NK cell engaging antibody fusion construct

Abstract

To provide multispecific antigen-binding proteins for engaging NK cells for triggering NK cell cytotoxicity by engaging CD16A (FcγRIIIA) expressed on the NK cells.SOLUTION: A multispecific antigen-binding protein comprises: (a) two target antigen-binding moieties each comprising a Fab fragment and an Fc portion, and (b) two CD16A antigen-binding moieties each in the format of a single chain variable fragment (scFv) comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the variable region at the N-terminus of each of the two scFvs is the VL, wherein the N-terminus of the first of the two scFvs is fused to the C-terminus of the Fc portion of the first target antigen-binding moiety of the two target antigen-binding moieties, and the N-terminus of the second of the two scFvs is fused to the C-terminus of the Fc portion of the second target antigen-binding moiety of the two target antigen-binding moieties.SELECTED DRAWING: Figure 1

Description

Cross - reference to related applications

[0001] This application claims priority to European Patent Application No. 18167384.9 filed on April 13, 2018, European Patent Application No. 18167385.6 filed on April 13, 2018, European Patent Application No. 18190661.1 filed on August 24, 2018, and European Patent Application No. 18190662.9 filed on August 24, 2018, the entire contents of each of which are incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing that was submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on April 10, 2019, has the name 087353_0101_SL.txt and is 137,653 bytes in size.

Technical Field

[0003] The present invention relates to a multispecific antigen - binding protein for binding to natural killer (NK) cells in order to induce the cytotoxicity of NK cells by binding to CD16A (FcγRIIIA) expressed on the NK cells. Here, the antigen - binding protein comprises at least two CD16A antigen - binding portions and at least one additional target antigen - binding portion.

Background Art

[0004] WO 2006 / 125668 and Reusch et al., "MABS" (2014), Vol. 6, No. 3, pp. 728 - 739 describe bispecific tandem diabodies for binding CD16A and their use for NK cell immunotherapy.

[0005] Natural killer (NK) cells are cytotoxic IFN-γ natural lymphocytes and are considered to constitute the first choice of defense against virus-infected cells and cancer cells (Cerwenka and Lanier, "Nat Rev Immunol." (2001), Vol. 1, No. 1, pp. 41-49). The cytotoxic ability of NK cells can be utilized in cancer immunotherapy by re-inducing NK cell lysis in tumor cells and stimulating the activating receptor CD16A, also known as FcγRIIIA, expressed on the cell surface of NK cells.

[0006] The induction of NK cells for tumor cell lysis using multispecific antibodies is considered a powerful immunotherapy approach with low toxicity and a well-acceptable safety profile.

[0007] CD16A is an activating receptor that induces the cytotoxic activity of NK cells. The affinity of antibodies for CD16A is directly correlated with their ability to induce NK cell activation and thus reduces the antibody dose required for activation.

[0008] The cytotoxic activity of NK cells can be increased by increasing the binding force through multivalent binding to CD16A, for example, bivalent binding to CD16A.

[0009] However, the bivalency and multivalency of CD16A can result in antibody-mediated cross-linking of CD16A-expressing cells, including NK cells, γδ T cells, and subsets of monocytes, macrophages, and dendritic cells. Such interactions are expected to result in unwanted cell stimulation via CD16A. For example, cross-linking of NK cells with CD16A-expressing immune cells results in the induction of NK cell activation, cytokine release, and cell-directed cytotoxicity. This reduces NK cell activity and promotes NK cell depletion independent of the desired anti-tumor effect. Thus, NK cell cross-linking with CD16A-expressing immune cells is expected to reduce the therapeutic effect of NK cell involvement. Most importantly, as previously described using the CD16-directed murine IgG antibody (3G8) that is bivalent for CD16A in rhesus monkeys and tamarins, cross-linking of two or more NK cells via bivalent or multivalent interactions with CD16A can cause NK cell activation and fratricide (NK-NK cell lysis), ultimately resulting in efficient NK cell depletion in vivo (Choi et al., "Immunology" (2008), Vol. 124, pp. 215-222; Yoshida et al., "Frontier in Microbiology" (2010), Vol. 1, p. 128). Therefore, induction of NK lysis impairs the efficacy of therapeutic antibodies by reducing the number of effector cells available to mediate ADCC.

[0010] Thus, there is a need for antibodies that can enhance NK cell involvement without being reduced by fratricide (NK-NK cell lysis).

Summary of the Invention

[0011] The present invention provides a CD16A-binding antibody that is capable of at least bivalent interaction with CD16A on NK cells, and thus has increased binding affinity and cytotoxic ability, but preferably is unable to induce NK-NK cell lysis.

[0012] The present invention refers to the following.

[0013] Comprising at least one target antigen-binding portion and at least two CD16A antigen-binding portions, wherein the two CD16A antigen-binding portions are fused to a constant domain, such as a Fab fragment or an Fc portion, a multivalent and multispecific antigen-binding protein. Preferably, the target antigen-binding portion is also fused to a Fab fragment or an Fc portion.

[0014] For example, each of the antigen-binding portions can be selected from the group consisting of a single-chain diabody (scDb), a diabody (Db), a single-chain Fv (scFv), or a Fab fragment.

[0015] In some embodiments, the light and heavy chain variable regions of the CD16A antigen-binding portion are sequentially linked by a polypeptide such that the variable region at the N-terminus of the polypeptide chain is the light chain variable region, thereby preventing fratricide (NK-NK cell lysis) induction.

[0016] A number of protein constructs have antigen-binding portions fused at the N-terminus or C-terminus to various units containing constant domains such as F(ab)’, F(ab) 2 ’, CH2-CH3, hinge-CH2-CH3, Fc, CH1-hinge-CH2-CH3, or IgG.

[0017] In some embodiments, the antigen-binding protein comprises a silenced Fc portion that does not bind to FcγR but retains binding to FcRn and optionally at least one additional Fc functionality.

[0018] In some embodiments, the antigen-binding protein comprises an Fc portion that is silenced so as not to bind to FcγR on the one hand and contains further mutations that increase or decrease its ability to bind to FcRn, correlating with different in vivo pharmacokinetics based on different abilities for transcytosis and recycling on the other hand.

[0019] In certain embodiments, the antigen-binding protein comprises at least an HSA antigen-binding portion. For example, the antigen-binding protein may comprise two, three, or four HSA antigen-binding portions.

[0020] In certain embodiments, the antigen-binding protein comprises at least two different target antigen-binding portions, i.e., a first and a second target antigen-binding portion.

[0021] In certain embodiments, a CD16A antigen-binding portion with increased or decreased affinity for CD16A is applied.

[0022] In particular, the present invention is characterized by the following.

[0023] 1. The involved functions of NK cells are enhanced as follows. ·Two CD16A antigen-binding portions fused to a Fab fragment or an Fc portion. This increases the cytotoxic ability due to the divalent binding to CD16A. ·Use of an anti-CD16A antigen-binding portion that binds to CD16A with high affinity. ·Avoid induction of fratricide (NK-NK cell lysis) by arranging CD16A antigen-binding in a specific order, thereby preventing weakening of the cytotoxic activity of NK cells against target cells. ·Avoiding fratricide is independent of the binding affinity of the CD16A antigen-binding portion. ·Use a silenced Fc portion to avoid further binding to FcγR-positive monocytes or other cells expressing FcγR while maintaining binding to FcRn, thereby extending the serum half-life.

[0024] 2. This enhanced NK cell-binding functionality may be due to the modularity of the antigen-binding portions for a number of protein constructs that achieve adjustable cytotoxicity, target affinity, CD16A functionality, target biology, tissue penetration and distribution, half-life, and exposure.

[0025] By using the diverse antibody formats specifically described herein, as well as functions including various half-lives and different exposures, it becomes possible to adapt novel therapeutic agents to various symptoms with high medical needs, and targeted product development can be obtained for each setting.

[0026] Furthermore, the lack of fratricide in NK cells is an important feature of high-affinity and at least bivalent immunocyte engager formats that are characterized by longer cell retention times and are used for the involvement of endogenous NK cells or combined with NK cell therapy approaches. These include NK cells from different sources, such as umbilical cord blood-derived, healthy donor peripheral blood-derived, patient-derived for autologous treatment, or stem cell-derived. These engagers can be co-infused or pre-mixed with such NK cells.

[0027] Protein formats are presented herein that further enable an increase in target binding affinity by at least bivalent binding to the target, and / or an increase in target selectivity by dual targeting of the target by two different first and second target antigen-binding portions.

[0028] Additional functionality can be incorporated, for example, into antigen-binding proteins such as the human serum albumin (HSA)-binding portion. BRIEF DESCRIPTION OF THE DRAWINGS

[0029]

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Modes for Carrying Out the Invention

[0030] I. Definitions The term "multispecificity" refers to an antigen-binding molecule consisting of antigen-binding sites that bind to at least two different epitopes, particularly epitopes of different antigens. "Multispecificity" includes, but is not limited to, bispecificity, trispecificity, and tetravalent specificity.

[0031] The term "valency" refers to the presence of a defined number of antigen-binding moieties in an antigen-binding protein. Native IgG has two antigen-binding moieties and is divalent. The antigen-binding proteins according to the present invention are at least trivalent. Examples of tetravalent, pentavalent, and hexavalent antigen-binding proteins are described herein.

[0032] The term "polypeptide" refers to a polymer in which amino acid residues are continuously linked by amide bonds. In certain embodiments, the term "polypeptide" refers to a group of molecules typically consisting of 30 or more amino acids. Polypeptides may further form multimers, such as dimers, trimers, and higher oligomers, i.e., multimers consisting of two or more polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc. may be identical or non-identical. The corresponding higher-order structures of such multimers are consequently referred to as homodimers or heterodimers, homotrimers or heterotrimers, etc. Examples of heteromultimers include antibody molecules in their naturally occurring form, which consist of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "peptide", "polypeptide", and "protein" also refer to naturally modified peptides / polypeptides / proteins whose modifications are affected by post-translational modifications such as glycosylation, acetylation, and phosphorylation. The "peptide", "polypeptide", or "protein" as referred to herein may also be chemically modified such as pegylated. Such modifications are known in the art and are described hereinafter in this specification.

[0033] "Fv polypeptide" refers to a fusion polypeptide in which antibody variable (Fv) domains are sequentially linked. The polypeptide may have contiguous amino acid residues in addition to N-terminal and / or C-terminal sequence extensions. For example, the polypeptide may contain a tag sequence at the C-terminus, which may also be preferably useful for purification and detection of the polypeptide. Examples of tag sequences include a histidine tag, such as a His tag consisting of 6 His residues (SEQ ID NO: 58), or a C tag, such as the EPEA tetrapeptide (SEQ ID NO: 59). In the case of multimeric antigen-binding molecules, different Tag sequences may use different polypeptides, for example, a His tag for the first polypeptide and a C tag for the second polypeptide of a heterodimeric molecule. In certain embodiments, the polypeptide includes a variable domain that provides an antigen-binding site and a constant antibody domain, such as C L C H 1C H , and / or an Fc moiety (CH2-CH3). For example, such embodiments include an Fv polypeptide fused to at least one constant antibody domain, such as an Fc moiety. In further embodiments, the polypeptide may be conjugated to another agent such as a toxin, an immunomodulator, or a signaling agent.

[0034] The term "Fc portion" refers to a polypeptide that includes the C-terminal portion of the immunoglobulin H chain and retains at least one function of the Fc region of the IgG region, particularly the function of binding to FcRn. The effector function of an antibody is determined by the sequence of the Fc region. The Fc portion may be composed of a CH2 domain, a CH3 domain, or a CH2-CH3 polypeptide chain. The CH2-CH3 polypeptide chain may be combined with another CH2-CH3 polypeptide chain to form a dimer of two CH2-CH3 polypeptides bound to each other. Here, dimerization is promoted by a covalent bond to the CH2 domain at the N-terminus of the hinge region. Therefore, in some embodiments, the Fc portion consists of a dimer of two CH2-CH3 polypeptide chains and a hinge region. Preferably, the Fc portion consists of the constant domain of an Ig class, such as IgA, IgD, IgE, IgM, preferably IgG1, IgG2, IgG2, IgG4, particularly IgG1. The light chain constant region (CL) can be selected from kappa (κ), lambda (λ), and sigma (σ). Here, the human lambda class has subclasses of lambda 1 to 4.

[0035] The "hinge" domain may be of the same or a different IgG class as the Fc portion, or may be an engineered or non-naturally occurring hinge domain. An example of an IgG hinge region has an amino acid sequence as shown in SEQ ID NO: 23. Also included are variants of the wild-type hinge region, such as a shortened hinge region, for example, the hinge shown as a middle.hinge having the amino acid sequence of SEQ ID NO: 24.

[0036] The terms "antigen-binding protein" and "antigen-binding molecule" (with or without hyphens) are used interchangeably herein to refer to immunoglobulin derivatives having antigen-binding properties. That is, a binding protein is an antigen-binding molecule. A binding protein contains an immunologically functional immunoglobulin portion capable of binding to a target antigen. The immunologically functional immunoglobulin portion can include an immunoglobulin or a portion thereof, a fusion peptide derived from an immunoglobulin portion, or a complex that binds to an immunoglobulin portion forming an antigen-binding site. Each antigen-binding site consists of at least the CDRs of the immunoglobulin heavy or light chain from which the antigen-binding portion is derived. The terms "antigen-binding protein" and "antigen-binding molecule" (with or without hyphens) are used interchangeably herein to refer to antibody fragments, antibody derivatives, or antibody-like binding proteins that retain their specificity and affinity for those antigens, including, for example, IgG-like fusion polypeptides based on Fv domains with additional constant domains. Depending on desired characteristics such as valency, multispecificity, pharmacokinetic, and pharmacodynamic properties, the Fv and constant domains and / or additional functional domains can be modularly assembled in different molecular formats or protein scaffolds, as described, for example, in Brinkmann and Kontermann, "mAbs" (2017), Vol. 9, No. 2, pp. 182-192, or Spiess et al., "Molecular Immunology" (2015), Vol. 67, pp. 95-106.

[0037] In some embodiments, the antigen-binding protein consists of a single polypeptide chain. Such an antigen-binding protein is a monomer. In other embodiments, the antigen-binding protein consists of at least two polypeptide chains. Such an antigen-binding protein is a multimer, e.g., a dimer, trimer, or tetramer.

[0038] Preferably, the antigen-binding protein is human, and most preferably fully human.

[0039] The term "antigen-binding portion" refers to the antibody-antigen binding site or paratope of an antigen-binding protein that specifically binds to an antigenic determinant (epitope) of an antigen. An antigen binding site is the binding portion of an antigen-binding protein that can recognize and specifically bind to an antigen.

[0040] As used herein, "Fv" refers to an antigen-binding portion consisting of both the variable domains of the light chain (V L ) and heavy chain (V H ) of an antibody that recognizes an antigen, i.e., the portion that binds to the epitope of the antigen. In certain embodiments, the antigen-binding site may be a single domain (sdAb), such as a V H H fragment from camels, or a V NAR fragment from cartilaginous fish.

[0041] Each antigen-binding portion is formed by an antibody that binds to the same epitope, i.e., an immunoglobulin, a variable heavy chain domain (V H ), and an antibody variable light chain domain (V L ), while the variable heavy chain domain (V H ) includes three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3, and the variable light chain domain (V L ) includes three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3. The variable heavy and light chain domains of the antigen-binding site may be covalently linked to each other, e.g., by a peptide linker, or non-covalently bound to each other to form the antigen-binding site.

[0042] "Linker" refers to an amino acid sequence that binds two juxtaposed variable domains that form an antigen-binding portion, where the C-terminus of one domain is bound to the N-terminus of the other juxtaposed domain, or vice versa, with a linker peptide. For amino acid compositions, Fv, i.e., V H / V L, a peptide linker sequence is selected that does not inhibit the formation of the antigen-binding site and the antigen-recognition site and does not inhibit multimerization, for example, dimerization of the polypeptide of the multispecific antigen-binding protein. For example, a linker consisting of glycine residues and serine residues generally confers protease resistance. In some embodiments, (G 2 S) x peptide linkers are used, where, for example, x = 1 to 20, for example (G 2 S), (G 2 S) 2 , (G 2 S) 3 , (G 2 S) 4 , (G 2 S) 5 , (G 2 S) 6 , (G 2 S) 7 or (G 2 S) 8 , or (G 3 S) x peptide linkers are used, where, for example, x = 1 to 15, or (G 4 S) x peptide linkers are used, where, for example, x = 1 to 10, preferably 1 to 6. The amino acid sequence of the linker can be optimized, for example, by phage display methods, in order to improve the formation of the antigen-binding site and the production yield of the polypeptide.

[0043] "Connector" refers to a peptide that binds an antigen-binding moiety to a Fab fragment, hinge, or Fc portion. By definition, a connector is not a peptide that links two variable domains.

[0044] The length of the linker and the connector can affect the folding and flexibility of the Fv polypeptide of the antigen-binding portion. The desired flexibility of the Fv polypeptide depends on the target antigen density and accessibility to the target antigen, i.e., the epitope on the target antigen. A longer linker or connector can provide a more flexible Fv polypeptide with a more sensitive antigen-binding site. The effect of linker length on the formation of dimeric antigen-binding polypeptides is described, for example, in Todorovska et al., "Journal of Immunological Methods" (2001), Vol. 248, pp. 47-66; Perisic et al., "Structure" (1994), Vol. 2, pp. 1217-1226; Le Gall et al., "Protein Engineering" (2004), Vol. 17, pp. 357-366, and WO 94 / 13804.

[0045] A "single-chain variable fragment" or "scFv" comprises an antigen-binding site consisting of a heavy-chain variable domain (V L ) optionally fused via a peptide linker to a light-chain variable domain (V H ). The scFv may be the following polypeptide chain: V L -linker-V H or V H -linker-V L from the N-terminus to the C-terminus of the polypeptide chain (Huston et al., "Proc. Natl. Acad. Sci. USA" (1988), Vol. 85, pp. 5879-83). The linker between the V H domain and the V L domain can form the desired structure that enables antigen binding.

[0046] An "antigen-binding (Fab) fragment" or "Fab" comprises one constant (CH1, CL) domain and one variable domain (V H , V L ) formed by dimerization of each of the heavy (H)-chain-derived sequence and the light (L)-chain-derived sequence, where the variable domains V H and V Lconstitutes the antigen-binding site. The two Fab’ fragments are bound to the Fc portion via the hinge region as F(ab’) 2 fragments at the N-terminus.

[0047] A “diabody” (Db) consists of two pairs of variable heavy (V H ) and variable light (V L ) chain domains, a first pair and a second pair, and shows a bivalent Fv molecule in which the two V L / V H bind to the antigen-binding site. Each pair of variable domains is sequentially bound within the polypeptide. In certain embodiments, the bivalent Fv molecule consists of the first and second pairs of two juxtaposed variable domains, where in each pair, the two variable domains are fused by a short peptide linker that excludes intermolecular association between the variable domains linked by a short linker. The first pair of variable domains is forcedly associated with and crossover with the second pair of variable domains to form two Fv antigen-binding sites. Thus, each of the two antigen-binding portions is formed by one variable domain of the first pair of variable domains and one variable domain of the second pair of variable domains. Thus, such a diabody constitutes at least one antigen-binding site consisting of two variable domains that are not directly linked by a short linker. In a diabody, the variable domains of the first antigen-binding portion are linked with the variable domains of the second antigen-binding portion by a linker. For example, V H of the first antigen-binding portion is bound to V L of the second antigen-binding portion in the first polypeptide by a first linker, and V L of the first antigen-binding portion is bound to V H of the second antigen-binding portion in the second polypeptide by a second linker. In each pair of juxtaposed variable domains, the short first linker or the second linker connects the C-terminus of one variable domain and the N-terminus of the other variable domain, or vice versa. In each pair, the variable domains are V L -V H , V H -V L , V H -V H and VL -V L can be oriented from the N-terminus to the C-terminus, where the two variable domains of the pair can have different antigen epitope specificities or can have the same antigen epitope specificity. In certain examples, the two variable domains are directly linked by a peptide bond between the C-terminus of one variable domain and the N-terminus of the other variable domain of the pair. The length of the short peptide linker that links the two variable domains in each of the first and second pairs of variable domains of the diabody is such that intermolecular association between the variable domains linked by the linker is prevented. Such a linker is "short". That is, it consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or about 12 amino acid residues. When the amino acid residue is 0, the linker is a peptide bond. Such short linkers facilitate correct dimerization between the two pairs of variable domains and the formation of two Fv antigen-binding sites. Shortening the linker to about 12 or fewer amino acid residues generally prevents adjacent domains on the same polypeptide chain from interacting with each other. In embodiments of the present invention, these linkers consist of about 3 to about 12, such as 5 to 10, particularly 7 to 9 consecutive amino acid residues. The length of the linker may be adjusted to a particular domain orientation within the polypeptide of the diabody. Moreover, it is in principle possible for two polypeptides having linkers with more than 12 amino acid residues between the paired variable antibody domains to dimerize correctly with each other (see, for example, Le Gall et al., "Protein Engineering" (2004), Vol. 17, pp. 357-366).

[0048] "Single-chain diabody (scDb)" refers to a diabody derivative that has been converted into a single-chain polypeptide by adding an additional linker that fuses the first and second polypeptides (Kontermann et al., "Immunol. Methods" (1999), Vol. 226, pp. 179-188). Thus, the additional linker connects the C-terminus of the first polypeptide consisting of the variable domains of the first and second antigen-binding portions linked by the first linker, and the N-terminus of the second polypeptide consisting of the recognition variable domains of the first and second antigen-binding portions linked by the second linker. Therefore, scDb is a single polypeptide chain, i.e., an Fv polypeptide, where the four variable domains of the two antigen-binding portions are arranged as follows from the N-terminus to the C-terminus: - VL-linker1-VH-linker3-VL-linker2-VH, or - VL-linker1-VL-linker3-VH-linker2-VH, or - VH-linker1-VL-linker3-VH-linker2-VL, or - VH-linker1-VH-linker3-VL-linker2-VL, Linker1 and Linker2 are "short" linkers such as those used in diabodies, e.g., composed of less than 13, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues.

[0049] Linker3 is a more flexible linker, i.e., one used in scFv that promotes intramolecular folding and the association of the two N-terminal variable domains and the two C-terminal variable domains into two antigen-binding portions. Linker3 consists of 13 or more amino acid residues, e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues. In some embodiments, Linker3 is 15-25, preferably 15-20 or 13-18 amino acids.

[0050] The term "tandem diabody" refers to an antigen-binding molecule constructed by linking at least four variable domains (two heavy-chain variable domains (VH) and two light-chain variable domains (VL)) in a single gene construct that enables the homodimerization of two of the translated polypeptide chains. In such a tandem diabody, the length of the linker is such that it prevents the pairing within the molecule of the variable domains so that the molecule cannot fold back on itself to form a monomeric single-chain molecule, but rather is forced to pair with complementary domains on another chain. The variable domains are also arranged such that the corresponding variable domains pair with each other during this dimerization (Weichel et al., "European Pharmaceutical Review" (2015), Vol. 20, No. 1, pp. 27 - 32; Reusch et al., "mAbs" (2014), Vol. 6, No. 3, pp. 728 - 739).

[0051] "Dual Affinity Retargeting Molecule (DART)" refers to a protein scaffold. Here, the VH of the first antigen-binding portion is linked to the VL of the second antigen-binding portion on a second polypeptide, and the VH of the second antigen-binding portion is linked to the VL on the first polypeptide in the sequence VL(A)-VH(B)+VL(B)-VH(A), where intermolecular disulfide bonds are introduced to stabilize the molecule.

