Agents that bind to cd16a and uses thereof

Abstract

The present disclosure provides binding agents that bind to CD 16a (e.g., antibodies, multispecific antibodies, antigen binding fragments) and uses thereof.

Description

AGENTS THAT BIND TO CD16A AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 508,869, filed June 16, 2023 and U.S. Provisional Patent Application No. 63 / 465,785, filed May 11, 2023, the disclosure of each of which is incorporated by reference herein it its entirety.SEQUENCE LISTING

[0002] This application contains a computer readable Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “ 13366-014-228_SEQ_LISTING.xmr, was created on May 8, 2024, and is 389,040 bytes in size.1. FIELD

[0003] The present disclosure relates generally to binding agents (such as antibodies, multispecific antibodies, and antigen binding fragments thereof) that bind to CD 16a, and methods of use thereof.2. BACKGROUND

[0004] Natural killer (NK) cells are part of the innate immune system and play a key role in the first line of self-defense against viral infections and transformed, malignant cells both through direct cytotoxic effects and through production of pro-inflammatory cytokines. NK cells express a variety of activating and inhibitory receptors and the overall balance of the two results in either a response to or tolerance of the target cells (Bryceson et al., Immunol Rev. 214:73-91 (2006); Myers and Miller: Nat. Rev.Clin. Oncol. 18:85-100 (2021)).

[0005] CD 16 is the most potent activating receptor expressed by NK cells (Bryceson et al., Immunol Rev. 214:73-91 (2006)). Two forms of CD16 exist: CD16a (UniProt #P08637), a transmembrane protein that is expressed largely on NK, monocytic cells, macrophages and dendritic cells and CD16b (UniProt # 075015), a GPI-anchored membrane protein that is expressed on granulocytes (Ueda et al., J Immunol. 143(4): 1274-7 (1989); Ravetch and Perussia, J Expt. Med. 170: 481-497 (1989); Selvaraj et al., J Immunol. 143:3283-3288 (1989)).

[0006] One hurdle to generating effective NK cell engager bispecific antibodies is that the extracellular domains (ECDs) of CD 16a and CD 16b are highly homologous and shareapproximately 97% identity and 98% similarity. Therefore, a highly specific antibody against CD 16a that can distinguish between the ECDs of these two forms is needed to avoid untoward activation of neutrophils and other granulocytes (Wei et al., Exp, Mol. Pathol. 101 :281-289 (2016); Koch and Tesar, Trans Med. Hemotherapy 44:337-350 (2017); US 9,035,026; US 9,701,750).

[0007] One additional hurdle to generating effective anti-CD16 antibody clones suitable for an NK cell engager platform is that two different alleles occur in humans for CD 16a, a 176F and a 176V (numbering based on the full-length UniProt entry) variant that differ in affinity for IgG (Wu et al., J Clin. Invest. 100: 1059-1070 (1997); Koene et al., Blood 90: 1109-1114 (1997)), and two different alleles exist for CD16b, NA1 and NA2 (Nagarajan et al., J Biol. Chem. 270:25762-70 (1995)).3. SUMMARY

[0008] The present disclosure provides an antibody or antigen binding fragment thereof that binds to CD 16a. In some embodiments, the antibody binds to both CD16a-F and CD 16a- V.

[0009] The present disclosure provides an antibody or antigen binding fragment thereof that binds to CD 16a but does not bind to CD 16b. In some embodiments, the antibody or antigen binding fragment thereof does not bind to CD16b-NAl and CD16b-NA2.

[0010] The present disclosure provides an antibody or antigen binding fragment thereof that binds CD16a, e.g., an antibody or antigen binding fragment provided in Tables 1-9.

[0011] The present disclosure further provides a multispecific antibody or antigen binding fragment thereof comprising a first binding arm that binds to CD 16a and a second binding arm that binds to a second antigen. In some embodiments, the second antigen is expressed on a tumor cell. In some embodiments, the second antigen is CD19. In some embodiments, the second antigen is BCMA. The first binding domain contains a VH and a VL comprising CDRs identical to any antibody or fragment thereof provided herein, for example, in Tables 1-9. The second binding domain contains a VH and a VL comprising CDRs identical to any antibody or fragment thereof provided herein, for example, in Table 10.

[0012] In some embodiments of the multispecific antibody or fragment thereof provided herein, the first binding domain and the second binding domain can be linked through a peptide bond or a peptide linker. In some embodiments, the multispecific antibody or fragment thereof comprises one polypeptide chain comprising the first binding domain and the second binding domain, wherein the first binding domain comprises a first scFv that bindsto CD 16, and the second binding domain comprises a second scFv that binds to CD 19. In some embodiments, the first scFv is linked to the N terminal of the second scFv. In some embodiments, the first scFv is linked to the C terminal of the second scFv. In some embodiments, the polypeptide chain further comprise a first CH2 and a first CH3 of an IgG Fc region. In some embodiments, the multispecific antibody or fragment comprises two identical polypeptide chains as provided herein. In some embodiments, the first polypeptide and the second polypeptide form a bivalent format through dimerization of the CH2 and / or CH3 regions. In some embodiments, the multispecific antibody or fragment thereof comprises a mutated Fcyl region to eliminate Fc binding activity to Fey receptors.

[0013] In addition, the present disclosure provides nucleic acids encoding such a binding agent provided herein (e.g., an antibody or fragment thereof), vectors comprising one or more of such nucleic acids, and cells expressing the same, and compositions comprising any one or more of the above. Methods or uses of such binding agents, nucleic acids, vectors, cells, and compositions are also provided herein.4. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows the sequence homology for the ECD of the two alleles for CD 16a and the two alleles of CD 16b (CD16a-176F: SEQ ID NO: 360, CD 16a- 176V: SEQ ID NO:361, CD16b-NAl : SEQ ID NO:362, CD16b-NA2: SEQ ID NO:363).

[0015] FIGs. 2A and 2B illustrate the removal of CD16b-NAl reactive clones from a population of CD16a-F and CD16a-V binding clones followed by positive selection for yeast clones binding biotinylated CD 16a detected with APC-labeled streptavidin. FIG. 2A. FACS3 where the Pl non-CD16b binders (Pl gate of the right most panel in the upper row) were collected and used for a positive sort performed with 100 nM CD16a-F. FIG. 2B. FACS4 performed with the P3 gate from the final step of FACS3 (P3 gate of the bottom right panel of FIG. 2 A), where the positive sort of the CD 16b subtracted population (Pl gate of the right most panel in the upper row) was performed with 100 nM CD16a-V.

[0016] FIGs. 3A and 3B illustrate specific binding of exemplary clones to recombinant CD16a-V, CD16a-F, CD 16b, and a negative control antigen by ELISA (FIG. 3 A) and to CD 16a-, CD 16b- expressing 293 cells or untransfected 293 cells (FIG. 3B).

[0017] FIGs. 4A-4L. FIG. 4 A shows binding affinity of biotinylated CD16a-F (solid square), CD16a-V (-up-pointing triangle) and CD16-NA2 (-down-pointing triangle) recombinant protein to an exemplary clone in IgG format coated on the wells of an ELISA plate. FIG. 4B shows binding kinetics of exemplary clones #1 A2, #2-2 A2, and the referenceclone in IgGl format (with silenced Fc) to CD16a-F, CD16a-V, Cyno-CD16, CD16b-NAl, and CD16b-NA2 determined by BLI. FIG. 4C shows the binding curves of clone #2-2 A2 in IgG format (with silenced Fc) to recombinant CD 16 a(F / V) and CD 16b (NA1 / NA2) determined by ELISA. FIG. 4D shows binding kinetics of exemplary clones #1 A2, #2-2 A2, and reference clone in Fab format to CD16a-F, CD16a-V in presence and absence of human IgG at 10 mg / ml determined by BLI. FIGs. 4E-4J show binding affinity of exemplary clones #1 A2, #2-2A2, the reference clone in IgGl format (with silenced Fc), an irrelevant negative control clone (CTL), and the positive control clone (3G8), to CD16a-F expressing 293 cells (FIG. 4E), CD16a-V expressing 293 cells (FIG. 4F), cyno-CD16 expressing 293 cells (FIG. 4G), CD16b-NAl expressing 293 cells (FIG. 4H), CD16b-NA2 expressing 293 cells (FIG. 41), and primary NK cells (FIG. 4J). FIG. 4K shows binding affinity of exemplary clone #55, the reference clone, and the negative control clone (CTL) in IgGl format (with silenced Fc), to primary NK cells. FIG. 4L shows Tm determination for IgGl and Fab format for clones #1 A2, #2-2A2, and the reference clone.

[0018] FIGs. 5A-5H. FIG. 5A shows competition assay for binding epitope determination of exemplary clones #1 A2, #2-2A2, 3G8, and the reference clone. FIGs. 5B- 5E show the binding curves of the exemplary clones #1 A2 (FIG. 5B), #2-2A (FIG. 5C), #55 (FIG. 5E), and the reference clone (FIG. 5D), binding to CD 16(F) mutants. FIG. 5F shows competition assay for binding epitope determination of exemplary clone #55, 3G8, and the reference clone. FIG. 5G shows the alignment of CD16a(F) with CD16b(NAl), CD16b(NA2) and cynomolgus CD 16a. FIG. 5H shows the impact of deglycosylation by PNGase-F on binding curves of two exemplary clones, A2 and #55, to CD16a(F), CD16a(V), CD16b(NAl) and CD16b(NA2).

[0019] FIGs. 6A-6H. FIGs. 6A-6C illustrate the structure (FIG. 6A), purity after preparative size exclusion chromatography (SEC) (FIG. 6B), and activity (FIG. 6C) of an exemplary anti-CD16a antibody clone A2 coupled to an exemplary CD 19 clone in an scFv- scFv antibody format in a CD 16a activation assay using CD16a-V-expressing Jurkat NFAT luciferase reporter cells in the presence of CD19-expressing Raji cells or non-target expressing MDA-MD-231 breast carcinoma cells used as controls (Symbols: squares represent Raji cells, triangles represent MDA-MD-231 cells). FIGs. 6D and 6E show the reporter cell assay results (EC50 values) of exemplary anti-CD16a antibody clones #1A2, #2- 2A2, and the reference clone in scFv-scFv antibody format in presence of human IgG (FIG. 6E) or in absence of human IgG (FIG. 6D). FIG. 6F shows the results (EC50 values) of activation of reporter cell assay without Raji cells. FIGs. 6G and 6H show the reporter cellassay results of exemplary anti-CD16a antibody clone #55, and the reference clone in scFv- scFv antibody format in presence of human IgG (FIG. 6H) or in absence of human IgG (FIG. 6G).

[0020] FIGs. 7A-7E FIGs. 7A-7C illustrate the structure (FIG. 7A), purity after preparative SEC (FIG. 7B), and activity (FIG. 7C) of an exemplary anti-CD16a antibody clone coupled to an exemplary CD 19 clone in an IgG4 bivalent format in a CD 16a activation assay using CD16a-V expressing Jurkat NFAT luciferase reporter cells in the presence of CD19-expressing Raji cells or non-target expressing MDA-MD-231 breast carcinoma cells (Symbols: triangles represent Raji cells, squares represent MDA-MD-231 cells). FIGs. 7D and 7E show the reporter cell assay results (EC50 values) of exemplary anti-CD16a antibody clones #1 A2, #2-2 A2, and the reference clone in scFv-scFv-IgG4 bivalent antibody format, CD19-IgGl with Fc enhanced mutations (S239D and I332E) in presence of human IgG (FIG. 7E) or in absence of human IgG (FIG. 7D).

[0021] FIG. 8 illustrates the binding of an exemplary anti-CD16a clone coupled to an exemplary anti-CD19 clone in a scFv-scFv-IgG4 bivalent antibody format to human NK cells shown by flow cytometry.

[0022] FIGs. 9A-9G FIGs. 9A-9C illustrate the cytotoxicity of an exemplary anti-CD16a clone A2 coupled to an exemplary anti-CD19 clone in a scFv-scFv-IgG4 bivalent antibody format to human NK cells isolated from patients with different CD 16a allelotypes. FIG. 9A: cell killing induced by an exemplary clone using NK cells from patient #1 with a CD16a-176 F / V genotype. The * indicates baseline level of cell death in the absence of the scFv-scFv- IgG4 construct. FIG. 9B: cell killing induced by an exemplary clone using NK cells from patient #2 with a CD16a-176 F / F genotype. FIG. 9C: cell killing induced by an exemplary clone using NK cells from patient #3 with a CD16a-176 F / V genotype. FIG. 9D: cell killing induced by a variant of the exemplary clone variant using NK cells from patient #3 with a CD16a-176 F / V genotype. FIGs. 9E-9G illustrate the cytotoxicity of other exemplary antiCD 16a clones coupled to an exemplary anti-CD19 clone in a scFv-scFv-IgG4 bivalent antibody format to human NK cells.

[0023] FIG. 10 illustrates the binding activity or lack thereof of an exemplary antiCD 16a clone, and two variant derivatives as full-length IgGl to neutrophils at a concentration of 200 nM compared to the binding activity of the APC-labeled secondary detection reagent alone (middle panel of the upper row) shown by flow cytometry. The presence of CD 16b was confirmed by staining for CD 16b with a pan CD 16 clone, 3G8, labeled with phycoerythrin(PE; X-axis, top right-most panel) and an APC labeled anti-CD15, clone HI98, (Y axis top right panel)

[0024] FIGs. 11A-11C show lack of binding activity of exemplary anti-CD16a clones A2 (FIG. 11 A), #2-2A2 (FIG. 1 IB), #55 (FIG. 11C) in IgG format (with silenced Fc) to human neutrophils with either an NA1 / NA1, NA1 / NA2 or NA2 / NA2 genotype compared to the binding activity of the anti-CD16 clone (3G8), the reference clone in IgG format (with silenced Fc), and / or the negative control clone (CTL) in IgG format (with silenced Fc).5. DETAILED DESCRIPTION

[0025] The present disclosure is based, at least in part, on novel binding agents that bind to CD 16a. In certain aspects, such binding agents can bind both CD16a-F and CD16a-V but do not bind the two CD 16b allelic forms, CD16b-NAl and CD16b-NA2. Such agents include antibodies (e.g., monospecific or multispecific, including bispecific) that bind to CD16a, including antibodies that bind to human CD 16a. In certain aspects, such binding agents are multispecific antibodies that bind to both CD 16a and a tumor antigen (such as CD 19) activating an anti -turn or response mediated by an immune cell. In addition, the binding agents provided herein are useful for the killing and / or removal of tumor cells. The binding agents provided herein are useful in compositions and in methods for treating a disease or disorder such as cancer.

[0026] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting and / or separating the subject matter described.5.1. Definitions

[0027] Techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et cd.. Molecular Cloning: A Laboratory Manual (3d ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Diibel eds., 2d ed. 2010). Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa.In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0028] The term “CD16a” refers to low affinity immunoglobulin gamma Fc region receptor III-A, a protein that in humans is encoded by the FCGR3A gene (UniProt # P08637). It is part of the cluster of differentiation cell surface molecules. Two functional polymorphisms of the CD16a have been described, 176 V and 176 F, sometimes called 158 V and 158 F, respectively, depending on when counted from the N-terminal of the mature protein or not. These two polymorphisms are also referred herein as “CD16a-V” and “CD16a-F.”

[0029] The term “CD16b” refers to low affinity immunoglobulin gamma Fc region receptor III-B, a protein that in humans is encoded by the FCGR3B gene (UniProt # 075015). It is part of the cluster of differentiation cell surface molecules. Two different alleles exist for CD16b, NA1 and NA2, which are also referred herein as “CD16b-NAl” and “CD16b- NA2.”

[0030] The term “CD 19” refers to Cluster of Differentiation 19 molecule, also known as B-lymphocyte antigen CD 19, B-Lymphocyte Surface Antigen B4, T-Cell Surface Antigen Leu-12 and CVID3. It is a transmembrane protein that in humans is encoded by the gene CD19 (UniProt # P15391).

[0031] As used herein, the term “binding agent” or a grammatical equivalent thereof refers to a molecule (e.g., antibody) with one or more antigen-binding sites that binds an antigen. In some embodiments, a binding agent as described herein is an antibody (including a multispecific antibody and an antibody fragment, such as an antigen-binding fragment or an epitope-binding fragment) or other peptide-based molecule as well as a conjugate of an antibody, antibody fragment, or peptide-based molecule (e.g., an antibody-drug conjugate) that binds to CD 16a (such as human CD 16a) and / or tumor antigens (such as CD 19).

[0032] The terms “antibody,” “immunoglobulin,” and “Ig” are used interchangeably herein, and are used in the broadest sense and specifically cover, for example polyclonal antibodies, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, recombinantly produced antibodies, single domain (e.g., VHH) antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies having full-length heavy and / or light chains. VHH as used herein refers to a domain antibody derived from a variable region of a heavy chain only antibody. Exemplary single domainantibodies include, but are not limited to, antibodies naturally devoid of light chains such as those from Camelidae species (e.g., llama), single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, and bovine. VHH can also be derived from other species besides Camelidae that may produce heavy chain antibodies naturally devoid of light chain. Antibodies also include antibody fragments (and / or polypeptides that comprise antibody fragments) that retain CD 16a binding characteristics. Non-limiting examples of antibody fragments include antigen-binding regions and / or effector regions of the antibody, e.g., Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, single chain antibody molecule, dual variable domain antibody, single variable domain, linear antibody, V region, a multispecific antibody formed from antibody fragments, F(ab)2, Fd, Fc, diabody, di-diabody, disulfide-linked Fvs (dsFv), single-domain antibody (e.g., nanobody) or other fragments (e.g., fragments consisting of the variable regions of the heavy and light chains that are non-covalently coupled). In general terms, a variable (V) region domain may be any suitable arrangement of immunoglobulin heavy (VH) and / or light (VL) variable domains. For example, antibodies also include tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, and an antibody heavy chain monomer. Thus, for example, the V region domain may be dimeric and contain VHH- VHH, VH-VH, VH-VL, or VL-VL dimers that bind CD 16a and / or CD 19. If desired, the VH and VL may be covalently coupled either directly or through a linker to form a single chain Fv (scFv). For ease of reference, scFv proteins are referred to herein as included in the category “antibody fragments.” Another form of an antibody fragment is a peptide comprising one or more complementarity determining regions (CDRs) of an antibody. CDRs (also termed “minimal recognition units” or “hypervariable regions”) can be obtained by constructing polynucleotides that encode one or more CDRs of interest. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA of antibody-producing cells as a template (see, for example, Larrick et aL, Methods: A Companion to Methods in Enzymology, 2:106 (1991); Courtenay -Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press (1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137, Wiley-Liss, Inc. (1995)). Antibody fragments may be incorporated, for example,into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable domains of new antigen receptors (v-NAR), and bis-single chain Fv regions (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136, 2005). In some embodiments, antibodies comprising a VH and / or VL further contain a light chain and / or a heavy chain constant region, such as one or more constant regions, including one or more IgGl, IgG2, IgG3 and / or IgG4 constant regions. In some embodiments, antibodies can include epitope-binding fragments of any of the above. The antibodies described herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule.

[0033] The term “monospecific” when used in reference to a binding agent (e.g., an antibody) as used herein denotes a binding agent that has one or more binding sites each of which binds to the same epitope of the same antigen.

[0034] The term “multispecific” when used in reference to a binding agent (e.g, an antibody) means that the binding agent is able to specifically bind to at least two distinct epitopes, for example two binding sites each formed by a pair of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) or each formed by a pair of VHH domains binding to different antigens or to different epitopes on the same antigen. Such a bispecific binding agent (e.g., an antibody) may have a 1+1 format (comprising one binding site for a first antigen or epitope and one binding site for a second antigen or epitope). Other bispecific binding agent (e.g., an antibody) formats may be 2+1 or 1+2 formats (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 format (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a bispecific binding agent (e.g., an antibody) comprises two antigen-binding sites, each may bind to a different epitope. Such a bispecific binding agent (e.g., an antibody) may bind to two different epitopes on the same antigen (e.g., epitopes on CD 16a).

[0035] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are notlimited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full-length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.

[0036] A “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a side chain with similar chemical characteristics. Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, soluble proteins, and / or antibodies of the disclosure do not abrogate the binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence, to the target binding site. Methods of identifying amino acid conservative substitutions which do not eliminate binding are well-known in the art.

[0037] The term “polypeptide” refers to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can includee.g., be interrupted by) non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as linkage to or conjugation with (directly or indirectly) a moiety such as a labeling component or a drug (e.g., toxin). Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure can be based upon antibodies or other members of the immunoglobulin superfamily, in some embodiments, the polypeptides can occur as single chains or dimers of single chains.

[0038] As used herein, an “antigen” is a moiety or molecule that contains an epitope to which a binding agent (e.g., an antibody) can bind. As such, an antigen can be bound by an antibody. In some embodiments, the antigen, to which a binding agent (e.g., an antibody) described herein binds, is CD 16a (e.g., human CD 16a), or a fragment thereof, including a fragment that comprises one or more domains of CD 16a. In some embodiments, the antigen, to which a binding agent (e.g., an antibody) described herein binds, is a tumor antigen, such as CD 19 (e.g., human CD 19), or a fragment thereof, including a fragment that comprises one or more domains of the tumor antigen, such as CD 19.

