of the anti-CD16A antibody or antigen-binding fragment to the CD16A polypeptide, and comparing the expression level by the test cells with the CD16A expression level in control cells (e.g., normal cells or non-CD16A-expressing cells of the same tissue origin as the test cells), wherein a higher CD16A expression level in the test cells compared to the control cells indicates the presence of a disorder associated with CD16A expression.

[0127] Pharmaceutical Compositions and Formulations Also provided are compositions, including pharmaceutical formulations, comprising an anti-CD16A antibody, an antigen-binding fragment thereof, a multispecific antibody, or a polynucleotide comprising a sequence encoding an anti-CD16A antibody, an antigen-binding fragment thereof, or a multispecific antibody. These compositions can further comprise suitable carriers, e.g., pharmaceutically acceptable excipients such as buffers, which are well known in the art.

[0128] Pharmaceutical formulations of the anti-CD16A antibodies or antigen-binding fragments thereof described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or antigen-binding fragments having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides, proteins, e.g., Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Nos. US 7,871,607 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0129] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0130] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0131] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0132] equivalent While the anti-human 4Ig-B7H3 antibody and antigen-binding fragments thereof have been described in connection with the detailed description thereof, it should be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0133] It should be understood that one, some, any, or all of the features of the various embodiments disclosed herein may be combined to form additional embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art.

[0134] Sequence Listing The sequence listings of the present disclosure are shown in Tables 1 and 2 below. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Example]

[0135] Example 1. Generation of anti-CD16A VHHs Human CD16A recombinant protein and cell lines for immunization and assays A recombinant 6-histidine-tagged extracellular domain (ECD) fragment of human CD16A protein (V158) (SEQ ID NO: 101) (referred to as human CD16A-His6(V158)) was purchased from a commercial source (Sino Biologics) and used as an antigen to immunize alpacas. Recombinant hexa-histidine-tagged ECD fragments of human CD16A(F158) (SEQ ID NO: 102), human CD16B(NA1) (SEQ ID NO: 103), human CD16B(NA2) (SEQ ID NO: 104), human CD16B(SH) (SEQ ID NO: 105), and cynomolgus CD16 (SEQ ID NO: 106) (referred to as human CD16A-His6(F158), human CD16B-His6(NA1), human CD16B-His6(NA2), human CD16B-His6(SH), and cynomolgus CD16-His6, respectively) were purchased from a commercial source (Sino Biologics) and used in various in vitro assays.

[0136] To facilitate screening and detection, a DNA fragment of human CD16A (V158) ECD (AA1-208 of SEQ ID NO: 101) was fused to a C-terminal human IgG1 mf Fc tag (SEQ ID NO: 107), a mouse IgG2a Fc tag, or an alpaca IgG2b Fc tag and transiently expressed in Expi293 cells (Thermofisher Scientific). Culture supernatants were collected, clarified, and affinity-purified using a Protein A column (Cytiva). The final product was buffer-exchanged into DPBS by ultrafiltration / diafiltration (UF / DF) and stored at -80°C.

[0137] To evaluate the binding activity of antibodies against CD16A expressed in live cells, NK92mi (ATCC, CRL-2407) cells were engineered to overexpress human CD16A by cotransfecting them with expression plasmids containing CD16A (F158 or V158) and FcRγ cDNAs (NK92mi / CD16A F158 and NK92mi / CD16A V158). NK92mi / CD16B(NA1)- and NK92mi / CD16B(NA2)-expressing cell lines were similarly prepared from CD16B(NA1)- or CD16B(NA2)-expressing plasmids.