[0052] The term "target" or "target antigen" refers to the type of cell that NK cells should be directed to induce or trigger the cytotoxicity of NK cells, that is, an antigen expressed by or associated with target cells or virus-infected cells. Examples of target antigens may be tumor antigens or tumor-associated antigens (Tumor-Associated Antigen: TAA). Tumor antigens or TAAs may be expressed on the surface of target cells or presented by MHC complexes as MHC-restricted peptides. Examples of tumor antigens include CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CCR4, CS-1, GD2, matrix metalloproteinase 1 (MMP1), laminin receptor precursor protein, BCMA, EGFR, EGFRvIII, Ep-CAM, gpA33, AMHRII, PDGFRα, SLAMF7, PLAP, Thomsen-Friedenreich (TF) antigen, MUC-1 (mucin), IGFR, IL4-Rα, IL13-R, HER2 / neu, HER3, PSMA, CEA, TAG-72, HPV E6, HPV E7, BING-4, cyclin-B 1 , 9D7, EphA2, EphA3, telomerase, mesothelin, survivin, SAP-1, cancer testis antigens (BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family), NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gp100 / pmel17, tyrosinase, TRP-1 / -2, MC1R, β-catenin, BRCA1 / 2, CDK4, CML66, MART-2, p53, Ras, TGF-βRII, and TCR ("Categories of Tumor Antigens, Holland-Frei Cancer Medicine.", 6th edition, Kufe DW, Pollock RE, Weichselbaum RR., eds. Hamilton (ON): Becker; from 2003). "Target" and "target antigen" also include serum albumin, particularly human serum albumin (HSA). In certain embodiments, the target antigen is BCMA.

[0053] In other embodiments, the target antigen may be, for example, an infectious agent such as a virus or bacterial pathogen from dengue virus, herpes simplex, cytomegalovirus, hepatitis virus, human T-cell lymphoma virus, influenza virus, RSV, papillomavirus (including antigens in addition to the above E6 and E7), influenza virus, or HIV. Peptide targets presented by the MHC complex as MHC-restricted peptides are also included.

[0054] "CD16A" refers to the activating receptor CD16A, also known as FcγRIIIA, which is expressed on the cell surface of NK cells. CD16A is an activating receptor that induces the cytotoxic activity of NK cells. The affinity of an antibody for CD16A is directly correlated with the ability to induce NK cell activation; thus, the higher the affinity for CD16A, the less amount of antibody required for activation. The antigen-binding site of the antigen-binding protein binds to CD16A but not to CD16B. For example, an antigen-binding site consisting of the variable domains of the heavy chain (VH) and light chain (VL) that binds to CD16A but not to CD16B may be provided by an antigen-binding site that specifically binds to an epitope of CD16A consisting of the C-terminal sequence SFFPPGYQ (SEQ ID NO: 57) and / or amino acid residues of residues G130 and / or Y141 (SEQ ID NO: 48) of CD16A, which is not present in CD16B.

[0055] "Myeloma cell" refers to a malignant (cancerous) plasma cell in which plasma cells in the bone marrow have arisen by malignant transformation. In myeloma, the malignant plasma cells produce large amounts of abnormal antibodies that are not capable of fighting infection. These abnormal antibodies are so-called monoclonal proteins or M proteins that function as tumor markers of myeloma. Myeloma cells are CD19 - / CD38 + / CD138 + / BCMA +has such a phenotype. Therefore, CD38, CD138, and BCMA represent antigens expressed on myeloma cells. Also included are malignant phenotypes of the B-cell lineage that are positive for CD19 / CD20 / CD22 / BCMA and other antigens (this should include phenotypes that may be an evolution from memory B cells or plasmablast lineages and are not classically understood as plasma cells).

[0056] "EGFR" refers to the epidermal growth factor receptor (epidermal growth factor receptor: EGFR; ErbB-1; HER1) in humans and includes all isoforms or variants described as being involved in activation, mutation, and pathophysiological processes. The EGFR antigen-binding site recognizes epitopes in the extracellular domain of EGFR. In certain embodiments, the antigen-binding site specifically binds to human and cynomolgus EGFR.

[0057] The epidermal growth factor receptor (EGFR) is a member of the HER family of receptor tyrosine kinases and is composed of four members: EGFR (ErbB1 / HER1), HER2 / neu (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Stimulation of the receptor via ligand binding (e.g., EGF, TGFa, HB-EGF, neuregulin, betacellulin, amphiregulin) activates the intrinsic receptor tyrosine kinase in the intracellular domain via tyrosine phosphorylation and promotes homodimerization or heterodimerization of the receptor with HER family members. These intracellular phosphotyrosines function as docking sites for various adapter proteins or enzymes, including MAPK and PI(3)K / Akt, and simultaneously initiate multiple signaling cascades that affect cell proliferation, angiogenesis, apoptosis resistance, invasion, and metastasis.

[0058] "EGFRvIII" refers to an extracellular domain variant of EGFR caused by an in-frame deletion of base pairs spanning exons 2-7 of the EGFR-encoding sequence (Gan HK et al., "FEBS" (2013), Vol. 280, pp. 5350-5370).

[0059] The term "binding domain," in the context of the present invention, characterizes a domain that (specifically) binds / interacts with / recognizes a given target epitope or a given target side on a target molecule (antigen), for example, CD16 and a target cell surface antigen, respectively. The structure and function of the first binding domain (e.g., recognizing CD16), and preferably the structure and / or function of the second binding domain (recognizing the target cell surface antigen), are based on the structure and / or function of an antibody, for example, the structure and / or function of a full-length or whole immunoglobulin molecule, and / or are derived from the variable heavy chain (V H ) and / or variable light chain (V L ) domains. Preferably, the first binding domain is characterized by the presence of three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the V L region), and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the V H region). The second binding domain also preferably includes the minimum structural requirements of an antibody that enable binding to the target. More preferably, the second binding domain includes at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the V L region), and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the V H region). The first and / or second binding domain is envisioned to be produced or obtained by phage display methods or library screening methods, rather than by grafting CDR sequences from existing (monoclonal) antibodies as a scaffold.

[0060] The terms “(specifically) bind”, “(specifically) recognize”, “(specifically) direct”, or “(specifically) react” each mean, according to the present invention, that the binding domain interacts or specifically interacts with a given epitope on a target molecule (antigen), such as CD16a, and a target cell surface antigen, such as BCMA, or a given target side.

[0061] The terms “essentially / substantially do not bind” or “are unable to bind” mean that the binding domain of the present invention does not bind to proteins or antigens other than CD16a, and the target cell surface antigen, i.e., proteins or antigens other than CD16a, and the target cell surface antigen show a reactivity of about 30% or more, preferably about 20% or less, more preferably about 10% or less, particularly preferably about 9% or less, about 8% or less, about 7% or less, about 6% or less, or about 5% or less, whereby the binding to CD16a and the target cell surface antigen is about 100% respectively.

[0062] Specific binding is thought to be affected by specific motifs in the amino acid sequences of the binding domain and the antigen. Thus, binding is achieved as a result of their primary structure, secondary structure, and / or tertiary structure, as well as the secondary modification of the structure. Specific interaction between the antigen interaction side and its specific antigen may result in simple binding between the side and the antigen. Further, specific interaction between the antigen interaction side and its specific antigen may alternatively or additionally initiate a signal, for example, due to induction of a conformational change of the antigen, oligomerization of the antigen, etc.

[0063] The term “variable” refers to the part of an antibody or immunoglobulin domain (i.e., “variable domain(s)”) that shows variability in its sequence and is involved in determining the specificity and binding affinity of a particular antibody. The variable heavy chain (V H ) pairs with the variable light chain (V L ) to form a single antigen-binding site.

[0064] Variability is not uniformly distributed across the entire variable region of an antibody but is concentrated in each of the subdomains of the heavy and light chain variable regions. These subdomains are referred to as “hypervariable regions” or “complementary determining regions” (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domain are called the “framework” regions (FRM or FR) and provide a scaffold for the six CDRs in three-dimensional space to form the antigen-binding surface. The variable domains of the heavy and light chains that exist in nature each contain four FRM regions (FR1, FR2, FR3, and FR4), mainly adopt a β-sheet conformation, are linked by three hypervariable regions, form loops that link to the β-sheet structure and in some cases form part of it. The hypervariable regions within each chain are held together in close proximity by the FRM and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding side (see Kabat et al. above).

[0065] The terms “CDR” and its plural form “CDRs” refer to the complementary determining regions, three of which constitute the binding characteristics of the light chain variable region (CDR-L1, CDR-L2, and CDR-L3) and three of which constitute the binding characteristics of the heavy chain variable region (CDR-H1, CDR-H2, and CDR-H3). CDRs contain most of the residues involved in the specific interaction between the antibody and the antigen and thus contribute to the functional activity of the antibody molecule. CDRs are the major determinants of antigen specificity.

[0066] The exact definitional boundaries and lengths of the CDRs follow different classification and numbering systems. Thus, a CDR can be referred to by any of the Kabat, Chothia, contact, or any other boundary definition including the numbering systems described herein. Despite the different boundaries, each of these systems has some overlap in what constitutes the so-called "hypervariable regions" within the variable arrays. Thus, the CDR definitions according to these systems may differ in length and boundary regions with respect to the adjacent framework regions. See, e.g., Kabat (an approach based on interspecies sequence variation), Chothia (an approach based on crystallographic studies of antigen-antibody complexes), MacCallum, Honegger, and IMGT (Wu and Kabat, "J. Exp. Med." (1970), vol. 132, pp. 211-250; Chothia et al., "J. Mol. Biol." (1987), vol. 196, pp. 901-917; MacCallum et al., "J. Mol. Biol." (1996), vol. 262, p. 732; Lefranc et al., "Dev. Comp. Immunol." (2003); vol. 27, pp. 55-77; Honegger et al., "J. Mol. Bol." (2001) vol. 309, pp. 657-670). Another criterion used to characterize the antigen-binding side is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, e.g., "Protein Sequence and Structure Analysis of Antibody Variable Domains" (Antibody Engineering Lab Manual (Duebel, S. and Kontermann, R., eds., Springer-Verlag, Heidelberg). Two residue identification techniques can be combined to define hybrid CDRs in the context of defining regions that overlap but are not identical. However, numbering according to the so-called Kabat system is preferred. In certain embodiments, the CDRs disclosed herein are identified according to the Kabat numbering system. In certain embodiments, the CDRs disclosed herein are identified according to the IMGT approach.In certain embodiments, the CDRs disclosed herein are identified according to the Honegger approach.

[0067] CDR3 of the light chain, particularly CDR3 of the heavy chain, can constitute the most important determinants in antigen binding within the variable regions of the light and heavy chains. In some antibody constructs, the heavy chain CDR3 appears to constitute the major contact area between the antigen and the antibody. In vitro selection schemes that vary CDR3 alone can be used to vary the binding properties of the antibody or to determine the residues that contribute to antigen binding. Thus, CDR3 is typically the largest source of molecular diversity on the antibody-binding side. H3 can, for example, be as short as two amino acid residues or as long as 26 or more amino acids.

[0068] In classical full-length antibodies or immunoglobulins, each light (L) chain is linked to the heavy (H) chain by one disulfide covalent bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. The CH domain closest to VH is usually called CH1. The constant ("C") domain is not directly involved in antigen binding but exhibits various effector functions such as antibody-dependent, cell-mediated cytotoxicity, and complement activation. The Fc region of the antibody is composed within the heavy chain constant domain and can interact, for example, with Fc receptors located on the cell surface.

[0069] Modifications of the amino acid sequences of the antibody-binding molecules described herein are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody construct. Amino acid sequence variants of the present antibody construct are prepared by introducing appropriate nucleotide changes into the antibody construct nucleic acid or by peptide synthesis. All of the amino acid sequence modifications described below should result in antibody constructs that still retain the desired biological activity (binding to CD16a and the target cell surface antigen) of the unmodified parent molecule.

[0070] Examples of amino acid modifications include, for example, deletions from residues within the amino acid sequence of the antibody binding molecule, and / or insertions into residues within the amino acid sequence of the antibody binding molecule, and / or substitutions of residues within the amino acid sequence of the antibody binding molecule. Any combination of deletions, insertions, and substitutions is made to reach the final molecule, provided that the final molecule has the desired characteristics. Amino acid changes can also modify post-translational processing of the antibody binding molecule, for example, the number or position of glycosylation sites can be altered.

[0071] For example, 1, 2, 3, 4, 5, or 6 amino acids may be inserted, substituted, or deleted in each of the CDRs (of course, depending on their length), while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids may be inserted, substituted, or deleted in each of the FRs. Amino acid sequence insertions into the antibody construct include amino-terminal and / or carboxyl-terminal fusions in the range of polypeptides containing from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues to 100 or more residues, as well as in-sequence insertions of single or multiple amino acid residues. Corresponding modifications may be made within the third domain of the antibody construct as defined in the context of the present invention. Insertion variants of the antibody construct as defined in the context of the present invention include fusions to the N-terminus or C-terminus of the antibody binding molecule of an enzyme, or fusions to a polypeptide.

[0072] The sites of greatest interest for substitution mutagenesis include, but are not limited to, the CDRs of the heavy and / or light chains, particularly the hypervariable regions, although modifications to the FRs in the heavy and / or light chains are also contemplated. The substitutions are preferably conservative substitutions as described herein. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted in the CDRs, while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids may be substituted in the framework region (FR), depending on the length of the CDR or FR. For example, if a CDR sequence comprises 6 amino acids, it is contemplated that 1, 2, or 3 of these amino acids may be substituted. Similarly, if a CDR sequence comprises 15 amino acids, it is contemplated that 1, 2, 3, 4, 5, or 6 of these amino acids may be substituted.

[0073] Generally, when an amino acid is substituted in one or more or all of the CDRs of the heavy and / or light chains, the resulting "substituted" sequence is preferably at least about 60% or about 65%, more preferably about 70% or about 75%, even more preferably about 80% or about 85%, and particularly preferably about 90% or about 95% identical to the "original" CDR sequence. That is, the degree of identity with the "substituted" sequence depends on the length of the CDR. For example, a CDR having 5 amino acids is preferably about 80% identical to its substituted sequence due to at least 1 amino acid being substituted. Thus, the CDRs of an antibody construct may have different degrees of identity to their substituted sequences; for example, CDRL1 may have about 80%, while CDRL3 may have about 90%.

[0074] Preferred substitutions (or exchanges) are conservative substitutions. However, any substitution (including non-conservative substitutions or one or more from the "Exemplary Substitutions" described in Table A below) is envisioned as long as the antibody construct retains its ability to bind to CD16a via the first domain and to a target cell surface antigen via the second domain, and / or its CDRs have an identity (at least about 60% or about 65%, more preferably about 70% or about 75%, even more preferably about 80% or about 85%, and particularly preferably about 90% or about 95% identity to the "original" CDR sequences) to the sequences that are substituted at that time.

[0075] Conservative substitutions are shown under the heading "Preferred Substitutions" in Table A. If such substitutions result in a change in biological activity, they are designated as "Exemplary Substitutions" in Table A, or more substantial changes are introduced with reference to amino acid classes as further described below, and the product can be screened for the desired characteristics.

[0076] TIFF2025081307000002.tif122170

[0077] Substantial modification of the biological properties of the antibody constructs of the present invention is achieved by selecting substitutions that differ significantly in terms of (a) the structure of the polypeptide backbone in the substitution region, e.g., sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the impact of the substitution on maintaining the volume of the side chain. Naturally occurring residues are classified according to their general side chain characteristics: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr, asn, gin; (3) acidic: asp, glu; (4) basic: his, lys, arg; (5) residues affecting strand orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

[0078] Non-conservative substitutions will generally involve exchanging a member of one of these classes for another. Any cysteine residues not involved in maintaining the proper conformation of the antibody construct are generally replaced with serine, which may improve the oxidative stability of the molecule and prevent abnormal crosslinking. Conversely, cysteine bonds (s) can be added to the antibody to improve its stability (especially if the antibody is an antibody fragment such as an Fv fragment).

[0079] For amino acid sequences, sequence identity and / or similarity is determined using standard techniques known in the art, including those described in Smith and Waterman's local sequence identity algorithm (1981), "Adv. Appl. Math.", Vol. 2, p. 482; Needleman and Wunsch's sequence identity alignment algorithm (1970), "J. Mol. Biol.", Vol. 48, p. 443; Pearson and Lipman's similarity search method (1988), "Proc. Nat. Acad. Sci. U.S.A.", Vol. 85, p. 2444; computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, made by the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI); the Best Fit sequence program, Devereux et al. (1984), "Nucl. Acid Res.", Vol. 12, pp. 387-395, preferably using default settings, or determined by inspection. Preferably, percent identity is calculated by FastDB based on the following parameters: mismatch penalty 1; gap penalty 1; gap size penalty 0.33; and linkage penalty 30, "Current Methods in Sequence Comparison and Analysis", Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc.

[0080] An example of a useful algorithm is PILEUP. PILEUP creates multiple sequence alignments from a group of related sequences using progressive pair - wise alignment. It can also plot a phylogenetic tree showing the clustering relationships used to create the alignment. PILEUP uses a simplified version of the progressive alignment method of Feng & Doolittle (1987), "J. Mol. Evol.", Vol. 35, pp. 351 - 360. This method is similar to the method described in Higgins & Sharp (1989), "CABIOS5", pp. 151 - 153. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and an end - gap weight.

[0081] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al. (1990), "J. Mol. Biol.", Vol. 215, pp. 403 - 410; Altschul et al. (1997), "Nucleic Acids Res.", Vol. 25, pp. 3389 - 3402; and Karin et al. (1993), "Proc. Natl. Acad. Sci. U.S.A.", Vol. 90, pp. 5873 - 5787. A particularly useful BLAST program is the WU - BLAST - 2 program obtained from Altschul et al. (1996), "Methods in Enzymology", Vol. 266, pp. 460 - 480. WU - BLAST - 2 uses several search parameters, most of which are set to default values. The adjustable parameters are set to the following values: overlap span = 1, overlap rate = 0.125, word threshold (T)=11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself according to the composition of the specific sequence and the composition of the specific database in which the sequence of interest is searched. However, the values may be adjusted to increase sensitivity.

[0082] An additional useful algorithm is the gapped BLAST described in Altschul et al. (1993), "Nucl. Acids Res.", Vol. 25, pp. 3389-3402. Gapped BLAST uses the BLOSUM-62 substitution score; the threshold T parameter is set to 9; the two-hit method for triggering gapless extension imposes a cost of 10 + k on the gap length k; Xu is set to 16; Xg is set to 40 in the database search stage and 67 in the output stage of the algorithm. Gap alignment is triggered by a score corresponding to about 22 bits.

[0083] Generally, the amino acid homology, similarity, or identity between individual variant CDRs or V H / V L sequences is at least about 60% relative to the sequences depicted herein, more typically, preferably at least about 65% or about 70%, more preferably at least about 75% or about 80%, even more preferably at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and nearly about 100% increase in homology or identity. Similarly, "percent (%) nucleic acid sequence identity" with respect to the nucleic acid sequences of the binding proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical to the nucleotide residues in the coding sequence of the antibody construct. The specific method is to use the BLASTN module of WU-BLAST-2 with the default parameters set such that the overlap span and overlap fraction are set to 1 and 0.125, respectively.

[0084] Generally, the individual variant CDRs or V H / V LThe homology, similarity, or identity of a nucleotide sequence encoding an array to the nucleotide sequences described herein is at least about 60%, more typically preferably at least about 65%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and has increased homology or identity of nearly 100%. Thus, a "variant CDR" or "variant V H / V L region" has a defined homology, similarity, or identity to the parental CDR / VH / VL as defined in the context of the present invention, and shares a biological function including, but not limited to, the specificity and / or activity of the parental CDR or V H / V L at at least about 60%, about 65%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0085] In one embodiment, the percentage of identity of the antibody construct according to the present invention with respect to the human germ cells is ≧ about 70% or ≧ about 75%, more preferably ≧ about 80% or ≧ about 85%, even more preferably ≧ about 90%, and most preferably ≧ about 91%, ≧ about 92%, ≧ about 93%, ≧ about 94%, ≧ about 95%, or even ≧ about 96%. The identity with the germline gene product of the human antibody is considered an important feature for reducing the risk of a therapeutic protein that elicits an immune response in a patient during treatment. Hwang & Foote (「Immunogenicity of engineered antibodies」; 「Methods」, Vol. 36 (2005), pp. 3-10) demonstrated that the reduction of the non-human portion of a drug antibody construct leads to a reduction in the risk of inducing anti-drug antibodies in the treated patient. By comparing a vast number of clinically evaluated antibody pharmaceuticals with their respective immunogenicity data, it has been shown that by humanizing the V region of an antibody, the immunogenicity of the protein is lower (average about 5.1% in patients) than that of an antibody with an unchanged non-human V region (average about 23.59% in patients). Therefore, a higher degree of identity to the human sequence is desirable for V region-based protein therapeutics in the form of antibody constructs. For this purpose of determining germline identity, the V L V region can be aligned with the amino acid sequences of the human germline V and J segments (http: / / vbase.mrc-cpe.cam.ac.uk / ) using Vector NTI software, and the amino acid sequence calculated by dividing by the total number of amino acid residues of V L . The same applies to the VH segment (http: / / vbase.mrc-cpe.cam.ac.uk / ), with the exception that V H CDR3 can be excluded due to its high diversity and the lack of existing human germline V H CDR3 alignment partners. Subsequently, recombinant techniques can be used to increase the sequence identity to the human antibody germline gene.

[0086] As used herein, the term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0087] As used herein, the term "about" or "approximately" means an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., on the limitations of the measurement system. For example, "about" can mean within three or more standard deviations per implementation in the art. Alternatively, "about" can mean within up to 20%, preferably up to 10%, more preferably up to 5%, even more preferably up to 1% of a given value. Alternatively, especially with respect to biological systems or processes, this term can mean within one order of magnitude, preferably within five-fold, more preferably within two-fold of the value.

[0088] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents, and reference to "a method" includes reference to equivalent steps and methods known to one of ordinary skill in the art that can be modified or substituted for the methods described herein.

[0089] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0090] As used herein, the term "and / or" includes the meanings of "and", "or", and "all or any other combination of the elements connected by said term".

[0091] The terms "less than" or "more than" include the specific number. For example, "less than 20" means less than or equivalent. Similarly, "greater than" or "more than" means greater than or equivalent, or more than or equivalent, respectively.

[0092] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are understood to mean the inclusion of the stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps. As used herein, the term "comprising" can be replaced by the term "containing" or "including" when used together with the term "having" herein.

[0093] As used herein, "consisting of" excludes any element, step, or component not specified in the claim. As used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the basic and novel features of the claim.

[0094] In each example herein, any one of the terms "comprising", "consisting essentially of", and "consisting of" may be replaced by any one of the other two terms.

[0095] II. CD16A antigen-binding protein In a first aspect, the present invention provides a CD16A antigen-binding protein for a natural killer (NK) cell-based targeting approach, namely: a multispecific antigen-binding protein, - at least a first target antigen-binding portion; - at least two CD16A antigen-binding portions; - and at least one antibody constant domain, and provides a CD16A antigen-binding protein.

[0096] In some embodiments, the constant domain is part of a Fab fragment or an Fc portion.

[0097] In some embodiments, the CD16A antigen-binding portion is fused to the constant domain, where the constant domain can be part of a Fab fragment (CH1 or CL) or an Fc portion.