[0039] As used herein, an “epitope” is a term in the art and refers to a localized region of an antigen to which an antibody can bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous, epitope. In the case of a polypeptide antigen, for example, an epitope can be contiguous amino acids of the polypeptide (a “linear” epitope) or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide (a “conformational,” “non-linear” or “discontinuous” epitope), e.g., CD 16a or CD 19. It will be appreciated by one of skill in the art that, in general, a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure. For example, in some embodiments, an antibody binds to a group of amino acids regardless of whether they are folded in a natural three-dimensional protein structure. In other embodiments, an antibody requires amino acid residues making up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.

[0040] An antibody binds “an epitope” or “essentially the same epitope” or “the same epitope” as a reference antibody, when the two antibodies recognize identical, overlapping or adjacent epitopes in a three-dimensional space. The most widely used and rapid methods for determining whether two antibodies bind to identical, overlapping or adjacent epitopes in athree-dimensional space are competition assays, which can be configured in a number of different formats, for example, using either labeled antigen or labeled antibody. In some assays, the antigen is immobilized on a 96-well plate, or expressed on a cell surface, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive, fluorescent or enzyme labels.

[0041] “Epitope binning” is the process of grouping antibodies based on the epitopes they recognize. More particularly, epitope binning comprises methods and systems for discriminating the epitope recognition properties of different antibodies, using competition assays combined with computational processes for clustering antibodies based on their epitope recognition properties and identifying antibodies having distinct binding specificities.

[0042] As used herein, the terms “specifically binds,” “specifically recognizes,” “immunospecifically binds,” “selectively binds,” “immunospecifically recognizes” and “immunospecific” are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., epitope) as such binding is understood by one skilled in the art. In some embodiments, “specifically binds” means, for instance that a polypeptide or molecule interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including related and unrelated proteins. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, generally with lower affinity as determined by, e.g., immunoassays, BIACORE™, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), the OctetQK384 system (ForteBio, Menlo Park, CA), or other assays known in the art. In some embodiments, an antibody or antigen-binding domain binds to or specifically binds to an antigen when it binds to the antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIAs) and enzyme linked immunosorbent assays (ELISAs). Typically a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion regarding binding specificity. In some embodiments, the extent of binding of an antibody or antigen-binding domain to a “non-targef ’ protein is less than about 10% of the binding of the antibody or antigen-binding domain to its particular target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIAs. In some embodiments, molecules that specifically bind to an antigen bind to the antigen with a Ka that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the Ka when the molecules bind to another antigen. In some embodiments, molecules that specifically bind to an antigendo not cross react with other proteins. In another specific embodiment, molecules that specifically bind to CD16a do not bind to CD16b. In some embodiments “specifically binds” means, for instance, that a polypeptide or molecule binds a protein or target with a KD of about O.lmM or less, but more usually less than about IpM. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a KD of at least about 0.1 pM or less, at least about 0.01 pM or less, or at least about InM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include a polypeptide or molecule that recognizes a protein or target in more than one species. Likewise, because of homology within certain regions of polypeptide sequences of different proteins, specific binding can include a polypeptide or molecule that recognizes more than one protein or target. It is understood that, in some embodiments, a polypeptide or molecule that specifically binds a first target may or may not specifically bind a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding, e.g., binding to a single target. Thus, a polypeptide or molecule can, in some embodiments, specifically bind more than one target. In some embodiments, multiple targets can be bound by the same antigen-binding site on the polypeptide or molecule. For example, an antibody can, in certain instances, comprise two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins. In certain alternative embodiments, an antibody can be bispecific and comprise at least two antigenbinding sites with differing specificities. Generally, but not necessarily, reference to “binding” means “specific binding”.

[0043] “Binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding agent such as an antibody) and its binding partner (e.g., an antigen such as CD16a). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. In one embodiment, the “KD” or “KD value” may be measured by biolayer interferometry (BLI) using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA). Alternatively, theKD may also be measured in a radiolabeled antigen-binding assay (RIA), for example, performed with the Fab version of an antibody of interest and its antigen (Chen, et al., (1999) J. Mol Biol 293:865-881) or using surface plasmon resonance (SPR) assays by BIACORE™, using, for example, a BIACORE™-2000 or a BIACORE™-3000 (BIACORE™, Inc., Piscataway, NJ). An “on-rate” or “rate of association” or “association rate” or “kon,” as well as an “off-rate” or “rate of dissociation” or “dissociation rate” or “kOff,” can also be determined with the same SPR or BLI techniques described above using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA) or a BIACORE™-2000 or a BIACORE™- 3000 (BIACORE™, Inc., Piscataway, NJ), respectively.

[0044] The term “compete” or any grammatical variation thereof when used in the context of binding agents (e.g, antibodies) means binding agents that compete for the same epitope or binding site on a target, which includes competition between such binding agents as determined by an assay in which the binding agent under study prevents or inhibits the specific binding of a reference molecule (e.g, a reference ligand, or reference antigenbinding protein, such as a reference antibody) to a common antigen (e.g., CD 16a or CD 19). Numerous types of competitive binding assays can be used to determine if a test binding agent competes with a reference molecule for binding to CD 16a (e.g., human CD 16a) or CD 19 (e.g., human CD 19). Examples of assays that can be employed include solid phase direct or indirect radioimmunoassay (RIA); solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli etal., (1983) Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol. 137:3614-3619 or Cheung, et al., (1990) Virology 176:546-552); solid phase direct labeled assay; solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., (1988) Molec. Immunol. 25:7-15); and direct labeled RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of a purified antigen (e.g., CD 16a, such as human CD 16a, or CD 19, such as human CD 19) bound to a solid surface or cells bearing either of an unlabeled test antigen-binding protein (e.g., test CD 16a antibody or test CD 19 antibody) or a labeled reference antigen-binding protein (e.g., reference CD 16a antibody or reference CD 19 antibody). Competitive inhibition may be measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein.Usually, the test antigen-binding protein is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope asthe reference antibody and / or antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference for antibodies steric hindrance to occur (e.g., similar epitope or overlapping epitope). Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 20%, for example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.

[0045] As used herein, the term “constant region” or “constant domain” is a well-known antibody term of art and refers to an antibody portion, for example, a carboxyl terminal portion of a light and / or heavy chain which is not directly involved in binding of an antibody to an antigen but which can exhibit various effector functions, such as interaction with an Fc receptor. The term includes the portion of an immunoglobulin molecule having a generally more conserved amino acid sequence relative to an immunoglobulin variable domain.

[0046] Antibody “effector functions” refer to those biological activities attributable to the Fc region (e.g., a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibodydependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0047] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226 (according to the EU numbering system), or from Pro230 (according to the EU numbering system), to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.

[0048] A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (such as antibody-dependent cellular phagocytosis, z.e., ADCP); down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functionsgenerally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays as disclosed.

[0049] A “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature, and not manipulated, modified, and / or changed (e.g., isolated, purified, selected, including or combining with other sequences such as variable region sequences) by a human. Native sequence human Fc regions include a native sequence human IgGl Fc region (non- A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.

[0050] A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., substituting, addition, or deletion), preferably one or more amino acid substitution(s). In some embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region described herein can possess at least about 80% homology with a native sequence Fc region and / or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith. The variant Fc region herein described herein may have a loss of an effector function (e.g., silent Fc). An exemplary variant Fc region comprises LALA (Leu234Ala plus Leu235Ala) and Pro329Gly mutations to eliminate the Fcgrl binding site for CD16.

[0051] As used herein, the term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy -terminal portion includes one or more constant regions. The “heavy chain” can refer to any distinct types, e.g., for example, alpha (a), delta (5), epsilon (a), gamma (y) and mu (p), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgGl, IgG2, IgG3 and IgG4.

[0052] As used herein, the term “light chain” when used in reference to an antibody can refer to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy -terminal portion includes a constant region. The approximate length of a light chain is 211 to 217amino acids. There are two distinct types, e.g., kappa (K) or lambda (X) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art.

[0053] The terms “antigen-binding fragment,” “antigen-binding domain,” “antigenbinding region,” and similar terms refer to that portion of an antibody, which comprises the amino acid residues that interact with an antigen and confer on the binding fragment, domain, or region its specificity and affinity for the antigen (e.g., the CDRs). “Antigen-binding fragment” as used herein includes “antibody fragment,” which comprises a portion of an antibody including one or more CDRs, such as the antigen-binding or variable region of the antibody.

[0054] Antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab’) fragments, disulfide-linked Fvs (sdFv), anti -idiotypic (anti- id) antibodies, and epitope-binding fragments of any of the above.

[0055] In some embodiments, antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, including molecules that contain one or more antigen-binding sites that bind to a CD 16a antigen and / or a CD 19 antigen.

[0056] Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In some embodiments, antibodies described herein are IgG antibodies (e.g., human IgG), or a class (e.g., human IgGl, IgG2, IgG3 or IgG4) or a subclass thereof.

[0057] In some embodiments, an antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs. In further embodiments, the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical. In other embodiments, the amino acid sequences of the H chains are different from each other. Additionally or alternatively, the amino acid sequences of the L chains are different from each other. For example, an antibody comprises a first H / L chain pair and a second H / L chain pair, wherein the first H / L chain pair binds to a CD 16a antigen and the second H / L chain pair binds to a non-CD16a tumor-target antigen (such as CD 19). In some embodiments, an antibody is a 2-chain antibody unit comprising a scFv-scFv pair. In further embodiments,the amino acid sequences of the scFv are identical. In other embodiments, the amino acid sequence of the scFv are different from each other. For example, an antibody comprises a first scFv and a second scFv, wherein the first scFv binds to a CD 16a antigen and the second scFv binds to a non-CD16a tumor target antigen (such as CD 19). In some embodiments, the scFv-scFv chains comprise constant regions, for example, human constant regions. In some embodiments, an antibody is a 2-chain antibody unit comprising a VHH-VHH pair. In further embodiments, the amino acid sequences of the VHH are identical. In other embodiments, the amino acid sequence of the VHH are different from each other. For example, an antibody comprises a first VHH and a second VHH, wherein the first VHH binds to a CD 16a antigen and the second VHH binds to a non-CD16a tumor target antigen (such as CD 19). In some embodiments, the H and / or L chains comprise constant regions, for example, human constant regions. In some embodiments, the L chain constant region of such antibodies is a kappa or lambda light chain constant region, for example, a human kappa or lambda light chain constant region. In some embodiments, the H chain constant region of such antibodies comprises a gamma heavy chain constant region, for example, a human gamma heavy chain constant region. In some embodiments, such antibodies comprise IgG constant regions, for example, human IgG constant regions (e.g., IgGl, IgG2, IgG3, and / or IgG4 constant regions).

[0058] An antibody or fragment thereof may preferentially bind to CD 16a (such as human CD 16a) and / or tumor target (such as human CD 19), meaning that the antibody or fragment thereof binds CD 16a and / or a tumor target (such as CD 19) with greater affinity than it binds to a control protein (e.g., unrelated control proteins such as hen egg white lysozyme, or CD 16b) and / or binds human CD 16a and / or a tumor target (such as CD 19) with greater affinity than it binds to an unrelated control protein. For example, the antibody or fragment thereof may specifically recognize and bind CD 16a and / or a tumor target (such as CD 19), or a portion of each thereof. “Specific binding” means that the antibody or fragment thereof binds to CD 16a and / or a tumor target (such as CD 19) with an affinity that is at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times greater than the affinity for an unrelated control protein (e.g., hen egg white lysozyme). In some embodiments, the antibody or fragment thereof may bind CD 16a and / or a tumor target (such as CD 19) substantially exclusively (e.g, is able to distinguish CD 16a and / or the tumor target (such as CD 19) from other known polypeptides, for example, by virtue of measurable differences in binding affinity). In some embodiments, a CD 16a binding agent (e.g, an antibody) may react with CD 16a sequences other than human CD 16a sequences (e.g., cynomolgous monkey CD 16a sequences). In other embodiments, a CD 16a binding agent (e.g., an antibody) does not reactwith non-human (such as cynomolgous monkey) CD 16a sequences. In some embodiments, a CD 19 binding agent (e.g., an antibody) may react with CD 19 sequences other than human CD 19 sequences (e.g., cynomolgous monkey CD 19 sequences). In other embodiments, a CD 19 binding agent (e.g., an antibody) does not react with non-human (such as cynomolgous monkey) CD 19 sequences.

[0059] The term “variable region” or “variable domain” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain, has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and is used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as “VH .” The variable region of the light chain may be referred to as “VL.” The term “variable” refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (c.g, extreme variability) called “hypervariable regions” or alternatively called “complementarity determining regions (CDRs).” The variable regions of heavy and light chains each comprise four frameworks (FR1, FR2, FR3 and FR4), largely adopting a P sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the P sheet structure. The hypervariable regions in each chain are held together in close proximity by the frameworks and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, (1991)). The constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). The variable regions differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable region are referred to as framework regions (FR). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with its antigen. In specific embodiments, the variable region is a human variable region.

[0060] The term “hypervariable region,” “HVR,” “HV,” “complementarity determining region,” or “CDR” when used herein refers to the regions of an antibody variable region that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six hypervariable regions: three in the VH (Hl or VH CDR1, H2 or VH CDR2, and H3 or VH CDR3), and three in the VL (LI or VL CDR1, L2 or VL CDR2, and L3 or VL CDR3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat CDRs are based on sequence variability and are the most commonly used (see, e.g., Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions or CDRs are noted below.

[0061] A universal numbering system has been developed and widely adopted, ImMunoGeneTics (IMGT®) Information System (Lefranc et al., Dev. Comp. Immunol. 27(l):55-77 (2003)). IMGT is an integrated information system specializing in immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates. Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the “location” of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues and are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pliickthun, J. Mol. Biol. 309: 657-670 (2001). Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numberingsystem, is well known to one skilled in the art (see, e.g., Kabat, supra, Chothia and Lesk, supra, Martin, supra, Lefranc et al., supra) and is also illustrated below. Various systems known in the art or described herein represent different ways of delineating CDRs, and when they are used to define the same antibody, they are often considered equivalent. An Exemplary system, shown herein, combines Kabat and Chothia. The residues from each of these hypervariable regions or CDRs are exemplified in the table below.Exemplary CDRs According to Various Numbering Systems

[0062] Hypervariable regions may comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 or 26- 35A (Hl), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. As used herein, the terms “hypervariable region,” “HVR,” “HV,” “complementarity determining region,” or “CDR” are used interchangeably.

[0063] “Polynucleotide” or “nucleic acid,” as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. A cell that produces a binding molecule of the present disclosure may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced. Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred toas “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3 ’ to the 3 ’ end of the RNA transcript are referred to as “downstream sequences.”

[0064] The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including for example, in order to introduce a nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and / or inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., both an antibody heavy and light chain or an antibody VH and VL) both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product (e.g., a binding agent as described herein), and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0065] The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

[0066] “Excipient” means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipientsinclude, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof. The term “excipient” can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete)) or vehicle. In some embodiments, excipients are pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants, such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington’s Pharmaceutical Sciences (18th ed. 1990). In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005;Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, pharmaceutically acceptable excipients are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. In some embodiments, a pharmaceutically acceptable excipient is an aqueous pH buffered solution.In some embodiments, excipients are sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary excipient when a composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly forinjectable solutions. An excipient can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral compositions, including formulations, can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Compositions, including pharmaceutical compounds, may contain a prophylactically or therapeutically effective amount of an CD 16a and / or CD 19 binding agent (e.g., an antibody), for example, in isolated or purified form, together with a suitable amount of excipient so as to provide the form for proper administration to the subject (e.g., patient). The formulation should suit the mode of administration.

[0067] An “effective amount” is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate the symptoms and / or underlying cause, prevent or delay the occurrence of symptoms and / or their underlying cause, and / or improve or remediate the damage that results from or is associated with a disease, disorder, or condition. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0068] The term “therapeutically effective amount” as used herein refers to the amount of an agent (e.g., an antibody described herein or any other agent described herein) that is sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder or condition, and / or a symptom related thereto. A therapeutically effective amount of an agent, including a therapeutic agent, can be an amount necessary for (i) reduction, delay or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction, delay or amelioration of the recurrence, development or onset of a given disease, disorder or conditions, and / or (iii) to improve or enhance the prophylactic or therapeutic effect of another therapy (e.g, a therapy other than the administration of an agent described herein). A “therapeutically effective amount” of a substance / molecule / agent of the present disclosure (e.g, a bispecific antibody) may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule / agent, to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance / molecule / agent are outweighed by the therapeutically beneficial effects. In certain embodiments, the term“therapeutically effective amount” refers to an amount of a multispecific binding agent effective to “treat” a disease, disorder, or condition, in a subject or mammal.

[0069] The term “treating” or any grammatical variation thereof refers to reducing and / or ameliorating the severity and / or duration of a given disease, disorder or condition, and / or a symptom related thereto, such as (i) reduction, delay or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction, delay or amelioration of the recurrence, development or onset of a given disease, disorder or conditions, and / or (iii) to improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than the administration of a multispecific binding agent described herein).

[0070] A “prophylactically effective amount” is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of a disease, disorder or condition, or reducing the likelihood of the onset (or reoccurrence) of a disease, disorder, or condition or associated symptom(s).

[0071] The full therapeutic or prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

[0072] The terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less variation of a given value or range.

[0073] As used herein, comparative terms as used herein, such as reduce, decrease, increase, or any grammatical variation thereof, can refer to certain variation from the reference. In some embodiments, such variation can refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1 fold, or about 2 fold, or about 3 fold, or about 4 fold, or about 5 fold, or about 10 fold, or about 20 fold, or about 30 fold, or about 40 fold, or about 100 fold or higher than the reference. In some embodiments, such variation can refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the reference.

[0074] As used in the present disclosure and claims, the singular forms “a”, “an” and“the” include plural forms unless the context clearly dictates otherwise.

[0075] In some embodiments, the terms “first,” “second,” “third,” “fourth” and similar in a component name are used to distinguish and identify more than one component sharing certain identity in their names. For example, “first antibody” and “second antibody” are used to distinguish two antibodies.

[0076] It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. It is also understood that wherever embodiments are described herein with the phrase “consisting essentially of’ otherwise analogous embodiments described in terms of “consisting of’ are also provided.

[0077] The term “between” as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B.

[0078] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0079] The term “optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances wherein the circumstance occurs, and the instances wherein the circumstance does not occur.5.2. Binding Agents5.2.1. CD16a Binding Agents

[0080] In some embodiments, the present disclosure provides CD 16a binding agents that can be used herein as an agent for enhancing immune responses and / or therapeutic agents. Such agents include antibodies (e.g., monospecific or multispecific, including bispecific) that bind to CD 16a. Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heteroconjugate antibodies, as well as variants thereof having increased or decreased affinity or other properties.

[0081] In some embodiments, described herein are CD 16a binding agents (e.g., antibodies) that bind to CD 16a, including a CD 16a polypeptide, a CD 16a polypeptide fragment, a CD 16a peptide or a CD 16a epitope. In some embodiments, the CD 16a binding agents are human or humanized antibodies (e.g., comprising human constant regions) that bind CD 16a, including a CD 16a polypeptide, a CD 16a polypeptide fragment, a CD 16a peptide or a CD 16a epitope. In some embodiments, a CD 16a binding agent (e.g., an antibody), such as a human CD 16a binding agent, can bind to CD 16a expressed on thesurface of a mammalian (e.g., human) cell, including a CD 16a expressing immune cell (e.g., an NK cell). In some embodiments, a CD 16a binding agent (e.g., an antibody) binds a CD 16a extracellular epitope exposed on a cell such as an immune cell. In some embodiments, described herein is a CD 16a binding agent e.g., an antibody) that binds to CD 16a, such as human CD 16a or a portion thereof. In some embodiments, CD 16a is a human CD 16a. In some embodiments, a CD 16a binding agent is a human CD 16a binding agent e.g., an antibody that binds to human CD 16a). In some embodiments, CD 16a binding agents e.g., antibodies) bind to both human and cyno CD 16a. In other embodiments, CD 16a binding agents e.g., antibodies) bind to human CD 16a but not to cyno CD 16a.

[0082] In some embodiments, described herein is a CD 16a binding agent e.g., an antibody) that binds to CD16a-F. In some embodiments, described herein is a CD16a binding agent e.g., an antibody) that binds to CD16a-V. In some embodiments, described herein is a CD 16a binding agent e.g., an antibody) that binds to CD16a-F and CD16a-V.

[0083] In some embodiments, the CD 16a binding agent e.g., an antibody) provided herein binds to CD 16a e.g., human CD 16a) with a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM e.g. 10'8M or less, e.g. from 10'8M to 10'13M, e.g., from 10'9M to 10'13M). A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure, including by RIA, for example, performed with the Fab version of an antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); by biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by OCTET®, using, for example, an OCTET®Red96 system, or by BIACORE®, using, for example, a BIACORE®TM-2000 or a BIACORE®TM-3000. An “on-rate” or “rate of association” or “association rate” or “kon” may also be determined with the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above using, for example, the OCTET®Red96, the BIACORE®TM-2000, the BIACORE®TM-3000 system, the BIACORE®TM-8K, or the BIACORE®TM-8K+ system.