[0138] Immunization and Screening An alpaca was immunized by an external research institute using recombinant human CD16A-His6 (V158) protein as an antigen, and after the third immunization, an immune VHH phage library was constructed from isolated alpaca peripheral blood mononuclear cells (PBMCs) (Pardon Els et al. (2014) Nature Protocols). Phage display selection was performed using standard protocols (Silacci et al. (2005) Proteomics, 5, 2340-50; Zhao et al. (2014) PLoS One, 9, e111339). Briefly, 10 mg / ml of immobilized human CD16A-V158-alpaca IgG2b in immunotubes was used to enrich for human CD16A (V158)-specific binders in panning rounds 1 and 2. The immunotubes were blocked for 1 hour with 5% milk powder (w / v) in PBS supplemented with 1% Tween® 20 (MPBST). After washing with PBST (PBS buffer supplemented with 0.05% Tween® 20), 1 × 10 13 (Round 1) or 2 x 10 12 Phages (round 2) were first depleted with human CD16B-His6(NA2) in MPBST for 1 hour and then incubated with antigen for 1 hour. After washing with PBST, bound phages were eluted with 100 mM triethylamine (Sigma-Aldrich). The eluted phages were used to infect mid-logarithmic phase E. coli TG1 bacteria, which were then plated onto 2xYT (yeast extract tryptone)-agar plates supplemented with 2% glucose and 100 μg / mL ampicillin. After three rounds of selection, individual clones were picked and phage-containing supernatants were prepared using standard protocols. Anti-human CD16A antibodies were screened using phage ELISA.

[0139] For phage ELISA, Maxisorp immunoplates were coated with recombinant human CD16A-His6(V158) protein as the antigen and blocked with 5% milk powder (w / v) in PBS buffer. Phage supernatants were blocked with MPBST for 30 min and added to the wells of the ELISA plate for 1 h. After washing with PBST, bound phages were detected using HRP-conjugated anti-M13 antibody (GE Healthcare) and 3,3',5,5'-tetramethylbenzidine substrate (catalog: 00-4201-56, eBioscience, USA).

[0140] Positive clones from the phage ELISA were sequenced and recovered. Six anti-CD16A VHH variants were constructed by fusing their open reading frames to a C-terminal human IgG1 mf Fc (SEQ ID NO: 107) tagged eukaryotic expression vector. Plasmids were transfected into ExpiCHO-s cells (Thermofisher Scientific) using the MAX Titer protocol. Fc-tagged VHH variants (VHH-Fc) were purified by MabSelect SuRe (Cytiva) followed by an SPHP column (Cytiva). The final product was buffer exchanged into DPBS by UF / DF and stored at -80°C for later use, including binding analysis.

[0141] For antigen ELISA, Maxisorp immunoplates were coated with antigen (human CD16A(V158), human CD16A(F158), human CD16B(NA1), human CD16B(NA2), human CD16B(SH), or cynomolgus monkey CD16) and blocked with 3% BSA (w / v) in PBS buffer (blocking buffer). Monoclonal VHH-Fc antibodies were blocked with blocking buffer for 30 min and added to the ELISA plate wells for 1 h. After washing with PBST, bound antibodies were detected using an HRP-conjugated anti-human IgG antibody (Sigma, A0170) and 3,3',5,5'-tetramethylbenzidine substrate (catalog: 00-4201-56, eBioscience, USA).

[0142] Flow cytometry revealed that NK92mi / CD16A V158 cells, NK92mi / CD16B(NA1) cells, and NK92mi / CD16B(NA2) cells (10 5 Cells (per well) were incubated with various concentrations of IgG-like antibodies, which were then bound with Alexa Fluro-647-labeled anti-human IgG Fc antibodies (catalog: 409320, BioLegend, USA). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte™ 8HT, Merck-Millipore, USA).