[0098] Thus, in a further embodiment, the multispecific antigen-binding protein is - at least a first target antigen-binding portion; - and at least two CD16A antigen-binding portions fused to a constant domain, such as a Fab fragment or an Fc portion.

[0099] This antigen-binding protein acts on NK cells so as to re-induce NK cell-mediated cytotoxicity to a target, such as a tumor antigen-positive cell, a virus-infected cell, or a pathogen.

[0100] The binding strength of NK cell binding via CD16A is increased by the bivalent binding by two CD16A binding portions, and NK cell effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) can be enhanced as compared with a monoclonal antibody or an antibody fragment.

[0101] NK cells are bound via their CD16A receptor by a multispecific antigen-binding protein, thereby being redirected to target, e.g., tumor associated antigen (TAA) expressing target cells. Thus, such multispecific antigen-binding proteins can selectively redirect NK cells and mediate lysis of tumor cells, virus-infected cells, or pathogens. In contrast, full-length antibodies of the IgG isotype bind, via the Fc region, to activating and inhibitory Fcγ receptors including CD16A, CD16B (FcγRIIIB), CD32A (FcγRIIA), CD32B (FcγRIIB), and CD64 (FcγRI). However, antigen-binding proteins having specificity for CD16A selectively target the activating subtype CD16A found on NK cells and macrophages, but not on neutrophils. Furthermore, NK cells that bind the antigen-binding protein interact bivalently with CD16A, resulting in an affinity approximately 1,000-fold higher compared to normal antibodies.

[0102] CD16A is an activating receptor that induces the cytotoxic activity of NK cells. The affinity of antibodies for CD16A directly correlates with their ability to induce NK cell activation. The antigen-binding protein binds bivalently to CD16A, i.e., binds with two antigen-binding moieties, increasing the affinity due to the higher binding force to CD16A.

[0103] Furthermore, by fusing the antigen-binding moiety to an Fc portion, or an IgG-based antibody having FcRn-binding ability, the serum half-life can be extended compared to an scFv-based antigen-binding scaffold, which makes therapeutic application in vivo more convenient. Thereby, such Fc portions can be modified for increased or decreased binding to FcRn. This further regulates the serum half-life and / or the transport of bound IgG via cells. The latter is also called transcytosis (Dickinson et al., 1999).

[0104] Furthermore, the serum half-life can be extended by fusion of the antigen-binding protein with human serum albumin (HSA) or an HSA-binding moiety. For example, while the serum half-life of a Fab fragment can be extended by an HSA-binding moiety in the Fv region of the Fab fragment, the CD16A-binding moiety and the target antigen-binding moiety are fused to the C-terminus of the Fab fragment. Alternatively, the HSA-binding moiety may be fused to the C-terminus of the Fab fragment, and the Fv region of the Fab fragment may provide the target antigen or CD16A-binding moiety.

[0105] The antigen-binding moiety can be fused to the Fab fragment or the Fc moiety in many ways, and the modular structure of the antigen-binding proteins described herein makes available a number of scaffolds that can be readily designed and selected according to the desired application.

[0106] Preferably, V H and V L variable regions are linked by a peptide linker that allows the variable regions to bind in a desired conformation for antigen binding. A polypeptide comprising an extra variable domain region is fused to the constant domain or Fc moiety of the Fab fragment by a linker.

[0107] The antigen-binding moiety is provided by an antigen-binding molecular format comprising one or more Fv polypeptides, such as, for example, single-chain Fv (scFv), tandem single-chain Fv consisting of two scFvs linked in a single polypeptide ((scFv) 2 ), diabody (Db), single-chain diabody (scDb), tandem diabody (TandAb®), Fab, F(ab’) 2 , or dual-affinity retargeting antibody (DART TM ). Particularly preferred as the CD16A antigen-binding protein is scFv, Db, or scDb. Each scFv provides a single antigen-binding moiety, while the bivalent Db, scDb, and (scFv) 2 provide two antigen-binding moieties for the antigen-binding protein. Preferably, the bivalent Db, scDb or (scFv) 2Alternatively, the two scFvs are monospecific and provide two antigen-binding moieties of the same antigen specificity, namely CD16A or the target antigen.

[0108] Each antigen-binding moiety is attached to the constant domain or Fc portion of the Fab by a "connector" or a peptide bond. Preferably, the connector is a peptide. In particular, the connector is a continuous chain of 1 to 30 amino acid residues, for example, a peptide consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acid residues. More specifically, the connector consists of 3 to 30, 6 to 20, 6 to 18, or 6 to 15 amino acid residues. In some embodiments, the connector is composed of G and S amino acid residues, for example, the connector is (G 2 S) x peptide, where x = 1 to 10 or (G 4 S) y and y = 1 to 6, for example, a connector having an amino acid sequence as shown in SEQ ID NO: 19, 20, 21, or 22.

[0109] In certain embodiments, the antigen-binding moiety is fused to the N-terminus of the CH2 domain of the Fc portion via a hinge region. The hinge region may be of the same or a different IgG class as the Fc portion, or it may be a engineered, non-naturally occurring hinge domain. An example of an IgG wild-type hinge region has an amino acid sequence as shown in SEQ ID NO: 23. An example of a modified (shortened) hinge region (middle hinge) has an amino acid sequence as shown in SEQ ID NO: 24.

[0110] In some embodiments, two CD16A antigen-binding portions are fused to the N-terminus of the Fc portion, while one or two target antigen-binding portions are fused to the C-terminus of the Fc portion; or, two CD16A antigen-binding portions are fused to the C-terminus of the Fc portion and one or two target antigen-binding portions are fused to the N-terminus of the Fc portion (e.g., KiH-scDb-Fc; FIGS. 3-6; Db-Fc, FIGS. 9-10; Bi-scFv-Fc, FIGS. 11-12, and KiH-scFv-Fc, FIGS. 15A and 15B).

[0111] In further embodiments, the antigen-binding portion is incorporated or fused into an Ig antibody, e.g., IgG. Each of the CD16A antigen-binding portions can be fused as an scFv to the C-terminus of the Fc portion and to a target antigen-binding portion incorporated into each of the two Fab arms (scFv-IgAb, FIG. 13). Alternatively, each of the CD16A antigen-binding portions can be incorporated as an Fv domain in each of the Fab arms while one or two target antigen-binding portions can be fused to the C-terminus of the Fc portion (scFv-IgAb, FIG. 16). In certain embodiments, additional target antigen-binding portions can be fused to the C-terminus of each of the CL domains (Bi-scFv-IgAb, FIG. 14).

[0112] In further embodiments, two CD16A antigen-binding portions and the target antigen portion are fused to the C-terminus of the Fab while the Fv domain of the Fab provides a second or third target antigen-binding portion (scDb-TriB(-scFv), FIGS. 7 and 8). Alternatively, two CD16A antigen-binding portions can be fused to the C-terminus of the Fab and the target antigen-binding portion is incorporated into the Fv domain of the Fab.

[0113] The antigen-binding protein is at least bivalent with respect to CD16A, i.e., it contains at least two CD16A antigen-binding portions.

[0114] In some embodiments, the antigen-binding protein is a tetravalent bispecific that contains at least two target antigen-binding portions for the same antigen and two CD16A antigen-binding portions.

[0115] In a further embodiment, the antigen-binding portion is a tetravalent triple-specificity comprising a first and a second target antigen-binding portion, and two CD16A antigen-binding portions. Such an antigen-binding protein can be used for simultaneous or dual targeting. For example, such an antigen-binding protein comprises a first tumor antigen-binding portion (TAA1) and a second tumor antigen-binding portion (TAA2), and two CD16A antigen-binding portions for re-inducing the cytotoxicity of NK cells against cells presenting the first (TAA1) and second (TAA2) tumor antigens. Alternatively, using such a triple-specific antigen-binding protein comprising a first (TAA1) tumor antigen-binding portion and a second (TAA2) tumor antigen-binding portion, and two CD16A antigen-binding portions, the cytotoxicity of NK cells can be re-induced in phenotypically different cell types expressing either the first (TAA1) or the second (TAA2) tumor antigen.

[0116] In certain embodiments, the antigen-binding protein can be trivalent and bispecific, trivalent and trispecific, tetravalent and bispecific, tetravalent and trispecific, tetravalent and quadraspecific, hexavalent and trispecific, or hexavalent and bispecific.

[0117] In a second aspect, the present invention provides a specific antigen-binding protein format having at least two CD16A antigen-binding portions and a defined anti-CD16A domain sequence within a polypeptide of a CD16A antigen-binding portion that prevents NK cell fratricide and enables efficient NK cell binding and recruitment for enhanced immune effector functions such as enhanced ADCC.

[0118] A CD16A antigen-binding protein is considered negative for inducing NK-NK lysis if there is no or only slight lysis measurable at antibody concentrations up to 30 μg / mL in an assay where daratumumab induces 50% or more NK lysis.

[0119] The effect of inhibiting fratricide of NK cells should be related to the specific three-dimensional structure characteristics of the antigen-binding proteins described herein, while the binding and mobilization for effector functions of NK cells are enhanced.

[0120] Binding of CD16A at the N-terminus of the antigen-binding proteins disclosed herein (e.g., as described in Example 7) generally resulted in less fratricide of NK cells than binding of CD16A at the C-terminus. However, the present disclosure encompasses antigen-binding proteins that bind CD16A at either the N-terminus or the C-terminus (e.g., see Examples 7, 8, and 9 for examples of binding at either terminus). With regard to N-terminal CD16 binding, more specifically, as disclosed in Example 8, more significant NK cell depletion was observed using Fab-based CD16A binding. Further, by analyzing the domain order of CD16 Fv, an antibody format containing the VL-VH domain order induced a clearly reduced occurrence of NK cell fratricide compared to an antibody format containing the VH-VL order.

[0121] In certain embodiments, the rate of fratricide of NK cells induced by the antibody and the efficacy of NK cell killing are improved by avoiding Fab-based CD16A binding and focusing on the beneficial VL-VH order of the CD16-binding Fv domain.

[0122] In certain embodiments, the variable regions within the polypeptide of the CD16A antigen-binding portion must be sequentially linked such that the light chain variable region (V L ) is located at the N-terminus of the polypeptide, i.e., the Fv polypeptide. The variable region adjacent to the N-terminally located V L may be V L or V H . Notably, fratricide can be avoided independently of the binding affinity of the CD16A antigen-binding portion when the variable regions of the CD16A antigen-binding portion are positioned such that V L is at the N-terminus of the polypeptide of the CD16A antigen-binding portion.

[0123] In particular, when the CD16A antigen-binding portion is a scFv, the variable regions are from the N-terminus to the C-terminus of the polypeptide: V L -V H and are located therein.

[0124] In particular, when the CD16A antigen-binding portion is a Db, the variable regions are from the N-terminus to the C-terminus in each of the two polypeptides forming the Db: V L -V H and are located therein.

[0125] In particular, when the CD16A antigen-binding portion is a scDb, the variable regions are (i) V L -V H -V L -V H or (ii) V L -V L -V H -V H or (iii) V H -V H -V L -V L and are located from the N-terminus to the C-terminus of any one of the polypeptides.

[0126] In particular, when the CD16A antigen-binding portion is (scFv) 2 the variable regions are from the N-terminus to the C-terminus of the polypeptide: V L -V H -V L -V H and are located therein.

[0127] The polypeptide of the CD16A antigen-binding portion, such as scFv, Db, or scDb, is fused via a linker to (i) the N-terminus of the CH2 domain of the Fc portion or to the hinge / mid-hinge region by its C-terminus, or (ii) the C-terminus of the CH3 domain of the Fc portion by its N-terminus, or to the C-terminus of the CL or CH1 domain.

[0128] Preferably, both of the CD16A antigen-binding portions are located within the antigen-binding protein at either the N-terminus or the C-terminus.

[0129] In some embodiments, the CD16A antigen-binding portion is fused to the Fc portion. Preferably, both CD16A-binding portions are fused to the Fc portion either at the N-terminus or the C-terminus. The Fc portion contains a part of a constant region that retains at least one functionality of the IgG Fc region. "Fc portion" includes the native sequence Fc region and variant Fc regions.

[0130] Preferably, the Fc portion is "silenced". "Silenced Fc portion" refers to a modified Fc portion that does not bind to Fc gamma receptors (FcγRs) including CD16A (FcγRIIIA) per se, but retains binding to the neonatal Fc receptor (FcRn). FcRn binding enables transcytosis and recycling across epithelial barriers to protect IgG, or a fusion protein containing an FcRn-binding Fc portion, from lysosomal degradation, resulting in an extended serum half-life and longer persistence in circulation. The antigen-binding protein is designed to specifically bind to CD16A on NK cells via the CD16A antigen-binding portion, and thus, in preferred embodiments, Fc binding to other Fcγ receptors should be prevented. Thus, modifications in the Fc portion of the Fc fusion antigen-binding protein that retain or enhance FcRn binding are preferred.

[0131] Several sets of mutations or changes that generate IgG1 Fc portions with reduced or no binding to Fcγ receptors (referred to as "silencing mutations" or "effectorless mutations") are as follows: mutations in the group consisting of C220S, C229S, E233P, L234A, L234V, L234F, L235A, L235E, P238S, D265A, N297A, N297Q, P331S; or mutations for generating IgG2 Fc portions with reduced binding to Fcγ receptors, which may be selected from the group consisting of H268Q, V309L, A330S, A331S; or mutations for generating IgG4 Fc portions with reduced binding to Fcγ receptors, which may be selected from the group consisting of L235A, G237A, E318A, as described (Strohl W., "Current Opinion in Biotechnology" (2009), Vol. 20, pp. 1-7; Kaneko E and Niwa R, "Biodrugs" (2011), Vol. 25, No. 1, pp. 1-11; Baudino L., "J. Immunology" (2008), Vol. 181, pp. 6664-6669).

[0132] Furthermore, the Fc portion can be engineered, for example, to increase or decrease, to regulate the serum half-life. The following mutations in the IgG1 Fc portion that increase the serum half-life of the antigen-binding protein have been described: T250Q, M252Y, S254T, T256E, T307A, E380A, M428L, H433K, N434A, N434Y (Srohl W., "Current Opinion in Biotechnology" (2009) Vol. 20, pp. 1-7; Borrok MJ et al., "J. Pharmaceutical Sciences" (2017), Vol. 106, No. 4, pp. 1008-1017).

[0133] In some embodiments, the IgG, particularly IgG1, Fc portion includes a combination of mutations at positions 234, 235, and 265 according to Kabat EU numbering, and in particular this combination of mutations is selected from L234F / V / A, L235A / E, and D265A. In particular, an IgG1 Fc portion comprising the combination of mutations L234F, L235E, and D265A is preferred. Thus, in some embodiments, the antigen-binding protein includes a silenced IgG1 Fc portion having the combination of mutations L234F, L235E, and D265A. All mutations described herein correspond to the Kabat EU numbering system (Kabat, E.A. et al., "Sequences of proteins of immunological interest.", 5th Edition, US Department of Health and Human Services, NIH publication, No. 91-3242, pp. 662, 680, 689 (1991)). In certain embodiments, the antigen-binding protein includes a silenced IgG1 Fc portion having the combination of mutations L234F and L235E.

[0134] In some embodiments, the CD16A antigen-binding portion is fused to a homodimeric Fc portion comprising two identical CH2-CH3 polypeptides bound to each other, wherein covalent dimerization is promoted by the hinge region N-terminus to the CH2 domain.

[0135] The antigen-binding portion can be fused N-terminally to the Fc portion via the hinge region or C-terminally to the CH3 domain. When the CD16A antigen-binding portion is fused N-terminally, the first target antigen-binding portion is preferably fused C-terminally to the Fc region.

[0136] In certain embodiments, the two CD16A antigen-binding portions are fused such that the Db is formed at the N-terminus or C-terminus of the Fc portion (Db-Fc) by dimerization, where the CD16A antigen-binding VL-VH polypeptide is fused to the N-terminus of the CH2-CH3 polypeptide or the C-terminus of CH3 via a linker and a hinge region, and the target antigen-binding portion is fused to opposite ends as scFv (Figure 9, Figure 10). Preferably, the hinge is the middle hinge (SEQ ID NO: 24). For example, such an antigen-binding protein comprises two polypeptide chains dimerized in the Fc region, where each polypeptide chain comprises, from N-terminus to C-terminus: (i) V L (CD16A)-V H (CD16A)-hinge-CH2-CH3-V H (target)-V L (target) (Figure 9); (ii) V L (CD16A)-V H (CD16A)-hinge-CH2-CH3-V L (target)-V H (target) (Figure 9); (iii) V L (target)-V H (target)-hinge-CH2-CH3-V L (CD16A)-V H (CD16A) (Figure 10); or, (iv) V H (target)-V L (target)-hinge-CH2-CH3-V L (CD16A)-V H (CD16A) (Figure 10).

[0137] In another embodiment, the two CD16A antigen-binding portions are fused to an Fc portion (Bi-scFv-Fc) as a scFv, where the scFv of the first specificity (target or CD16A binding) is fused to one end of the CH2-CH3 polypeptide and the scFv of the second specificity (CD16A or target binding) is fused to the other end of the CH2-CH3 polypeptide that forms the dimeric Fc portion. Thereby, each of the two target antigen-binding portions is fused as a scFv to the opposing ends of each of the two CH2-CH3 polypeptides (FIGS. 11, 12). For example, such an antigen-binding protein comprises two polypeptide chains that homodimerize in the hinge region and the Fc region, where each polypeptide chain comprises scFvs at the N-terminus and C-terminus of the CH2-CH3 polypeptide, and the domains are located from the following N-terminus to C-terminus: (i) V L (CD16A)-V H (CD16A)-hinge-CH2-CH3-V H (target)-V L (target) (FIG. 11); (ii) V L (CD16A)-V H (CD16A)-hinge-CH2-CH3-V L (target)-V H (target) (FIG. 11); (iii) V L (target)-V H (target)-hinge-CH2-CH3-V L (CD16A)-V H (CD16A) (FIG. 12); or, (iv) V H (target)-V L (target)-hinge-CH2-CH3-V L (CD16A)-V H (CD16A) (FIG. 12).

[0138] In some embodiments, the CD16A antigen-binding portion is fused to a heterodimeric (asymmetric) Fc portion. Such a heterodimeric Fc portion comprises modifications of each of two CH2-CH3 polypeptides of the Fc region that promote dimerization of the two polypeptides (heterodimerization). These comprise separate modifications in each of the polypeptides that are complementary to one another to promote assembly of the two CH2-CH3 polypeptides. The modifications can be nucleic acid mutations that translate to amino acid substitutions. Such variants are known as "knob-into-hole" (KiH), and the construction of numerous variants has been described, for example, by Ridgway et al. ("Protein Engineering", Vol. 9, No. 7, pp. 617-621 (1996)). For example, the substitution T366Y in one CH2-CH3 polypeptide, and the substitution Y407T in the other polypeptide, follow the EU numbering system (Kabat et al., "Sequences of Proteins of Immunological Interest", 5th Edition, NIH (1991)). Examples of Fc portions containing the knob-into-hole substitution T366Y in the first IgG1 CH2-CH3 polypeptide, and the Y407T substitution in the second IgG1 CH2-CH3 polypeptide, have amino acid sequences as shown in SEQ ID NOs: 31, 32. In addition, further strategies for generating complementary interfaces that promote heterodimerization have been developed (reviewed in Brinkmann and Kontermann, "MABS" (2017), Vol. 9, No. 2, pp. 182-212). In further embodiments, disulfide bridges can be incorporated to further stabilize the heterodimer and increase yields (Merchant et al., "Nature Biotech." (1998), Vol. 16, pp. 677-681; Atwell et al., "J. Mol. Biol.", Vol. 270 (1997), pp. 26-35).

[0139] In certain embodiments, the two CD16A antigen-binding portions are fused as scDb to a heterodimeric Fc portion (KiH-scDb-Fc), where a single polypeptide of the scDb is fused to a first CH2-CH3 polypeptide and a single target antigen-binding portion is fused as scFv to opposite ends of the same or a second CH2-CH3 polypeptide (FIGS. 3, 5). For example, such an antigen-binding protein comprises two polypeptide chains that are heterodimerized at the center. A hinge or hinge and Fc region, where the first polypeptide chain is, from the N-terminus to the C-terminus: V L (CD16A)-V H (CD16A)-V L (CD16A)-V H (CD16A)-hinge-CH2-CH3 or V L (CD16A)-V L (CD16A)-V H (CD16A)-V H (CD16A)-hinge-CH2-CH3, and the second polypeptide is, from the N-terminus to the C-terminus: hinge-CH2-CH3-V H (target)-V L (target) or hinge-CH2-CH3-V L (target)-V H (target), (FIG. 3); or the first polypeptide chain is, from the N-terminus to the C-terminus: hinge-CH2-CH3-V L (CD16A)-V H (CD16A)-V L (CD16A)-V H (CD16A) or hinge-CH2-CH3-V L (CD16A)-V L (CD16A)-V H (CD16A)-V H (CD16A), and the second polypeptide is from the N-terminus to the C-terminus: V H (target)-V L (target)-hinge-CH2-CH3 or V L (target)-V H(Target)-hinge-CH2-CH3 containing (Figure 5); or, a single polypeptide of the scDb is fused to a first CH2-CH3 polypeptide, and a single target antigen-binding portion is fused as an scFv to the same end of a second CH2-CH3 polypeptide (Figure 4). For example, such an antigen-binding protein comprises two polypeptide chains, wherein the first polypeptide chain is, from the following N-terminus to C-terminus: V L (CD16A)-V H (CD16A)-V L (CD16A)-V H (CD16A)-hinge-CH2-CH3 or V L (CD16A)-V L (CD16A)-V H (CD16A)-V H (CD16A)-hinge-CH2-CH3 containing, and the second polypeptide is, from the N-terminus to C-terminus: V H (Target)-V L (Target)-hinge-CH2-CH3 or V L (Target)-V H (Target)-hinge-CH2-CH3 containing (Figure 4).

[0140] In another embodiment, two CD16A antigen-binding portions are fused as an scDb to a heterodimeric Fc portion, a single polypeptide of the scDb is fused to a first CH2-CH3 polypeptide, a single first target antigen-binding portion (TAA1) is fused as an scFv to the respective opposite ends of this first CH2-CH3 polypeptide, and a second target antigen-binding portion (TAA2) is fused as an scFv to the second CH2-CH3 polypeptide at the same end as the first target antigen-binding portion (Figure 6). For example, such an antigen-binding protein comprises two polypeptide chains that heterodimerize in the hinge or middle hinge and the Fc region, wherein the first polypeptide chain is, from the N-terminus to C-terminus: V H (Target 1)-V L (Target 1)-hinge-CH2-CH3-V L (CD16A)-V H (CD16A)-V L (CD16A)-V H (CD16A)) or VH (Target 1)-V L (Target 1)-hinge-CH2-CH3-V L (CD16A)-V L (CD16A)-V H (CD16A)-V H (CD16A) or V L (Target 1)-V H (Target 1)-hinge-CH2-CH3-V L (CD16A)-V H (CD16A)-V L (CD16A)-V H (CD16A) or V L (Target 1)-V H (Target 1)-hinge-CH2-CH3-V L (CD16A)-V L (CD16A)-V H (CD16A)-V H (CD16A) contains, and the second polypeptide is from N-terminus to C-terminus: V H (Target 2)-V L (Target 2)-hinge-CH2-CH3 or V L (Target 2)-V H (Target 2)-hinge-CH2-CH3 is included (Figure 6).