[0084] In some embodiments, the CD 16a binding agent e.g., an antibody) provided herein does not bind to CD 16b e.g., human CD 16b and / or cyno CD 16b). In some embodiments, the CD 16a binding agent e.g., an antibody) provided herein does not bind to CD16b-NAl. In some embodiments, the CD 16a binding agent e.g., an antibody) provided herein does not bind to CD16b-NA2. In some embodiments, the CD 16a binding agent e.g., an antibody) provided herein does not bind to CD16b-NAl or CD16b-NA2. In some embodiments, the CD 16a binding agent e.g., an antibody) provided herein does not bind tohuman CD 16b. In some embodiments, the CD 16a binding agent (e.g., an antibody) provided herein does not bind to human CD 16b or cyno CD 16b. In other embodiments, the CD 16a binding agent (e.g., an antibody) provided herein binds to CD 16a (e.g., human CD 16a) with higher affinity than to CD 16b (e.g., human CD 16b). In some embodiments, the binding affinity of the CD 16a binding agent (e.g., an antibody) provided herein to CD 16a (e.g., human CD 16a) is at least 2 fold of that to CD 16b (e.g., human CD 16b). In some embodiments, the binding affinity of the CD 16a binding agent (e.g., an antibody) provided herein to CD 16a (e.g., human CD 16a) is at least 5 fold of that to CD 16b (e.g., human CD 16b). In some embodiments, the binding affinity of the CD 16a binding agent (e.g., an antibody) provided herein to CD 16a (e.g., human CD 16a) is at least 10 fold of that to CD 16b (e.g., human CD 16b). In some embodiments, the binding affinity of the CD 16a binding agent (e.g., an antibody) provided herein to CD 16a (e.g., human CD 16a) is at least 100 fold of that to CD 16b (e.g., human CD 16b). In some embodiments, the binding affinity of the CD 16a binding agent (e.g., an antibody) provided herein to CD 16a (e.g., human CD 16a) is at least 1000 fold of that to CD 16b (e.g., human CD 16b).

[0085] In some embodiments, the CD 16a binding agents (e.g., antibodies) described herein comprise a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of any one of the antibodies described herein, such as an amino acid sequence of a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 depicted in Tables 1-9. Accordingly, in some embodiments, a CD 16a binding agent (e.g., an antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from: (a) the antibody designated AvGn-CD16a-A2; (b) the antibody designated AvGn-CD16a- #1A2; (c) the antibody designated AvGn-CD16a-#2-lA2; (d) the antibody designated AvGn- CD16a-#2-2A2; (e) the antibody designated AvGn-CD16a-#2-3A2; (f) the antibody designated AvGn-CD16a-#3A2; (g) the antibody designated AvGn-CD16a-#6A2; (h) the antibody designated AvGn-CD16a-#10A2; and (i) the antibody designated AvGn-CD16a- #55, as shown in Tables 1-9. In some embodiments, a CD16a binding agent (e.g., an antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and any one, any two, and / or all three light chain CDRs from: (a) the antibody designated AvGn-CD16a-A2; (b) the antibody designated AvGn-CD16a-#lA2; (c) the antibody designated AvGn-CD16a-#2-lA2; (d) the antibody designated AvGn-CD16a-#2-2A2; (e) the antibody designated AvGn-CD16a-#2-3A2; (f) the antibody designated AvGn-CD16a-#3A2;(g) the antibody designated AvGn-CD16a-#6A2; (h) the antibody designated AvGn-CD16a- #10A2; and (i) the antibody designated AvGn-CD16a-#55, as shown in Tables 1-9.

[0086] In some embodiments, a CD 16a binding agent (e.g., an antibody) comprises a VH region, which comprises a VH CDR1, a VH CDR2, and / or a VH CDR3, and / or a VL region, which comprises a VL CDR1, a VL CDR2, and / or a VL CDR3, of any one of the binding agents described herein (see, e.g., any one of Tables 1-9). Accordingly, in some embodiments, a CD 16a binding agent (e.g., an antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from Table 1. In some embodiments, a CD 16a binding agent (e.g., an antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from Table 2. In some embodiments, a CD 16a binding agent (e.g., an antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from Table 3. In some embodiments, a CD 16a binding agent (e.g., an antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from Table 4. In some embodiments, a CD 16a binding agent (e.g., an antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from Table 5. In some embodiments, a CD 16a binding agent (e.g., an antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from Table 6. In some embodiments, a CD 16a binding agent (e.g., an antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from Table 7. In some embodiments, a CD 16a binding agent (e.g., an antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from Table 8. In some embodiments, a CD 16a binding agent (e.g., an antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from Table 9.

[0087] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises (i) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:37, SEQ ID NO:75, SEQ ID NO: 113, SEQ ID NO: 151, SEQ ID NO: 189, SEQ ID NO:227, SEQ ID NO:265, SEQ ID NO:303, or SEQ ID NO:341, and / or (ii) a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:38, SEQ ID NO:76, SEQ ID NO: 114,SEQ ID NO: 152, SEQ ID NO: 190, SEQ ID NO:228, SEQ ID NO:266, SEQ ID NO:304, or SEQ ID NO:342.

[0088] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:37 and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:37, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:38. CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDR sequences are determined according to a combination of any two or more of the above-mentioned numbering systems, for example, a combination of Kabat and Chothia. Various exemplary CDR numbering systems are described and illustrated above in Section 5.1.

[0089] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, and 31; a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, and 32; and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, and 33; and / or (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, and 34; a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, and 35; and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, and 36.

[0090] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:4, a VLCDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0091] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a VH CDR3 comprising the amino acid sequence of SEQ ID NOV; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 12.

[0092] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 18.

[0093] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:20, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:21; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:22, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:23, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:24.

[0094] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:25, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:26, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:27; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:30.

[0095] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:32, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:33; and a VLregion comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:34, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:35, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:36.

[0096] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:75 and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:75 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:76. CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDR sequences are determined according to a combination of any two or more of the above-mentioned numbering systems, for example, a combination of Kabat and Chothia.

[0097] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 45, 51, 57, 63, and 69; a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 40, 46, 52, 58, 64, and 70; and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 47, 53, 59, 65, and 71; and / or (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 48, 54, 60, 66, and 72; a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 49, 55, 61, 67, and 73; and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 50, 56, 62, 68, and 74.

[0098] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:40, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:41; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:42, a VLCDR2 comprising the amino acid sequence of SEQ ID NO:43, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:44.

[0099] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:45, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:46, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:47; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:48, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:49, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:50.

[0100] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:51, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:52, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:53; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:54, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:55, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:56.

[0101] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:57, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:58, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:59; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:60, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:61, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:62.

[0102] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 63, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:64, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:65; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:66, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:67, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 68.

[0103] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 69, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:70, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:71; and a VLregion comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:72, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:73, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:74.

[0104] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 113 and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 114. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 113 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 114. CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDR sequences are determined according to a combination of any two or more of the above-mentioned numbering systems, for example, a combination of Kabat and Chothia.

[0105] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 83, 89, 95, 101, and 107; a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 78, 84, 90, 96, 102, and 108; and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 79, 85, 91, 97, 103, and 109; and (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 80, 86, 92, 98, 104, and 110; a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 81, 87, 93, 99, 105, and 111; and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 82, 88, 94, 100, 106, and 112.

[0106] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:77, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:78, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:79; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:80, a VLCDR2 comprising the amino acid sequence of SEQ ID NO:81, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:82.

[0107] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 83, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:84, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:85; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:86, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:87, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:88.

[0108] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 89, a VH CDR2 comprising the amino acid sequence of SEQ ID NOVO, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:91; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:92, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:93, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:94.

[0109] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:95, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:96, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:97; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:98, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:99, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 100.

[0110] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 101, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 102, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 103; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 104, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 105, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 106.

[0111] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 107, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 108, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 109; and a VLregion comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 110, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 111, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 112.

[0112] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 151, and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 152. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 151 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 152. CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDR sequences are determined according to a combination of any two or more of the above-mentioned numbering systems, for example, a combination of Kabat and Chothia.

[0113] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 121, 127, 133, 139, and 145; a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 116, 122, 128, 134, 140, and 146; and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 117, 123, 129, 135, 141, and 147; and (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 118, 124, 130, 136, 142, and 148; a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 125, 131, 137, 143, and 149; and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 120, 126, 132, 138, 144, and 150.

[0114] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 115, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 116, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 117; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 118, aVL CDR2 comprising the amino acid sequence of SEQ ID NO: 119, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 120.

[0115] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 121, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 122, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 123; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 124, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 125, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 126.

[0116] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 127, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 128, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 129; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 130, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 131, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 132.

[0117] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 133, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 134, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 135; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 136, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 137, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 138.

[0118] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 139, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 140, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 141; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 142, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 143, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 144.

[0119] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 145, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 146, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 147; and a VLregion comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 148, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 149, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 150.

[0120] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 189, and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 190. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 190. CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDR sequences are determined according to a combination of any two or more of the above-mentioned numbering systems, for example, a combination of Kabat and Chothia.

[0121] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 153, 159, 165, 171, 177, and 183; a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 154, 160, 166, 172, 178, and 184; and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 155, 161, 167, 173, 179, and 185; and (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 156, 162, 168, 174, 180, and 186; a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 157, 163, 169, 175, 181, and 187; and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 164, 170, 176, 182, and 188.

[0122] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 153, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 154, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 155; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 156, aVL CDR2 comprising the amino acid sequence of SEQ ID NO: 157, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 158.

[0123] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 159, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 160, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 161; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 162, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 163, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 164.

[0124] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 165, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 166, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 167; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 168, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 169, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 170.

[0125] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 171, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 172, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 173; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 174, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 175, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 176.

[0126] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 177, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 178, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 179; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 180, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 181, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 182.

[0127] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 183, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 184, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 185; and a VLregion comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 186, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 187, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 188.

[0128] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:227, and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:228. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:227 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:228. CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDR sequences are determined according to a combination of any two or more of the above-mentioned numbering systems, for example, a combination of Kabat and Chothia.

[0129] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 197, 203, 209, 215, and 221; a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 192, 198, 204, 210, 216, and 222; and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 193, 199, 205, 211, 217, and 223; and (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 194, 200, 206, 212, 218, and 224; a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 195, 201, 207, 213, 219, and 225; and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 196, 202, 208, 214, 220, and 226.

[0130] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 191, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 192, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 193; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 194, aVL CDR2 comprising the amino acid sequence of SEQ ID NO: 195, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 196.

[0131] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 197, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 198, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 199; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:200, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:201, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:202.

[0132] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:203, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:204, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:205; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:206, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:207, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:208.

[0133] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:209, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:210, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:211; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:212, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:213, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:214.

[0134] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:215, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:216, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:217; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:218, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:219, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:220.

[0135] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:221, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:222, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:223; and a VLregion comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:224, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:225, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:226.

[0136] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:265, and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:266. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:265 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:266. CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDR sequences are determined according to a combination of any two or more of the above-mentioned numbering systems, for example, a combination of Kabat and Chothia.

[0137] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 229, 235, 241, 247, 253, and 259; a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 230, 236, 242, 248, 254, and 260; and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 231, 237, 243, 249, 255, and 261; and (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 232, 238, 244, 250, 256, and 262; a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 233, 239, 245, 251, 257, and 263; and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 234, 240, 246, 252, 258, and 264.

[0138] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:229, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:230, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:231; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:232, aVL CDR2 comprising the amino acid sequence of SEQ ID NO:233, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:234.

[0139] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:235 a VH CDR2 comprising the amino acid sequence of SEQ ID NO:236, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:237; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:238, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:239, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:240.

[0140] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:241, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:242, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:243; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:244, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:245, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:246.

[0141] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:247, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:248, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:249; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:250, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:251, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:252.

[0142] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:253, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:254, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:255; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:256, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:257, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:258.

[0143] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:259, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:260, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:261; and a VLregion comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:262, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:263, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:264.

[0144] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:303, and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:304. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:303 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:304. CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDR sequences are determined according to a combination of any two or more of the above-mentioned numbering systems, for example, a combination of Kabat and Chothia.

[0145] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 267, 273, 279, 285, 291, and 297; a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 268, 274, 280, 286, 292, and 298; and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 269, 275, 281, 287, 293, and 299; and (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 270, 276, 282, 288, 294, and 300; a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 271, 277, 283, 289, 295, and 301; and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 272, 278, 284, 290, 296, and 302.

[0146] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:267, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:268, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:269; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:270, aVL CDR2 comprising the amino acid sequence of SEQ ID NO:271, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:272.

[0147] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:273 a VH CDR2 comprising the amino acid sequence of SEQ ID NO:274, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:275; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:276, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:277, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:278.

[0148] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:279, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:280, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:281; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:282, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:283, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:284.

[0149] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:285, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:286, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:287; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:288, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:289, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:290.

[0150] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:291, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:292, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:293; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:294, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:295, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:296.

[0151] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:297, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:298, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:299; and a VLregion comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:300, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:301, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:302.

[0152] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:341, and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:342. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:341 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:342. CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDR sequences are determined according to a combination of any two or more of the above-mentioned numbering systems, for example, a combination of Kabat and Chothia.

[0153] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 305, 311, 317, 323, 329, and 335; a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 306, 312, 318, 324, 330, and 336; and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307, 313, 319, 325, 331, and 337; and (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 308, 314, 320, 326, 332, and 338; a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 309, 315, 321, 327, 333, and 339; and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 310, 316, 322, 328, 334, and 340.

[0154] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:305, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:306, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:307; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:308, aVL CDR2 comprising the amino acid sequence of SEQ ID NO:309, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:310.

[0155] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:3 I l a VH CDR2 comprising the amino acid sequence of SEQ ID NO:312, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:313; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:314, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:315, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:316.

[0156] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 317, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:318, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:319; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:320, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:321, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:322.

[0157] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:323, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:324, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:325; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:326, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:327, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:328.

[0158] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:329, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:330, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:331; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:332, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:333, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:334.

[0159] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:335, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:336, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:337; and a VLregion comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:338, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:339, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:340.

[0160] In some embodiments, the antibody further comprises one or more framework regions of SEQ ID NOs: 37, 38, 75, 76, 113, 114, 151, 152, 189, 190, 227, 228, 265, 266, 303, 304, 341, and / or 342. In some embodiments, the antibody or fragment thereof further comprises a framework 1 (FR1), a framework 2 (FR2), a framework 3 (FR3) and / or a framework 4 (FR4) sequence as set forth in any one of SEQ ID NOs:37, 38, 75, 76, 113, 114, 151, 152, 189, 190, 227, 228, 265, 266, 303, 304, 341, and / or 342. In some embodiments, the antibody provided herein is a humanized antibody. Framework regions described herein are determined based upon the boundaries of the CDR numbering system. In other words, if the CDRs are determined by, e.g., Kabat, IMGT, or Chothia, then the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format, from the N- terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residues N-terminal to the CDR1 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR2 is defined as the amino acid residues between CDR1 and CDR2 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR3 is defined as the amino acid residues between CDR2 and CDR3 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, and FR4 is defined as the amino acid residues C-terminal to the CDR3 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0161] In some embodiments, CD 16a binding agents (e.g., antibodies such as monospecific or bispecific antibodies), including human CD16a binding agents, described herein comprise a VH region or VH domain. Additionally or alternatively, in some embodiments, CD 16a binding agents (e.g., antibodies such as monospecific or bispecific antibodies), including human CD 16a binding agents, described herein comprise a VL region or VL domain. In some embodiments, CD 16a binding agents (e.g., antibodies such as monospecific or bispecific antibodies), including human CD16a binding agents, described herein have a combination of (i) a VH domain or VH region; and (ii) a VL domain or VL region.

[0162] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:37. In someembodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VL comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO: 37, and a VL comprising the amino acid sequence of SEQ ID NO:38.

[0163] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:75. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VL comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:75, and a VL comprising the amino acid sequence of SEQ ID NO:76.

[0164] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO: 113. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VL comprising the amino acid sequence of SEQ ID NO: 114. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO: 113 and a VL comprising the amino acid sequence of SEQ ID NO: 114.

[0165] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO: 151. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VL comprising the amino acid sequence of SEQ ID NO: 152. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO: 151 and a VL comprising the amino acid sequence of SEQ ID NO: 152.

[0166] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO: 189. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VL comprising the amino acid sequence of SEQ ID NO: 190. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL comprising the amino acid sequence of SEQ ID NO: 190.

[0167] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:227. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VL comprising the amino acid sequence of SEQ ID NO:228. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:227 and a VL comprising the amino acid sequence of SEQ ID NO:228.

[0168] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:265. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VL comprising the amino acid sequence of SEQ ID NO:266. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:265 and a VL comprising the amino acid sequence of SEQ ID NO:266.

[0169] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:303. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VL comprising the amino acid sequence of SEQ ID NO:304. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:303 and a VL comprising the amino acid sequence of SEQ ID NO:304.

[0170] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:341. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) comprises a VL comprising the amino acid sequence of SEQ ID NO:342. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:341 and a VL comprising the amino acid sequence of SEQ ID NO:342.

[0171] In certain embodiments, the CD 16a binding agent (e.g., an antibody of a fragment thereof) provided herein comprises amino acid sequences with certain percent identity (such as at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or as at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at leastabout 96%, or at least about 97%, or at least about 98%, or at least about 99%, or higher) relative to any antibody or fragment thereof provided herein, for example, a CDR, VH or VL in Tables 1-9, or any full-length antibody chain as disclosed herein. In some embodiments, the CD 16a binding agent (e.g., an antibody or a fragment thereof) provided herein comprises CDRs of any antibody or fragment thereof provided herein, for example in Tables 1-9. In further embodiments, the CD 16a binding agent provided herein comprises amino acid sequences with certain percent identity (such as at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or as at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or higher) relative to any antibody or fragment thereof provided herein, for example, a VH or VL in Tables 1-9, or any full-length antibody chain as disclosed herein.

[0172] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 87:2264 2268 (1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 90:5873 5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, word length=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, word length=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul etal., Nucleic Acids Res. 25:3389 3402 (1997). In some embodiments, the percent identity between two sequences is calculated by dividing the number of residue(s) varied (excluding or including conservative amino acid substitution(s) or degenerate nucleotide substitution(s)) between the two sequences in the alignment with the residue number of any one of the following: (i) full length of the shorter sequence, (ii) full length of the longer sequence, (iii) mean length of the two sequences, (iv) total length of the non-gap portion of the alignment, (v) length of the alignment excluding overhangs, or (vi) length of the alignment including overhangs. Overhangs as used herein with respect to asequence alignment refer to either or both ends of the alignment where residues of one sequence are considered as aligning to no residues (e.g., gap) in the other sequence. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CAB IOS 4: 11-17 (1998). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0173] In some embodiments, the binding agent e.g., an antibody) provided herein contains substitutions (e.g, conservative substitutions), insertions, or deletions relative to the reference sequence, but the binding agent comprising that sequence retains the ability to bind to CD 16a. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in a reference amino acid sequence. In some embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (e.g, in the FRs and / or constant regions).

[0174] In some embodiments, the position of one or more CDRs along the VH (e.g, CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) region of a CD16a binding agent (e.g., an antibody), including a human CD 16a binding agent, described herein may vary by one, two, three, four, five, or six amino acid positions so long as binding to CD16a (e.g., human CD16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, the position defining a CDR of any of Table 1, 2, 3, 4, 5, 6, 7, 8, or 9 may vary by shifting the N-terminal and / or C-terminal boundary of the CDR by one, two, three, four, five, or six amino acids, relative to the current CDR position, so long as binding to CD16a (e.g., human CD16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Additionally or alternatively, in some embodiments, the length of one or more CDRs alongthe VH (e.g, CDR1, CDR2, or CDR3) and / or VL (e.g, CDR1, CDR2, or CDR3) region of an CD 16a binding agent (e.g., an antibody), including a human CD 16a binding agent, described herein may vary (e.g., be shorter or longer) by one, two, three, four, five, or more amino acids, so long as binding to CD16a (e.g, human CD16a) is maintained (e.g, substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, a VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be one, two, three, four, five or more amino acids shorter than one or more of the CDRs described by SEQ ID NOs: l-36, 39-74, 77-112, 115-150, 153-188, 191-226, 229-264, 267-302, or 305-340, so long as binding to CD16a (e.g., human CD16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). In other embodiments, a VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be one, two, three, four, five or more amino acids longer than one or more of the CDRs described by SEQ ID NOs: 1-36, 39-74, 77- 112, 115-150, 153-188, 191-226, 229-264, 267-302, or 305-340, so long as binding to CD16a (e.g., human CD16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). In some embodiments, the amino terminus of a VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be extended or shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOs: 1-36, 39-74, 77-112, 115- 150, 153-188, 191-226, 229-264, 267-302, or 305-340, so long as binding to CD16a (e.g., human CD16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Additionally or alternatively, in some embodiments, the carboxy terminus of a VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be extended or shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOs: 1-36, 39- 74, 77-112, 115-150, 153-188, 191-226, 229-264, 267-302, or 305-340, so long as binding to CD16a (e.g., human CD16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Any method known in the art can be used to ascertain whether binding to CD 16a (e.g., human CD 16a) is maintained, for example, the binding assays and conditions described in the “Examples” section described herein.