[0143] Following the procedures disclosed above, 44 positive clones were sequenced and recovered, and one representative positive anti-CD16A variant, BG523P (VHH AA SEQ ID NO: 112, VHH DNA SEQ ID NO: 113), was obtained from six VHH-Fc fusion clones. The binding affinity of BG523P to CD16A was also confirmed by antigen ELISA. The results of ELISA and FACS analysis of BG523P relative to the positive control LS21 are shown in Tables 3, 4, and 5, as well as Figures 1 and 2A-2C. More specifically, the FACS binding data of BG523P to the NK92mi / CD16A cell line compared to the human CD16A-specific binder LS21 (SEQ ID NO: 108, patent EP1888645B1) as a positive control are shown in Table 3, demonstrating the specificity of BG523P for binding to NK92mi / CD16A cells. Figure 1 shows that BG523P exhibits higher binding to human CD16A, CD16B (NA1), and cynomolgus monkey CD16 at 1 μg / ml compared to LS21. Figure 2A shows that BG523P specifically binds to NK92mi / CD16A cells. Figures 2A and 2B show that BG523P exhibits weak binding to NK92mi / CD16B at high concentrations. BG523P exhibits slight binding activity to CD16B (NA1) in ELISA and FACS assays (Figure 1, Table 4), but its binding affinity was significantly reduced (calculated EC 50(The values ​​were 40-fold reduced compared to those of CD16A binding in the FACS assay.) This result suggests that BG523P can selectively bind to CD16A over CD16B. Figure 1 also shows that BG523P hardly binds to the CD16B SH allotype. Considering that the predominant variants of human CD16B are the NA1 and NA2 allotypes and that the frequency of the SH allotype is rare, reported to be less than 0.05 in Caucasians, the binding properties of CD16B to the SH allotype were not further characterized. [Table 3] [Table 4] [Table 5]

[0144] Example 2. Humanization of anti-human CD16A VHH BG523P For humanization of BG523P, human germline IgG genes were searched for sequences sharing high homology with the cDNA sequence of the variable region of BG523P by blasting the human immunoglobulin gene databases on the IMGT (http: / / www.imgt.org / IMGT_vquest / share / textes / index.html) and NCBI (http: / / www.ncbi.nlm.nih.gov / igblast / ) websites. Human IGVH genes, which are frequently present in the human antibody repertoire (Glanville 2009 PNAS 106:20216-20221) and share high homology with BG523P, were selected as templates for humanization.

[0145] Humanization was performed by CDR grafting (Methods in Molecular Biology, Vol. 248: Antibody Engineering, Methods and Protocols, Humana Press), and humanized VHH variants were engineered as VHH-Fc using an in-house developed expression vector for subsequent binding and biophysical stability analysis. In the first round of humanization, mutations from camelid amino acid residues to human amino acid residues in the framework regions were guided by simulated 3D structures, and structurally important camelid framework residues for maintaining the canonical structure of the CDRs were retained in the first version of humanized VHH, BG523P. Among the many variants, BG524P is the preferred humanized VHH with the most Camelid residues retained. Specifically, HCDR1 (SEQ ID NO: 109) and HCDR3 (SEQ ID NO: 111) of BG523P were grafted onto the framework of the human germline variable gene IGVH3-7, with five Camelidae framework residues (F37, R45, V78, P84, and A94 according to Kabat numbering) retained, while one mutation was introduced into HCDR2 to remove a potential isomerization site. The sequence of BG524P is presented in Table 2 as SEQ ID NOs: 109, 114, 111, and 115-116.

[0146] The humanized BG523P variant was fused to the N-terminus of the Fc in a VHH-Fc format using an in-house developed expression vector containing the Fc region of the human IgG1 variant (SEQ ID NO: 107) with an adaptable subcloning site. Expression and preparation of the humanized BG523P VHH-Fc antibody was achieved by transfection of the construct into ExpiCHO-s cells and purification using a Protein A column. The purified VHH-Fc antibody was concentrated to 0.5-5 mg / mL in PBS and stored in aliquots at -80°C in a freezer.