[0141] In a further embodiment, two CD16A antigen-binding portions are each fused as scFvs to a heterodimeric Fc portion (KiH-scFv-Fc), and each of the two scFvs is fused to one of the first and second CH2-CH3 polypeptides, a single first target antigen-binding portion (TAA1) is fused as an scFv to opposite ends of the first CH2-CH3 polypeptide, and a second target antigen-binding portion (TAA2) is fused as an scFv to the second CH2-CH3 polypeptide at the same end as the first target antigen-binding portion (Figure 15). For example, such an antigen-binding protein comprises two polypeptide chains heterodimerized in the Fc region, where the first polypeptide chain is from the following N-terminus to C-terminus: V H (Target 1)-V L (Target 1)-hinge-CH2-CH3-V L (CD16A)-V H(CD16A) or V L (Target 1)-V H (Target 1)-hinge-CH2-CH3-V L (CD16A)-V H (CD16A)-containing, the second polypeptide is from N-terminus to C-terminus: V H (Target 2)-V L (Target 2)-hinge-CH2-CH3-V L (CD16A)-V H (CD16A) or V L (Target 2)-V H (Target 2)-hinge-CH2-CH3-V L (CD16A)-V H (CD16A)-containing (Figure 15b); alternatively, such an antigen-binding protein comprises two polypeptide chains hetero-dimerized in the Fc region, where the first polypeptide chain is from N-terminus to C-terminus: V L (CD16A)-V H (CD16A)-hinge-CH2-CH3-V H (Target 1)-V L (Target 1) or V L (CD16A)-V H (CD16A)-hinge-CH2-CH3-V L (Target 1)-V H (Target 1)-containing, the second polypeptide is from N-terminus to C-terminus: V L (CD16A)-V H (CD16A)-hinge-CH2-CH3-V H (Target 2)-V L (Target 2) or V L (CD16A)-V H (CD16A)-hinge-CH2-CH3-V L (Target 2)-V H (Target 2)-containing (Figure 15a).

[0142] In a further embodiment, the two CD16A antigen-binding moieties are fused as scDb to a monomeric Fc portion (scDb-mFc, FIGS. 1 and 2). Such a monomeric Fc portion (mFc) contains a mutation in the CH3 domain that prevents dimerization with another CH2-CH3 polypeptide, lacks a hinge region, maintains binding to the FcRn receptor, and does not bind to other FcγRs. Such mutations are described in Ying et al. ("MABS" (2014), Vol. 6, No. 5, pp. 1201-1210; or WO 2013 / 138643). An example of such a monomeric Fc portion has an amino acid sequence as shown in SEQ ID NO: 30. Such an antigen-binding protein consists of a single polypeptide chain, where the scDb containing the two CD16A antigen-binding moieties is fused to one end of the monomeric Fc portion, and the target antigen-binding moiety is fused to the other end of the monomeric Fc portion. Such an antigen-binding protein has, from the N-terminus to the C-terminus: V L (CD16A)-V H (CD16A)-V L (CD16A)-V H (CD16A)-CH2-CH3-V H (Target)-V L (Target) or V L (CD16A)-V H (CD16A)-V L (CD16A)-V H (CD16A)-CH2-CH3-V L (Target)-V H (Target), or V L (CD16A)-V L (CD16A)-V H (CD16A)-V H (CD16A)-CH2-CH3-V H (Target)-V L (Target), or V L (CD16A)-V L (CD16A)-V H (CD16A)-V H (CD16A)-CH2-CH3-V L (Target)-V H (Target) (FIG. 1), or V H (Target)-V L(Target)-CH2-CH3-V L (CD16A)-V H (CD16A)-V L (CD16A)-V H (CD16A), or V L (Target)-V H (Target)-CH2-CH3-V L (CD16A)-V H (CD16A)-V L (CD16A)-V H (CD16A), or V H (Target)-V L (Target)-CH2-CH3-V L (CD16A)-V L (CD16A)-V H (CD16A)-V H (CD16A), or V L (Target)-V H (Target)-CH2-CH3-V L (CD16A)-V L (CD16A)-V H (CD16A)-V H (CD16A) (Figure 2) consists of a single polypeptide chain.

[0143] In other embodiments, the N-terminus of the polypeptide of the antigen-binding portion fuses to the C-terminus of the Fab fragment. For example, one Db, scDb, or two scFvs containing two CD16A antigen-binding portions fuse to the C-terminus of the Fab fragment, and the Fv of the Fab fragment contains the target antigen-binding portion.

[0144] In other embodiments, the antigen-binding protein is trispecific (scDb-TriB(-scFv), where the scDb containing two CD16A antigen-binding moieties is fused C-terminally to the first of the two polypeptides of the Fab fragment, and the first target antigen-binding moiety (TAA1) is fused C-terminally to the second polypeptide of the Fab fragment as an scFv or as a bivalent scDb, and the Fv of the Fab fragment provides the second target antigen-binding moiety (TAA2). In certain embodiments, the second target antigen-binding moiety of the Fv is a human serum albumin (HSA) antigen-binding moiety. Such an antigen-binding protein comprises two polypeptide chains heterodimerized in a Fab fragment fusion having two scDbs fused C-terminally to the Fab, where the first polypeptide chain is, from N-terminus to C-terminus: V L (HSA)-CL-V L (CD16A)-V H (CD16A)-V L (CD16A)-V H (CD16A), and the second polypeptide is, from N-terminus to C-terminus: V H (HSA)-CH1-V H (Target 1)-V L (Target 1)-V H (Target 1)-V L (Target 1) (Figure 7). In another embodiment, the scDb containing two CD16A antigen-binding moieties and the scFv containing the first target antigen-binding moiety are fused C-terminally to the Fab fragment, where the first polypeptide chain is, from N-terminus to C-terminus: V L (HSA)-CL-V H (Target 1)-V L (Target 1), and the second polypeptide is, from N-terminus to C-terminus: V H (HSA)-CH1-V L (CD16A)-V H (CD16A)-V L (CD16A)-V H (CD16A) (Figure 8).

[0145] In a further embodiment, the two CD16A antigen-binding portions are fused to the C-terminus of IgG, where the two Fab arms of the IgG provide two additional antigen-binding portions (scFv-IgAb, FIG. 13). In such an antigen-binding protein, the first scFv containing the CD16A antigen-binding portion is fused to the CH3 of the first heavy chain at the C-terminus, and the second scFv containing the CD16A antigen-binding portion is fused to the CH3 of the second heavy chain, and the two Fab arms provide a target antigen-binding portion together with each Fv. Both Fv's can have the same target antigen-binding portion, or the two Fv's can provide the first and second target antigen-binding portions. For example, such an antigen-binding protein contains two identical heavy chains and two identical light chains, where the heavy chain is from N-terminus to C-terminus: V H (target)-CH1-hinge-CH2-CH3-V L (CD16A)-V H (CD16A), and the light chain is from N-terminus to C-terminus: V L (target)-CL (FIG. 13).

[0146] Alternatively, the scFv-IgAb of the two CD16A antigen-binding portions is provided by the two Fv's of the Fab arms of IgG, and one or two additional antigen-binding portions are fused to the C-terminus of IgG. In such an antigen-binding protein, each of the two Fab arms provides the CD16A antigen-binding portion with each Fv, the first scFv containing the target antigen-binding portion is fused to the CH3 of the first heavy chain at the C-terminus, and optionally, the second scFv containing a further first or second target antigen-binding portion is fused to the CH3 of the second heavy chain. For example, such an antigen-binding protein contains two identical heavy chains and two identical light chains, where the heavy chain is from N-terminus to C-terminus: V H (CD16A)-CH1-hinge-CH2-CH3-V L (target)-V H (target), and the light chain is from N-terminus to C-terminus: V L (CD16A)-CL (FIG. 16), or the heavy chain is from N-terminus to C-terminus: V H (CD16A)-CH1-hinge-CH2-CH3-V H (target)-V L(Target), and the L chain is from the N-terminus to the C-terminus: V L (CD16A)-CL

[0147] In a further embodiment, two CD16A antigen-binding portions are fused at the C-terminus to an IgG, and the two Fab arms of the IgG provide two first target antigen-binding portions (TAA1), and a further scFv second target antigen-binding portion (TAA2) is fused at the C-terminus to each of the two CL domains (Bi-scFv-IgAb, Figure 14). In such an antigen-binding protein, a first scFv comprising a CD16A antigen-binding portion is fused at the C-terminus to the CH3 of the first H chain, a second scFv comprising a CD16A antigen-binding portion is fused to the CH3 of the second H chain, the two Fab arms provide a first target antigen-binding portion (TAA1) having each Fv, and two further second target antigen-binding portions (TAA2) are provided by each of the scFv second target antigen-binding portions fused to each of the CL domains. For example, such an antigen-binding protein comprises two identical H chains and two identical L chains, where the H chain is from the N-terminus to the C-terminus: V H (Target 1)-CH1-hinge-CH2-CH3-V L (CD16A)-V H (CD16A), and the L chain is from the N-terminus to the C-terminus: V L (Target 1)-CL-V L (Target 2)-V H (Target 2) or V L (Target 1)-CL-V H (Target 2)-V L (Target 2) (Figure 14).

[0148] According to the present invention, a multispecific antigen-binding protein comprises a CD16A antigen-binding portion that specifically binds to CD16A and at least a target antigen (TA) different from CD16A.

[0149] In some embodiments, the CD16A-binding portion binds to human and cynomolgus CD16A.

[0150] In some embodiments, the CD16A antigen-binding portion comprises a heavy chain variable domain and a light chain variable domain that are specific for CD16A, where (i) the heavy chain variable domain (V H ) specific for CD16A comprises a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 50; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 51 or SEQ ID NO: 56; a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable domain (V L ) specific for CD16A comprises a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 53; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 54; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 55; or (ii) the heavy chain variable domain (V H ) specific for CD16A has the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, and / or (iii) the light chain variable domain (V L ) specific for CD16A has the amino acid sequence shown in SEQ ID NO: 2.

[0151] In some embodiments, the CD16A antigen-binding portion comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ), where (a) V H comprises a CDR1 comprising or having the amino acid sequence shown in SEQ ID NO: 73, a CDR2 comprising or having the amino acid sequence shown in SEQ ID NO: 74, and a CDR3 comprising or having the amino acid sequence shown in SEQ ID NO: 75; and (b) V L comprises a CDR1 comprising or having the amino acid sequence shown in SEQ ID NO: 76, a light chain CDR2 comprising or having the amino acid sequence shown in SEQ ID NO: 77, and a CDR3 comprising or having the amino acid sequence shown in SEQ ID NO: 78.

[0152] In certain embodiments, the V H of the CD16A antigen-binding portion comprises or has the amino acid sequence shown in SEQ ID NO: 3. In certain embodiments, the VL comprises or has the amino acid sequence shown in SEQ ID NO: 2.

[0153] In some embodiments, the CD16A antigen-binding portion comprises V H and V L wherein: (a) V H comprises a CDR1 comprising or having the amino acid sequence shown in SEQ ID NO: 82, a CDR2 comprising or having the amino acid sequence shown in SEQ ID NO: 83, and a CDR3 comprising or having the amino acid sequence shown in SEQ ID NO: 52; and (b) V L comprises a CDR1 comprising or having the amino acid sequence shown in SEQ ID NO: 84, a CDR2 comprising or having the amino acid sequence shown in SEQ ID NO: 85, and a CDR3 comprising or having the amino acid sequence shown in SEQ ID NO: 86.

[0154] In certain embodiments, the V H of the CD16A antigen-binding portion comprises or has the amino acid sequence shown in SEQ ID NO: 4. In certain embodiments, the V L of the CD16A antigen-binding portion comprises or has the amino acid sequence shown in SEQ ID NO: 5.

[0155] In some embodiments, the CD16A antigen-binding portion comprises V H and V L wherein: (a) V H comprises a CDR1 comprising or having the amino acid sequence shown in SEQ ID NO: 87, a CDR2 comprising or having the amino acid sequence shown in SEQ ID NO: 88, and a CDR3 comprising or having the amino acid sequence shown in SEQ ID NO: 52; and (b) V L comprises a CDR1 comprising or having the amino acid sequence shown in SEQ ID NO: 89, a CDR2 comprising or having the amino acid sequence shown in SEQ ID NO: 95, and a CDR3 comprising or having the amino acid sequence shown in SEQ ID NO: 90.

[0156] In certain embodiments, the V Hcomprises or has the amino acid sequence shown in SEQ ID NO: 6. In certain embodiments, the V L of the CD16A antigen-binding portion comprises or has the amino acid sequence shown in SEQ ID NO: 7.

[0157] In some embodiments, the CD16A antigen-binding portion comprises V H and V L wherein (a) V H comprises CDR1 comprising or having the amino acid sequence shown in SEQ ID NO: 91, CDR2 comprising or having the amino acid sequence shown in SEQ ID NO: 92, and CDR3 comprising or having the amino acid sequence shown in SEQ ID NO: 52; and (b) V L comprises CDR1 comprising or having the amino acid sequence shown in SEQ ID NO: 93, CDR2 comprising or having the amino acid sequence shown in SEQ ID NO: 85, and CDR3 comprising or having the amino acid sequence shown in SEQ ID NO: 94.

[0158] In certain embodiments, the V H of the CD16A antigen-binding portion comprises or has the amino acid sequence shown in SEQ ID NO: 8. In certain embodiments, the V L of the CD16A antigen-binding portion comprises or has the amino acid sequence shown in SEQ ID NO: 9.

[0159] In some embodiments, the CD16A antigen-binding portion comprises V H and V L wherein (a) V H comprises CDR1 comprising or having the amino acid sequence shown in SEQ ID NO: 82, CDR2 comprising or having the amino acid sequence shown in SEQ ID NO: 95, and CDR3 comprising or having the amino acid sequence shown in SEQ ID NO: 52; and (b) V L comprises CDR1 comprising or having the amino acid sequence shown in SEQ ID NO: 96, CDR2 comprising or having the amino acid sequence shown in SEQ ID NO: 97, and CDR3 comprising or having the amino acid sequence shown in SEQ ID NO: 98.

[0160] In certain embodiments, the V of the CD16A antigen-binding portion H comprises or has the amino acid sequence shown in SEQ ID NO: 10. In certain embodiments, the V of the CD16A antigen-binding portion L comprises or has the amino acid sequence shown in SEQ ID NO: 11.

[0161] In some embodiments, the CD16A antigen-binding portion comprises V H and V L wherein: (a) V H comprises CDR1 comprising or having the amino acid sequence shown in SEQ ID NO: 82, CDR2 comprising or having the amino acid sequence shown in SEQ ID NO: 99, and CDR3 comprising or having the amino acid sequence shown in SEQ ID NO: 52; and (b) V L comprises CDR1 comprising or having the amino acid sequence shown in SEQ ID NO: 100, CDR2 comprising or having the amino acid sequence shown in SEQ ID NO: 101, and CDR3 comprising or having the amino acid sequence shown in SEQ ID NO: 102.

[0162] In certain embodiments, the V of the CD16A antigen-binding portion H comprises or has the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, the V of the CD16A antigen-binding portion L comprises or has the amino acid sequence shown in SEQ ID NO: 13.

[0163] In some embodiments, the CD16A antigen-binding portion comprises V H and V L wherein: (a) V H comprises CDR1 comprising or having the amino acid sequence shown in SEQ ID NO: 50, CDR2 comprising or having the amino acid sequence shown in SEQ ID NO: 103, and CDR3 comprising or having the amino acid sequence shown in SEQ ID NO: 52; and (b) V L comprises CDR1 comprising or having the amino acid sequence shown in SEQ ID NO: 53, CDR2 comprising or having the amino acid sequence shown in SEQ ID NO: 54, and CDR3 comprising or having the amino acid sequence shown in SEQ ID NO: 104.

[0164] In some embodiments, the CD16A antigen-binding portion comprises V H and V L wherein (a) V H comprises CDR1 comprising or having the amino acid sequence set forth in SEQ ID NO: 50, CDR2 comprising or having the amino acid sequence set forth in SEQ ID NO: 103, and CDR3 comprising or having the amino acid sequence set forth in SEQ ID NO: 52; and (b) V L comprises CDR1 comprising or having the amino acid sequence set forth in SEQ ID NO: 53, CDR2 comprising or having the amino acid sequence set forth in SEQ ID NO: 54, and CDR3 comprising or having the amino acid sequence set forth in SEQ ID NO: 105.

[0165] This CD16A antigen-binding portion to CD16A does not bind to CD16B and binds to known CD16A allotypes F158 and V158 with similar affinities. Two allelic single nucleotide polymorphisms that alter the amino acid at position 158, which is important for the interaction with the hinge region of IgG, have been identified in human CD16A. The allelic frequencies of the homozygous 158F / F and heterozygous 158V / F alleles were similar within the Caucasian population, in the ranges of 35 - 52% and 38 - 50%, respectively, while the homozygous 158V / V allele was found only in 10 - 15% (Lopez - Escamez JA et al., "BMC Med Genet" (2011), Vol. 12, p. 2). Thus, binding and activation of NK cells by this anti - CD16A domain in all patient populations with similar affinities is advantageous. This CD16A antigen-binding portion binds to human and cynomolgus CD16A. Further, a CD16A antigen-binding portion comprising heavy and light chain variable domains that binds to CD16A but not to CD16B is described in WO 2006 / 125668.

[0166] In some embodiments, the multispecific antigen-binding molecule comprises an anti-CD16A Fv domain with improved binding to CD16A compared to anti-CD16A having SEQ ID NOs: 1 and 2, does not bind to CD16B, and recognizes the known CD16A allotypes F158 and V158 with a similar affinity. In certain embodiments, the anti-CD16A Fv domain with improved affinity comprises a VCDR having a sequence as shown in SEQ ID NOs: 53, 54, 55, or a V having a sequence as shown in SEQ ID NO: 2 and having at least one modification in the sequence as shown in SEQ ID NO: 2. L , and a V consisting of H and having at least one modification in the sequence as shown in SEQ ID NO: 2.

[0167] Such improved binding agents can be affinity-matured CD16A Fv antigen-binding moieties.

[0168] Accordingly, in some embodiments, the CD16A antigen-binding moiety, when tested by ELISA on wells coated with 0.16 μg / mL of human CD16A (SEQ ID NO: 48) measured with a purified scFv molecule in a Biacore T200 system, exhibits a higher affinity (e.g., improvement by affinity maturation) and / or selectivity for human CD16A than the anti-CD16A Fv of SEQ ID NOs: 1 and 2, and comprises an Fv domain. For example, such a higher affinity for human CD16A can exceed 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 150-fold. For example, the monovalent binding affinity measured on a Biacore T200 system can be K D = 0.27 nM to 1.91 nM, which is 21 to 150-fold higher than 40.5 nM measured for the binding of SEQ ID NOs: 1 and 2 to human CD16A.

[0169] Preferably, such affinity-matured Fv domains further show similarly improved affinity for cynomolgus CD16A when tested by ELISA on wells coated with 0.16 μg / mL cynomolgus CD16A (SEQ ID NO: 49) measured with an scFv molecule purified on a Biacore T200 system X100. For example, such higher affinity for cynomolgus CD16A can exceed 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 150, 200, 250, 300, 400, 500, 541-fold. For example, the monovalent binding affinity measured on the Biacore T200 system can be K D = 0.24 - 3.63 nM, which is 36 - 541-fold higher than 132 nM measured for binding to cynomolgus CD16A of SEQ ID NOs: 1, 2.

[0170] In the anti-CD16 Fvs of SEQ ID NOs: 1, 2, specific sequence positions (X37, X44, X46) have been identified that are beneficial for specific CD16A binding in the V L domain (SEQ ID NO: 2). See Table 1.

[0171] TIFF2025081307000003.tif65170

[0172] Not all amino acid substitutions shown in Table 1 are equally important for binding to CD16A. Substitutions at positions X44 and X46 may be more important, and preferably, the affinity-matured clone conserves the amino acids of SEQ ID NO: 2.

[0173] In certain embodiments, further involved in CD16A binding are the sequence of LCDR3, and the NI N-terminal residue of LCDR1 or the QDX sequence motif in LCDR2.

[0174] In certain embodiments, the V of the CD16A antigen-binding portion Lcomprises light chain CDR1, light chain CDR2, and light chain CDR3 that contain 1, 2, 3, 4, or 5 amino acid substitutions as compared to the sequences set forth in SEQ ID NOs: 53, 54, and 55, and V L retains the binding specificity to CD16A. In certain embodiments, one or more substitutions in the sequence of SEQ ID NO: 53 are at positions 3 to 6. In certain embodiments, the substitution in the sequence of SEQ ID NO: 54 is at position 3. In certain embodiments, the residue Y at position 6 and the residue V at position 8 of the sequence of SEQ ID NO: 55 are not substituted, and. Preferably, LCDR3 has the sequence set forth in SEQ ID NO: 55.

[0175] In a further embodiment, the V of the CD16A antigen-binding portion L has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity as compared to the sequence of SEQ ID NO: 2, wherein the residues GGHNI from positions 23 to 27 of SEQ ID NO: 2 and the sequence motif QDXK from positions 49 to 52 of SEQ ID NO: 2 are not mutated, and V L retains the binding specificity to CD16A.

[0176] The V of the CD16A antigen-binding portion H in which, as compared to the sequence of SEQ ID NO: 1, substitutions at position 9 of HCDR1 and / or position 11 of HCDR2 are beneficial for improving the binding to CD16A. In particular, the residue at position 9 of HCDR1 is Q and / or the residue at position 11 of HCDR2 is V.

[0177] Thus, in a further embodiment, the V of the CD16A antigen-binding portion H has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity as compared to the sequence of SEQ ID NO: 1, the residue at position 9 of HCDR1 is Q, and / or the residue at position 11 of HCDR2 is V.

[0178] Accordingly, in one embodiment, the CD16A antigen-binding portion comprises V L and V H wherein the V L of CD16A has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity compared to the sequence of SEQ ID NO: 2, wherein the residues GGHNI at positions 23-27 of SEQ ID NO: 2 and the sequence motif QDXK at positions 49-52 of SEQ ID NO: 2 are not mutated, and V L retains the binding specificity to CD16A, and the V H of the CD16A antigen-binding portion has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity compared to the sequence of SEQ ID NO: 1, the residue at position 9 of HCDR1 is Q, and / or the residue at position 11 of HCDR2 is V.

[0179] In a further embodiment, the V L of the CD16A antigen-binding portion has at least about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, or about 80% sequence identity compared to the sequence of SEQ ID NO: 2, wherein the residues GGHNI at positions 23-27 of SEQ ID NO: 2 and the sequence motif QDXK at positions 49-52 of SEQ ID NO: 2 are not mutated, and V L retains the binding specificity to CD16A.

[0180] In the V H of the CD16A antigen-binding portion, the substitutions at position 9 of HCDR1 and / or position 11 of HCDR2 are beneficial for improving the binding to CD16A compared to the sequence of SEQ ID NO: 1. In particular, the residue at position 9 of HCDR1 is Q, and / or the residue at position 11 of HCDR2 is V.

[0181] Thus, in a further embodiment, the V of the CD16A antigen-binding portion H has at least about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, or about 80% sequence identity compared to the sequence of SEQ ID NO: 1, the residue at position 9 of HCDR1 is Q, and / or the residue at position 11 of HCDR2 is V.

[0182] Thus, in one embodiment, the CD16A antigen-binding portion is V L and V H wherein the V of CD16A L has at least about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, or about 80% sequence identity compared to the sequence of SEQ ID NO: 2, wherein the residues GGHNI from positions 23 to 27 of SEQ ID NO: 2 and the sequence motif QDXK from positions 49 to 52 of SEQ ID NO: 2 are not mutated, V L retains the binding specificity to CD16A, and the V of the CD16A antigen-binding portion H has at least about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, or about 80% sequence identity compared to the sequence of SEQ ID NO: 1, the residue at position 9 of HCDR1 is Q, and / or the residue at position 11 of HCDR2 is V.