[0175] In other embodiments, the CD 16a binding agents (e.g., antibodies), including human CD 16a binding agents, presented herein that bind to CD 16a, further comprise conservative sequence modifications. With respect to polypeptides that are CD 16a bindingagents (e.g., antibodies), such as human CD 16a binding agents, conservative sequence modifications include conservative amino acid substitutions that include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Thus, in some embodiments, a predicted nonessential amino acid residue in an CD 16a is replaced with another amino acid residue from the same side chain family. Methods of identifying amino acid conservative substitutions which do not eliminate antigen binding and nucleotides encoding thereof are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1 ISO- 1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). In some embodiments, the conservative sequence modifications described herein modify the amino acid sequences of the CD 16a binding agents (e.g., antibodies), including human CD16a binding agents, by 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%. In some embodiments, the amino acid sequence modifications refer to at most 1, 2, 3, 4, 5, or 6 amino acid substitutions to the CDRs, such as those described in any one of Tables 1-9. Thus, for example, each such CDR may contain up to 5 conservative amino acid substitutions, for example up to (not more than) 4 conservative amino acid substitutions, for example up to (not more than) 3 conservative amino acid substitutions, for example up to (not more than) 2 conservative amino acid substitutions, or no more than 1 conservative amino acid substitution. In some embodiments, a CD 16a binding agent (e.g., an antibody), including a human CD 16a binding agent, contains one or more, including six, CDRs having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the CDRs of AvGn-CD16a-A2, AvGn-CD16a-#lA2, AvGn-CD16a-#2-lA2, AvGn-CD16a-#2-2A2, AvGn-CD16a-#2-3A2, AvGn-CD16a-#3A2, AvGn-CD16a-#6A2, AvGn-CD16a-#10A2, or AvGn-CD16a-#55 (see, e.g., Tables 1, 2, 3, 4, 5, 6, 7, 8, or 9).

[0176] In some embodiments, a CD 16a binding agent (e.g., an antibody), including a human CD 16a binding agent, contains a VH and a VL comprising CDRs identical to those of AvGn-CD16a-A2, AvGn-CD16a-#lA2, AvGn-CD16a-#2-lA2, AvGn-CD16a-#2-2A2, AvGn-CD16a-#2-3A2, AvGn-CD16a-#3A2, AvGn-CD16a-#6A2, AvGn-CD16a-#10A2, or AvGn-CD16a-#55 (see, e.g., Tables 1, 2, 3, 4, 5, 6, 7, 8, or 9). In some embodiments, the amino acid sequence modifications do not include any modification within an SDR. In some embodiments, the amino acid sequence modifications do not include any modification within a CDR (such as CDR1, CDR2, CDR3, or any combination thereof). Additionally oralternatively, the amino acid sequence modifications are in the framework, constant region, and / or fragment crystallizable region (Fc).

[0177] In some embodiments, the antibody or fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:37, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:38, and the binding of the antibody or fragment thereof to CD 16a (e.g., human CD 16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0178] In some embodiments, the antibody or fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:75, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:76, and the binding of the antibody or fragment thereof to CD 16a (e.g., human CD 16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0179] In some embodiments, the antibody or fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 113, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 114, and the binding of the antibody or fragment thereof to CD16a (e.g., human CD16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0180] In some embodiments, the antibody or fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 151, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 152, and the binding of the antibody or fragment thereof to CD16a (e.g., human CD16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0181] In some embodiments, the antibody or fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 189, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 190, and the binding of the antibody or fragment thereof to CD16a (e.g., human CD16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0182] In some embodiments, the antibody or fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:227, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:228, and the binding of the antibody or fragment thereof to CD16a (e.g., human CD16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0183] In some embodiments, the antibody or fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:265, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to the amino acid sequence of SEQ ID NO:266, and the binding of the antibody or fragment thereof to CD16a (e.g., human CD16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0184] In some embodiments, the antibody or fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:303, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:304, and the binding of the antibody or fragment thereof to CD16a (e.g., human CD16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0185] In some embodiments, the antibody or fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:341, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:342, and the binding of the antibody or fragment thereof to CD16a (e.g., human CD16a) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0186] In some embodiments, functional epitopes can be mapped, e.g., by combinatorial alanine scanning, to identify amino acids in the CD 16a protein (including CD16a-F and / or CD16a-V) that are necessary for interaction with CD 16a binding agents (such as antibodies) provided herein. In some embodiments, conformational and crystal structure of CD 16a binding agents (such as antibodies) bound to CD 16a may be employed to identify the epitopes. In some embodiments, the present disclosure provides an antibody that specifically binds to the same epitope as any of the CD 16a binding agents (such as antibodies or fragments thereof) provided herein.

[0187] For example, in some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VHCDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:37 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti- CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:37, and a VL comprising the amino acid sequence of SEQ ID NO:38.

[0188] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:75 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:75, and a VL comprising the amino acid sequence of SEQ ID NO:76.

[0189] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 113 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 114. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 113, and a VL comprising the amino acid sequence of SEQ ID NO:114.

[0190] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 151 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 152. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 151, and a VL comprising the amino acid sequence of SEQ ID NO: 152.

[0191] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprisingthe amino acid sequence of SEQ ID NO: 190. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 189, and a VL comprising the amino acid sequence of SEQ ID NO: 190.

[0192] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:227 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:228. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:227, and a VL comprising the amino acid sequence of SEQ ID NO:228.

[0193] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:265 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:266. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:265, and a VL comprising the amino acid sequence of SEQ ID NO:266.

[0194] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:303 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:304. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:303, and a VL comprising the amino acid sequence of SEQ ID NO:304.

[0195] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:341 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:342. In some embodiments, the CD16a binding agent provided herein (e.g., an antibody) binds to the same epitope as an anti-CD16aantibody comprising a VH comprising the amino acid sequence of SEQ ID NO:341, and a VL comprising the amino acid sequence of SEQ ID NO:342.

[0196] In some embodiments, the CD 16a binding agent provided herein recognizes an epitope comprising the residue Asp 82 of human CD 16a (numbering based on UniProt propeptide sequence P08637). In some embodiments, the CD16a binding agent provided herein recognizes an epitope comprising the residue Asp 82 of human CD 16a with an affinity for CD 16a at least 10-fold higher than for CD 16b. In some embodiments, the CD 16a binding agent provided herein recognizes an epitope comprising the residue Tyr 158 of human CD16a (numbering based on UniProt propeptide sequence P08637). In some embodiments, the CD16a binding agent provided herein recognizes an epitope comprising the residue Tyr 158 of human CD 16a with an affinity for CD 16a at least 10-fold higher than for CD 16b.

[0197] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with any one of the anti-CD16a antibodies or fragments thereof described herein.

[0198] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:37 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 37, and a VL comprising the amino acid sequence of SEQ ID NO:38.

[0199] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:75 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:75, and a VL comprising the amino acid sequence of SEQ ID NO:76.

[0200] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprisinga VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 113 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:114. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 113, and a VL comprising the amino acid sequence of SEQ ID NO: 114.

[0201] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 151 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 152. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 151, and a VL comprising the amino acid sequence of SEQ ID NO: 152.

[0202] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 190. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 189, and a VL comprising the amino acid sequence of SEQ ID NO: 190.

[0203] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:227 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:228. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:227, and a VL comprising the amino acid sequence of SEQ ID NO:228.

[0204] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:265 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:266. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:265, and a VL comprising the amino acid sequence of SEQ ID NO:266.

[0205] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 303 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:304. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 303, and a VL comprising the amino acid sequence of SEQ ID NO:304.

[0206] In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:341 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:342. In some embodiments, the CD 16a binding agent provided herein (e.g., an antibody) specifically binds to CD 16a competitively with an anti-CD16a antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:341, and a VL comprising the amino acid sequence of SEQ ID NO:342.

[0207] In some embodiments, the CD 16a binding agent binds to CD16a-F and CD16a-V, but not binds to CD16b-NAl or CD16b-NA2. In further embodiments, the CD 16a binding agent comprises six CDRs of the antibody designated AvGn-CD16a-A2. In yet further embodiments, the CD 16a binding agent comprises six CDRs as listed in one column of Table 1. In some embodiments, the CD 16a binding agent comprises three CDRs of the heavy chain variable region as set forth in SEQ ID NO:37 and three CDRs of the light chain variable regions as set forth in SEQ ID NO:38. In some embodiments, the CD 16a binding agentcomprises the heavy chain variable region as set forth in SEQ ID NO:37 and the light chain variable regions as set forth in SEQ ID NO:38.

[0208] In some embodiments, the CD 16a binding agent binds to CD16a-F and CD16a-V, but not binds to CD16b-NAl or CD16b-NA2. In further embodiments, the CD 16a binding agent comprises six CDRs of the antibody designated AvGn-CD16a-#lA2. In yet further embodiments, the CD 16a binding agent comprises six CDRs as listed in one column of Table2. In some embodiments, the CD 16a binding agent comprises three CDRs of the heavy chain variable region as set forth in SEQ ID NO: 75 and three CDRs of the light chain variable regions as set forth in SEQ ID NO:76. In some embodiments, the CD16a binding agent comprises the heavy chain variable region as set forth in SEQ ID NO:75 and the light chain variable regions as set forth in SEQ ID NO:76.

[0209] In some embodiments, the CD 16a binding agent binds to CD16a-F and CD16a-V, but not binds to CD16b-NAl or CD16b-NA2. In further embodiments, the CD 16a binding agent comprises six CDRs of the antibody designated AvGn-CD16a-#2-l A2. In yet further embodiments, the CD 16a binding agent comprises six CDRs as listed in one column of Table3. In some embodiments, the CD 16a binding agent comprises three CDRs of the heavy chain variable region as set forth in SEQ ID NO: 113 and three CDRs of the light chain variable regions as set forth in SEQ ID NO: 114. In some embodiments, the CD16a binding agent comprises the heavy chain variable region as set forth in SEQ ID NO: 113 and the light chain variable regions as set forth in SEQ ID NO: 114.

[0210] In some embodiments, the CD 16a binding agent binds to CD16a-F and CD16a-V, but not binds to CD16b-NAl or CD16b-NA2. In further embodiments, the CD 16a binding agent comprises six CDRs of the antibody designated AvGn-CD16a-#2-2A2. In yet further embodiments, the CD 16a binding agent comprises six CDRs as listed in one column of Table4. In some embodiments, the CD 16a binding agent comprises three CDRs of the heavy chain variable region as set forth in SEQ ID NO: 151 and three CDRs of the light chain variable regions as set forth in SEQ ID NO: 152. In some embodiments, the CD16a binding agent comprises the heavy chain variable region as set forth in SEQ ID NO: 151 and the light chain variable regions as set forth in SEQ ID NO: 152.

[0211] In some embodiments, the CD 16a binding agent binds to CD16a-F and CD16a-V, but not binds to CD16b-NAl or CD16b-NA2. In further embodiments, the CD 16a binding agent comprises six CDRs of the antibody designated AvGn-CD16a-#2-3A2. In yet further embodiments, the CD 16a binding agent comprises six CDRs as listed in one column of Table5. In some embodiments, the CD 16a binding agent comprises three CDRs of the heavy chainvariable region as set forth in SEQ ID NO: 189 and three CDRs of the light chain variable regions as set forth in SEQ ID NO: 190. In some embodiments, the CD16a binding agent comprises the heavy chain variable region as set forth in SEQ ID NO: 189 and the light chain variable regions as set forth in SEQ ID NO: 190.

[0212] In some embodiments, the CD 16a binding agent binds to CD16a-F and CD16a-V, but not binds to CD16b-NAl or CD16b-NA2. In further embodiments, the CD 16a binding agent comprises six CDRs of the antibody designated AvGn-CD16a-#3A2. In yet further embodiments, the CD 16a binding agent comprises six CDRs as listed in one column of Table6. In some embodiments, the CD 16a binding agent comprises three CDRs of the heavy chain variable region as set forth in SEQ ID NO:227 and three CDRs of the light chain variable regions as set forth in SEQ ID NO:228. In some embodiments, the CD16a binding agent comprises the heavy chain variable region as set forth in SEQ ID NO:227 and the light chain variable regions as set forth in SEQ ID NO:228.

[0213] In some embodiments, the CD 16a binding agent binds to CD16a-F and CD16a-V, but not binds to CD16b-NAl or CD16b-NA2. In further embodiments, the CD 16a binding agent comprises six CDRs of the antibody designated AvGn-CD16a-#6A2. In yet further embodiments, the CD 16a binding agent comprises six CDRs as listed in one column of Table7. In some embodiments, the CD 16a binding agent comprises three CDRs of the heavy chain variable region as set forth in SEQ ID NO:265 and three CDRs of the light chain variable regions as set forth in SEQ ID NO:266. In some embodiments, the CD16a binding agent comprises the heavy chain variable region as set forth in SEQ ID NO:265 and the light chain variable regions as set forth in SEQ ID NO:266.

[0214] In some embodiments, the CD 16a binding agent binds to CD16a-F and CD16a-V, but not binds to CD16b-NAl or CD16b-NA2. In further embodiments, the CD 16a binding agent comprises six CDRs of the antibody designated AvGn-CD16a-#10A2. In yet further embodiments, the CD 16a binding agent comprises six CDRs as listed in one column of Table8. In some embodiments, the CD 16a binding agent comprises three CDRs of the heavy chain variable region as set forth in SEQ ID NO: 303 and three CDRs of the light chain variable regions as set forth in SEQ ID NO:304. In some embodiments, the CD16a binding agent comprises the heavy chain variable region as set forth in SEQ ID NO:303 and the light chain variable regions as set forth in SEQ ID NO:304.

[0215] In some embodiments, the CD 16a binding agent binds to CD16a-F and CD16a-V, but not binds to CD16b-NAl or CD16b-NA2. In further embodiments, the CD 16a binding agent comprises six CDRs of the antibody designated AvGn-CD16a-#55. In yet furtherembodiments, the CD 16a binding agent comprises six CDRs as listed in one column of Table 9. In some embodiments, the CD 16a binding agent comprises three CDRs of the heavy chain variable region as set forth in SEQ ID NO:341 and three CDRs of the light chain variable regions as set forth in SEQ ID NO:342. In some embodiments, the CD16a binding agent comprises the heavy chain variable region as set forth in SEQ ID NO:341 and the light chain variable regions as set forth in SEQ ID NO:342.

[0216] In some embodiments, the CD 16a binding agent provided herein is in a scFv format. In some embodiments, the CD 16a binding agent comprises a scFv having a combination of (i) a VH described herein and (ii) a VL described herein. In some embodiments, the CD 16a binding agent comprises an amino acid sequence of SEQ ID NO: 346.

[0217] In some embodiments, the CD16a binding agent provided herein is in a Fab format. In some embodiments, CD 16a binding agents (e.g., antibodies such as monospecific or bispecific antibodies), including human CD 16a binding agents, described herein comprise a heavy chain having a combination of (i) a VH described herein, such as in any one of Tables 1-9; and (ii) a heavy chain constant domain (e.g., CHI). In further embodiments, the carboxyl terminus (C terminus) of the VH is conjugated directly or indirectly to the amino terminus (N terminus) of the heavy chain constant domain (e.g., CHI). In some embodiments, CD 16a binding agents (e.g., antibodies such as monospecific or bispecific antibodies), including human CD 16a binding agents, described herein comprise a light chain having a combination of (i) a VL domain described herein, such as in any one of Tables 1-9; and (ii) a light chain constant domain (CL). In further embodiments, the C terminus of the VL is conjugated directly or indirectly to the N terminus of the CL. In some embodiments, CD 16a binding agents (e.g., antibodies such as monospecific or bispecific antibodies), including human CD 16a binding agents, described herein comprise (a) a heavy chain having a combination of (i) a VH described herein, such as in any one of Tables 1-9, and (ii) a heavy chain constant domain (e.g., CHI); and (b) a light chain having a combination of (i) a VL described herein, such as in any one of Tables 1-9, and (ii) a light chain constant domain (CL).

[0218] In some embodiments, the binding agents are superior developability based on a known assay in the art, for example, various chromatographic methods, including size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and standup monolayer adsorption chromatography (SMAC). In some embodiments, the bindingagents are superior developability based on measurement of monomer percentage, solubility, and / or antibody aggregation or precipitation.

[0219] In some embodiments, CD 16a binding agents (e.g., antibodies such as monospecific or bispecific antibodies), including human CD16a binding agents, described herein comprise a heavy chain having a combination of (i) a VH described herein, such as in any one of Tables 1-9; and (ii) one or more heavy chain constant domains (e.g., CHI, Hinge, CH2, and CH3). In further embodiments, the carboxyl terminus (C terminus) of the VH is conjugated directly or indirectly to the amino terminus (N terminus) of the one or more heavy chain constant domains.

[0220] In some embodiments, CD 16a binding agents (e.g., antibodies such as monospecific or bispecific antibodies), including human CD16a binding agents, described herein comprise a light chain having a combination of (i) a VL domain described herein, such as in any one of Tables 1-9; and (ii) a light chain constant domain (CL). In further embodiments, the C terminus of the VL is conjugated directly or indirectly to the N terminus of the CL.

[0221] In some embodiments, CD 16a binding agents (e.g., antibodies such as monospecific or bispecific antibodies), including human CD16a binding agents, described herein comprise (a) a heavy chain having a combination of (i) a VH described herein, such as in any one of Tables 1-9, and (ii) one or more heavy chain constant domains (e.g., CHI, Hinge, CH2, and CH3); and (b) a light chain having a combination of (i) a VL described herein, such as in any one of Tables 1-9, and (ii) a light chain constant domain in an IgG format (CL or CL1).

[0222] In some embodiments, the CD 16a binding agent provided herein is in an IgG format. In some embodiments, the CD 16a binding agent provided herein is in an IgGl format, the CD 16a binding agent provided herein is in an IgG4 format In some embodiments, the CD16a binding agent provided herein is in an IgG format with mutated Fcyl region to eliminate Fc binding activity to Fey receptors.

[0223] In some embodiments, the CD 16a binding agent provided herein is in an IgG format comprising Leu234Ala, Leu235Ala and / or Pro329Gly mutations (Fc silencing mutations) to remove the Fc binding site for CD 16a. In some embodiments, the CD 16a binding properties of the CD 16a binding agents are not significantly impacted by the Fc silencing mutations (e.g. Leu234Ala, Leu235Ala and / or Pro329Gly). In some embodiments, the CD 16a binding agent the Fc silencing mutations provided herein binds to CD 16a e.g.,human CD 16a) with a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10'8M or less, e.g. from 10'8M to 10'13M, e.g., from 10'9M to 10'13M).

[0224] In some embodiments, the CD 16a binding properties of the CD 16a binding agents are not significantly impacted by the presence of human IgG.

[0225] In some embodiments, provided herein is an CD 16a binding protein comprising any one of the anti-CD16a antibodies described herein. In some embodiments, the CD 16a binding protein is an antibody comprising two heavy chains and two light chains. In some embodiments, the CD 16a binding protein is an antibody comprising two heavy chains comprising a same VH region and two light chains comprising a same VL region.

[0226] In some embodiments, the CD 16a binding protein provided herein is a monoclonal antibody, including a mouse, chimeric, humanized, or human antibody. In some embodiments, the anti-CD16a antibody is an antibody fragment, e.g., an scFv. In some embodiments, the CD 16a binding protein is a fusion protein comprising the anti-CD16a antibody provided herein. In other embodiments, the CD 16a binding protein is a multispecific antibody comprising the anti-CD16a antibody or fragment thereof provided herein.

[0227] In some embodiments, the CD 16a binding agent provided herein is a monoclonal antibody, including a mouse, chimeric, humanized, or human antibody. In some embodiments, the anti-CD16a antibody is an antibody fragment, e.g., an scFv. In some embodiments, the CD 16a binding agent is a fusion protein comprising the anti-CD16a antibody provided herein. In other embodiments, the CD 16a binding agent is a multispecific antibody comprising the anti-CD16a antibody or fragment thereof provided herein. In some embodiments, the CD 16a binding agent described herein is a multispecific antibody comprising a first binding domain comprising any one of the anti-CD16a antibodies described herein, and a second binding domain that binds to a second antigen. In some embodiments, the second antigen is expressed on a tumor cell. In some embodiments, the second antigen is CD 19.