[0147] For affinity measurements, VHH-Fc antibodies were captured by anti-human Fc surfaces and used in affinity assays based on surface plasmon resonance (SPR) technology. The results of the SPR-measured binding profiles of anti-CD16A VHHs are summarized in Table 6. BG524P showed slightly improved binding affinity to CD16A 158V and CD16A 158F, with dissociation constants of 0.08 nM and 0.08 nM, respectively, compared to the binding affinity of BG523P. Meanwhile, BG524P maintained selectivity for CD16A over CD16B, as characterized by SPR. [Table 6]

[0148] Live NK92mi / CD16A 158F cells were seeded in 96-well plates and incubated with serial dilutions of anti-CD16A VHH-Fc. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. EC 50 Values ​​were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in Figure 3A and Table 7, BG524P showed improved binding affinity to native CD16A 158F, but exhibited a decreased Emax. [Table 7]

[0149] To determine whether humanized BG523P maintained the optimal biophysical stability of BG523P, the melting temperature (Tm) and aggregation temperature (Tagg) of BG524P were determined and compared to those of BG523P. Both the Tm and Tagg of BG524P were reduced compared to those of BG523P (Table 8).

[0150] Melting temperatures (Tm) were measured using a high-throughput MicroCal™ VP-Capillary DSC (Malvern Instruments, Northampton, MA). Thermograms of each protein (350 μL at 0.5 mg / mL) were acquired from 20°C to 100°C using a scan rate of 60°C / hr. A buffer-alone thermogram was subtracted from each protein sample. Results indicate the transition temperature (Tm) midpoint and calorimetric enthalpy (ΔH) values ​​for the samples.

[0151] The aggregation temperature Tagg (°C) represents the colloidal stability of the sample and was obtained by monitoring the onset of aggregation with an SLS266 using UNCLE™ (Unchained Lab, Pleasanton, CA). The sample was loaded into a Uni and the temperature was increased from 15°C to 95°C. Back-reflecting optics cannot detect near-ultraviolet light scattering by protein aggregates, and therefore only unscattered light reaches the detector. Therefore, the reduction in back-reflected light is a direct measure of aggregation in the sample. [Table 8]

[0152] BG524P was further engineered for therapeutic use in humans by introducing mutations in the CDRs and backmutations in the framework regions to improve biophysical properties, remove PTM sites, and restore binding Emax to native CD16A.

[0153] Taken together, successfully engineered versions of humanized monoclonal antibodies, BG525P (SEQ ID NOs: 109-111 and 117-118) and BG526P (SEQ ID NOs: 109, 114, 111, and 119-120), were derived from the above mutational process, as characterized in detail in Tables 9-11 and Figure 3B, which retained both the binding affinity for CD16A, selectivity over CD16B, and optimal biophysical stability of the parent clones. [Table 9] [Table 10] [Table 11]

[0154] Example 3. Native CD16B binding of anti-CD16A VHHs To evaluate the ability of anti-CD16A VHHs to bind native CD16B on live cells, NK92mi cells were engineered to overexpress human CD16B NA1 or NA2. Live NK92mi / CD16B cells were seeded into 96-well plates and incubated with 300 nM anti-CD16A VHH-Fc. Goat anti-human IgG was used as a secondary antibody to detect binding of anti-CD16A VHH-Fc to the cell surface. The binding signals of the humanized VHH-Fc to CD16B were comparable to or lower than those of the parent clone and significantly lower than the corresponding binding signals to CD16A (Table 10, Figures 3A-B), as shown in Figure 4 and Table 12. [Table 12]

[0155] Example 4. Human IgG competition for VHH binding to native CD16A To assess the effect of human IgG competition on the ability of anti-CD16A VHH-Fc to bind native CD16A on live cells, a FACS-based assay was performed in the presence or absence of human IgG. Live NK92mi / CD16A cells were seeded into 96-well plates and incubated at 37°C with serial dilutions of biotinylated anti-CD16A VHH-Fc alone or with 10 mg / ml of human IgG1 antibody CB6 (anti-SARS-Covid19 antibody) (SEQ ID NOs: 121-122). Streptavidin-AF647 was used as a secondary antibody to detect binding of biotinylated anti-CD16A VHH-Fc to the cell surface. EC2 values ​​for dose-dependent binding to human native CD16A were calculated. 50 Values ​​were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in Figures 5A-5C and Table 13, binding of BG525P and BG526P to CD16A 158F was similarly affected by the presence of human IgG compared to that of the parental BG523P. [Table 13]