[0183] In a particular embodiment, the V of the CD16A antigen-binding portion H has at least about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, or about 80% sequence identity compared to the sequence shown in SEQ ID NO: 3.

[0184] In certain embodiments, the CD16A antigen-binding portion comprises an amino acid sequence that is at least about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, or about 80% identical or homologous to the amino acid sequence shown in SEQ ID NO: 2, and a V H that, and an amino acid sequence that is at least about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, or about 80% identical or homologous to the amino acid sequence shown in SEQ ID NO: 3, and a V L comprising.

[0185] In certain embodiments, the CD16A antigen-binding portion is V L and V H comprising, wherein V L is selected from the group consisting of regions having SEQ ID NOs: 2, 5, 7, 9, 11, and 13, and V H is selected from the group consisting of SEQ ID NOs: 1, 3, 4, 6, 8, 10, and 12.

[0186] III. Target Antigen-Binding Portion In certain embodiments, the target antigen of the target antigen-binding portion is an antigen presented on myeloma cells or plasma cells. Myeloma cells are malignant (cancerous) plasma cells that arise from plasma cells in the bone marrow. In myeloma, the malignant plasma cells produce large amounts of abnormal antibodies that are unable to fight infection. These abnormal antibodies are monoclonal proteins or M proteins that function as tumor markers of myeloma. Myeloma cells have the phenotype of CD19 - / CD38 + / CD138 + / BCMA + Thus, CD38, CD138, and BCMA represent antigens expressed on myeloma cells.

[0187] A. BCMA B cell maturation antigen (BCMA, CD269, or TNFRSF17) is a protein of the TNF receptor superfamily that is important for the long-term survival of plasma cells through binding of B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) (O’Connor, B.P. et al., “BCMA is essential for the survival of long-lived bone marrow plasma cells.”, J. Exp. Med. (2004), vol. 199, pp. 91-96). Human BCMA is an 184 amino acid (aa) protein consisting of a 54aa extracellular domain, a 23aa transmembrane domain, and a 107aa intracellular domain (Entrez Gene ID: 608 (human), 102145399 (cynomolgus monkey); UniProt Q02223 (human)).

[0188] BCMA plays a role in the long-term cell survival of plasma cells (O’Connor, “JEM” (2004), vol. 199, no. 1, pp. 91-98). BCMA is expressed in normal plasma cells, upregulated in multiple myeloma, and has a high morbidity rate.

[0189] In certain embodiments, the target antigen-binding portion specifically binds to BCMA, such as the extracellular domain of BCMA.

[0190] Preferably, such an anti-BCMA Fv used in the antigen-binding protein of the invention has an equilibrium dissociation constant (K -7 less than 10 -8 M, preferably less than 10 -9 M, most preferably less than 10 -10 M (measured by Biacore) and interacts with BCMA. Such an anti-BCMA Fv domain incorporated into the target antigen-binding portion can re-direct CD16A-expressing NK cells and induce ADCC in the presence of BCMA D MM (multiple myeloma) cells. BCMA can be used both in vitro and in vivo + and +Proof-of-concept for bispecific antibodies that bind T cells via CD3 to myeloma cells has been reported (e.g., Hipp S. et al., "Leukemia." (August 2017); Vol. 31, No. 8, pp. 1743-1751 (Epub Dec 27, 2016)). In certain embodiments, the target antigen-binding portion binds to BCMA with a K -9 of about 1×10 -9 M to about 5×10 D M. In certain embodiments, the target antigen-binding portion binds to BCMA with a K -9 of about 2×10 D M.

[0191] Such BCMA antigen-binding portions can be obtained, for example, by phage or ribosome library screening methods, or by immunization of non-human animals with the extracellular domain of BCMA, as described, for example, in Ryan M.C. et al., "Antibody targeting of B-cell maturation antigen on malignant plasma cells" ( "Mol Cancer Ther." (2007), Vol. 6, pp. 3009-3018).

[0192] Ryan et al. describe the production of anti-BCMA antibodies having cytotoxic activity either as IgG or as antibody-drug conjugates. Ryan M.C. et al., which is incorporated by reference, describe the generation of human BCMA-selective antibodies for tumor cell targeting. Antibodies against the human BCMA extracellular domain were generated (ECD, amino acids 5-51; NP_001183). This antibody induced potent ADCC against MM cells in vitro and was increased by an Fc mutation that enhanced CD16A binding. The binding affinity K D of SG1 for H929 cells was 51 nmol / L by saturation binding. These antibodies showed in vitro antitumor activity against MM cell lines, and thus their Fv domains can be used as the BCMA antigen-binding portion in the antigen-binding proteins according to the present invention.

[0193] Furthermore, WO 02 / 066516 describes BCMA antibodies that cross-react with TACI. Anti-BCMA / TACI bispecific antibodies are described as binding to residues 1-48 of BCMA and residues 30-67 and 68-154 of TACI. Their variable heavy chain and variable light chain domains can be used as the BCMA antigen-binding portion in the antigen-binding proteins of the present invention and are incorporated by reference.

[0194] Ramadoss et al., "J. Am. Chem. Soc." (2015), vol. 137, pp. 5288-5291, incorporated by reference, describes bispecific (BiFab-BCMA) antibodies that re-direct T cells to lyse malignant MM cells. Bispecific antibodies are described as potentially useful in the treatment of MM because they target both quiescent cancer stem cells and a small number of tumor-associated antigens.

[0195] WO 2014 / 122144 describes bispecific antibodies that specifically bind to human BCMA and CD3 in a bispecific format. The disclosed anti-BCMA variable domains are suitable for the BCMA antigen-binding portion according to the present invention.

[0196] WO 2013 / 072406 discloses anti-BCMA Fv domains designated as BCMA-1 to BCMA-108 in bispecific single-chain antibodies having a second specificity for CD3 for binding T cells. The anti-tumor efficacy of these BCMA / CD3 bispecific single-chain antibodies in a human tumor xenograft model is described, and thus these anti-BCMA Fv domains can be used for the BCMA antigen-binding portion of the present invention.

[0197] Additional anti-BCMA antibodies that can be used in bispecific antibodies that bind to immune effector cells such as T cells or NK cells are disclosed in International Publication Nos. WO 2010 / 104949, WO 2012 / 163805, WO 2013 / 072415, WO 2014 / 140248, and WO 2014 / 068079. These references also describe anti-BCMA Fv domains that specifically target BCMA with high affinity and bispecific antibodies that use such anti-BCMA Fv domains to induce potent and effective myeloma cell lysis. Thus, proof of concept has been shown for anti-BCMA Fv domains in bispecific antibodies that re-direct T cells to lyse MM cells.

[0198] Additional antigen-binding portions that bind to antigens expressed on myeloma cells, particularly BCMA, can be derived from other known or commercially available antibodies or can be newly generated by methods known in the art. For example, variable domains specific for BCMA can be obtained by selecting variable fragments (Fv) specific for BCMA. This can be achieved, for example, by screening a single-chain Fv (scFv) phage display library or via hybridoma technology. For example, an IgM-based phage display library of human scFv sequences can be subjected to several rounds of in vitro selection to enrich for BCMA-specific binders (Example 1). The affinity of the selected scFv can be further increased by affinity maturation.

[0199] In certain embodiments, the BCMA targeting moiety comprises a V H CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 67, a V H CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 68, a V H CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 69, a V L CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 70, a V L CDR2, and a V comprising the amino acid sequence set forth in SEQ ID NO: 71L It includes CDR3.

[0200] In certain embodiments, the V of the BCMA targeting moiety H includes an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence shown in SEQ ID NO: 65.

[0201] In certain embodiments, the V of the BCMA targeting moiety L includes an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence shown in SEQ ID NO: 66.

[0202] In certain embodiments, the V of the BCMA targeting moiety H includes or has the amino acid sequence shown in SEQ ID NO: 65. In certain embodiments, the V of the BCMA targeting moiety L includes or has the amino acid sequence shown in SEQ ID NO: 66.

[0203] In certain embodiments, the V of the BCMA targeting moiety H includes an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence shown in SEQ ID NO: 37.

[0204] In certain embodiments, the V of the BCMA targeting moiety Lcomprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical or the same as the amino acid sequence set forth in SEQ ID NO: 38.

[0205] In certain embodiments, the V of the BCMA targeting moiety H comprises or has the amino acid sequence set forth in SEQ ID NO: 65. In certain embodiments, the V of the BCMA targeting moiety L comprises or has the amino acid sequence set forth in SEQ ID NO: 66.

[0206] In certain embodiments, the CD16A / BCMA binding antibody is an scFv-IgAb antigen binding protein having the structure shown in FIG. 13, which comprises (a) two CD16A antigen binding portions in the format of an scFv fused to the C-terminus of a homodimeric human IgG CH2-CH3 Fc portion in the order of V L -V H and (b) two target antigen binding portions provided by each Fv within the Fab arm of the IgG, wherein the target antigen binding portions bind to BCMA.

[0207] In certain embodiments, each of the CD16A antigen binding portions comprises a V H CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, a V H CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, a V H CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75, a V L CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, a V L CDR2, and a V L CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77. In certain embodiments, the CDRs are identified according to the Kabat numbering system. Each of the CD16A antigen binding portions has a V having the amino acid sequence set forth in SEQ ID NO: 3 Hand V having the amino acid sequence shown in SEQ ID NO: 2 L and, including.

[0208] In certain embodiments, the CD16A antigen-binding portion is at the C-terminus of the Fc portion connected via a linker having the amino acid sequence shown in SEQ ID NO: 22, in the following order: V L (CD16A)-linker L3-V L (CD16A) is fused, and linker L3 has the amino acid sequence shown in SEQ ID NO: 18.

[0209] In certain embodiments, each of the BCMA-targeting portions has a V containing the amino acid sequence shown in SEQ ID NO: 67 H CDR1, V containing the amino acid sequence shown in SEQ ID NO: 68 H CDR2, V containing the amino acid sequence shown in SEQ ID NO: 69 H CDR3, V containing the amino acid sequence shown in SEQ ID NO: 70 L CDR1, V containing the amino acid sequence shown in SEQ ID NO: 71 L CDR2, and V containing the amino acid sequence shown in SEQ ID NO: 72 L CDR3. In certain embodiments, the CDRs are identified according to the Kabat numbering system. Each of the BCMA-targeting portions has a V having the amino acid sequence shown in SEQ ID NO: 65 H and V having the amino acid sequence shown in SEQ ID NO: 66 L and, including.

[0210] In certain embodiments, the Fc portion of the CD16A / BCMA antibody is a silenced Fc portion comprising the human IgG1 CH2 and CH3 heavy chain constant domains containing the amino acid sequence shown in SEQ ID NO: 29. The CH2 heavy chain constant domain has two silencing mutations (also referred to as "effectorless mutations"): L234F and L235E. The CH2 heavy chain constant domain contains the amino acid sequence shown in SEQ ID NO: 79. The CH3 heavy chain constant domain contains the amino acid sequence shown in SEQ ID NO: 109. The CH1 heavy chain constant domain linked to the BCMA-targeting portion contains the amino acid sequence shown in SEQ ID NO: 33.

[0211] In certain embodiments, CD16A / BCMA antibody I comprises polypeptide chain 1 having the amino acid sequence shown in SEQ ID NO: 61 and polypeptide chain 2 having the amino acid sequence shown in SEQ ID NO: 62.

[0212] In certain embodiments, the CD16A / BCMA antibody is a KiH-scDb-Fc having the structure shown in FIG. 5, which comprises (a) two CD16A antigen-binding portions in the format of an scDb fused to the C-terminus of an IgG CH2-CH3 Fc portion in the order of V L -V H -V L -V H and (b) a single target antigen-binding portion in the format of an scFv fused to the N-terminus of the Fc portion, wherein the target antigen-binding portion binds to BCMA.

[0213] In certain embodiments, each of the CD16A antigen-binding portions comprises a V H CDR1 comprising the amino acid sequence shown in SEQ ID NO: 73, a V H CDR2 comprising the amino acid sequence shown in SEQ ID NO: 74, a V H CDR3 comprising the amino acid sequence shown in SEQ ID NO: 75, a V L CDR1 comprising the amino acid sequence shown in SEQ ID NO: 76, a V L CDR2 comprising the amino acid sequence shown in SEQ ID NO: 77, and a V L CDR3 comprising the amino acid sequence shown in SEQ ID NO: 78. In certain embodiments, the CDRs are identified according to the Kabat numbering system. Each of the CD16A antigen-binding portions comprises a V H having the amino acid sequence shown in SEQ ID NO: 3 and a V L having the amino acid sequence shown in SEQ ID NO: 2.

[0214] In certain embodiments, the CD16A antigen-binding portion is fused to a heterodimeric Fc portion and is connected, via a linker having the amino acid sequence shown in SEQ ID NO: 20, in the following order: V L(CD16A)-Linker L1-V H (CD16A)-Linker L2-V L (CD16A)-Linker L1-V H (CD16A), wherein Linker L1 has the amino acid sequence shown in SEQ ID NO: 16 and Linker L2 has the amino acid sequence shown in SEQ ID NO: 17.

[0215] In certain embodiments, each of the BCMA targeting moieties is a V comprising the amino acid sequence shown in SEQ ID NO: 67. H a V comprising the amino acid sequence shown in SEQ ID NO: 68 for CDR1 H a V comprising the amino acid sequence shown in SEQ ID NO: 69 for CDR2 H a V comprising the amino acid sequence shown in SEQ ID NO: 70 for CDR3 L a V comprising the amino acid sequence shown in SEQ ID NO: 71 for CDR1 L a V comprising the amino acid sequence shown in SEQ ID NO: 72 for CDR2, and L including CDR3. In certain embodiments, the CDRs are identified according to the Kabat numbering system. Each of the BCMA targeting moieties is a V having the amino acid sequence shown in SEQ ID NO: 65 H and a V having the amino acid sequence shown in SEQ ID NO: 66 L and.

[0216] In certain embodiments, the Fc portion of the CD16A / BCMA antibody III is a silent Fc portion comprising the human IgG1 CH2 and CH3 heavy chain constant domains having the amino acid sequence shown in SEQ ID NO: 31. The CH2 heavy chain constant domain has two silent mutations or effectorless mutations: L234F and L235E. The CH2 heavy chain constant domain comprises the amino acid sequence shown in SEQ ID NO: 79. The CH3 heavy chain constant domain has one silent mutation or effectorless mutation D265A. The CH3 heavy chain constant domain comprises the amino acid sequence shown in SEQ ID NO: 81.

[0217] In certain embodiments, the CD16A / BCMA antibody comprises a polypeptide chain 1 having the amino acid sequence shown in SEQ ID NO: 63 and a polypeptide chain 2 having the amino acid sequence shown in SEQ ID NO: 64.

[0218] In one embodiment of the multispecific antigen-binding protein of the present invention, the protein is a tetramer comprising a polypeptide having an amino acid sequence as shown in SEQ ID NOs: 61 and 62 or SEQ ID NOs: 63 and 64.

[0219] B.EGFR An EGFR / CD16A antigen-binding protein for a natural killer (NK) cell-based EGFR targeting approach is also provided.

[0220] In some embodiments, the antigen-binding portion of EGFR described herein also binds to EGFRvIII. Thus, the EGFR / CD16A antigen-binding protein can be used for the treatment of both EGFR-expressing cancers and EGFRvIII-expressing cancers. In contrast to EGFR, EGFRvIII is expressed only in cancer cells and not in healthy tissues. Thus, a broader range of EGFR and / or EGFRvIII positive tumors, and also a broader patient population, can be targeted with the EGFR / CD16A antigen-binding proteins described herein. An example of an EGFR binding domain suitable for the antigen-binding proteins described herein comprises the amino acid sequence shown in SEQ ID NO: 40 as VH and the amino acid sequence shown in SEQ ID NO: 41 as VL.

[0221] IV. Polynucleotide The antigen-binding proteins according to any one of the embodiments described herein can be produced by expressing a polynucleotide encoding the individual polypeptide chains that form the antigen-binding molecule. Accordingly, a further embodiment of the invention is a polynucleotide, such as DNA or RNA, encoding a polypeptide of an antibody molecule as described above herein.

[0222] The polynucleotide can be constructed by operably linking a gene encoding a variable domain, which is either separated by, for example, a peptide linker or directly linked by a peptide bond of a polypeptide chain, to a gene construct operably linked to a suitable promoter and optionally a suitable transcription terminator, and expressing it in bacteria or other suitable expression systems such as, for example, CHO cells. Depending on the vector system and host used, any number of suitable transcription and translation elements can be used, including constitutive and inducible promoters. The promoter is selected so that it drives the expression of the polynucleotide in each host cell.

[0223] The polynucleotide can be inserted into a vector, preferably an expression vector, representing a further embodiment of the invention.

[0224] Various expression vector / host systems can be utilized to contain and express the polynucleotide encoding the polypeptide chain of the invention. Examples of expression vectors for expression in E. coli include pSKK (LeGall et al., "J Immunol Methods" (2004), Vol. 285, No. 1, pp. 111 - 27) or pcDNA5 (Invitrogen) for expression in mammalian cells.

[0225] V. Treatment Methods The invention further provides a composition comprising a multispecific antigen - binding protein, particularly a multispecific antigen - binding protein as described herein and at least one further component.

[0226] The invention further provides a multispecific antigen - binding protein or a composition comprising a multispecific antigen - binding protein as described herein for use in NK cell - based immunotherapy. NK cell - based immunotherapy includes active NK cell - based treatment methods in which either endogenous or adoptively transferred NK cells are activated via binding by the antigen - binding molecule of the invention. In particular, the ability of NK cells to attack and kill abnormal cells such as cancer cells is enhanced.

[0227] Furthermore, current perspectives in immuno-oncology (IO) include commercially available checkpoint regulators such as anti-CTLA4 and anti-PD-1 antibodies. However, these agents have shown clinical efficacy (with an ORR of usually 20 - 30% in various indications), and medical need and opportunity remain. Thus, despite such recent advances in treatment, new therapies are needed to achieve increased patient response rates and long-term remissions in a greater number of patients. For example, NK cells play an important role in the immune response against MM and are involved in the clinical efficacy of current standard therapeutic interventions, including IMiD, proteasome inhibitors, recently approved immunotherapies, and autologous stem cell transplantation (ASCT). Many strategies have been developed to enhance the cytotoxicity of natural NK cells against myeloma cells, which are often dysregulated in MM. Approaches include modulation of activity via cytokine stimulation or immune checkpoint targeting, and adoptive transfer of cultured expanded NK cells in ASCT-eligible MM. Although these approaches are very attractive, they rely on the natural cytotoxicity of NK cells and are not targeted, and thus may not benefit from antigen-specific retargeting and effector activation.

[0228] Furthermore, a rational combination in IO should be based on agents that can exert maximal immune effects while managing safety. A promising approach goes beyond adaptive immunity (most checkpoint antibodies are active only on T cells) and includes activation of innate immunity, thus generating an integrated immune response. Based on an extended serum half-life, the antibody formats described herein are ideally suited for these purposes, enabling convenient administration similar to currently marketed monoclonal antibodies and, ideally, can be combined with checkpoint regulators such as anti-LAG3 or anti-PD1 antibodies.

[0229] In certain embodiments, the present invention provides a multispecific antigen-binding protein that specifically binds to CD16A and an antigen expressed on myeloma cells or plasma cells selected from the group consisting of BCMA, CS-1, CD19, CD20, CD22, CD38, and CD138, for use in the treatment of multiple myeloma, and provides a method of use comprising the step of administering the multispecific antigen-binding protein. In certain embodiments, the present invention provides a multispecific antigen-binding molecule that specifically binds to a target cell antigen, such as a tumor antigen, and CD16A for use in NK cell immunotherapy, wherein the multispecific antigen-binding protein is mixed with NK cells ex vivo and a composition of NK cells and the multispecific antigen-binding molecule is administered to a patient.

[0230] In certain embodiments, the tumor antigen is BCMA and the composition is used for the treatment of plasma cell disorders or autoimmune diseases, particularly multiple myeloma.

[0231] Plasma cell diseases include, for example, multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance (MGUS), and smoldering myeloma.

[0232] Autoimmune diseases are, for example, systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA).

[0233] Accordingly, in certain embodiments herein, medical uses and methods are provided, where an antigen-binding protein specific for BCMA and CD16A, such as scDb-mFc, KiH-scDb-Fc, scDb-Trib(-scFv), scFv-IgAb, Bi-scFv-IgAb, KiH-scFv-Fc, Db-Fc, or Bi-scFv-Fc as described above, is administered to a subject in an effective amount for the treatment of BCMA + cancer or an autoimmune disease, such as multiple myeloma.

[0234] Administration is performed by different methods, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. The dosage is determined by the attending physician and other clinical factors. The dosage for any one subject depends on many factors, including the patient's weight, body surface area, age, gender, the particular compound being administered, the time and route of administration, the type of treatment, general health status, and other drugs being co-administered. "Effective amount" refers to the amount of the active ingredient sufficient to affect the course and severity of the disease and to bring about such a reduction or alleviation of such pathology. BCMA + An "effective amount" useful for the treatment and / or prevention of a disease can be determined using known methods.

[0235] Furthermore, the present invention provides a method for the treatment or amelioration of a disease, the method comprising administering to a subject in need thereof a multispecific antigen-binding protein of the present invention.

[0236] In a preferred embodiment of the method for the treatment or amelioration of a disease, the disease is multiple myeloma.

[0237] The present disclosure provides the use of an antigen-binding molecule of the present disclosure, such as a CD16A / BCMA antigen-binding molecule, for the treatment and / or prevention of cancer, such as multiple myeloma. In certain embodiments, the method comprises administering a currently disclosed antigen-binding molecule, such as a CD16A / BCMA antigen-binding molecule, or a pharmaceutical composition comprising the same, to a subject suffering from multiple myeloma ("MM").

[0238] In certain embodiments, the subject being treated with an antigen-binding molecule, such as a CD16A / BCMA antigen-binding molecule, or a pharmaceutical composition comprising the same, is a patient with relapsed / refractory ("R / R") multiple myeloma. In certain embodiments, the subject has received daratumumab. In certain embodiments, the subject is daratumumab-naive. In certain embodiments, the subject is daratumumab-resistant. In certain embodiments, the subject is daratumumab-refractory. In certain embodiments, the subject is daratumumab-relapsed.

[0239] In certain embodiments, the subject being treated with an antigen-binding molecule, such as a CD16A / BCMA antigen-binding molecule, or a pharmaceutical composition comprising the same, expresses a CD16A polymorphism. For example, but not limited to, the CD16A polymorphism is the CD16A-158V / F polymorphism. See Example 8 (e.g., FIGS. 31A and 31B).

[0240] In certain embodiments, the treatment methods described herein further comprise administering a second treatment method, such as a T cell-based treatment method or a checkpoint inhibitor-based treatment method. Such second treatment methods include, but are not limited to, treatment methods comprising administration of an anti-TIGIT antibody, treatment methods comprising administration of an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab), treatment methods comprising administration of an anti-PD-L1 antibody (e.g., atezolizumab), treatment methods comprising administration of an anti-VEGF antibody (e.g., bevacizumab), and treatment methods comprising administration of a CD3 bispecific antibody (e.g., an anti-FcRH5 / CD3 antibody (including such antibodies disclosed in International Application No. PCT / US2016 / 037879, which is incorporated by reference in its entirety, but not limited thereto)).