[0228] In some embodiments, the CD 16a binding agents (e.g. antibodies) can activateCD 16a mediated cell signaling pathway. In some embodiments, the antibodies described herein activate CD 16a mediated cell signaling pathway by at least about 10%. In some embodiments, the antibodies described herein activate CD 16a mediated cell signaling pathway by at least about 20%. In some embodiments, the antibodies described herein activate CD 16a mediated cell signaling pathway by at least about 30%. In some embodiments, the antibodies described herein activate CD 16a mediated cell signalingpathway by at least about 40%. In some embodiments, the antibodies described herein activate CD 16a mediated cell signaling pathway by at least about 50%. In some embodiments, the antibodies described herein activate CD 16a mediated cell signaling pathway by at least about 60%. In some embodiments, the antibodies described herein activate CD 16a mediated cell signaling pathway by at least about 70%. In some embodiments, the antibodies described herein activate CD 16a mediated cell signaling pathway by at least about 80%. In some embodiments, the antibodies described herein activate CD 16a mediated cell signaling pathway by at least about 90%. In some embodiments, the antibodies described herein activate CD 16a mediated cell signaling pathway by at least about 95%. In certain embodiments, the antibodies described herein activate CD 16a mediated cell signaling pathway by at least about 15% to about 65%. In certain embodiments, the antibodies described herein activate CD 16a mediated cell signaling pathway by at least about 20% to about 65%. In certain embodiments, the antibodies described herein activate CD 16a mediated cell signaling pathway by at least about 30% to about 65%. In some embodiments, the CD 16a binding agent does not activate CD 16b mediated cell signaling pathway.

[0229] In some embodiments, the CD 16a binding agents (e.g. antibodies) can modulate NK cell activity. In some embodiments, the antibodies described herein can activate an NK cell. In some embodiments, the antibodies described herein activate an NK cell activity by at least about 10%. In some embodiments, the antibodies described herein activate an NK cell activity by at least about 20%. In some embodiments, the antibodies described herein activate an NK cell activity by at least about 30%. In some embodiments, the antibodies described herein activate an NK cell activity by at least about 40%. In some embodiments, the antibodies described herein activate an NK cell activity by at least about 50%. In some embodiments, the antibodies described herein activate an NK cell activity by at least about 60%. In some embodiments, the antibodies described herein activate an NK cell activity by at least about 70%. In some embodiments, the antibodies described herein activate an NK cell activity by at least about 80%. In some embodiments, the antibodies described herein activate an NK cell activity by at least about 90%. In some embodiments, the antibodies described herein activate an NK cell activity by at least about 95%. In certain embodiments, the antibodies described herein activate an NK cell activity by at least about 15% to about 65%. In certain embodiments, the antibodies described herein activate an NK cell activity by at least about 20% to about 65%. In certain embodiments, the antibodies described herein activate an NK cell activity by at least about 30% to about 65%. In specific embodiments, theNK cells are human NK cells. In specific embodiments, the NK cells express CD16a-F. In specific embodiments, the NK cells express CD16a-V. In specific embodiments, the NK cells are NK cells from a subject with a CD16a-F / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD16a-V / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD 16a- V / V genotype.

[0230] In some embodiments, the CD 16a binding agents (e.g. antibodies) described herein can induce ADCC activity of an NK cell. In some embodiments, the antibodies described herein induce ADCC activity of an NK cell by at least about 10%. In some embodiments, the antibodies described herein induce ADCC activity of an NK cell by at least about 20%. In some embodiments, the antibodies described herein induce ADCC activity of an NK cell by at least about 30%. In some embodiments, the antibodies described herein induce ADCC activity of an NK cell by at least about 40%. In some embodiments, the antibodies described herein induce ADCC activity of an NK cell by at least about 50%. In some embodiments, the antibodies described herein induce ADCC activity of an NK cell at least about 60%. In some embodiments, the antibodies described herein induce ADCC activity of an NK cell by at least about 70%. In some embodiments, the antibodies described herein induce ADCC activity of an NK cell by at least about 80%. In some embodiments, the antibodies described herein induce ADCC activity of an NK cell by at least about 90%. In some embodiments, the antibodies described herein induce ADCC activity of an NK cell by at least about 95%. In certain embodiments, the antibodies described herein induce ADCC activity of an NK cell by at least about 15% to about 65%. In certain embodiments, the antibodies described herein induce ADCC activity of an NK cell by at least about 20% to about 65%. In certain embodiments, the antibodies described herein induce ADCC activity of an NK cell by at least about 30% to about 65%. In specific embodiments, the NK cells are human NK cells. In specific embodiments, the NK cells express CD16a-F. In specific embodiments, the NK cells express CD16a-V. In specific embodiments, the NK cells are NK cells from a subject with a CD 16a F / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD16a-V / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD 16a- V / V genotype.

[0231] In some embodiments, the CD 16a binding agents (e.g. antibodies) described herein can induce cytokine secretion of an NK cell. In some embodiments, the antibodies described herein induce cytokine secretion of an NK cell by at least about 10%. In some embodiments, the antibodies described herein induce cytokine secretion of an NK cell by at least about 20%. In some embodiments, the antibodies described herein induce cytokinesecretion of an NK cell by at least about 30%. In some embodiments, the antibodies described herein induce cytokine secretion of an NK cell by at least about 40%. In some embodiments, the antibodies described herein induce cytokine secretion of an NK cell by at least about 50%. In some embodiments, the antibodies described herein induce cytokine secretion of an NK cell at least about 60%. In some embodiments, the antibodies described herein induce cytokine secretion of an NK cell by at least about 70%. In some embodiments, the antibodies described herein induce cytokine secretion of an NK cell by at least about 80%. In some embodiments, the antibodies described herein induce cytokine secretion of an NK cell by at least about 90%. In some embodiments, the antibodies described herein induce cytokine secretion of an NK cell by at least about 95%. In certain embodiments, the antibodies described herein induce cytokine secretion of an NK cell by at least about 15% to about 65%. In certain embodiments, the antibodies described herein induce cytokine secretion of an NK cell by at least about 20% to about 65%. In certain embodiments, the antibodies described herein induce cytokine secretion of an NK cell by at least about 30% to about 65%. In specific embodiments, the NK cells are human NK cells. In specific embodiments, the NK cells express CD16a-F. In specific embodiments, the NK cells express CD16a-V. In specific embodiments, the NK cells are NK cells from a subject with a CD 16a- F / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD16a-V / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD 16a- V / V genotype.

[0232] In some embodiments, the CD 16a binding agents (e.g. antibodies) in an IgG format described herein can activate NK cell activity even with one or more Fc silencing mutations (e.g. Leu234Ala, Leu235Ala and / or Pro329Gly). In some embodiments, the antibodies in an IgG format described herein can activate NK cell activity even in an Fc silenced format with Leu234Ala, Leu235Ala and / or Pro329Gly mutations. In some embodiments, the antibodies in an IgG format described herein can activate NK cell activity with mutated Fcyl region to eliminate Fc binding activity to Fey receptors.

[0233] In some embodiments, the CD 16a binding agents (e.g. antibodies) described herein do not bind to cells expressing CD16b. In some embodiments, the antibodies described herein do not bind to cells expressing CD16b-NAl. In some embodiments, the antibodies described herein do not bind to cells expressing CD16b-NA2. In some embodiments, the antibodies described herein do not bind to cells derived from a subject with CD16b-NAl / CD16b-NAl genotype. In some embodiments, the antibodies described hereindo not bind to cells derived from a subject with CD16b-NAl / CD16b-NA2 genotype. In some embodiments, the antibodies described herein do not bind to cells derived from a subject with CD16b-NA2 / CD16b-NA2 genotype. In some embodiments, the antibodies described herein do not bind to neutrophils. In some embodiments, the antibodies described herein do not bind to neutrophils derived from a subject with CD16b-NAl / CD16b-NAl genotype. In some embodiments, the antibodies described herein do not bind to neutrophils derived from a subject with CD16b-NAl / CD16b-NA2 genotype. In some embodiments, the antibodies described herein do not bind to neutrophils derived from a subject with CD16b-NA2 / CD16b- NA2 genotype. In some embodiments, the antibodies described herein do not induce activation of neutrophils. In some embodiments, the antibodies described herein induce activation of NK cells and do not induce activation of neutrophils. In some embodiments, the antibodies described herein do not induce activation of neutrophils and other granulocytes. In some embodiments, the antibodies described herein induce activation of NK cells and do not induce activation of neutrophils and other granulocytes.

[0234] Other exemplary CD 16a binding molecules are described in more detail in the following sections. In some embodiments, the anti-CD16a antibody or antigen-binding protein according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 5.2.4 to 5.2.7 below.Table 1: AvGn-CD16a-A272NAI-1540022551V1Table 2: AvGn-CD16a-#lA273NAI-1540022551V1Table 3: AvGn-CD16a-#2-lA274NAI-1540022551V1Table 4: AvGn-CD16a-#2-2A275NAI-1540022551V1Table 5: AvGn-CD16a-#2-3A276NAI-1540022551V1Table 6: AvGn-CD16a-#3A277NAI-1540022551V1Table 7: AvGn-CD16a-#6A278NAI-1540022551V1Table 8: AvGn-CD16a-#10A279NAI-1540022551V1Table 9: AvGn-CD16a-#5580NAI-1540022551V15.2.2. CD19 Binding Domains

[0235] Also provided herein is a binding agent that binds to CD 19. In some embodiments, the binding agent comprises one or more CD 19 binding domains. In some embodiments, the multispecific binding agents provided herein further comprise one or more CD 19 binding domains. In some embodiments, described herein are multispecific binding agents (e.g., bispecific antibodies) that bind to CD 19. As used herein, CD 19 refers to a CD 19 polypeptide, a CD 19 polypeptide fragment, a CD 19 peptide or a CD 19 epitope. In some embodiments, the CD 19 binding domains in the present binding agents, such as multispecific binding agents, are derived from human or humanized antibodies (e.g., comprising human framework regions) that bind CD 19, including a CD 19 polypeptide, a CD 19 polypeptide fragment, a CD 19 peptide or a CD 19 epitope. In some embodiments, the binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) can bind to CD 19 expressed on the surface of a mammalian (e.g., human) cell, including a CD 19 expressing cancer or tumor cell. In some embodiments, the binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) provided herein binds a CD 19 extracellular epitope exposed on a cell such as a cancer or tumor cell. In some embodiments, described herein is a binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) that binds to CD 19, such as human CD 19 or portions thereof. In some embodiments, CD 19 is a human CD 19. In some embodiments, the binding agent (such as an antibody, a multispecific antibody, or antibody fragment) provided herein is a human CD 19 binding agent (e.g., an antibody that binds to human CD 19). In some embodiments, the binding agent provided herein binds to both human CD 19 and cyno CD 19. In other embodiments, the binding agent provided herein binds to human CD 19 but not cyno CD 19.

[0236] In some embodiments, the binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) provided herein binds to CD 19 (e.g., human CD 19) with a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10'8M or less, e.g. from 10'8M to 10'13M, e.g., from 10'9M to 10'13M). A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure, including those described herein, e.g., in Section 5.2.1 above.

[0237] In some embodiments, the binding agent provided herein is obtained from Drug Bank accession number DB09052. In some embodiments, the binding agents (e.g., an antibody, a multispecific antibody, or antibody fragment) described herein comprise a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VLCDR3 of any anti-CD19 antibody, such as an amino acid sequence of a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 depicted in Table 10. Accordingly, in some embodiments, a binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from the antibody as shown in Table 10. In some embodiments, a binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) described herein comprises any one, any two, and / or all three heavy chain CDRs and any one, any two, and / or all three light chain CDRs from the antibody as shown in Table 10.

[0238] In some embodiments, a binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) comprises a VH region, which comprises a VH CDR1, a VH CDR2, and / or a VH CDR3, and / or a VL region, which comprises a VL CDR1, a VL CDR2, and / or a VL CDR3, of any one of the binding agents described herein see, e.g., Table 10). Accordingly, in some embodiments, a binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from Table 10.

[0239] In some embodiments, the binding agent provided herein (e.g., an antibody, a multispecific antibody, or antibody fragment) comprises (i) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:343 and / or (ii) a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, the binding agent provided herein (e.g., an antibody, a multispecific antibody, or antibody fragment) comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:343 and / or a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344. CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDR sequences are determined according to a combination of any two or more of the above-mentioned numbering systems, for example, a combination of Kabat and Chothia. Various exemplary CDR numbering systems are described and illustrated above in Section 5.1.

[0240] In some embodiments, the CD 19 binding domain in the binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) provided herein further comprises one or more framework regions of SEQ ID NOs:343 and 344. In some embodiments, the CD19 binding domain further comprises a framework 1 (FR1), a framework 2 (FR2), a framework 3 (FR3) and / or a framework 4 (FR4) sequence as set forth in any one of SEQ ID NOs:343 and 344. In some embodiments, the CD19 binding domain provided herein is derived from a humanized antibody. Framework regions described herein are determined based upon the boundaries of the CDR numbering system as described in Section 5.2.1 above.

[0241] In some embodiments, the CD 19 binding domains described herein comprise a VH region or VH domain. Additionally or alternatively, in some embodiments, the CD 19 binding domains described herein comprise a VL region or VL domain. In some embodiments, the CD 19 binding domains described herein have a combination of (i) a VH domain or VH region; and (ii) a VL domain or VL region.

[0242] In some embodiments, the binding agent provided herein (e.g., an antibody, a multispecific antibody, or antibody fragment) comprises a VH comprising the amino acid sequence of SEQ ID NO:343. In some embodiments, the binding agent provided herein (e.g., an antibody, a multispecific antibody, or antibody fragment) comprises a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, the binding agent provided herein (e.g., an antibody, a multispecific antibody, or antibody fragment) comprises a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL comprising the amino acid sequence of SEQ ID NO:344.

[0243] In certain embodiments, the binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment ) provided herein comprises amino acid sequences with certain percent identity (such as at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about86%, or at least about 87%, or at least about 88%, or at least about 89%, or as at least about90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about98%, or at least about 99%, or higher) relative to any antibody or fragment thereof provided herein, for example, a CDR, VH or VL in Table 10, or a full-length antibody chain as disclosed herein. In some embodiments, the binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) provided herein comprises CDRs of any antibody or fragment thereof provided herein, for example in Table 10. In further embodiments, thebinding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) provided herein comprises amino acid sequences with certain percent identity (such as at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or as at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or higher) relative to any antibody or fragment thereof provided herein, for example, a VH or VL in Table 10, or a full-length antibody chain as disclosed herein. The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a method as described in Section 5.2.1 above.

[0244] In some embodiments, the binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) provided herein contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) comprising that sequence retains the ability to bind to CD 19. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in a reference amino acid sequence. In some embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs, constant regions, and / or Fc regions).

[0245] In some embodiments, the position of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) region of a CD19 binding domain described herein may vary by one, two, three, four, five, or six amino acid positions so long as binding to CD19 (e.g., human CD19) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, the position defining a CDR of Table 10 may vary by shifting the N-terminal and / or C-terminal boundary of the CDR by one, two, three, four, five, or six amino acids, relative to the current CDR position, so long as binding to CD19 (e.g., human CD19) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).Additionally or alternatively, in some embodiments, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) region of a CD 19 binding domain described herein may vary (e.g., be shorter or longer) by one, two, three, four, five, or more amino acids, so long as binding to CD 19 (e.g., human CD 19) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, or at least 95%). Any method known in the art can be used to ascertain whether binding to CD 19 (e.g, human CD 19) is maintained, for example, the binding assays and conditions described in the “Examples” section described herein.

[0246] In other embodiments, the binding agents (e.g., an antibody, a multispecific antibody, or antibody fragment) further comprise conservative sequence modifications (e.g., in a CD 19 binding domain). Conservative sequence modifications are described in more detail in Section 5.2.1 above. In some embodiments, the conservative sequence modifications described herein modify the amino acid sequences of the binding agents (e.g., an antibody, a multispecific antibody, or antibody fragment), including human CD 19 binding agents, by 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%. In some embodiments, the amino acid sequence modifications refer to at most 1, 2, 3, 4, 5, or 6 amino acid substitutions to the CDRs, such as those described in Table 10. Thus, for example, each such CDR may contain up to 5 conservative amino acid substitutions, for example up to (not more than) 4 conservative amino acid substitutions, for example up to (not more than) 3 conservative amino acid substitutions, for example up to (not more than) 2 conservative amino acid substitutions, or no more than 1 conservative amino acid substitution. In some embodiments, the CD 19 binding domain contains one or more, including six, CDRs having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the CDRs of the antibody disclosed in Table 10.

[0247] In some embodiments, a CD 19 binding domain contains a VH and a VL comprising CDRs identical to those of Table 10. In some embodiments, the amino acid sequence modifications do not include any modification within an SDR. In some embodiments, the amino acid sequence modifications do not include any modification within a CDR (such as CDR1, CDR2, CDR3, or any combination thereof). In further embodiments, the amino acid sequence modifications are in the framework, constant region, and / or Fc region.

[0248] In some embodiments, the binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:343, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:344, and the binding of the binding agent to CD19 (e.g,human CD19) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0249] In some embodiments, functional epitopes can be mapped, e.g., by combinatorial alanine scanning, to identify amino acids in the CD 19 protein that are necessary for interaction with a multispecific binding agent, a CD 19 binding domain thereof, and / or an anti-CD19 antibody provided herein (e.g., in the following paragraphs). In some embodiments, conformational and crystal structure of a multispecific binding agent, a CD 19 binding domain thereof, and / or an anti-CD19 antibody (e.g., provided in the following paragraphs) bound to CD 19 may be employed to identify the epitopes. In some embodiments, the present disclosure provides a binding agent (e.g., an antibody, a multispecific antibody, or antibody fragment) comprising a CD 19 binding domain that specifically binds to the same epitope as any of the multispecific binding agents as disclosed herein, CD 19 binding domains thereof, and / or the anti-CD19 antibodies or fragments thereof provided herein (e.g., provided in the following paragraphs).

[0250] For example, in some embodiments, the CD 19 binding domain provided herein binds to the same epitope as an anti-CD19 antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, the CD19 binding domain provided herein binds to the same epitope as an anti-CD19 antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:343, and a VL comprising the amino acid sequence of SEQ ID NO:344.

[0251] In some embodiments, the binding agent provided herein (e.g., an antibody, a multispecific antibody, or antibody fragment) specifically binds to CD 19 competitively with any one of the anti-CD19 antibodies or fragments thereof described herein.

[0252] In some embodiments, the binding agent provided herein (e.g., an antibody, a multispecific antibody, or antibody fragment) specifically binds to CD 19 competitively with an anti-CD19 antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 343 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, the binding agent provided herein (e.g., an antibody, a multispecific antibody, or antibody fragment) specifically binds to CD 19 competitively with an anti-CD19 antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:343, and a VL comprising the amino acid sequence of SEQ ID NO:344.

[0253] In yet another aspect, provided herein is a binding agent (e.g., an antibody or fragment thereof) that binds to CD 19 comprising one or more CDR(s) described above. In some embodiments, provided herein is a binding agent e.g., an antibody or fragment thereof) that binds to CD 19 comprising the VH and / or VL described above. In some embodiments, the binding agent comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344.

[0254] In some embodiments, the binding agent provided herein comprises a VH region comprising the amino acid sequence of SEQ ID NO:343 and a VL region comprising the amino acid sequence of SEQ ID NO:344.

[0255] In some embodiments, the CD 19 binding agent comprises a scFv having a combination of (i) a VH described herein and (ii) a VL described herein. In some embodiments, the CD19 binding agent comprises an amino acid sequence of SEQ ID NO: 345 obtained from Drug Bank accession number DB09052.Table 105.2.3. Multispecific Antibodies

[0256] Provided herein is a multispecific binding agent that binds to CD 16a and a nonCD 16a antigen (such as a tumor antigen). In some embodiments, the multispecific binding agent comprises one or more CD 16a binding domains and one or more non-CD16a antigen binding domains (such as one or more tumor antigen binding domains). In certain embodiments, the present multispecific binding agent is a multispecific antibody comprising one or more CD 16a binding domains each independently selected from the CD 16a binding domains described in Section 5.2.1 above and one or more tumor antigen binding domain. In some embodiments, the tumor antigen is CD 19. In some embodiments, the tumor antigen isBCMA. In certain embodiments, the present multispecific binding agent is a multispecific antibody comprising one or more CD 16a binding domains each independently selected from the CD16a binding domains described in Section 5.2.1 above and one or more CD19 binding domains each independently selected from the CD 19 binding domains described in Section 5.2.2 above.

[0257] In certain embodiments, the present multispecific binding agent is a multispecific antibody comprising a first binding domain that binds to CD 16a and a second binding domain that binds to a second antigen. In some embodiments, the second antigen is a tumor antigen. In some embodiments, the second antigen is CD 19.

[0258] In some embodiments of the multispecific antibody provided herein, the first binding domain that binds to CD 16a comprises any antibody or antigen binding fragment described in Section 5.2.1 above. In some embodiments, the second antigen is a tumor antigen. In some embodiments, the second antigen is CD 19. In some embodiments of the multispecific antibody provided herein, the second binding domain that binds to CD 19 comprises any antibody or antigen binding fragment described in Section 5.2.2 above.

[0259] In some embodiments of the multispecific antibody provided herein, the first binding domain comprises six CDRs of any anti-CD16a antibody or antigen binding fragment described in Section 5.2.1 above. In some embodiments, the second antigen is a tumor antigen. In some embodiments, the second antigen is CD 19. In some embodiments of the multispecific antibody provided herein, the second binding domain comprises six CDRs of any anti-CD19 antibody or antigen binding fragment described in Section 5.2.2 above.