[0156] Example 5. Binding affinity of humanized CD16A to cynomolgus monkey CD16 by SPR For affinity measurements, VHH-Fc was captured by anti-human Fc surface and used in affinity assays based on surface plasmon resonance (SPR) technology. The binding profile of anti-CD16A VHH-Fc determined by SPR is summarized in Table 14. Humanized anti-CD16A VHH-Fc retained cross-reactivity to cynomolgus CD16. [Table 14]

Claims

1. An anti-CD16A antibody or antigen-binding fragment thereof, including an antibody or binding fragment thereof that specifically binds to human CD16A.

2. The anti-CD16A antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment is more selective for human CD16A than for human CD16B.

3. The anti-CD16A antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment has cross-binding affinity for both human CD16A and cynomolgus monkey CD16A.

4. The antibody or antigen-binding fragment may be a monoclonal antibody, a single-chain antibody (scFv), a Fab fragment, a F(ab') ２ The anti-CD16A antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is a fragment, a heavy chain antibody (HcAb), or a VHH.

5. 10. The anti-CD16A antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antigen-binding fragment is a VHH.

6. The anti-CD16A antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is a chimeric antibody, a humanized antibody, or a human engineered antibody.

7. the antibody or antigen-binding fragment thereof a) the heavy chain CDR1 sequence of SEQ ID NO: 109; b) a heavy chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 110 and 114, and c) the heavy chain CDR3 sequence of SEQ ID NO: 111 10. The anti-CD16A antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising at least one heavy chain CDR selected from the group consisting of:

8. the antibody comprising the following heavy chain CDRs: a) the heavy chain CDR1 sequence of SEQ ID NO: 109; b) a heavy chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 110 and 114, and c) the heavy chain CDR3 sequence of SEQ ID NO: 111 10. The anti-CD16A antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising each of:

9. the antibody or antigen-binding fragment thereof a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 110, and (c) an HCDR3 of SEQ ID NO: 111, or (b) HCDR1 of SEQ ID NO: 109, (b) HCDR2 of SEQ ID NO: 114, and (c) HCDR3 of SEQ ID NO: 111 10. The anti-CD16A antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising a heavy chain variable region comprising:

10. 10. The anti-CD16A antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) comprising at least one amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 112, 115, 117, or 119.

11. 10. The anti-CD16A antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment comprises at least one amino acid sequence selected from SEQ ID NOs: 112, 115, 117, and 119, and wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in at least one of SEQ ID NOs: 112, 115, 117, and 119 have been inserted, deleted, or substituted.

12. An anti-CD16A antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 112, 115, 117, and 119.

13. A construct comprising an anti-CD16A antibody or antigen-binding fragment thereof according to any one of the preceding claims.

14. The construct of claim 13 , wherein the antibody or the antigen-binding fragment thereof is a VHH.

15. The construct according to claim 13 or 14, wherein the construct is a multispecific antibody, a heavy chain antibody, a bivalent VHH, a double paratopic VHH, a bispecific VHH, or a VHH-scFv.

16. The construct of claim 15, wherein the construct is a multispecific antibody.

17. The construct of claim 16 , wherein the multispecific antibody is a bispecific antibody.

18. A pharmaceutical composition comprising an anti-CD16A antibody or antigen-binding fragment thereof according to any one of the preceding claims and a pharmaceutically acceptable carrier.

19. An isolated nucleic acid encoding the anti-CD16A antibody or antigen-binding fragment thereof or construct of any one of claims 1 to 17.

20. A vector comprising the nucleic acid of claim 19.

21. 21. A host cell comprising the nucleic acid of claim 19 or the vector of claim 20.

22. A process for producing an anti-CD16A antibody or its antigen-binding fragment, comprising culturing a host cell described in claim 21 and recovering the antibody or antigen-binding fragment or the construct from the culture.

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