[0241] In certain embodiments, the treatment methods described herein further include the administration of cytokine therapy. As used herein, "cytokine therapy" refers to any treatment, including but not limited to, pegylated cytokines and Fc-fusion cytokines, that includes any derivative or modification of a cytokine. Non-limiting examples of cytokines that can be used in connection with such embodiments include IL-15, IL-2, IL-12, IL-21, IL-17, IL-18, IL-23, IL-27, and IL-6. In certain embodiments, the method further includes administering IL-15. In certain embodiments, the method further includes administering IL-2. As shown in Example 8, IL-15 and IL-2 can increase the activity of currently disclosed CD16A / BCMA antigen-binding molecules.

[0242] In certain embodiments, the administration of the antigen-binding molecules disclosed herein, such as CD16A / BCMA antigen-binding molecules, or pharmaceutical compositions containing them, is the first-line (1L) treatment. For example, but not limited to, such first-line administration can occur in subjects presenting with a CD16A polymorphism that exhibits a decrease in the efficacy of therapeutic functions via an Fc-based MOA, such as daratumumab therapy. In certain embodiments, the first-line administration of the antigen-binding molecules of the present disclosure can occur in subjects in whom fratricide of NK cells is contraindicated. See Example 8 (e.g., FIGS. 40A-40E). In certain embodiments, the first-line administration of the antigen-binding molecules of the present disclosure can occur in subjects in whom avoidance of CRS is desired.

[0243] In certain embodiments, administration of the antigen-binding molecules disclosed herein, such as the CD16A / BCMA antigen-binding molecules, or pharmaceutical compositions comprising them, is a second-line (2L) or third-line (3L) therapeutic. For example, without limitation, such second-line or third-line administration can occur in subjects who have already received therapeutic benefit via an Fc-based MOA, such as daratumumab therapy. In certain embodiments, such second-line or third-line therapy can occur in subjects refractory or resistant to daratumumab therapy.

[0244] In certain embodiments, one or more of the following criteria are used to select an NK cell inducer for use in the treatment of multiple myeloma: · The NK cell inducer can bind to two targets: CD16A on NK cells and BCMA on multiple myeloma cells. · The NK cell inducer is at least bivalent with respect to CD16A, for example, comprises at least two CD16A antigen-binding portions. · The NK cell inducer is an NK cell bispecific antibody that is not cross-reactive with CD16B and is preferably cross-reactive with non-human primates. · The NK cell inducer is potent and general, and + target-dependently kills tumor cells and primary myeloma in vitro (e.g., kills about 60% or more of cells with an EC50 ≦ 5 nM). · The NK cell inducer does not significantly kill NK cells, for example, reduces or avoids fratricide of NK cells. · The NK cell inducer has, for example, a superior in vitro cytokine release profile compared to other T cell inducers (e.g., the NK cell inducer lacks CRS), and adverse events are monitorable, manageable, and reversible. · The NK cell inducer can be administered intravenously to multiple myeloma subjects, and · The NK cell inducer needs to be administered at a frequency of once a week (QW) or less.

[0245] In certain embodiments, the following pharmacodynamic (PD) biomarkers can be used in connection with the administration of the antigen-binding proteins of the present disclosure: the concentrations of serum M protein and free light chain (FLC), the reduction of monoclonal plasma cells, the activation and mobilization of NK cells in bone marrow samples after treatment, and the activation of peripheral NK cells. In certain embodiments, the treatment method can include BMCA-dependent diagnosis, but based on the high prevalence of BMCA in multiple myeloma, such a diagnosis is not necessarily required.

[0246] VI. Exemplary Embodiments In certain embodiments, the present disclosure is directed to a multispecific antigen-binding protein comprising (a) at least one first target antigen-binding moiety and (b) at least two CD16A antigen-binding moieties, wherein the at least two CD16A antigen-binding moieties are Fab fragments comprising at least one constant domain, or are fused to an Fc portion. In certain embodiments, at least one of the two CD16A antigen-binding moieties is an antigen-binding molecule selected from the group consisting of single-chain Fv (scFv), single-chain diabody (scDb), and diabody Db. In certain embodiments, each of the at least two CD16A antigen-binding moieties is an scFv. In certain embodiments, the at least two CD16A antigen-binding moieties are in the format of scDb. In certain embodiments, each of the at least two CD16A antigen-binding moieties comprises a light chain variable region (V L ) and a heavy chain variable region (V H ) sequentially linked by a polypeptide chain, and the variable region at the N-terminus of the polypeptide chain is the V L . In certain embodiments, the variable regions of the at least two CD16A antigen-binding moieties in the polypeptide chain are V L -V H , V L -V L -V H -V H , or V L -V H -V L -V Hare located from the N-terminus to the C-terminus in the order of. In certain embodiments, the variable regions of the at least two CD16A antigen-binding portions in the polypeptide chain are V L -V H are located from the N-terminus to the C-terminus in the order of. In certain embodiments, the variable regions of the at least two CD16A antigen-binding portions in the polypeptide chain are V L -V H -V L -V H are located from the N-terminus to the C-terminus in the order of. In certain embodiments, (i) the C-terminus of the polypeptide chain fuses to the N-terminus of the CH2 domain of the Fc portion; or (ii) the C-terminus of the polypeptide chain fuses to the N-terminus of the hinge of the Fc portion; (iii) the N-terminus of the polypeptide chain fuses to the C-terminus of the CH3 domain of the Fc portion, or (iv) the N-terminus of the polypeptide chain fuses to the C-terminus of the Fab fragment. In certain embodiments, the N-terminus of the polypeptide chain fuses to the C-terminus of the CH3 domain of the Fc portion. In certain embodiments, the Fc portion is selected from the group consisting of a monomeric CH2-CH3 fragment, a heterodimeric Fc region, and a homodimeric Fc region. In certain embodiments, the Fc portion does not bind to the Fcγ receptor but retains binding to the neonatal Fc receptor. In certain embodiments, an scDb or Db comprising two CD16 antigen-binding portions fuses to the C-terminus of one chain of the Fab fragment, and the first target antigen-binding portion fuses to the C-terminus of the other chain of the Fab fragment. In certain embodiments, the Fab fragment comprises an HSA antigen-binding Fv at the N-terminus.

[0247] In certain embodiments, the antigen-binding protein described herein is tetravalent.

[0248] In certain embodiments, the multispecific antigen-binding protein described herein comprises at least two target antigen-binding portions.

[0249] In certain embodiments, the multispecific antigen-binding protein described herein comprises a first target antigen-binding portion, a second target antigen-binding portion, and at least two CD16A antigen-binding portions fused to a dimeric Fc portion.

[0250] In certain embodiments, the multispecific antigen-binding protein described herein comprises a CD16A antigen-binding portion fused to the C-terminus of each heavy chain of IgG, wherein IgG comprises at the N-terminus a first target antigen-binding Fv portion in each of the two Fabs, and the second target antigen-binding portion is fused at the C-terminus to each of the CL domains.

[0251] In certain embodiments, the CD16A antigen-binding portion described herein comprises (i) a heavy chain variable region comprising a CDR1 having the amino acid sequence shown in SEQ ID NO: 50; a CDR2 having the amino acid sequence shown in SEQ ID NO: 51; and a CDR3 having the amino acid sequence shown in SEQ ID NO: 52, and / or (ii) a light chain variable region comprising a CDR1 having the amino acid sequence shown in SEQ ID NO: 53; a CDR2 having the amino acid sequence shown in SEQ ID NO: 54; and a CDR3 having the amino acid sequence shown in SEQ ID NO: 55.

[0252] In certain embodiments, the multispecific antigen-binding protein described herein comprises a CD16A antigen-binding portion comprising (i) a heavy chain variable region comprising a CDR1 having the amino acid sequence shown in SEQ ID NO: 73; a CDR2 having the amino acid sequence shown in SEQ ID NO: 74; and a CDR3 having the amino acid sequence shown in SEQ ID NO: 75, and / or (ii) a light chain variable region comprising a CDR1 having the amino acid sequence shown in SEQ ID NO: 76; a CDR2 having the amino acid sequence shown in SEQ ID NO: 77; and a CDR3 having the amino acid sequence shown in SEQ ID NO: 78.

[0253] In certain embodiments, the multispecific antigen-binding protein described herein comprises a CD16A antigen-binding portion that comprises (i) a heavy-chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 3, and / or (ii) a light-chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0254] In certain embodiments, the multispecific antigen-binding protein described herein comprises a CD16A antigen-binding portion that comprises (i) a heavy-chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3, and / or (ii) a light-chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0255] In certain embodiments, the multispecific antigen-binding protein described herein binds to a first target antigen selected from BCMA and EGFR. In certain embodiments, the multispecific antigen-binding protein described herein binds to a first target antigen that is BCMA.

[0256] In certain embodiments, the multispecific antigen-binding protein described herein comprises a tetramer comprising a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 61 or 63 and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 62 or 64. In certain embodiments, the multispecific antigen-binding protein described herein comprises a tetramer comprising a first and a second polypeptide selected from the following: (i) a first polypeptide having the amino acid sequence set forth in SEQ ID NO: 61 and a second polypeptide having the amino acid sequence set forth in SEQ ID NO: 62; (ii) a first polypeptide having the amino acid sequence set forth in SEQ ID NO: 61 and a second polypeptide having the amino acid sequence set forth in SEQ ID NO: 64; (iii) a first polypeptide having the amino acid sequence set forth in SEQ ID NO: 63 and a second polypeptide having the amino acid sequence set forth in SEQ ID NO: 62; and (iv) a first polypeptide having the amino acid sequence set forth in SEQ ID NO: 63 and a second polypeptide having the amino acid sequence set forth in SEQ ID NO: 64.

[0257] In certain embodiments, the multispecific antigen-binding protein described herein comprises a BCMA antigen-binding portion comprising: (i) a heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 67, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 68, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 69; and / or (ii) a light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 70, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 71, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 72.

[0258] In certain embodiments, the multispecific antigen-binding protein described herein comprises a BCMA antigen-binding portion comprising: (i) a heavy chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 65; and / or (ii) a light chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 66.

[0259] In certain embodiments, the multispecific antigen-binding protein described herein comprises a BCMA antigen-binding portion comprising: (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65; and / or (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66.

[0260] In certain embodiments, the multispecific antigen-binding protein described herein comprises: (i) a CD16A antigen-binding portion comprising: (a) a heavy chain variable region comprising a CDR1 having the amino acid sequence shown in SEQ ID NO: 73, a CDR2 having the amino acid sequence shown in SEQ ID NO: 74, and a CDR3 having the amino acid sequence shown in SEQ ID NO: 75, and (b) a light chain variable region comprising a CDR1 having the amino acid sequence shown in SEQ ID NO: 76, a CDR2 having the amino acid sequence shown in SEQ ID NO: 77, and a CDR3 having the amino acid sequence shown in SEQ ID NO: 78; and (ii) a BCMA antigen-binding portion comprising: (a) a heavy chain variable region comprising a CDR1 having the amino acid sequence shown in SEQ ID NO: 67, a CDR2 having the amino acid sequence shown in SEQ ID NO: 68, and a CDR3 having the amino acid sequence shown in SEQ ID NO: 69, and (ii) a light chain variable region comprising a CDR1 having the amino acid sequence shown in SEQ ID NO: 70, a CDR2 having the amino acid sequence shown in SEQ ID NO: 71, and a CDR3 having the amino acid sequence shown in SEQ ID NO: 72; and, an antigen-binding protein.

[0261] In certain embodiments, the present disclosure is directed to a pharmaceutical composition comprising the multispecific antigen-binding protein described herein and a pharmaceutically acceptable carrier.

[0262] In certain embodiments, the multispecific antigen-binding protein described herein is for use as a medicament.

[0263] In certain embodiments, the present disclosure is directed to a method for treating or ameliorating a disease, the method comprising administering to a subject in need thereof the multispecific antigen-binding protein described herein, or the pharmaceutical composition described herein. In certain embodiments, the disease is cancer. In certain embodiments, the disease is a blood cancer. In certain embodiments, the disease is multiple myeloma.

[0264] In certain embodiments, the present disclosure is directed to a method for treating and / or preventing a disease in a subject, the method comprising administering to a subject in need thereof a bispecific antigen-binding protein described herein, or a pharmaceutical composition described herein. In certain embodiments, the multispecific antigen-binding protein is administered intravenously. In certain embodiments, the multispecific antigen-binding protein is administered subcutaneously. In certain embodiments, the method disclosed herein comprises administering a second therapy. In certain embodiments, the second therapy is selected from the group consisting of a therapy comprising an anti-PD-1 antibody, a therapy comprising an anti-PD-L1 antibody, a therapy comprising a CD3 bispecific antibody, a therapy comprising an anti-TIGIT antibody, a therapy comprising an anti-VEGF antibody, and a therapy comprising an anti-FcRH5 antibody. In certain embodiments, the second therapy is cytokine therapy. In certain embodiments, the cytokine therapy comprises administration of a cytokine selected from the group consisting of IL-15, IL-2, IL-12, IL-21, and IL6. In certain embodiments, the cytokine therapy comprises administration of IL-2. In certain embodiments, the cytokine therapy comprises administration of IL-15.

[0265] In certain embodiments, the subject to be treated as described herein is a cancer patient. In certain embodiments, the subject is a blood cancer patient. In certain embodiments, the subject is a multiple myeloma patient. In certain embodiments, the subject is a relapsed / refractory multiple myeloma patient. In certain embodiments, the subject has received anti-CD38 therapy. In certain embodiments, the subject has received daratumumab. In certain embodiments, the subject is daratumumab-naïve, daratumumab-resistant, daratumumab-refractory, or daratumumab-relapsed. In certain embodiments, the subject is daratumumab-resistant, daratumumab-refractory, or daratumumab-relapsed. In certain embodiments, the subject expresses a CD16A polymorphism. In certain embodiments, the CD16A polymorphism is the CD16A-158V / F polymorphism.

[0266] In certain embodiments, the present disclosure is directed to a bispecific antigen-binding protein comprising (a) at least one BCMA-binding moiety and (b) at least two CD16A antigen-binding moieties, wherein the at least two CD16A antigen-binding moieties are Fab fragments comprising at least one constant domain or are fused to an Fc moiety. In certain embodiments, at least one of the two CD16A antigen-binding moieties is an antigen-binding molecule selected from the group consisting of single-chain Fv (scFv), single-chain diabody (scDb), and diabody Db. In certain embodiments, each of the at least two CD16A antigen-binding moieties is a scFv. In certain embodiments, the at least two CD16A antigen-binding moieties are in the format of scDb. In certain embodiments, each of the at least two CD16A antigen-binding moieties is sequentially linked by a polypeptide chain to a variable light chain region (V L ) and a variable heavy chain region (V H ), and the variable regions at the N-terminus of the polypeptide chain are the V L . In certain embodiments, the variable regions of the at least two CD16A antigen-binding moieties in the polypeptide chain are V L -V H , V L -V L -V H -V H , or V L -V H -V L -V H , located from the N-terminus to the C-terminus in that order.

[0267] In certain embodiments, the variable regions of the at least two CD16A antigen-binding moieties in the polypeptide chain are V L -V H , located from the N-terminus to the C-terminus in that order. In certain embodiments, the variable regions of the at least two CD16A antigen-binding moieties in the polypeptide chain are V L -V H -V L -V H , located from the N-terminus to the C-terminus in that order.

[0268] In certain embodiments, the present disclosure is directed to bispecific antigen-binding proteins, where (i) the C-terminus of the polypeptide chain fuses to the N-terminus of the CH2 domain of the Fc portion; or (ii) the C-terminus of the polypeptide chain fuses to the N-terminus of the hinge of the Fc portion; (iii) the N-terminus of the polypeptide chain fuses to the C-terminus of the CH3 domain of the Fc portion, or (iv) the N-terminus of the polypeptide chain fuses to the C-terminus of the Fab fragment. In certain embodiments, the N-terminus of the polypeptide chain fuses to the C-terminus of the CH3 domain of the Fc portion. In certain embodiments, the Fc portion is selected from the group consisting of monomeric CH2-CH3 fragments, heterodimeric Fc regions, and homodimeric Fc regions. In certain embodiments, the Fc portion does not bind to Fcγ receptors but retains binding to the neonatal Fc receptor. In certain embodiments, the Fc portion contains at least one effectorless mutation. In certain embodiments, the at least one effectorless mutation is selected from the group consisting of C220S, C229S, E233P, L234A, L234V, L234F, L235A, L235E, P238S, D265A, N297A, N297Q, and P331S. In certain embodiments, the at least one effectorless mutation is selected from the group consisting of L234A, L234V, L234F, L235A, L235E, P238S, and D265A. In certain embodiments, the at least one effectorless mutation is selected from the group consisting of L234F, L235E, and D265A. In certain embodiments, the Fc portion has two effectorless mutations. In certain embodiments, the two effectorless mutations are L234F and L235E. In certain embodiments, the Fc portion has three effectorless mutations. In certain embodiments, the three effectorless mutations are L234F, L235E, and D265A.

[0269] In certain embodiments, the bispecific antigen-binding protein of the present disclosure has at least two CD16A antigen-binding portions fused to the Fc portion. In certain embodiments, the bispecific antigen-binding protein of the present disclosure comprises two BCMA targeting portions. In certain embodiments, the bispecific antigen-binding protein of the present disclosure comprises two CD16A antigen-binding portions and two BCMA targeting portions. In certain embodiments, each of the two CD16A antigen-binding portions is fused to the C-terminus of each heavy chain of IgG, and each of the two BCMA targeting portions is fused to the N-terminus of the IgG.

[0270] In certain embodiments, the bispecific antigen-binding protein of the present disclosure comprises a bispecific antigen-binding protein having the structure shown in FIG. 13. In certain such embodiments, the bispecific antigen-binding protein comprises one BCMA targeting portion. In certain such embodiments, the BCMA targeting portion is fused to the N-terminus of the Fc portion to which the at least two CD16A antigen-binding portions are fused.

[0271] In certain embodiments, the bispecific antigen-binding has the structure shown in FIG. 5.

[0272] In certain embodiments, the bispecific antigen-binding protein of the present disclosure comprises a CD16A antigen-binding portion comprising: (i) a CDR1 having the amino acid sequence shown in SEQ ID NO: 50; a CDR2 having the amino acid sequence shown in SEQ ID NO: 51 or 56; and a CDR3 having the amino acid sequence shown in SEQ ID NO: 52 heavy chain variable region, and / or (ii) a CDR1 having the amino acid sequence shown in SEQ ID NO: 53; a CDR2 having the amino acid sequence shown in SEQ ID NO: 54; and a CDR3 having the amino acid sequence shown in SEQ ID NO: 55 light chain variable region. In certain embodiments, the heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 51. In certain embodiments, the heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 56.

[0273] In certain embodiments, the bispecific antigen-binding protein of the present disclosure includes a CD16A antigen-binding portion comprising: (i) a heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75, and / or (ii) a light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 78. In certain embodiments, the CD16A antigen-binding portion comprises: (i) a heavy chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 3, and / or (ii) a light chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the CD16A antigen-binding portion comprises: (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3, and / or (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0274] In certain embodiments, the bispecific antigen-binding protein of the present disclosure comprises a BCMA antigen-binding portion comprising: (i) a heavy-chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 67, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 68, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 69, and / or (ii) a light-chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 70, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 71, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 72. In certain embodiments, the BCMA antigen-binding portion comprises: (i) a heavy-chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 65, and / or (ii) a light-chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 66. In certain embodiments, the BCMA antigen-binding portion comprises: (i) a heavy-chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65, and / or (ii) a light-chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66.

[0275] In certain embodiments, the bispecific antigen-binding protein of the present disclosure comprises two CD16A antigen-binding portions and two BCMA targeting portions, wherein (a) each CD16A antigen-binding portion comprises a V H CDR1 having the amino acid sequence set forth in SEQ ID NO: 73, a V H CDR2 having the amino acid sequence set forth in SEQ ID NO: 74, a V H CDR3 having the amino acid sequence set forth in SEQ ID NO: 75, a V L CDR1 having the amino acid sequence set forth in SEQ ID NO: 76, a V L CDR2, and a V L CDR3 having the amino acid sequence set forth in SEQ ID NO: 78, and (b) each BCMA targeting portion comprises a V H CDR1 having the amino acid sequence set forth in SEQ ID NO: 67, a V HCDR2, CDR3 having the amino acid sequence shown in SEQ ID NO: 69, V having the amino acid sequence shown in SEQ ID NO: 70 L CDR1, V having the amino acid sequence shown in SEQ ID NO: 71 L CDR2, and V having the amino acid sequence shown in SEQ ID NO: 72 L It includes CDR3. In certain embodiments, (a) each of the CD16A antigen-binding portions comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2; (b) each of the BCMA-targeting portions comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 65 and (ii) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 66.

[0276] In certain embodiments, the bispecific antigen-binding protein of the present disclosure is a tetramer comprising a first polypeptide having the amino acid sequence shown in SEQ ID NO: 61 and a second polypeptide having the amino acid sequence shown in SEQ ID NO: 62. In certain embodiments, the bispecific antigen-binding protein of the present disclosure is a tetramer comprising a first polypeptide having the amino acid sequence shown in SEQ ID NO: 63 and a second polypeptide having the amino acid sequence shown in SEQ ID NO: 64.

[0277] In certain embodiments, the bispecific antigen-binding protein of the present disclosure comprises two CD16A antigen-binding portions and two BCMA antigen-binding portions, wherein (i) each of the CD16A antigen-binding portions comprises: (a) a heavy chain variable region comprising a CDR1 having the amino acid sequence shown in SEQ ID NO: 73; a CDR2 having the amino acid sequence shown in SEQ ID NO: 74; and a CDR3 having the amino acid sequence shown in SEQ ID NO: 75, and (b) a light chain variable region comprising a CDR1 having the amino acid sequence shown in SEQ ID NO: 76; a CDR2 having the amino acid sequence shown in SEQ ID NO: 77; and a CDR3 having the amino acid sequence shown in SEQ ID NO: 78, and (ii) each of the BCMA antigen-binding portions comprises: (a) a heavy chain variable region comprising a CDR1 having the amino acid sequence shown in SEQ ID NO: 67; a CDR2 having the amino acid sequence shown in SEQ ID NO: 68; and a CDR3 having the amino acid sequence shown in SEQ ID NO: 69, and (ii) a light chain variable region comprising a CDR1 having the amino acid sequence shown in SEQ ID NO: 70; a CDR2 having the amino acid sequence shown in SEQ ID NO: 71; and a CDR3 having the amino acid sequence shown in SEQ ID NO: 72.

[0278] In certain embodiments, the present disclosure is directed to a pharmaceutical composition comprising the bispecific antigen-binding protein described herein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition is for use as a medicament.