[0260] In some embodiments of the multispecific antibody provided herein, the first binding domain comprises a VH and a VL of any anti-CD16a antibody or antigen binding fragment described in Section 5.2.1 above. In some embodiments, the second antigen is a tumor antigen. In some embodiments, the second antigen is CD 19. In some embodiments of the multispecific antibody provided herein, the second binding domain comprises a VH and a VL of any anti-CD19 antibody or antigen binding fragments as described in Section 5.2.2 above.

[0261] In some embodiments of the multispecific antibody provided herein, the first binding domain comprises six CDRs of any anti-CD16a antibody or antigen binding fragment described in Tables 1-9. In some embodiments, the second antigen is a tumor antigen. In some embodiments, the second antigen is CD 19. In some embodiments of the multispecific antibody provided herein, the second binding domain comprises six CDRs of any anti-CD19 antibody or antigen binding fragment described in Table 10.

[0262] In some embodiments of the multispecific antibody provided herein, the first binding domain comprises a VH and a VL of any anti-CD16a antibody or antigen binding fragment described in Tables 1-9. In some embodiments, the second antigen is a tumor antigen. In some embodiments, the second antigen is CD 19. In some embodiments of the multispecific antibody provided herein, the second binding domain comprises a VH and a VL of any anti-CD19 antibody or antigen binding fragments as described in Table 10.

[0263] In some specific embodiments, provided herein is a multispecific binding agent comprising one or more CD 16a binding domains each independently selected from CD 16a binding domains comprising: (i) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:37 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:38; (ii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 75 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:76; (iii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 113 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 114; (iv) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 151 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 152; (v) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 190; (vi) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:227 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:228; (vii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:265 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:266; (viii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:303 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:304; or (ix) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:341 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:342; and one or more CD 19 binding domains comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth ina VH comprising the amino acid sequence of SEQ ID NO:343 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344.

[0264] In some embodiments, provided herein is a multispecific binding agent comprising one or more CD 16a binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:37 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:38; and one or more CD19 binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19).

[0265] In some embodiments, provided herein is a multispecific binding agent comprising one or more CD 16a binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:75 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:76; and one or more CD19 binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with twoidentical pairs of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19).

[0266] In some specific embodiments, provided herein is a multispecific binding agent comprising one or more CD 16a binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 113 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 114; and one or more CD19 binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19).

[0267] In some specific embodiments, provided herein is a multispecific binding agent comprising one or more CD 16a binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 151 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 152; and one or more CD19 binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acidsequence of SEQ ID NO:343 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19).

[0268] In some specific embodiments, provided herein is a multispecific binding agent comprising one or more CD 16a binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 190; and one or more CD19 binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19).

[0269] In some specific embodiments, provided herein is a multispecific binding agent comprising one or more CD 16a binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:227 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 228; and one or more CD19 binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19).

[0270] In some specific embodiments, provided herein is a multispecific binding agent comprising one or more CD 16a binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:265 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:266; and one or more CD19 binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, themultispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19).

[0271] In some specific embodiments, provided herein is a multispecific binding agent comprising one or more CD 16a binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:303 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:304; and one or more CD19 binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19).

[0272] In some specific embodiments, provided herein is a multispecific binding agent comprising one or more CD 16a binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:341 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:342; and one or more CD19 binding domains, each comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, themultispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and one CD 19 binding domain (e.g., with a pair of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 16a) and two CD 19 binding domains (e.g., with two identical pairs of VH and VL regions that bind to CD 19).

[0273] In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH comprising the amino acid sequence of SEQ ID NO:37 and a VL comprising the amino acid sequence of SEQ ID NO:38; and a second binding domain that binds to CD19 comprising a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH having at least 90% sequence identity to SEQ ID NO: 37 and a VL having at least 90% sequence identity to SEQ ID NO:38; and a second binding domain that binds to CD19 comprising a VH having at least 90% sequence identity to SEQ ID NO:343 and a VL having at least 90% sequence identity to SEQ ID NO:344.

[0274] In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH comprising the amino acid sequence of SEQ ID NO:75 and a VL comprising the amino acid sequence of SEQ ID NO:76; and a second binding domain that binds to CD19 comprising a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH having at least 90% sequence identity to SEQ ID NO: 75 and a VL having at least 90% sequence identity to SEQ ID NO:76; and a second binding domain that binds to CD19 comprising a VH having at least 90% sequence identity to SEQ ID NO:343 and a VL having at least 90% sequence identity to SEQ ID NO:344.

[0275] In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH comprising the amino acid sequence of SEQ ID NO: 113 and a VL comprising the amino acid sequence of SEQ ID NO: 114; and a second binding domain that binds to CD19 comprising a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH having at least 90% sequence identity to SEQ ID NO: 113 and a VL having at least 90% sequence identity to SEQ ID NO:114; and a second binding domain that binds to CD19 comprising a VH having at least 90% sequence identity to SEQ ID NO:343 and a VL having at least 90% sequence identity to SEQ ID NO:344.

[0276] In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH comprising the amino acid sequence of SEQ ID NO: 151 and a VL comprising the amino acid sequence of SEQ ID NO: 152; and a second binding domain that binds to CD19 comprising a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH having at least 90% sequence identity to SEQ ID NO: 151 and a VL having at least 90% sequence identity to SEQ ID NO: 152; and a second binding domain that binds to CD 19 comprising a VH having at least 90% sequence identity to SEQ ID NO:343 and a VL having at least 90% sequence identity to SEQ ID NO:344.

[0277] In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL comprising the amino acid sequence of SEQ ID NO: 190; and a second binding domain that binds to CD19 comprising a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH having at least 90% sequence identity to SEQ ID NO: 189 and a VL having at least 90% sequence identity to SEQ ID NO: 190; and a second binding domain that binds to CD 19 comprising a VH having at least 90% sequence identity to SEQ ID NO:343 and a VL having at least 90% sequence identity to SEQ ID NO:344.

[0278] In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH comprising the amino acid sequence of SEQ ID NO:227 and a VL comprising the amino acid sequence of SEQ ID NO:228; and a second binding domain that binds to CD19 comprising a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH having at least 90% sequence identity to SEQ ID NO: 227 and a VL having at least 90% sequence identity to SEQ ID NO:228; and a second binding domain that binds to CD 19 comprising a VH having at least 90% sequence identity to SEQ ID NO:343 and a VL having at least 90% sequence identity to SEQ ID NO:344.

[0279] In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH comprising the amino acid sequence of SEQ ID NO:265 and a VL comprising the amino acid sequence of SEQ ID NO:266; and a second binding domain that binds to CD19 comprising a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH having at least 90% sequence identity to SEQ ID NO: 265 and a VL having at least 90% sequence identity to SEQ ID NO:266; and a second binding domain that binds to CD 19 comprising a VH having at least 90% sequence identity to SEQ ID NO:343 and a VL having at least 90% sequence identity to SEQ ID NO:344.

[0280] In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH comprising the amino acid sequence of SEQ ID NO:303 and a VL comprising the amino acid sequence of SEQ ID NO:304; and a second binding domain that binds to CD19 comprising a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH having at least 90% sequence identity to SEQ ID NO: 303 and a VL having at least 90% sequence identity to SEQ ID NO:304; and a second binding domain that binds to CD19 comprising a VH having at least 90% sequence identity to SEQ ID NO:343 and a VL having at least 90% sequence identity to SEQ ID NO:344.

[0281] In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH comprising the amino acid sequence of SEQ ID NO:341 and a VL comprising the amino acid sequence of SEQ ID NO:342; and a second binding domain that binds to CD19 comprising a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL comprising the amino acid sequence of SEQ ID NO:344. In some embodiments, provided herein is a multispecific binding agent comprising a first binding domain that binds to CD 16a comprising a VH having at least 90% sequence identity to SEQ ID NO: 341 and a VL having at least 90% sequence identity to SEQ ID NO:342; and a second binding domain that binds to CD 19 comprising a VH having at least 90% sequence identity to SEQ ID NO:343 and a VL having at least 90% sequence identity to SEQ ID NO:344.

[0282] In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domains (e.g., with a pair of VH and VL regions that bind to CD 16a) and one tumor antigen binding domain (e.g., with a pair of VH and VL regions that bind to a tumor antigen). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical or different pairs of VH and VL regions that bind to CD 16a) and one tumor antigen binding domain (e.g., with a pair of VH and VL regions that bind to a tumor antigen). In some embodiments, the multispecific binding agent provided herein comprises one CD 16a binding domain (e.g., with a pair of VH and VL regions that bind to CD 16a) and two tumor antigen binding domains (e.g., with two identical or different pairs of VH and VL regions that bind to one or more tumor antigen). In some embodiments, the multispecific binding agent provided herein comprises two CD 16a binding domains (e.g., with two identical or different pair of VH and VL regions that bind to CD 16a) and two tumor antigen binding domains (e.g, with two identical or different pairs of VH and VL regions that bind to one or more tumor antigen). In some embodiments, the multispecific binding agent further comprises one or more heavy chain constant regions (such as CHI, CH2, and / or CH3) and / or one or more light chain constant regions (such as CL). In some embodiments, the tumor antigen is CD 19.

[0283] In some embodiments, the multispecific binding agent provided herein has an antibody format shown in FIG. 6A. In some embodiments, the multispecific binding agent provided herein has an antibody format shown FIG. 6A, but its CD 19 binding arm is switched with its CD 16a binding arm. In some embodiments, the multispecific binding agent provided herein has an antibody format shown in FIG. 7A. In some embodiments, themultispecific binding agent provided herein has an antibody format shown FIG. 7A, but its CD 19 binding arm is switched with its CD 16a binding arm.

[0284] Although specific exemplary multispecific antibody formats are described in the Example section below and in any of FIG. 6A or FIG. 7A, it is contemplated that any multispecific antibody formats known in the art can be used and are included in the present disclosure.

[0285] Multispecific binding agents (e.g., bispecific antibodies) described herein may be bispecific, trispecific or of greater multispecificity. Such agents may include multispecific antibodies. In some embodiments, multispecific antibodies, such as bispecific antibodies, are monoclonal antibodies that have binding specificities for at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., a bispecific antibody directed to CD 16a with a first binding domain for a first epitope of an CD 16a, and a second binding domain for a second epitope of CD 16a). In some embodiments, the multispecific (e.g., bispecific) antibodies can be constructed based on the sequences of the antibodies described herein, e.g., the CDR sequences listed in Tables 1-9. In some embodiments, the multispecific antibodies described herein are bispecific antibodies. In some embodiments, the multispecific antibody described herein (e.g., a bispecific antibody) has binding specificities for two targets, for example, CD 16a and a tumor antigen (such as CD 19). In some embodiments, bispecific antibodies are mouse, chimeric, human or humanized antibodies.

[0286] In some embodiments, one of the binding specificities of the multispecific antibody provided herein is for CD 16a, a second binding specificity of the multispecific antibody provided herein is a tumor antigen (such as CD 19), and yet one or more additional binding specificities are for any other target(s) (e.g., antigen). In some embodiments, a multispecific antibody can comprise more than one target (e.g., antigen) binding domain, in which different binding domains are specific for different targets. In some embodiments, the additional target is an immune checkpoint regulator (e.g., a negative checkpoint regulator). In some embodiments, the additional target is expressed on an immune cell. In some embodiments, the additional target is expressed on a tumor or cancer cell.

[0287] In some embodiments, multispecific (e.g, bispecific) antibody molecules can bind more than one (e.g, two or more) epitopes on the same target (e.g., antigen). In some embodiments, a multispecific (e.g., bispecific) binder as disclosed herein can bind to one or more epitopes on a first target (such as CD 16a) and one or more epitopes on a second target (such as a tumor antigen).

[0288] Methods for making multispecific antibodies are known in the art, such as, by coexpression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For further details of generating multispecific antibodies (e.g., bispecific antibodies), see, for example, Bispecific Antibodies (Kontermann ed., 2011).

[0289] Exemplary structures of multispecific antibodies are known in the art and are further described in Weidle et al., 2013, Cancer Genomics & Proteomics 10: 1-18; Brinkman et al., 2017, MABS, 9:2, 182-212; Godar etal., 2018, Expert Opinion on Therapeutic Patents, 28:3, 251-276; and Spiess et al., 2015, Mol. Immunol. 67 95-106.

[0290] For example, bispecific antibody molecules can be classified into different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG appended with an additional antigen-binding moiety; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates. As a non-limiting example, BsIgG formats can include crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in- holes common LC, knobs-in-holes assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, rA-body, and / or orthogonal Fab.

[0291] In some embodiments, BsIgG comprises heavy chains that are engineered for heterodimerization. For example, heavy chains can be engineered for heterodimerization using a “knobs-into-holes” strategy, a SEED platform, a common heavy chain (e.g., in rA- bodies), and use of heterodimeric Fc regions. Strategies are known in the art to avoid heavy chain pairing of homodimers in BsIgG, including knobs-into-holes, duobody, azymetric, charge pair, HA-TF, SEEDbody, and differential protein A affinity.

[0292] Another bispecific antibody format is IgG appended with an additional antigenbinding moiety. For example, monospecific IgG can be engineered to have bispecificity by appending an additional antigen-binding unit onto the monospecific IgG, e.g., at the N- or C- terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g, variable heavy chain or variable light chain), engineered protein scaffolds, and paired antibody variable domains (e.g, single chain variable fragments or variable fragments). Non-limiting examples of appended IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv- (L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv- IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four- in-one). See Spiess et al. Mol. Immunol. 67(2015):95-106. In some embodiments, an exemplary antibody format is a B- Body format for monospecific or multispecific (e.g., bispecific antibodies) as described ine.g. WO 2018 / 075692, U.S. Pat. Publ. No. 2018 / 0118811, and U.S. Pat. Publ. No. 2021 / 0155692.

[0293] Bispecific (Bs) antibody (BsAb) fragments are a format of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some BsAb lack an Fc region. In embodiments, bispecific antibody fragments include heavy and light chain regions that are connected by a peptide linker that permits efficient expression of the BsAb in a single host cell. Non-limiting examples of bispecific antibody fragments include, but are not limited to, nanobody, nanobody -HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HC Ab, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, and intrabody.

[0294] Bispecific fusion proteins include antibody fragments linked to other proteins. For example, bispecific fusion proteins can be linked to other proteins to add additional specificity and / or functionality. In some embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with higher valency. For example, bispecific antibody fusions to albumin binding proteins or human serum albumin can be constructed to extend the serum half-life of antibody fragments. In some embodiments, chemical conjugation, e.g., chemical conjugation of antibodies and / or antibody fragments, can be used to create BsAb molecules. An exemplary bispecific antibody conjugate includes the CovX-body format, in which a low molecular weight drug is conjugated site-specifically to a single reactive lysine in each Fab arm or an antibody or fragment thereof. In some embodiments, the conjugation improves the serum half-life.

[0295] Methods of production of multispecific antibodies, including bispecific antibodies, are known in the art. For example, multispecific antibodies, including bispecific antibodies, can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly or by expression of the component antibodies in a single host cell. Purification of multispecific (e.g., bispecific) antibody molecules can be performed by various methods known in the art, including affinity chromatography.

[0296] In some embodiments, multispecific binding agents (e.g., bispecific antibodies) disclosed herein can be provided in any antibody format disclosed herein or known in the art. As a non-limiting example, in some embodiments, the multispecific binding agents (e.g., bispecific antibodies) can be selected from Fabs-in-tandem-lg (FIT-lg); DVD-lg; hybrid hybridoma (quadroma or tetradoma); anticalin platform (Pieris); diabodies; single chain diabodies; tandem single chain Fv fragments; TandAbs, Trispecific Abs (Affimed); Dartsdual affinity retargeting (Macrogenics); Bispecific Xmabs (Xencor); Bispecific T cell engagers (Bites; Amgen; 55kDa); Triplebodies; Tribody = Fab-scFv Fusion Protein multifunctional recombinant antibody derivates (CreativeBiolabs); Duobody platform (Genmab); dock and lock platform; knobs-into-holes (KIH) platform; Humanized bispecific IgG antibody (REGN1979) (Regeneron); Mab2 bispecific antibodies (F-Star); DVD-lg = dual variable domain immunoglobulin (Abbott); kappa-lambda bodies; TBTI = tetravalent bispecific tandem Ig; B-Body; and CrossMab (Roche).

[0297] In some specific embodiments, the bispecific antibody provided herein is in a format depicted in any of FIG. 6A or FIG. 7A. In some embodiments, the bispecific antibody provided herein is in the 1x1 format as depicted in FIG. 6A. In some embodiments, the bispecific antibody provided herein is in the 2x2 format as depicted in FIG. 7A.

[0298] In some embodiments, the multispecific antibody provided herein comprises one polypeptide chain comprising the first binding domain and the second binding domain. In some embodiment, the first binding domain comprises a first scFv that binds to CD 16a, and the second binding domain comprises a second scFv that binds to a tumor antigen (such as CD 19). In some embodiment, the multispecific antibody provided comprises an antibody format of ScFv - ScFv. “ScFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. In some embodiments, the linker comprises amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 358). In some embodiment, the first scFv is linked to the N terminal of the second scFv. In some embodiment, the first scFv is linked to the C terminal of the second scFv. In some embodiments, the scFv-scFv construct comprises amino acid sequence of SEQ ID NO: 357.

[0299] In some specific embodiments, the multispecific antibody provided herein comprises one polypeptide chain from N-terminus to C-terminus: a first VL, a first VH, a second VL, a second VH. In some specific embodiments, the multispecific antibody provided herein comprises one polypeptide from N-terminus to C-terminus: a first VH, a first VL, a second VL, a second VH. In some specific embodiments, the multispecific antibody provided herein comprises one polypeptide from N-terminus to C-terminus: a first VL, a first VH, a second VH, a second VL. In some specific embodiments, the multispecific antibody provided herein comprises one polypeptide from N-terminus to C-terminus: a first VH, a first VL, a second VH, a second VL . In some embodiments, the first VL and the first VH form a first scFv binding domain that binds to CD 16a, and the second VL and the second VH form asecond scFv binding domain that binds to a tumor antigen (such as CD 19). In some embodiments, the first VL and the first VH form a first scFv binding domain that binds to a tumor antigen (such as CD 19), and the second VL and the second VH form a second scFv binding domain that binds to CD 16a. In some embodiments, the first scFv and the second scFv is linked through a polypeptide linker. In some embodiments, the polypeptide linker comprises GGSGGSGGS (SEQ ID NO: 359).

[0300] In some specific embodiments, the polypeptide chain described above further comprises a first CH2 and a first CH3 of an IgG Fc region. In some specific embodiments, the multispecific antibody provided herein comprises two identical polypeptide chains as described herein. In some specific embodiments, the first polypeptide and the second polypeptide form a bivalent format through dimerization of the CH2 and / or CH3 regions (e.g. the first CH2 is paired with the second CH2, and / or the first CH3 is paired with the second CH2). In some specific embodiments, the two polypeptide chains are not identical, wherein amino acid sequences of the first CH2 and the second CH2 are different from each other. In some specific embodiments, the two polypeptide chains are not identical, wherein amino acid sequences of the first CH3 and the second CH3 are different from each other. In some specific embodiments, the two polypeptide chains are not identical, wherein amino acid sequences of the first CH2 and the second CH2, and the first CH3 and the second CH3 are different from each other.

[0301] In some embodiments of the multispecific antibody provided herein, one or more scFvs are linked to an Fc region of an IgG. In some embodiments, one or more scFvs are linked to N terminal of the Fc region of the IgG. In some embodiments, one or more scFvs are linked to C terminal of the Fc region of the IgG. In some embodiments, one or more scFvs are linked to both N terminal and C terminal of the Fc region of the IgG. In some embodiments, at least one scFv binds to CD 16a, and at least one scFv binds to a tumor antigen (such as CD 19). In some embodiments, the IgG is IgG4. In some embodiments, the IgG is IgGl.