[0279] In certain embodiments, the present disclosure is directed to a method of treating and / or preventing a disease, the method comprising administering a bispecific antigen-binding protein described herein or a pharmaceutical composition described herein. In certain embodiments, the disease is cancer. In certain embodiments, the disease is a blood cancer. In certain embodiments, the disease is multiple myeloma. In certain embodiments, the bispecific antigen-binding protein is administered intravenously. In certain embodiments, the bispecific antigen-binding protein is administered subcutaneously. In certain embodiments, the bispecific antigen-binding protein is administered in a second therapy. In certain embodiments, the second therapy is selected from the group consisting of a therapy comprising an anti-PD-1 antibody, a therapy comprising an anti-PD-L1 antibody, a therapy comprising a CD3 bispecific antibody, a therapy comprising an anti-TIGIT antibody, a therapy comprising an anti-VEGF antibody, and a therapy comprising an anti-FcRH5 antibody. In certain embodiments, the second therapy is cytokine therapy. In certain embodiments, the cytokine therapy is the administration of a cytokine selected from the group consisting of IL-15, IL-2, IL-12, IL-21, IL-17, IL-18, IL-23, IL-27, and IL-6. In certain embodiments, the cytokine is IL-15. In certain embodiments, the cytokine is IL-2. In certain embodiments, the subject is a blood cancer patient. In certain embodiments, the subject is a blood cancer patient. In certain embodiments, the subject is a multiple myeloma patient. In certain embodiments, the subject is a relapsed / refractory multiple myeloma patient. In certain embodiments, the subject has received anti-CD38 therapy. In certain embodiments, the subject has received daratumumab. In certain embodiments, the subject is daratumumab-naïve, daratumumab-resistant, daratumumab-refractory, or daratumumab-relapsed. In certain embodiments, the subject is daratumumab-resistant, daratumumab-refractory, or daratumumab-relapsed. In certain embodiments, the subject expresses the CD16A polymorphism. In certain embodiments, the CD16A polymorphism is the CD16A-158V / F polymorphism.

[0280] In certain embodiments, the present disclosure is directed to a multispecific antigen-binding protein or bispecific antigen-binding protein described herein, where the multispecific antigen-binding protein or bispecific antigen-binding protein, when administered to a subject, does not substantially deplete or reduce the subject's natural killer (NK) cell population.

[0281] In certain embodiments, the present disclosure is directed to a method of treating a subject having a depleted or reduced NK cell population, comprising administering a multispecific antigen-binding protein or bispecific antigen-binding protein described herein. In certain embodiments, the subject has been previously treated with an anti-CD38 therapy. In certain embodiments, the anti-CD38 therapy is daratumumab therapy.

[0282] In certain embodiments, the present disclosure is directed to a bispecific antigen-binding protein comprising two CD16A antigen-binding portions and two BCMA targeting portions, where each of the two CD16A antigen-binding portions is fused to the C-terminus of each heavy chain of an IgG, and each of the two BCMA targeting portions is fused to the N-terminus of each of the heavy chains of the IgG, where (i) the CD16A antigen-binding portion comprises, respectively: (a) a heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75; and (b) a light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 78; and (ii) the BCMA antigen-binding portion comprises, respectively: (a) a heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 67; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 68; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 69; and (ii) a light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 70; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 71; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 72.

[0283] In certain embodiments, the present disclosure is directed to a method of treating cancer in a subject, comprising administering to the subject a bispecific antigen-binding protein comprising two CD16A antigen-binding portions and two BCMA-targeting portions, wherein each of the two CD16A antigen-binding portions is fused to the C-terminus of each heavy chain of an IgG, and each of the two BCMA-targeting portions is fused to the N-terminus of each of the heavy chains of the IgG, and (i) the CD16A antigen-binding portion comprises, respectively: (a) a heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75, and (b) a light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 78, and (ii) the BCMA antigen-binding portion comprises, respectively: (a) a heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 67; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 68; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 69, and (ii) a light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 70; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 71; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 72. In certain embodiments, the cancer is multiple myeloma.

[0284] In certain embodiments, the present disclosure is directed to an antibody or antigen-binding protein that binds or is capable of binding to CD16A and BCMA, and includes the following: (i) a first heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75, (ii) a first light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 78; (iii) a second heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 67; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 68; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 69, and (iv) a second light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 70; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 71; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 72. For example, in certain embodiments, the antibody or antigen-binding protein that binds or is capable of binding to CD16A and BCMA includes (i) a first heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3; (ii) a first light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; (iii) a second heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65; (iv) a second light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66; (v) a first heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 and a first light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; (vi) a second heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65 and a second light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66; or, (vii) a first heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3; and a first light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; and a second heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65 and a second light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66.In certain embodiments, as described herein, an antibody or antigen - binding protein that binds to CD16A and BCMA, or that can bind to CD16A and BCMA, does not bind to CD16B. In certain embodiments, as described herein, an antibody or antigen - binding protein that binds to CD16A and BCMA, or that can bind to CD16A and BCMA, does not substantially deplete or reduce the population of natural killer (NK) cells in a subject when administered to the subject. In certain embodiments, as described herein, an antibody or antigen - binding protein that binds to CD16A and BCMA, or that can bind to CD16A and BCMA, binds to human CD16A. In certain embodiments, as described herein, an antibody or antigen - binding protein that binds to CD16A and BCMA, or that can bind to CD16A and BCMA, binds to human BCMA.

[0285] In certain embodiments, the present disclosure is directed to a pharmaceutical composition comprising an antibody or antigen - binding protein that binds to CD16A and BCMA, or that can bind to CD16A and BCMA, as described herein, and a pharmaceutically acceptable carrier.

[0286] In certain embodiments, as described herein, an antibody or antigen - binding protein that binds to CD16A and BCMA, or that can bind to CD16A and BCMA, is for use as a medicament.

[0287] In certain embodiments, the present disclosure is directed to methods of treating and / or preventing a disease, where the method comprises administering to a subject in need thereof an antibody or antigen-binding protein that binds to CD16A and BCMA, or can bind to CD16A and BCMA, as described herein, or a pharmaceutical composition comprising an antibody or antigen-binding protein that binds to CD16A and BCMA, or can bind to CD16A and BCMA, as described herein. In certain embodiments, the disease is cancer. In certain embodiments, the disease is a blood cancer. In certain embodiments, the disease is multiple myeloma. In certain embodiments, the antibody or antigen-binding protein is administered intravenously. In certain embodiments, the antibody or antigen-binding protein is administered subcutaneously. In certain embodiments, treating and / or preventing a disease, where the method comprises administering an antibody or antigen-binding protein that binds to CD16A and BCMA, or can bind to CD16A and BCMA, as described herein, or a pharmaceutical composition comprising an antibody or antigen-binding protein that binds to CD16A and BCMA, or can bind to CD16A and BCMA, as described herein further comprises administering a second therapy. In certain embodiments, the second therapy is selected from the group consisting of a therapy comprising an anti-PD-1 antibody, a therapy comprising an anti-PD-L1 antibody, a therapy comprising a CD3 bispecific antibody, a therapy comprising an anti-TIGIT antibody, a therapy comprising an anti-VEGF antibody, and a therapy comprising an anti-FcRH5 antibody. In certain embodiments, the second therapy is cytokine therapy. In certain embodiments, the cytokine therapy is administration of a cytokine selected from the group consisting of IL-15, IL-2, IL-12, IL-21, IL-17, IL-18, IL-23, IL-27, and IL-6. In certain embodiments, the cytokine is IL-15. In certain embodiments, the cytokine is IL-2. In certain embodiments, the subject is a blood cancer patient. In certain embodiments, the subject is a blood cancer patient. In certain embodiments, the subject is a multiple myeloma patient.In certain embodiments, the subject is a patient with relapsed / refractory multiple myeloma. In certain embodiments, the subject has received anti-CD38 therapy. In certain embodiments, the subject has received daratumumab. In certain embodiments, the subject is daratumumab-naïve, daratumumab-resistant, daratumumab-refractory, or daratumumab-relapsed. In certain embodiments, the subject is daratumumab-resistant, daratumumab-refractory, or daratumumab-relapsed. In certain embodiments, the subject expresses the CD16A polymorphism. In certain embodiments, the CD16A polymorphism is the CD16A-158V / F polymorphism.

[0288] In certain embodiments, the present disclosure is directed to a method of treating a subject having a depleted or decreased NK cell population, the method comprising administering a pharmaceutical composition comprising an antibody or antigen-binding protein that binds to CD16A and BCMA, or that is capable of binding to CD16A and BCMA, as described herein, or an antibody or antigen-binding protein that binds to CD16A and BCMA, or that is capable of binding to CD16A and BCMA, as described herein. In certain embodiments, the subject has been previously treated with anti-CD38 therapy. In certain embodiments, the anti-CD38 therapy is daratumumab therapy.

[0289] In certain embodiments, the present disclosure is directed to an antibody or antigen-binding protein comprising at least one arm that binds or is capable of binding to CD16A and at least one arm that binds or is capable of binding to BCMA, wherein (i) the at least one arm that binds or is capable of binding to CD16A comprises (a) a heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75, and (b) a light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 78, and (ii) the at least one arm that binds or is capable of binding to BCMA comprises (a) a heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 67, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 68, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 69, and (ii) a light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 70, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 71, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 72. In certain such embodiments, the at least one arm that binds or is capable of binding to CD16A is different from the at least one arm that binds or is capable of binding to BCMA.In certain such embodiments, (i) at least one arm that binds or is capable of binding to CD16A comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3; (ii) at least one arm that binds or is capable of binding to CD16A comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; (iii) at least one arm that binds or is capable of binding to BCMA comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65; (iv) at least one arm that binds or is capable of binding to BCMA comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66; (v) at least one arm that binds or is capable of binding to CD16A comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; (vi) at least one arm that binds or is capable of binding to BCMA comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66; or (vii) at least one arm that binds or is capable of binding to CD16A comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; and at least one arm that binds or is capable of binding to BCMA comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66. In certain such embodiments, the antibody or antigen-binding protein does not bind to CD16B. In certain such embodiments, the antibody or antigen-binding protein, when administered to the subject, does not substantially deplete or reduce the subject's natural killer (NK) cell population. In certain such embodiments, the antibody or antigen-binding protein binds to human CD16A. In certain such embodiments, the antibody or antigen-binding protein binds to human BCMA.

[0290] In certain embodiments, the present disclosure is directed to a pharmaceutical composition comprising an antibody or antigen-binding protein comprising at least one arm that binds or is capable of binding to CD16A and at least one arm that binds or is capable of binding to BCMA, and a pharmaceutically acceptable carrier, as disclosed herein.

[0291] In certain embodiments, the present disclosure is directed to an antibody or antigen-binding protein comprising at least one arm that binds or is capable of binding to CD16A and at least one arm that binds or is capable of binding to BCMA for use as a pharmaceutical.

[0292] In certain embodiments, the present disclosure is directed to methods of treating and / or preventing a disease, where the method comprises administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding protein, or the like, comprising at least one arm that binds or is capable of binding to CD16A and at least one arm that binds or is capable of binding to BCMA, as described herein. In certain embodiments, the disease is cancer. In certain embodiments, the disease is a blood cancer. In certain embodiments, the disease is multiple myeloma. In certain embodiments, the antibody or antigen-binding protein is administered intravenously. In certain embodiments, the antibody or antigen-binding protein is administered subcutaneously. In certain embodiments, a method of treating and / or preventing a disease, where the method comprises administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding protein, or the like, comprising at least one arm that binds or is capable of binding to CD16A and at least one arm that binds or is capable of binding to BCMA, as described herein, comprises administering a second therapy. In certain embodiments, the second therapy is selected from the group consisting of a therapy comprising an anti-PD-1 antibody, a therapy comprising an anti-PD-L1 antibody, a therapy comprising a CD3 bispecific antibody, a therapy comprising an anti-TIGIT antibody, a therapy comprising an anti-VEGF antibody, and a therapy comprising an anti-FcRH5 antibody. In certain embodiments, the second therapy is cytokine therapy. In certain embodiments, the cytokine therapy is administration of a cytokine selected from the group consisting of IL-15, IL-2, IL-12, IL-21, IL-17, IL-18, IL-23, IL-27, and IL-6. In certain embodiments, the cytokine is IL-15. In certain embodiments, the cytokine is IL-2. In certain embodiments, the subject is a blood cancer patient. In certain embodiments, the subject is a blood cancer patient. In certain embodiments, the subject is a multiple myeloma patient.In certain embodiments, the subject is a patient with relapsed / refractory multiple myeloma. In certain embodiments, the subject is receiving anti-CD38 therapy. In certain embodiments, the subject is receiving daratumumab. In certain embodiments, the subject is daratumumab-naive, daratumumab-resistant, daratumumab-refractory, or daratumumab-relapsed. In certain embodiments, the subject is daratumumab-resistant, daratumumab-refractory, or daratumumab-relapsed. In certain embodiments, the subject expresses the CD16A polymorphism. In certain embodiments, the CD16A polymorphism is the CD16A-158V / F polymorphism. In certain embodiments, the method includes treating a subject having a depleted or decreased NK cell population. In certain embodiments, the subject has been previously treated with anti-CD38 therapy. In certain embodiments, the anti-CD38 therapy is daratumumab therapy.

[0293] In certain embodiments, the present disclosure is directed to an antibody or antigen-binding protein that binds or is capable of binding to BCMA, the antibody or antigen-binding protein comprising: (i) a heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 67; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 68; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 69, and (ii) a light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 70; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 71; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 72. In certain embodiments, BCMA is human BCMA. In certain embodiments, the antibody or antigen-binding protein comprises at least one arm that binds or is capable of binding to CD16A. In certain embodiments, CD16A is human CD16A. In certain such embodiments, the at least one arm that binds or is capable of binding to CD16A comprises: (i) a heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75, and (ii) a light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 78.In certain such embodiments, the antibody or antigen-binding protein that binds or is capable of binding to BCMA comprises: (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3; (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; (iii) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65; (iv) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66; (v) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; (vi) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66; or, (vii) a first heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3, a first light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; and a second heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65 and a second light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66.

[0294] In certain embodiments, the present disclosure is directed to a pharmaceutical composition comprising an antibody or antigen-binding protein that binds or is capable of binding to BCMA as described herein, and a pharmaceutically acceptable carrier.

[0295] In certain embodiments, the present disclosure is directed to an antibody or antigen-binding protein that binds or is capable of binding to BCMA as described herein for use as a medicament.

[0296] In certain embodiments, the present disclosure is directed to methods of treating and / or preventing a disease, the method comprising administering to a subject in need thereof an antibody or antigen-binding protein that binds or is capable of binding to BCMA, or a pharmaceutical composition comprising the same. In certain embodiments, the disease is cancer. In certain embodiments, the disease is a blood cancer. In certain embodiments, the disease is multiple myeloma. In certain embodiments, the antibody or antigen-binding protein is administered intravenously. In certain embodiments, the antibody or antigen-binding protein is administered subcutaneously. In certain embodiments, the method of treating and / or preventing a disease, the method comprising administering to a subject in need thereof an antibody or antigen-binding protein that binds or is capable of binding to BCMA, or a pharmaceutical composition comprising the same, further comprises administering a second therapy. In certain embodiments, the second therapy is selected from the group consisting of a therapy comprising an anti-PD-1 antibody, a therapy comprising an anti-PD-L1 antibody, a therapy comprising a CD3 bispecific antibody, a therapy comprising an anti-TIGIT antibody, a therapy comprising an anti-VEGF antibody, and a therapy comprising an anti-FcRH5 antibody. In certain embodiments, the second therapy is cytokine therapy. In certain embodiments, the cytokine therapy is administration of a cytokine selected from the group consisting of IL-15, IL-2, IL-12, IL-21, IL-17, IL-18, IL-23, IL-27, and IL-6. In certain embodiments, the cytokine is IL-15. In certain embodiments, the cytokine is IL-2. In certain embodiments, the subject is a blood cancer patient. In certain embodiments, the subject is a blood cancer patient. In certain embodiments, the subject is a multiple myeloma patient. In certain embodiments, the subject is a relapsed / refractory multiple myeloma patient. In certain embodiments, the subject has received anti-CD38 therapy. In certain embodiments, the subject has received daratumumab. In certain embodiments, the subject is daratumumab-naive, daratumumab-resistant, daratumumab-refractory, or daratumumab-relapsed.In certain embodiments, the subject is daratumumab resistant, daratumumab refractory, or daratumumab relapsed. In certain embodiments, the subject expresses a CD16A polymorphism. In certain embodiments, the CD16A polymorphism is the CD16A-158V / F polymorphism. In certain embodiments, the antibody or antigen-binding protein is administered to a subject having a depleted or reduced NK cell population. In certain embodiments, the subject has been previously treated with anti-CD38 therapy. In certain embodiments, the anti-CD38 therapy is daratumumab therapy.

Example

[0297] Example 1: Structure of the BCMA Antigen-Binding Portion To construct an antigen-binding portion, for example, as described by Smith GP ("Science" (1985), Vol. 228, pp. 1315-7) and Clackson et al. ("Nature" (1991), Vol. 352, pp. 624-8), by expression and display of single-chain Fv domains (scFv) on filamentous fusion phage and by enrichment of phage particles encoding scFv that exhibit target binding by panning on recombinant target antigen or target antigen-positive cells, antibody fragments can be isolated from a human antibody library having selective binding to a selected target antigen. To isolate a BCMA-binding antibody fragment, recombinant human BCMA(1-54)-Fc, cynomolgus BCMA(1-53)-Fc, and CHO cells stably expressing cell surface anchor-type human BCMA(1-54) or cynomolgus BCMA(1-53) fused to the transmembrane region and cytoplasmic domain of human CD3 zeta can be used in subsequent rounds of panning to enrich bound phage particles. For this purpose, phage particles are incubated, for example, at room temperature for 2 hours with the recombinant Fc-fusion antigen in solution, then captured with protein G-coated beads and washed in PBS-Tween and PBS to remove unbound phage. The bound phage is eluted with glycine. For enrichment of bound phage on target antigen-expressing cells, phage is incubated, for example, at room temperature for 1 hour with stably transfected CHO cells, then washed with cell culture medium, and the bound phage is eluted with glycine. To reduce enrichment of phage particles encoding antibody fragments that selectively bind to Fc or non-transfected CHO cells, the phage pool is incubated with an irrelevant Fc-fusion antigen or target antigen-negative CHO cells. Following each round of panning and elution of bound phage, the eluted phage particles are used to infect Escherichia coli (XL1 Blue) to propagate the phage and scFv-encoding DNA.Phage panning was repeated, and after growing the enriched phage clones, the DNA was isolated from E. coli and recloned into a bacterial expression vector, such as pSKK2, using standard molecular biology techniques for subsequent production of the His-tagged (SEQ ID NO: 59) scFv antibody fragment in E. coli and preparation of bacterial periplasmic extracts. The periplasmic extracts containing the scFv antibody fragment are subjected to screening methods such as ELISA or flow cytometry to evaluate binding to the target antigen. For example, recombinant human BCMA(1 - 54)-Fc or cynomolgus BCMA(1 - 53)-Fc is bound by an anti-human Fc antibody coated on standard ELISA microtiter plates, then incubated with the bacterial periplasmic extract and washed extensively. scFv binding is detected using an anti-His-HRP conjugate. To evaluate binding of the scFv to cell-expressed BCMA, the bacterial periplasmic extract is incubated with recombinant CHO cells expressing human or cynomolgus BCMA anchored to the cell surface, then washed and the bound scFv is detected by flow cytometry using anti-His-R-PE. Plasmids encoding scFv antibody fragments that selectively bind to human and / or cynomolgus BCMA antigens are isolated from the respective bacterial clones and the DNA sequences are analyzed to obtain the DNA sequences encoding the scFv. For example, a BCMA antigen-binding portion having an amino acid sequence as described in SEQ ID NO: 39 is obtained. Here, VH is described in SEQ ID NO: 37 and VL is described in SEQ ID NO: 38.

[0298] Example 2: Generation of Different Antigen-Binding Protein Scaffolds 2.1 scDb-mFc (FIGS. 1 and 2): scDb-mFc refers to an antigen-binding protein that is monomeric and contains a bivalent CD16A antigen-binding portion in the format of an scDb fused to a monomeric Fc portion. Either of the scDbs consisting of two CD16A antigen-binding portions fuses to the N-terminus of the Fc portion consisting of a variant CH2-CH3 polypeptide and an scFv containing a single target antigen-binding portion fused to the C-terminus of the Fc portion (Figure 1). Alternatively, the scDb consisting of two CD16A antigen-binding portions fuses to the C-terminus of the Fc portion consisting of a variant CH2-CH3 polypeptide, and the scFv containing a single target antigen-binding portion fuses to the N-terminus of the Fc portion (Figure 2).

[0299] For the expression of the scDb-mFc antigen-binding protein in CHO cells, the coding sequence of the molecule was cloned into a mammalian expression vector system. Briefly, the gene sequences encoding the anti-TAA (tumor-associated antigen) Fv domain and the anti-CD16A Fv domain (SEQ ID NOs: 1-13) in an scFv format linked by a gene sequence encoding a short peptide linker (SEQ ID NOs: 16-18) in the scFv were synthesized by Thermo Fisher Scientific / Invitrogen GeneArt (Regensburg, Germany). PCR amplification products of monomeric Fc portions containing different variable domains and a silencing point mutation (SEQ ID NO: 30) were prepared using corresponding primers. Subsequently, different overlapping DNA fragments and a linearized backbone vector were combined in one isothermal reaction. The scDb-mFc expression constructs were designed to contain, respectively, the coding sequence of an N-terminal signal peptide to promote antibody secretion and an Fc portion to facilitate antibody secretion and purification. The sequences of all constructs were confirmed by DNA sequencing at GATC (Cologne, Germany) using custom primers. The expression cassette of scDb-mFc was cloned such that the anti-CD16A domain was placed at the N-terminus or C-terminus of the monomeric Fc portion and linked in the following possible orders via a linker sequence (SEQ ID NOs: 19-22).

[0300] 1.) VL(CD16A)-L1-VH(CD16A)-L2-VL(CD16A)-L1-VH(CD16A) 2.) VH(CD16A)-L1-VL(CD16A)-L3-VH(CD16A)-L1-VL(CD16A) 3.) VL(CD16A)-L4-VL(CD16A)-L3-VH(CD16A)-L4-VH(CD16A) 4.) VH(CD16A)-L4-VH(CD16A)-L2-VL(CD16A)-L4-VL(CD16A)

[0301] In the structure of the antigen-binding protein, scDb-mFc_01, scDb-mFc_02, scDb-mFc_05, and scDb-mFc_06 specific to SEQ ID NO: 16 are used for linker L1, SEQ ID NO: 17 is used for linker L2, and SEQ ID NO: 18 is used for L3. In the structure of the antigen-binding protein, scDb-mFc_03, scDb-mFc_04, scDb-mFc_07, and scDb-mFc_08 specific to SEQ ID NO: 60 are used for linker L4, SEQ ID NO: 17 is used for linker L2, and SEQ ID NO: 18 is used for L3.

[0302] 2.2 KiH-scDb-Fc (Figures 3 - 6): The KiH-scDb-Fc antigen-binding protein is a heterodimer and contains a bivalent CD16A antigen-binding portion in the format of scDb fused to the heterodimer (KiH) Fc portion. A single target antigen-binding portion is provided by the scFv.

[0303] The DNA expression construct encoding KiH-scDb-Fc is generated by cloning the coding sequence of the anti-CD16A Fv domain (SEQ ID NOs: 1-13) into a modified mammalian expression vector containing a CMV-controlled expression cassette comprising IgG1 constant domains CH2 and CH3 with Fc silencing and "knob-into-hole" point mutations (SEQ ID NOs: 31, 32) for co-expression of two gene cassettes from the same vector. The "knob-into-hole" mutation enables the generation of a heterodimeric trivalent bispecific (Figs. 3-5) or tetravalent trispecific (Fig. 6) Fc fusion construct. Subsequently, a PCR amplification product is generated from the gene sequence encoding the anti-TAA (tumor-associated antigen) Fv domain using corresponding primers. The resulting overlapping DNA fragment is inserted into the co-expression vector at the relevant positions. All necessary gene sequences encoding the variable and constant domains containing Fc silencing and "knob-into-hole" point mutations were synthesized by Thermo Fisher Scientific / Invitrogen GeneArt (Regensburg, Germany). The KiH-scDb-Fc expression construct is designed to contain the coding sequences of an N-terminal signal peptide, a C-tag, and / or a His-tag (6xHis) respectively, to facilitate antibody secretion and purification. The sequences of all constructs were confirmed by DNA sequencing at GATC (Cologne, Germany) using custom primers. The expression cassette of the KiH-scDb-Fc construct is cloned such that the CD16A antigen-binding portion is located at the N-terminus of the heterodimeric Fc portion in only the following possible order and is connected via a linker (SEQ ID NOs: 19-22) and a hinge (SEQ ID NOs: 23) or a middle hinge (SEQ ID NOs: 24), or is located at the C-terminus of the heterodimeric Fc portion and is connected via a linker (SEQ ID NOs: 19-22).