[0302] In some specific embodiments, the multispecific antibody provided herein comprises two polypeptides: a first polypeptide comprising from N-terminus to C-terminus: a first scFv, a second scFv, a first CH2, and a first CH3; a second polypeptide comprising from N-terminus to C-terminus: a third scFv, a forth scFv, a second CH2, and a second CH3. In some specific embodiments, the multispecific antibody provided herein comprises two polypeptides: a first polypeptide comprising from N-terminus to C-terminus: a first scFv, a second scFv, a first CH2, and a first CH3; a second polypeptide comprising from N-terminusto C-terminus: a second CH2, a second CH3, a third scFv, and a forth scFv. In some specific embodiments, the multispecific antibody provided herein comprises two polypeptides: a first polypeptide comprising from N-terminus to C-terminus: a first scFv, a second scFv, a first CH2, and a first CH3; a second polypeptide comprising from N-terminus to C-terminus: a third scFv, a second CH2, a second CH3, and a forth scFv. In some specific embodiments, the multispecific antibody provided herein comprises two polypeptides: a first polypeptide comprising from N-terminus to C-terminus: a first CH2, a first CH3, a first scFv, and a second scFv; a second polypeptide comprising from N-terminus to C-terminus: a second CH2, a second CH3, a third scFv, and a forth scFv. In some specific embodiments, the multispecific antibody provided herein comprises two polypeptides: a first polypeptide comprising from N-terminus to C-terminus: a first CH2, a first CH3, a first scFv, and a second scFv; a second polypeptide comprising from N-terminus to C-terminus: a third scFv, a forth scFv, a second CH2, and a second CH3. In some specific embodiments, the multispecific antibody provided herein comprises two polypeptides: a first polypeptide comprising from N-terminus to C-terminus: a first CH2, a first CH3, a first scFv, and a second scFv; a second polypeptide comprising from N-terminus to C-terminus: a third scFv, a second CH2, a second CH3, and a forth scFv. In some specific embodiments, the multispecific antibody provided herein comprises two polypeptides: a first polypeptide comprising from N-terminus to C-terminus: a first scFv, a first CH2, a first CH3, and a second scFv; a second polypeptide comprising from N-terminus to C-terminus: a third scFv, a second CH2, a second CH3, and a forth scFv. In some specific embodiments, the multispecific antibody provided herein comprises two polypeptides: a first polypeptide comprising from N-terminus to C-terminus: a first scFv, a first CH2, a first CH3, and a second scFv; a second polypeptide comprising from N-terminus to C-terminus: a third scFv, a forth scFv, a second CH2, and a second CH3. In some specific embodiments, the multispecific antibody provided herein comprises two polypeptides: a first polypeptide comprising from N-terminus to C-terminus: a first scFv, a first CH2, a first CH3, and a second scFv; a second polypeptide comprising from N-terminus to C-terminus: a second CH2, a second CH3, a third scFv, and a forth scFv. In some embodiments, the scFv domain is in the VL-VH orientation from N terminal to C terminal. In some embodiments the scFv domain is in the VH-VL orientation from N terminal to C terminal. In some embodiments, one scFv binds to CD 16a, three scFvs bind to one or more tumor antigen. In some embodiments, one scFv binds to a tumor antigen, three scFvs bind CD 16a. In some embodiments, two scFvs bind to CD 16a, two scFvs bind to one or more tumor antigen (suchas CD 19). In some embodiments, the first scFv and the second scFv bind to CD 16a, and the third scFv and the forth scFv bind to one or more tumor antigen (such as CD 19). In some embodiments, the first scFv and the third scFv bind to CD 16a, and the second scFv and the forth scFv bind to a tumor antigen (such as CD 19). In some embodiments, the first scFv and the forth scFv bind to CD 16a, and the second scFv and the third scFv bind to one or more tumor antigen (such as CD 19). In some embodiments, the second scFv and the third scFv bind to CD 16a, and the first scFv and the forth scFv bind to one or more tumor antigen (such as CD 19). In some embodiments, the second scFv and the forth scFv bind to CD 16a, and the first scFv and the third scFv bind to one or more tumor antigen (such as CD 19). In some embodiments, the second scFv and the third scFv bind to CD 16a, and the first scFv and the forth scFv bind to one or more tumor antigen (such as CD 19). In some embodiments, the third scFv and the forth scFv bind to CD 16a, and the first scFv and the second scFv bind to one or more tumor antigen (such as CD 19). In some specific embodiments, amino acid sequences of the first CH2 is paired with the second CH2. In some specific embodiments, amino acid sequences of the first CH3 is paired with the second CH3. In some specific embodiments, amino acid sequences of the first CH2 and the second CH2 are different from each other. In some specific embodiments, amino acid sequences of the first CH3 and the second CH3 are different from each other. In some specific embodiments, amino acid sequences of the first CH2 and the second CH2 are identical to each other. In some specific embodiments, amino acid sequences of the first CH3 and the second CH3 are identical to each other.

[0303] In some embodiments, the multispecific binding agent as disclosed herein is in an IgG format. In some embodiments, the multispecific binding agent as disclosed herein is in an IgG format with mutated Fcyl region to eliminate Fc binding activity to Fey receptors. In some embodiments, the IgG is IgG4. In some embodiments, the IgG is IgGl.

[0304] In some embodiments, the multispecific binding agent as disclosed herein is in an IgG format comprising Leu234Ala, Leu235Ala and / or Pro329Gly mutations (Fc silencing mutations) to remove the Fc binding site for CD 16a. In some embodiments, the CD 16a binding properties of the multispecific binding agent as disclosed herein are not significantly impacted by the Fc silencing mutations (e.g. Leu234Ala, Leu235Ala and / or Pro329Gly). In some embodiments, the multispecific binding agent with the Fc silencing mutations provided herein binds to CD 16a (e.g., human CD 16a) with a dissociation constant (KD) of < 1 pM,< 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10'8M or less, e.g. from 10'8M to 10'13M, e.g., from 10'9M to 10'13M).

[0305] In some embodiments, the multispecific binding agent as disclosed herein are modified by one or more insertions, one or more deletions, or one or more substitutions in the amino acid sequence. In further embodiments, these insertions, deletions, or substitutions are not within a CDR. In some embodiments, the modified polypeptide is at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the polypeptide from which it is modified.

[0306] In some embodiments, the multispecific antibody described herein activates an NK cell. In certain embodiments, the multispecific antibody described herein contacts or directs NK cells to a target cell. In certain embodiments, the multispecific antibody described herein contacts or directs NK cells to a target cell, wherein the multispecific antibody comprises a first binding domain that binds to CD 16a on an NK cell and a second binding domain that binds to a tumor antigen (such as CD 19) on a target cell. In specific embodiments, the target cells are tumor cells. In certain embodiments, the multispecific antibody described herein induces NK cell dependent cytotoxicity of the tumor cell, wherein the multispecific antibody comprises a first binding domain that binds to CD 16a on an NK cell and a second binding domain that binds to a tumor antigen (such as CD 19) on a target cell. In specific embodiments, the NK cells are human NK cells. In specific embodiments, the NK cells express CD16a-F. In specific embodiments, the NK cells express CD16a-V. In specific embodiments, the NK cells are NK cells from a subject with a CD16a-F / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD16a-V / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD 16a- V / V genotype.

[0307] In some embodiments, the multispecific antibodies can modulate NK cell activity. In some embodiments, the multispecific antibodies described herein can activate an NK cell. In some embodiments, the multispecific antibodies described herein activate an NK cell activity by at least about 10%. In some embodiments, the multispecific antibodies described herein activate an NK cell activity by at least about 20%. In some embodiments, the multispecific antibodies described herein activate an NK cell activity by at least about 30%. In some embodiments, the multispecific antibodies described herein activate an NK cell activity by at least about 40%. In some embodiments, the multispecific antibodies described herein activate an NK cell activity by at least about 50%. In some embodiments, themultispecific antibodies described herein activate an NK cell activity by at least about 60%. In some embodiments, the multispecific antibodies described herein activate an NK cell activity by at least about 70%. In some embodiments, the multispecific antibodies described herein activate an NK cell activity by at least about 80%. In some embodiments, the multispecific antibodies described herein activate an NK cell activity by at least about 90%. In some embodiments, the multispecific antibodies described herein activate an NK cell activity by at least about 95%. In certain embodiments, the multispecific antibodies described herein activate an NK cell activity by at least about 15% to about 65%. In certain embodiments, the multispecific antibodies described herein activate an NK cell activity by at least about 20% to about 65%. In certain embodiments, the multispecific antibodies described herein activate an NK cell activity by at least about 30% to about 65%. In specific embodiments, the NK cells are human NK cells. In specific embodiments, the NK cells express CD16a-F. In specific embodiments, the NK cells express CD16a-V. In specific embodiments, the NK cells are NK cells from a subject with a CD16a-F / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD16a-V / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD 16a- V / V genotype.

[0308] In some embodiments, the multispecific antibodies described herein can induce ADCC activity of an NK cell. In some embodiments, the multispecific antibodies described herein induce ADCC activity of an NK cell by at least about 10%. In some embodiments, the multispecific antibodies described herein induce ADCC activity of an NK cell by at least about 20%. In some embodiments, the multispecific antibodies described herein induce ADCC activity of an NK cell by at least about 30%. In some embodiments, the multispecific antibodies described herein induce ADCC activity of an NK cell by at least about 40%. In some embodiments, the multispecific antibodies described herein induce ADCC activity of an NK cell by at least about 50%. In some embodiments, the multispecific antibodies described herein induce ADCC activity of an NK cell at least about 60%. In some embodiments, the multispecific antibodies described herein induce ADCC activity of an NK cell by at least about 70%. In some embodiments, the multispecific antibodies described herein induce ADCC activity of an NK cell by at least about 80%. In some embodiments, the multispecific antibodies described herein induce ADCC activity of an NK cell by at least about 90%. In some embodiments, the multispecific antibodies described herein induce ADCC activity of an NK cell by at least about 95%. In certain embodiments, the multispecific antibodies described herein induce ADCC activity of an NK cell by at least about 15% to about 65%. In certainembodiments, the multispecific antibodies described herein induce ADCC activity of an NK cell by at least about 20% to about 65%. In certain embodiments, the multispecific antibodies described herein induce ADCC activity of an NK cell by at least about 30% to about 65%. In specific embodiments, the NK cells are human NK cells. In specific embodiments, the NK cells express CD16a-F. In specific embodiments, the NK cells express CD16a-V. In specific embodiments, the NK cells are NK cells from a subject with a CD 16a F / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD16a-V / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD 16a- V / V genotype.

[0309] In some embodiments, the multispecific antibodies described herein can induce cytokine secretion of an NK cell. In some embodiments, the multispecific antibodies described herein induce cytokine secretion of an NK cell by at least about 10%. In some embodiments, the multispecific antibodies described herein induce cytokine secretion of an NK cell by at least about 20%. In some embodiments, the multispecific antibodies described herein induce cytokine secretion of an NK cell by at least about 30%. In some embodiments, the multispecific antibodies described herein induce cytokine secretion of an NK cell by at least about 40%. In some embodiments, the multispecific antibodies described herein induce cytokine secretion of an NK cell by at least about 50%. In some embodiments, the multispecific antibodies described herein induce cytokine secretion of an NK cell at least about 60%. In some embodiments, the multispecific antibodies described herein induce cytokine secretion of an NK cell by at least about 70%. In some embodiments, the multispecific antibodies described herein induce cytokine secretion of an NK cell by at least about 80%. In some embodiments, the multispecific antibodies described herein induce cytokine secretion of an NK cell by at least about 90%. In some embodiments, the multispecific antibodies described herein induce cytokine secretion of an NK cell by at least about 95%. In certain embodiments, the multispecific antibodies described herein induce cytokine secretion of an NK cell by at least about 15% to about 65%. In certain embodiments, the multispecific antibodies described herein induce cytokine secretion of an NK cell by at least about 20% to about 65%. In certain embodiments, the multispecific antibodies described herein induce cytokine secretion of an NK cell by at least about 30% to about 65%. In specific embodiments, the NK cells are human NK cells. In specific embodiments, the NK cells express CD16a-F. In specific embodiments, the NK cells express CD16a-V. In specific embodiments, the NK cells are NK cells from a subject with a CD 16a- F / F genotype. In specific embodiments, the NK cells are NK cells from a subject with aCD16a-V / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD 16a- V / V genotype.

[0310] In certain embodiments, the multispecific antibody described herein induces NK cell dependent cytotoxicity of a tumor cell, wherein the multispecific antibody comprises a first binding domain that binds to CD 16a on an NK cell and a second binding domain that binds to a tumor antigen (such as CD 19) on the tumor cell. In specific embodiments, the NK cells are human NK cells. In some embodiments of the multispecific antibody provided herein, the multispecific antibody induces NK cell dependent cytotoxicity of the tumor cell by at least 10%. In some embodiments of the multispecific antibody provided herein, the multispecific antibody induces NK cell dependent cytotoxicity of the tumor cell by at least 20%. In some embodiments of the multispecific antibody provided herein, the multispecific antibody induces NK cell dependent cytotoxicity of the tumor cell by at least 30%. In some embodiments of the multispecific antibody provided herein, the multispecific antibody induces NK cell dependent cytotoxicity of the tumor cell by at least 40%. In some embodiments of the multispecific antibody provided herein, the multispecific antibody induces NK cell dependent cytotoxicity of the tumor cell by at least 50%. In some embodiments of the multispecific antibody provided herein, the multispecific antibody induces NK cell dependent cytotoxicity of the tumor cell by at least 60%. In some embodiments of the multispecific antibody provided herein, the multispecific antibody induces NK cell dependent cytotoxicity of the tumor cell by at least 70%. In some embodiments of the multispecific antibody provided herein, the multispecific antibody induces NK cell dependent cytotoxicity of the tumor cell by at least 80%. In some embodiments of the multispecific antibody provided herein, the multispecific antibody induces NK cell dependent cytotoxicity of the tumor cell by at least 90%. In some embodiments of the multispecific antibody provided herein, the multispecific antibody induces NK cell dependent cytotoxicity of the tumor cell by at least 95%. In certain embodiments of the multispecific antibody provided herein, the multispecific antibody induces NK cell dependent cytotoxicity of the tumor cell by at least about 15% to about 65%. In certain embodiments of the multispecific antibody provided herein, the multispecific antibody induces NK cell dependent cytotoxicity of the tumor cell by at least about 20% to about 65%. In certain embodiments of the multispecific antibody provided herein, the multispecific antibody induces NK cell dependent cytotoxicity of the tumor cell by at least about 30% to about 65%. In specific embodiments, the NK cells are human NK cells. In specific embodiments, the NK cells express CD16a-F. In specific embodiments, the NK cellsexpress CD16a-V. In specific embodiments, the NK cells are NK cells from a subject with a CD16a-F / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD16a-V / F genotype. In specific embodiments, the NK cells are NK cells from a subject with a CD 16a- V / V genotype.

[0311] In some embodiments, the multispecific antibodies in an IgG format described herein can activate NK cell activity even with mutated Fcyl region to eliminate Fc binding activity to Fey receptors.

[0312] In some embodiments, the multispecific antibodies in an IgG format described herein can activate NK cell activity even with one or more Fc silencing mutations. In some embodiments, the multispecific antibodies in an IgG format described herein can activate NK cell activity even in an Fc silenced format with Leu234Ala, Leu235Ala and Pro329Gly mutations.

[0313] In some embodiments, the multispecific antibodies described herein do not bind to cells expressing CD16b. In some embodiments, the multispecific antibodies described herein do not bind to cells expressing CD16b-NAl. In some embodiments, the multispecific antibodies described herein do not bind to cells expressing CD16b-NA2. In some embodiments, the multispecific antibodies described herein do not bind to cells derived from a subject with CD16b-NAl / CD16b-NAl genotype. In some embodiments, the multispecific antibodies described herein do not bind to cells derived from a subject with CD 16b- NAl / CD16b-NA2 genotype. In some embodiments, the multispecific antibodies described herein do not bind to cells derived from a subject with CD16b-NA2 / CD16b-NA2 genotype. In some embodiments, the multispecific antibodies described herein do not bind to neutrophils. In some embodiments, the multispecific antibodies described herein do not bind to neutrophils derived from a subject with CD16b-NAl / CD16b-NAl genotype. In some embodiments, the multispecific antibodies described herein do not bind to neutrophils derived from a subject with CD16b-NAl / CD16b-NA2 genotype. In some embodiments, the multispecific antibodies described herein do not bind to neutrophils derived from a subject with CD16b-NA2 / CD16b-NA2 genotype. In some embodiments, the multispecific antibodies described herein do not induce activation of neutrophils. In some embodiments, the multispecific antibodies described herein induce activation of NK cells and do not induce activation of neutrophils In some embodiments, the multispecific antibodies described herein induce activation of NK cells and do not induce activation of neutrophils and other granulocytes.

[0314] Other exemplary binding molecules are described in more detail in the following sections. In some embodiments, the multispecific binding agents according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 5.2.4 to 5.2.7 below.5.2.4. Antibody Fragments

[0315] Even though the term “antibody” is sometimes used in a phrase of “antibody or fragment thereof’ herein, it should be understood the term “antibody” as used herein also includes various antibody fragments, such as an antigen-binding fragment or epitope-binding fragment. Thus, when the term “antibody” is used alone without being followed by “fragment thereof’ or similar terms, it should be understood the term “antibody” includes an antibody fragment, such as an antigen-binding fragment or epitope-binding fragment. Antibodies provided herein include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. In some embodiments, the multispecific binding agents provided herein comprise one or more antibody fragments.

[0316] Variants and derivatives of antibodies include antibody functional fragments that retain the ability to bind to an antigen. Antibody fragments include but are not limited to those described in Section 5.1 above. Exemplary functional fragments include Fab fragments (e.g., an antibody fragment that contains the antigen-binding domain and comprises a light chain and part of a heavy chain bridged by a disulfide bond); Fab’ (e.g., an antibody fragment containing a single antigen-binding domain comprising an Fab and an additional portion of the heavy chain through the hinge region); F(ab’)2 (e.g., two Fab’ molecules joined by interchain disulfide bonds in the hinge regions of the heavy chains; the Fab’ molecules may be directed toward the same or different epitopes); a bispecific Fab (e.g., a Fab molecule having two antigen-binding domains, each of which may be directed to a different epitope); a single chain comprising a variable region, also known as, scFv (e.g., the variable, antigenbinding determinative region of a single light and heavy chain of an antibody linked together by a chain of, e.g., 10-25 amino acids); a disulfide-linked Fv, or dsFv (e.g., the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a disulfide bond); a bispecific scFv (e.g., an scFv or a dsFv molecule having two antigen-binding domains, each of which may be directed to a different epitope); a diabody (e.g., a dimerized scFv formed when the VH domain of a first scFv assembles with the VL domain of a second scFv and the VL domain of the first scFv assembles with the VH domain of the second scFv; the two antigen-binding regions of the diabody may be directed towards the same or different epitopes); a triabody (e.g., a trimerized scFv, formed in a mannerI l lsimilar to a diabody, but in which three antigen-binding domains are created in a single complex; the three antigen-binding domains may be directed towards the same or different epitopes); and a tetrabody (e.g., a tetramerized scFv, formed in a manner similar to a diabody, but in which four antigen-binding domains are created in a single complex; the four antigenbinding domains may be directed towards the same or different epitopes).

[0317] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al.. 1992, J. Biochem. Biophys. Methods 24: 107-17; and Brennan et aL, 1985, Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. For example, Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli, yeast or insect cells, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab’-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab’)2 fragments (Carter et al., 1992, Bio / Technology 10:163-67). According to another approach, F(ab’)2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab’)2 fragments with increased in vivo half-life comprising salvage receptor binding epitope residues are described in, for example, U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In certain embodiments, an antibody is a single chain Fv fragment (scFv) (see, e.g., WO 93 / 16185; U.S. Pat. Nos. 5,571,894 and 5,587,458). Fv and scFv have intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv (See, e.g., Borrebaeck ed., supra). The antibody fragment may also be a “linear antibody,” for example, as described in the references cited above. Such linear antibodies may be monospecific or multi-specific, such as bispecific.5.2.5. Humanized Antibodies

[0318] The present disclosure provides humanized antibodies that bind to CD 16a and / or the tumor target antigen, such as CD 19, including human CD 16a and / or human CD 19. Humanized antibodies of the present disclosure may comprise one or more CDRs from a VH and / or VL disclosed herein, such as those as shown in Tables 1-10. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which aretypically taken from an “import” variable domain. Humanized antibodies that bind CD 16a and / or the tumor target antigen, such as CD 19 may be produced using techniques known to those skilled in the art (Zhang et al., Molecular Immunology, 42(12): 1445-1451, 2005; Hwang et al, Methods, 36(1): 35-42, 2005; Dall’Acqua et al., Methods, 36(1): 43-60, 2005; Clark, Immunology Today, 21(8): 397-402, 2000, and U.S. Pat. Nos. 6,180,370; 6,054,927; 5,869,619; 5,861,155; 5,712,120; and 4,816,567).

[0319] In some cases, the humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of a VH and a VL of the parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework. For example, Padlan et al. (FASEB J. 9: 133-139, 1995) determined that only about one third of the residues in the CDRs actually contact the antigen, and termed these the “specificity determining residues,” or SDRs. In the technique of SDR grafting, only the SDR residues are grafted onto the human antibody framework (see, e.g., Kashmiri et al., Methods 36: 25-34, 2005).

[0320] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. For example, according to the so-called “best-fit” method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent may be selected as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151 :2296; Chothia c / a / . (1987) J. Mol. Biol. 196:901). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al. (1992) roc. Natl. Acad. Sci. USA, 89:4285; Presta c / o / . (1993) J. Immunol., 151 :2623). In some cases, the framework is derived from the consensus sequences of the most abundant human subclasses, VL6 subgroup I (VL6I) and VH subgroup III (VHIII). In another method, human germline genes are used at the source of the framework regions.

[0321] In an alternative paradigm based on comparison of CDRs, called Superhumanization, framework homology is irrelevant. The method consists of comparison of the non-human sequence with the functional human germline gene repertoire. Those genes encoding the same or closely related canonical structures to the murine sequences are then selected. Next, within the genes sharing the canonical structures with the non-human antibody, those with highest homology within the CDRs are chosen as framework donors.Finally, the non-human CDRs are grafted onto these frameworks (see, e.g., Tan et al, J. Immunol. 169: 1119-1125, 2002).

[0322] It is further generally desirable that antibodies be humanized with retention of their affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13: 819-824, 2000), Modeller (Sali and Blundell, J. Mol. Biol. 234: 779-815, 1993), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18: 2714-2713, 1997). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, framework residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0323] Another method for antibody humanization is based on a metric of antibody humanness termed Human String Content (HSC). This method compares the mouse sequence with the repertoire of human germline genes and the differences are scored as HSC. The target sequence is then humanized by maximizing its HSC rather than using a global identity measure to generate multiple diverse humanized variants. See, e.g., Lazar et al., Mol. Immunol. 44: 1986-1998, 2007.

[0324] In addition to the methods described above, empirical methods may be used to generate and select humanized antibodies. These methods include those that are based upon the generation of large libraries of humanized variants and selection of the best clones using enrichment technologies or high throughput screening techniques. Antibody variants may be isolated from phage, ribosome and yeast display libraries as well as by bacterial colony screening (see, e.g., Hoogenboom, Nat. Biotechnol. 23: 1105-1116, 2005; Dufner et al., Trends Biotechnol. 24: 523-529, 2006; Feldhaus et al, Nat. BiotechnoL 21 : 163-70, 2003; Schlapschy et al., Protein Eng. Des. SeL 17: 847-60, 2004).

[0325] In the framework library approach, a collection of residue variants are introduced at specific positions in the framework followed by selection of the library to select the framework that best supports the grafted CDR. The residues to be substituted may include some or all of the “Vernier” residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224: 487-499, 1992), or from the more limited set of target residues identified by Baca et al. (J. Biol. Chem. 272: 10678-10684, 1997).

[0326] In framework shuffling, whole frameworks are combined with the non-human CDRs instead of creating combinatorial libraries of selected residue variants (see, e.g., Dall’ Acqua et al, Methods 36: 43-60, 2005). The libraries may be screened for binding in a two-step selection process, first humanizing VL, followed by VH. Alternatively, a one-step framework shuffling process may be used. Such a process has been shown to be more efficient than the two-step screening, as the resulting antibodies exhibited improved biochemical and physico-chemical properties including enhanced expression, increased affinity and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44: 3049-60, 2007).

[0327] The “humaneering” method is based on experimental identification of essential minimum specificity determinants (MSDs) and is based on sequential replacement of non- human fragments into libraries of human frameworks and assessment of binding. It begins with regions of the CDR3 of non-human VH and VL chains and progressively replaces other regions of the non-human antibody into the human frameworks, including the CDR1 and CDR2 of both VH and VL. This methodology typically results in epitope retention and identification of antibodies from multiple sub-classes with distinct human V-segment CDRs. Humaneering allows for isolation of antibodies that are 91-96 % homologous to human germline gene antibodies. See, e.g., Alfenito, Cambridge Healthtech Institute’s Third Annual PEGS, The Protein Engineering Summit, 2007.

[0328] The "human engineering" method involves altering a non-human antibody or antibody fragment, such as a mouse or chimeric antibody or antibody fragment, by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies. Generally, the technique involves classifying amino acid residues of a non-human (e.g., mouse) antibody as “low risk”, “moderate risk”, or “high risk” residues. The classification is performed using a global risk / reward calculation that evaluates the predicted benefits of making a particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution willaffect the resulting antibody’s folding and / or are substituted with human residues. The particular human amino acid residue to be substituted at a given position (e.g., low or moderate risk) of a non-human (e.g., mouse) antibody sequence can be selected by aligning an amino acid sequence from the non-human antibody’s variable regions with the corresponding region of a specific or consensus human antibody sequence. The amino acid residues at low or moderate risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence according to the alignment. Techniques for making human engineered proteins are described in greater detail in Studnicka et al., Protein Engineering, 7: 805-814 (1994), U.S. Pat. Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and WO 93 / 11794.5.2.6. Antibody Variants

[0329] Modifications of the antibodies that bind to CD 16a and / or the tumor target antigen ( such as CD 19) described herein are contemplated. For example, it may be desirable to optimize the binding affinity and / or other biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector functions, glycosylation, reduced immunogenicity, or solubility. Thus, it is contemplated that variants of the antibodies described herein can be prepared and are included in the present disclosure. In some embodiments, antibody variants are antibodies with amino acid sequence variations as compared with the original antibody, for example, substitution, deletion, or insertion of one or more amino acid(s), as described above. For example, variations may be a substitution, deletion, or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence (e.g., a conservative substitution) as compared with the original antibody or polypeptide. Sites of interest for substitutional mutagenesis include the CDRs, FRs and / or constant regions. For example, antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and / or by synthesis of the desired antibody or polypeptide. Those skilled in the art who appreciate that amino acid changes may alter post-translational processes of the antibody.Chemical Modifications

[0330] Other exemplary modifications include chemical modifications, for example, by the covalent attachment of any type of molecule to the binding agent. Antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein,or conjugation to one or more immunoglobulin domains e.g., Fc or a portion of an Fc). Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody may contain one or more non-classical amino acids.

[0331] In some embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0332] When the antibody provided herein is fused to an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in the binding molecules provided herein may be made in order to create variants with certain improved properties.

[0333] In other embodiments, when the antibody provided herein is fused to an Fc region, antibody variants provided herein may have a carbohydrate structure that lacks fucose attached (directly or indirectly) to said Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Pat. Publ. Nos. 2003 / 0157108 and 2004 / 0093621. Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: U.S. Pat. Publ. No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Pat. Publ. No. 2003 / 0115614; U.S. Pat. Publ. No. 2002 / 0164328; U.S. Pat. Publ. No. 2004 / 0093621; U.S. Pat. Publ. No.2004 / 0132140; U.S. Pat. Publ. No. 2004 / 0110704; U.S. Pat. Publ. No. 2004 / 0110282; U.S. Pat. Publ. No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; W02005 / 053742; W02002 / 031140; Okazaki et al. J. Mol. Biol. 336: 1239- 1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells deficien...

Claims

WHAT IS CLAIMED IS:

1. An antibody or fragment thereof that binds to CD 16a, wherein the antibody or antigen binding fragment comprises:(i) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:37 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:38;(ii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:75 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:76;(iii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 113 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 114;(iv) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 151 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 152;(v) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 190;(vi) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:227 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:228;(vii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:265 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:266;(vii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:303 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:304; or(ix) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:341 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:342.

2. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises:(a) a VH region comprising:(1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, and 31;(2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, and 32; and(3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, and 33; and(b) a VL region comprising:(1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, and 34;(2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, and 35; and(3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, and 36.

3. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises any one or more of (i)-(vi):(i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NON, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6;(ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:8, a VH CDR3 comprising the amino acid sequence of SEQ ID NOV; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprisingthe amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 12;(iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 14, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 18;(iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:20, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:21; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:22, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:23, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:24;(v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:25, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:26, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:27; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30; or(vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:33; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:34, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:35, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:36.

4. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises(a) a VH region comprising:(1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 45, 51, 57, 63, and 69;(2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 40, 46, 52, 58, 64, and 70; and(3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 47, 53, 59, 65, and 71; and(b) a VL region comprising:(1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 48, 54, 60, 66, and 72;(2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 49, 55, 61, 67, and 73; and(3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 50, 56, 62, 68, and 74.

5. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises any one or more of (i)-(vi):(i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:39, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:40, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:41; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:42, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:43, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:44;(ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:45, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:46, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:47; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:48, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:49, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:50;(iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:51, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:52, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:53; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:54, a VL CDR2comprising the amino acid sequence of SEQ ID NO:55, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:56;(iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:57, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:58, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:59; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:60, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:61, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 62;(v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:63, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:64, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:65; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:66, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:67, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:68; or(vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:69, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:70, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:71; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:72, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:73, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:74.

6. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises:(a) a VH region comprising:(1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 83, 89, 95, 101, and 107;(2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 78, 84, 90, 96, 102, and 108; and(3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 79, 85, 91, 97, 103, and 109; and(b) a VL region comprising:(1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 80, 86, 92, 98, 104, and 110;(2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 81, 87, 93, 99, 105, and 111; and(3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 82, 88, 94, 100, 106, and 112.

7. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises any one or more of (i)-(vi):(i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:77, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:78, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:79; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:80, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:81, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 82;(ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:83, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:84, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:85; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:86, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:87, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:88;(iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:89, a VH CDR2 comprising the amino acid sequence of SEQ ID NOVO, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:91; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:92, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:93, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 94;(iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:95, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:96, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:97; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:98, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:99, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 100;(v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 101, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 102, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 103; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 104, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 105, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 106; or(vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 107, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 108, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 109; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 110, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 111, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 112.

8. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises:(a) a VH region comprising:(1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 121, 127, 133, 139, and 145;(2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 116, 122, 128, 134, 140, and 146; and(3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 117, 123, 129, 135, 141, and 147; and(b) a VL region comprising:(1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 118, 124, 130, 136, 142, and 148;(2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 125, 131, 137, 143, and 149; and(3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 120, 126, 132, 138, 144, and 150.

9. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises any one or more of (i)-(vi):(i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 115, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 116, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 117; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 118, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 119, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 120;(ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 121, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 122, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 123; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 124, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 125, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 126;(iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 127, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 128, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 129; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 130, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 131, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 132;(iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 133, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 134, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:135; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 136, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 137, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 138;(v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 139, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 140, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 141; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 142, a VL CDR2comprising the amino acid sequence of SEQ ID NO: 143, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 144; or(vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 145, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 146, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 147; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 148, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 149, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 150.

10. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises(a) a VH region comprising:(1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 153, 159, 165, 171, 177, and 183;(2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 154, 160, 166, 172, 178, and 184; and(3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 155, 161, 167, 173, 179, and 185; and(b) a VL region comprising:(1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 156, 162, 168, 174, 180, and 186;(2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 157, 163, 169, 175, 181, and 187; and(3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 164, 170, 176, 182, and 188.

11. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises any one or more of (i)-(vi):(i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 153, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 154, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 155; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 156, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 157, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 158;(ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 159, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 160, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 161; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 162, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 163, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 164;(iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 165, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 166, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:167; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 168, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 169, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 170;(iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 171, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 172, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 173; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 174, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 175, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 176;(v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 177, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 178, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 179; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 180, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 181, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 182; or(vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 183, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 184, and aVH CDR3 comprising the amino acid sequence of SEQ ID NO: 185; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 186, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 187, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 188.

12. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises:(a) a VH region comprising:(1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 197, 203, 209, 215, and 221;(2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 192, 198, 204, 210, 216, and 222; and(3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 193, 199, 205, 211, 217, and 223; and(b) a VL region comprising:(1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 194, 200, 206, 212, 218, and 224;(2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 195, 201, 207, 213, 219, and 225; and(3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 196, 202, 208, 214, 220, and 226.

13. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises any one or more of (i)-(vi):(i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 191, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 192, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 193; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 194, a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 195, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 196;(ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 197, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 198, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 199; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:200, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:201, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:202;(iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:203, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:204, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:205; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:206, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:207, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:208;(iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:209, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:210, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:211; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:212, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:213, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:214;(v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:215, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:216, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:217; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:218, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:219, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:220; or(vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:221, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:222, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:223; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:224, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:225, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:226.

14. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises:(a) a VH region comprising:(1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 229, 235, 241, 247, 253, and 259;(2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 230, 236, 242, 248, 254, and 260; and(3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 231, 237, 243, 249, 255, and 261; and(b) a VL region comprising:(1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 232, 238, 244, 250, 256, and 262;(2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 233, 239, 245, 251, 257, and 263; and(3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 234, 240, 246, 252, 258, and 264.

15. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises any one or more of (i)-(vi):(i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:229, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:230, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:231; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:232, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:233, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:234;(ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:235 a VH CDR2 comprising the amino acid sequence of SEQ ID NO:236, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:237; and a VL regioncomprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:238, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:239, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:240;(iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:241, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:242, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:243; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:244, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:245, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:246;(iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:247, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:248, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:249; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:250, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:251, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:252;(v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:253, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:254, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:255; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:256, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:257, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:258; or(vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:259, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:260, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:261; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:262, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:263, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:264.

16. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises:(a) a VH region comprising:(1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 267, 273, 279, 285, 291, and 297;(2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 268, 274, 280, 286, 292, and 298; and(3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 269, 275, 281, 287, 293, and 299; and(b) a VL region comprising:(1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 270, 276, 282, 288, 294, and 300;(2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 271, 277, 283, 289, 295, and 301; and(3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 272, 278, 284, 290, 296, and 302.

17. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises any one or more of (i)-( ix):(i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:267, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:268, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:269; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:270, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:271, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:272;(ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:273 a VH CDR2 comprising the amino acid sequence of SEQ ID NO:274, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:275; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:276, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:277, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:278;(iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:279, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:280, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:281; and a VL regioncomprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:282, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:283, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:284;(iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:285, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:286, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:287; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:288, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:289, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:290;(v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:291, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:292, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:293; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:294, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:295, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:296; or(vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:297, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:298, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:299; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:300, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:301, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:302.

18. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises:(a) a VH region comprising:(1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 305, 311, 317, 323, 329, and 335;(2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 306, 312, 318, 324, 330, and 336; and(3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307, 313, 319, 325, 331, and 337;(b) a VL region comprising:(1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 308, 314, 320, 326, 332, and 338;(2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 309, 315, 321, 327, 333, and 339; and(3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 310, 316, 322, 328, 334, and 340. .

19. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises any one or more of (i)-(vi):(i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:305, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:306, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:307; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:308, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:309, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 310;(ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:3 I l a VH CDR2 comprising the amino acid sequence of SEQ ID NO:312, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:313; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:314, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:315, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 316;(iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:317, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:318, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:319; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:320, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:321, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:322;(iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:323, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:324, and aVH CDR3 comprising the amino acid sequence of SEQ ID NO:325; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:326, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:327, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:328;(v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:329, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:330, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:331; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:332, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:333, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:334; or(vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:335, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:336, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:337; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:338, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:339, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:340.

20. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises:(i) a VH comprising the amino acid sequence of SEQ ID NO: 37 and a VL comprising the amino acid sequence of SEQ ID NO: 38;(ii) a VH comprising the amino acid sequence of SEQ ID NO: 75 and a VL comprising the amino acid sequence of SEQ ID NO: 76;(iii) a VH comprising the amino acid sequence of SEQ ID NO: 113 and a VL comprising the amino acid sequence of SEQ ID NO: 114;(iv) a VH comprising the amino acid sequence of SEQ ID NO: 151 and a VL comprising the amino acid sequence of SEQ ID NO: 152;(v) a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL comprising the amino acid sequence of SEQ ID NO: 190;(vi) a VH comprising the amino acid sequence of SEQ ID NO: 227 and a VL comprising the amino acid sequence of SEQ ID NO: 228;(vii) a VH comprising the amino acid sequence of SEQ ID NO: 265 and a VL comprising the amino acid sequence of SEQ ID NO: 266;(viii) a VH comprising the amino acid sequence of SEQ ID NO: 303 and a VL comprising the amino acid sequence of SEQ ID NO: 304; or(ix) a VH comprising the amino acid sequence of SEQ ID NO: 341 and a VL comprising the amino acid sequence of SEQ ID NO: 342.

21. An antibody or fragment thereof that binds to CD 16a, wherein the antibody or fragment thereof binds to both CD16a-F and CD16a-V.

22. The antibody or fragment thereof of claim 21, wherein the antibody or fragment thereof does not bind to CD 16b.

23. The antibody or fragment thereof of claim 21 or 22, wherein the antibody or fragment thereof does not bind to CD16b-NAl and / or CD16b-NA2.

24. A multispecific antibody or fragment thereof, comprising a first binding domain that binds to CD 16a and a second binding domain that binds to a second antigen, wherein the first binding domain comprises an antibody or fragment thereof according to any one of claims 1 to 23.

25. The multispecific antibody or fragment thereof of claim 24, wherein the second antigen is expressed on a tumor cell.

26. The multispecific antibody or fragment thereof of claim 24 or 25, wherein the second antigen is CD 19.

27. The multispecific antibody or fragment thereof of any one of claims 24 to 26, wherein the first binding domain comprises any one or more of (i)-(ix):(i) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:37 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:38;(ii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:75 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:76;(iii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 113 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 114;(iv) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 151 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 152;(v) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 190;(vi) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:227 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:228;(vii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:265 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:266;(vii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:303 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:304; or(ix) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:341 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:342; and wherein the second binding domain comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:343 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:344.

28. The multispecific antibody or fragment thereof of claim 27, wherein the first binding domain comprises any one or more of (i)-(ix):(i) a VH comprising the amino acid sequence of SEQ ID NO: 37 and a VL comprising the amino acid sequence of SEQ ID NO: 38;(ii) a VH comprising the amino acid sequence of SEQ ID NO: 75 and a VL comprising the amino acid sequence of SEQ ID NO: 76;(iii) a VH comprising the amino acid sequence of SEQ ID NO: 113 and a VL comprising the amino acid sequence of SEQ ID NO: 114;(iv) a VH comprising the amino acid sequence of SEQ ID NO: 151 and a VL comprising the amino acid sequence of SEQ ID NO: 152; a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL comprising the amino acid sequence of SEQ ID NO: 190;(vi) a VH comprising the amino acid sequence of SEQ ID NO: 227 and a VL comprising the amino acid sequence of SEQ ID NO: 228.(vii) a VH comprising the amino acid sequence of SEQ ID NO: 265 and a VL comprising the amino acid sequence of SEQ ID NO: 266;(viii) a VH comprising the amino acid sequence of SEQ ID NO: 303 and a VL comprising the amino acid sequence of SEQ ID NO: 304; or(ix) a VH comprising the amino acid sequence of SEQ ID NO: 341 and a VL comprising the amino acid sequence of SEQ ID NO: 342; and wherein the second binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 343 and a VL comprising the amino acid sequence of SEQ ID NO: 344;29. The multispecific antibody or fragment thereof of any one of claims 24-28, wherein the first binding domain and the second binding domain can be linked through a peptide bond or a peptide linker.

30. The multispecific antibody or fragment thereof of any one of claims 26-29, wherein the multispecific antibody or fragment thereof comprises one polypeptide chain comprising the first binding domain and the second binding domain, wherein the first binding domain comprises a first scFv that binds to CD 16, and the second binding domain comprises a second scFv that binds to CD 19.

31. The multispecific antibody or fragment thereof of claim 30, wherein the first scFv is linked to the N terminal of the second scFv.

32. The multispecific antibody or fragment thereof of claim 30, wherein the first scFv is linked to the C terminal of the second scFv.

33. The multispecific antibody or fragment thereof of any one of claims 30-32, wherein the polypeptide chain further comprises a CH2 and a CH3 of an IgG Fc region.

34. The multispecific antibody or fragment thereof of any one of claims 30-33, wherein the multispecific antibody or fragment comprises two identical polypeptide chains of claim 33.

35. The multispecific antibody or fragment thereof of claim 34, wherein the first polypeptide and the second polypeptide form a bivalent format through dimerization of the CH2 and / or CH3 regions.

36. An isolated nucleic acid comprising a nucleic acid sequence encoding the antibody of any one of claims 1-23, or the multispecific antibody of any one of claims 24-35.

37. A vector comprising the isolated nucleic acid of claim 36.

38. A pharmaceutical composition, comprising the antibody of any one of claims 1- 23, the multispecific antibody of any one of claims 24-35, or the vector of claim 37, and a pharmaceutically acceptable excipient.

39. A method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the antibody of any one of claims 1-23, the multispecific antibody of any one of claims 24-35, or the vector of claim 37, or the pharmaceutical composition of claim 38.

40. The method of claim 39, wherein the disease or disorder is a cancer.

41. The method of claim 39, wherein the disease or disorder is leukemia or lymphoma.

42. A method of activating NK cell activity in a subject, comprising administering to the subject an effective amount of the antibody of any one of claims 1-23, the multispecific antibody of any one of claims 24-35, or the vector of claim 37, or the pharmaceutical composition of claim 38.

43. A method of activating NK cell activity without activating neutrophils in a subject, comprising administering to the subject an effective amount of the antibody of any oneof claims 1-23, the multispecific antibody of any one of claims 24-35, or the vector of claim 37, or the pharmaceutical composition of claim 38.

44. The method of any one of claims 39 to 43, wherein the subject has CD16a-F / F, CD16a-F / V, or CD16a-V / V genotype.

45. The method of any one of claims 39 to 44, wherein the subject has CD16b-NAl / CD16b-NA2, CD16b-NAl / CD16b-NAl, or CD16b-NA2 / CD16b-NA2 genotype.

46. An antibody or fragment thereof which specifically recognizes an epitope comprising the residue Asp 82 of human CD 16a with an affinity for CD 16a at least 10-fold higher than for CD 16b.

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