[0304] 1.) VL(CD16A)-L1-VH(CD16A)-L2-VL(CD16A)-L1-VH(CD16A) 2.) VH(CD16A)-L1-VL(CD16A)-L3-VH(CD16A)-L1-VL(CD16A) 3.) VL(CD16A)-L4-VL(CD16A)-L3-VH(CD16A)-L4-VH(CD16A) 4.) VH(CD16A)-L4-VH(CD16A)-L2-VL(CD16A)-L4-VL(CD16A)

[0305] The sequence number 16 is used for linker L1, the sequence number 17 is used for linker L2, the sequence number 18 is used for linker L3, and the sequence number 60 is used for linker L4.

[0306] Examples of such antigen-binding proteins include KiH-scDb-Fc_08 and KiH-scDb-Fc_12 having the following structure from the N-terminus to the C-terminus.

[0307] The first polypeptide chain V L (CD16A)-V H (CD16A)-V L (CD16A)-V H (CD16A)-middle hinge-CH2-CH3 and the second polypeptide chain hinge-CH2-CH3-V H (target)-V L (target) (Figure 3); or, the first polypeptide chain middle hinge-CH2-CH3-V L (CD16A)-V H (CD16A)-V L (CD16A)-V H (CD16A) and the second polypeptide V H (target)-V L (target)-hinge-CH2-CH3 (Figure 5).

[0308] 2.3 scDb-TriB or scDb-TriB-scFv scDb-TriB is a trispecific antigen-binding protein, a heterodimeric Fab fragment comprising a CD16A, a target, and an HSA antigen-binding portion, where the antigen-binding portion is provided by two scDbs fused to the C-terminus of the Fab fragment (Figure 7), or by either of two scDbs comprising a CD16A antigen-binding protein, and the scFv comprising the target antigen-binding portion is fused to the C-terminus of the Fab fragment (Figure 8).

[0309] The DNA expression construct encoding scDb-TriB(-scFv) is prepared by cloning the coding sequences of the anti-HSA Fv domains (SEQ ID NOs: 14, 15) into a modified mammalian expression vector containing a CMV-controlled expression cassette comprising only the heavy and light chain constant domains of the Fab portion with a fusion (SEQ ID NOs: 33 - 35) for co-expression of two gene cassettes from the same vector. Subsequently, PCR amplification products are generated using corresponding primers from the gene sequences encoding the anti-TAA (tumor-associated antigen) Fv domain and the anti-CD16A Fv domain (SEQ ID NOs: 1 - 13). The resulting overlapping DNA fragments are inserted into the co-expression vector at the relevant positions. All necessary gene sequences encoding the variable domains and only the constant domains of the Fab portion were synthesized by Thermo Fisher Scientific / Invitrogen GeneArt (Regensburg, Germany). The scDb-TriB(-scFv) expression construct is designed to contain the coding sequences of an N-terminal signal peptide, a C-tag, and / or a His-tag (6xHis), respectively, to facilitate antibody secretion and purification. The sequences of all constructs were confirmed by DNA sequencing by GATC (Cologne, Germany) using custom primers. The expression cassette of the scDb-TriB(-scFv) construct is cloned such that the anti-CD16A domain is located at the C-terminus of CL or at CH1 of the Fab, connected via a linker (SEQ ID NOs: 19 - 22) to the heavy or light chain of the Fab in the following possible order.

[0310] 1.) VL(CD16A)-L1-VH(CD16A)-L2-VL(CD16A)-L1-VH(CD16A) 2.) VL(CD16A)-L1-VL(CD16A)-L2-VH(CD16A)-L1-VH(CD16A)

[0311] Examples of such antigen-binding proteins include scDb-TriB-scFv_01, which contains the following structure from the N-terminus to the C-terminus. The first polypeptide chain V H (HSA)-CH1-V L (CD16A)-V H (CD16A)-V L (CD16A)-V H (CD16A) and the second polypeptide chain V L (HSA)-CL-V H (target)-V L (target) (Figure 8). SEQ ID NO: 16 is used for linker L1, and SEQ ID NO: 17 or SEQ ID NO: 18 is used for linker L2.

[0312] 2.4 Db-Fc (Figures 9-10): Db-Fc is an antigen-binding protein that is a homodimer and contains a bivalent CD16A antigen-binding portion in the format of Db fused to the hinge at the N-terminus of the homodimeric Fc portion (CH2-CH3) and two target antigen-binding portions in the format of scFv fused to the other end of the Fc portion. For the expression of the Db-Fc antigen-binding protein in CHO cells, the coding sequence of the molecule was cloned into a mammalian expression vector system. Briefly, a gene sequence encoding an anti-TAA (tumor-associated antigen) Fv domain and an anti-CD16A Fv domain (SEQ ID NOs: 1-13) linked by a short peptide linker (SEQ ID NO: 16) was synthesized by Thermo Fisher Scientific / Invitrogen GeneArt (Regensburg, Germany). PCR amplification products of the Fc portion containing different variable domains and a silencing point mutation (SEQ ID NO: 29) were generated using corresponding primers. Subsequently, different overlapping DNA fragments and a linearized backbone vector were combined in one isothermal reaction. The Db-Fc expression construct was designed to contain the sequence of an N-terminal signal peptide to promote antibody secretion. The sequences of all constructs were confirmed by DNA sequencing by GATC (Cologne, Germany) using custom primers. The expression cassette of Db-Fc was cloned such that the anti-CD16A domain is located at the N-terminus or C-terminus of the homodimeric Fc portion (CH2-CH3) connected to the middle hinge (SEQ ID NO: 24) via a linker (SEQ ID NOs: 19-22) only in the following possible order, or is located at the C-terminus of CH3 via a linker (SEQ ID NOs: 19-22).

[0313] 1.) VL(CD16A)-L1-VH(CD16A) 2.) VH(CD16A)-L1-VL(CD16A)

[0314] Examples of such antigen-binding proteins include Db-Fc_02 and Db-Fc_04, which have the following structure from the N-terminus to the C-terminus: V L (CD16A)-V H (CD16A)-middle hinge-CH2-CH3-V H(Target)-V L (Target)(Fig. 9); or V H (Target)-V L (Target)-Middle hinge-CH2-CH3-V L (CD16A)-V H (CD16A) (Fig. 10). Linker L1 uses SEQ ID NO: 16.

[0315] 2.5 Bi-scFv-Fc (Fig. 11 - Fig. 12): Bi-scFv-Fc contains two CD16A antigen-binding parts and two target antigen-binding parts, each in the format of scFv, and is a homodimeric and tetravalent antigen-binding protein fused to an Fc homodimer.

[0316] For the expression of Bi-scFv-Fc antigen-binding protein in CHO cells, the coding sequence of the molecule was cloned into a mammalian expression vector system. Briefly, a gene sequence encoding an anti-TAA (tumor-associated antigen) Fv domain and an anti-CD16A Fv domain (SEQ ID NOs: 1 - 13) linked by a short peptide linker (SEQ ID NOs: 17 - 18) was synthesized by Thermo Fisher Scientific GeneArt (Regensburg, Germany). PCR amplification products of different variable domains with either an Fc portion containing a silencing point mutation (SEQ ID NO: 29) or a wild-type Fc portion (SEQ ID NO: 36) were generated using corresponding primers. Subsequently, different overlapping DNA fragments and a linearized backbone vector were combined in one isothermal reaction. The Bi-scFv-Fc expression constructs were designed to contain, respectively, the coding sequence of an N-terminal signal peptide and an Fc portion to facilitate antibody secretion and purification. The sequences of all constructs were confirmed by DNA sequencing at GATC (Cologne, Germany) using custom primers. The expression cassette of Bi-scFv-Fc was cloned such that the anti-CD16A domain is located at the N-terminus of the Fc portion connected via a linker (SEQ ID NOs: 19 - 22) or a hinge (SEQ ID NO: 23) only in the following possible order, or at the C-terminus of the Fc portion connected to CH3 via a linker (SEQ ID NOs: 19 - 22).

[0317] 1.) VL(CD16A)-L3-VH(CD16A) 2.) VH(CD16A)-L2-VL(CD16A)

[0318] Examples of such antigen-binding proteins include Bi-scFv-Fc_27 and Bi-scFv-Fc_26, which have the following structure from the N-terminus to the C-terminus. V L (CD16A)-V H (CD16A)-hinge-CH2-CH3-V H (target)-V L (target) (Figure 11); or V H (target)-V L (target)-hinge-CH2-CH3-V L (CD16A)-V H (CD16A) (Figure 12). SEQ ID NO: 17 is used for linker L2, and SEQ ID NO: 18 is used for linker L3.

[0319] 2.6 scFv-IgAb (Figure 13): The scFv-IgAb antigen-binding protein comprises an IgG antibody in scFv format and two CD16A antigen-binding moieties, where each of the scFv CD16A antigen-binding moieties is fused to one C-terminus of the H chain, and the two target antigen-binding moieties are provided to each Fv within the Fab arms of the IgG.

[0320] The DNA expression construct encoding scFv-IgAb is prepared by cloning the coding sequence of the anti-TAA Fv domain into a modified mammalian expression vector containing a CMV-controlled expression cassette containing heavy and light chain constant domains with an Fc silencing point mutation (SEQ ID NO: 25, 27-28) or wild-type Fc portion (SEQ ID NO: 26-28) for co-expression of both gene cassettes from the same vector. Subsequently, PCR amplification products are generated using corresponding primers from a gene sequence encoding an anti-CD16A Fv domain (SEQ ID NO: 1-13) separated by a short peptide linker (SEQ ID NO: 17-18). The resulting overlapping DNA fragments are inserted into the co-expression vector at the relevant positions. All necessary gene sequences encoding variable and constant domains containing the Fc silencing point mutation were synthesized by Thermo Fisher Scientific / Invitrogen GeneArt (Regensburg, Germany). The scFv-IgAb expression constructs were each designed to contain a coding sequence for an N-terminal signal peptide and an Fc portion to facilitate antibody secretion and purification. The sequences of all constructs were confirmed by DNA sequencing at GATC (Cologne, Germany) using custom primers. The expression cassette of scFv-IgAb is cloned such that the anti-CD16A domain is positioned at the C-terminus of the Fc portion and linked in the following possible order via a linker (SEQ ID NO: 19-22).

[0321] 1.) VL(CD16A)-L3-VH(CD16A) 2.) VH(CD16A)-L2-VL(CD16A)

[0322] An example of such an antigen-binding protein is scFv-IgAb_30, which contains the following structure from the N-terminus to the C-terminus.

[0323] The first polypeptide chain V H (target)-CH1-hinge-CH2-CH3-V L (CD16A)-V H (CD16A) and the second polypeptide chain V L(Target)-CL(Fig. 13). SEQ ID NO: 17 is used for linker L2, and SEQ ID NO: 18 is used for linker L3.

[0324] 2.7 Bi-scFv-IgAb(Fig. 14): The Bi-scFv-IgAb antigen-binding protein comprises an IgG antibody and four scFv antigen-binding moieties fused thereto, wherein each of the two CD16A antigen-binding moieties in the scFv format is fused to the C-terminus of one of the two H chains, and each of the two target antigen-binding moieties is fused to the C-terminus of one of the two CL regions.

[0325] The DNA expression construct encoding Bi-scFv-IgAb is prepared by cloning the coding sequence of the anti-TAA Fv domain into a modified mammalian expression vector containing a CMV-controlled expression cassette comprising heavy-chain and light-chain constant domains with Fc silencing point mutations (SEQ ID NOs: 25, 27-28) for co-expression of two gene cassettes from the same vector. Subsequently, PCR amplification products are generated using corresponding primers from gene sequences encoding anti-CD16A or target antigen-binding Fv domains (SEQ ID NOs: 1-13) separated by short peptide linkers. The resulting overlapping DNA fragments are inserted into the co-expression vector at the relevant positions. All necessary gene sequences encoding variable and constant domains containing Fc silencing point mutations were synthesized by Thermo Fisher Scientific / Invitrogen GeneArt (Regensburg, Germany). The Bi-scFv-IgAb expression construct was designed to contain the coding sequence of the N-terminal signal peptide and an Fc portion to facilitate antibody secretion and purification, respectively. The sequences of all constructs were confirmed by DNA sequencing at GATC (Cologne, Germany) using custom primers. The expression cassette of Bi-scFv-IgAb is cloned such that the anti-CD16A domain is placed at the C-terminus of the Fc portion and linked in the following possible order via a linker (SEQ ID NOs: 19-22).

[0326] 1.) VL(CD16A)-L3-VH(CD16A) 2.) VH(CD16A)-L2-VL(CD16A)

[0327] Examples of such antigen-binding proteins include Bi-scFv-IgAb_03, which contains the following structure from the N-terminus to the C-terminus.

[0328] The first polypeptide chain V H (Target 1)-CH1-Hinge-CH2-CH3-V L (CD16A)-V H (CD16A) and the second polypeptide chain V L (Target 1)-CL-V L (Target 2)-V H (Target 2) (Figure 14). SEQ ID NO: 17 is used for linker L2, and SEQ ID NO: 18 is used for linker L3.

[0329] 2.8 KiH-scFv-Fc (Figures 15a and 15b): The KiH-scFv-Fc antigen-binding protein contains four scFv antigen-binding portions fused to the heterodimeric (KiH) Fc portion, where the two CD16A antigen-binding portions are fused to the Fc portion either at the N-terminus or the C-terminus.

[0330] The DNA expression construct encoding KiH-scFv-Fc is generated by cloning the coding sequence of the anti-CD16A Fv domain (SEQ ID NOs: 1 to 13) into a modified mammalian expression vector containing a CMV-controlled expression cassette including IgG1 constant domains CH2 and CH3 with Fc silencing and "knob-into-hole" point mutations (SEQ ID NOs: 31 to 32) for co-expression of two gene cassettes from the same vector. The "knob-into-hole" mutation enables the generation of bispecific Fc fusion constructs. Subsequently, PCR amplification products are generated from the gene sequence encoding the anti-TAA (tumor-associated antigen) Fv domain using corresponding primers. The resulting overlapping DNA fragments are inserted into the co-expression vector at the relevant positions. All necessary gene sequences encoding variable and constant domains containing Fc silencing and "knob-into-hole" point mutations were synthesized by Thermo Fisher Scientific / Invitrogen GeneArt (Regensburg, Germany). The KiH-scFv-Fc expression construct is designed to contain the coding sequences of an N-terminal signal peptide, an Fc portion, a C-tag, and a His-tag (6xHis), respectively, to facilitate antibody secretion and purification. The sequences of all constructs were confirmed by DNA sequencing by GATC (Cologne, Germany) using custom primers. The expression cassette of the KiH-scFv-Fc construct is cloned such that the anti-CD16A domain is located at the N-terminus of the heterodimeric Fc portion connected via a linker (SEQ ID NOs: 19 to 22) and a hinge (SEQ ID NO: 23) only in the following possible order, or at the C-terminus of the Fc portion via the linker (SEQ ID NOs: 19 to 22).

[0331] 1.) VL(CD16A)-L3-VH(CD16A) 2.) VH(CD16A)-L2-VL(CD16A)

[0332] Examples of such antigen-binding proteins include KiH-scFv-Fc_09 and KiH-scFv-Fc_11, which consist of the following structure from the N-terminus to the C-terminus.

[0333] The first polypeptide chain V L (CD16A)-V H (CD16A)-hinge-CH2-CH3-V H (Target 1)-V L (Target 1) and the second polypeptide chain V L (CD16A)-V H (CD16A)-hinge-CH2-CH3-V H (Target 2)-V L (Target 2) (Figure 15a); or the first polypeptide chain V H (Target 1)-V L (Target 1)-hinge-CH2-CH3-V L (CD16A)-V H (CD16A) and the second polypeptide chain V H (Target 2)-V L (Target 2)-hinge-CH2-CH3-V L (CD16A)-V H (CD16A) (Figure 15b). SEQ ID NO: 17 is used for linker L2 and SEQ ID NO: 18 is used for linker L3.

[0334] Example 3: Production of an NK cell inducer antibody format using a stable CHO cell pool Host cell culture Flp-In CHO cells (Life Technologies), a derivative of CHO-K1 Chinese hamster ovary cells (ATCC, CCL-61) (Kao and Puck, 1968), were cultured in Ham’s F-12 Nutrient Mix supplemented with L-glutamine, 10% FCS, and 100 μg / mL zeocin. Adherent cells were detached with 0.25% trypsin-EDTA and subcultured according to the standard cell culture protocol from Life Technologies.

[0335] To adapt the cells for growth in suspension, the cells were detached from the tissue culture flask, placed in serum-free HyClone CDM4 CHO medium, and then in 5% CO at 37 °C 2And incubated in a shaking flask at 120 rpm. The standard medium for the culture of suspension-adapted Flp-In CHO host cells was HyClone CDM4 CHO supplemented with L-glutamine, HT supplement, penicillin / streptomycin, and 100 μg / mL zeocin. The suspension-adapted cells were cryopreserved in medium with 10% DMSO and tested for Mycoplasma negativity using the MycoAlert Mycoplasma detection kit (Lonza).

[0336] Generation of stably transfected cell pools A recombinant Flp-In CHO cell line that stably expresses a secreted recombinant antibody, Fc fusion construct, or comparative antibody was generated by transfection of a suspension-compatible host cell. For this, 1 day prior to co-transfection of an expression plasmid (2.5 μg) encoding the protein of interest (pcDNA5-FRT) and Flp recombinase (pOG44, Life Technologies) using polyethyleneimine (PEI), the cells were placed in standard medium without zeocin. Briefly, vector DNA and transfection reagent were mixed at a DNA:PEI ratio of 1:3 (μg / μg) in a total of 100 μL of OptiMEM I medium, incubated for 10 minutes, and then added to 2E+6 Flp-In CHO cells (Life Technologies) suspended in 1 mL of CHO-S-SFMII medium. Following 24 - 48 hours of incubation, selection of stably transfected cells was initiated by addition of 6 - 7 μg / mL of puromycin dihydrochloride, after which the culture was diluted to a density of 0.2E+6 viable cells / mL in CHO-S-SFMII medium. Flp recombinase mediates the insertion of the Flp-In expression construct into the genome at the FRT sites integrated by site-specific DNA recombination (O’Gorman et al., 1991). During selection, the viable cell density was measured twice a week, the cells were centrifuged, and re-suspended in fresh selection medium at a maximum density of 0.2E+6 viable cells / mL. A cell pool that stably expressed the recombinant protein product was recovered after 2 - 3 weeks of selection when the cells were transferred to standard culture medium in a shake flask. Expression of the recombinant secreted protein was confirmed by protein gel electrophoresis of the cell culture supernatant using the Criterion Stain-Free (Bio-Rad) technology (see below). Stable cell pools were cryopreserved in medium containing 7.5% DMSO.

[0337] Production of recombinant protein in a fed-batch CHO cell suspension culture The recombinant protein was produced in a fed-batch culture of CHO cells stably transfected by secretion into the cell culture supernatant for 10 or 11 days. For this purpose, cells stably expressing a recombinant antibody, an Fc fusion antigen, or a comparator antibody were seeded in a standard medium in a polycarbonate Erlenmeyer flask equipped with a gas-permeable cap (Corning) at an initial density of 6E+5 cells / mL and incubated at 37 °C and 5% CO 2 while stirring at 140 rpm. During the fed-batch culture, the medium was supplemented with 40 mL / L of ActiCHO Feed A (GE Healthcare) and 4 mL / L of ActiCHO Feed B (GE Healthcare) on day 0 (start day), and double the amounts were supplemented on days 3, 5, and 7. The cell culture supernatant was typically collected after 10 or 11 days with a culture viability of over 75%. Samples were taken from the production culture every other day before feeding to evaluate cell density and viability. On the day of collection, the cell culture supernatant was removed prior to further use by centrifugation and vacuum filtration (0.22 μm) using Millipore Express PLUS Membrane Filters (Millipore).

[0338] Expression titer quantification: The protein expression titer and product integrity in the cell culture supernatant (CSS) were analyzed by SDS-PAGE on days 5, 7, and 10 or 11 of the production culture. Samples were mixed with SDS PAGE sample buffer before loading onto 4–20% Criterion TGX Precast SDS PAGE Gels (Biorad). Total protein was visualized in the gel using a Criterion Stain-free Molecular Imaging System (Biorad). The product titer was determined semi-quantitatively by comparison with a reference antibody of known concentration.

[0339] Purification of antigen-binding protein 1. Antigen-binding proteins containing an Fc portion (scDb-mFc, Db-Fc, Bi-scFv-Fc, Bi-scFv-IgAb, scFv-Fc, scFv-IgAb as described in Example 2) The target protein was purified from clarified CHO cell culture supernatant in a two-step procedure involving Protein A and preparative SEC. For Protein A, the clarified supernatant was loaded onto a HiTrap MabSelectSuRe column (GE Healthcare). After washing with phosphate-buffered saline pH 7.4 and 10 mM sodium phosphate pH 7.0, the protein was eluted with a two-step gradient of 50 mM sodium acetate pH 3.5 and 10 mM glycine / HCL pH 2.0. The purity of the fractions was analyzed using SE-HPLC and SDS-PAGE. Fractions showing acceptable purity were pooled and subjected to preparative gel filtration using a Superdex 200 prep-grade column (GE-Healthcare). The eluted fractions containing the purified target molecule were pooled and buffer exchange was performed using a Sephadex G-25 column having 10 mM sodium acetate, 4.5% sorbitol pH 5.0, and concentrated to a typical concentration of approximately 1 mg / mL by ultrafiltration. The typical purity (measured by SE-HPLC) of these constructs ranged from 87.6% to 99.8%, and the homogeneity (non-reducing SDS-PAGE) was between 58.4% and 100%.

[0340] 2. Antigen-binding proteins KiH-scDb-Fc and scDb-tribody-scFv according to Example 2 containing a κ-light chain and His tag The target protein was purified from clarified CHO cell culture supernatant in a three-step procedure involving Protein L, IMAC, and preparative SEC. For Protein L, the clarified supernatant was loaded onto a HiTrap Protein L column (GE Healthcare). After washing with phosphate buffered saline pH 7.4 and 10 mM sodium phosphate pH 7.0, the protein was eluted with a two-step gradient of 10 mM glycine / HCL pH 3.0 and 10 mM glycine / HCL pH 2.0. Fractions were analyzed for purity using SE-HPLC and SDS-PAGE. Fractions showing acceptable purity were pooled and subjected to further IMAC purification. Thus, the sample was diluted (1:4) with equilibration buffer (50 mM Tris / HCL, 150 mM sodium chloride pH 7.5) and loaded onto a HisTrap FF column (GE Healthcare). After washing with equilibration buffer (50 mM Tris / HCL, 150 mM sodium chloride pH 7.5), the protein was eluted with a three-step gradient of 7% / 30% / 100% elution buffer (50 mM Tris / HCL, 0.4 M arginine, 500 mM imidazole pH 7.5). Fractions were analyzed for purity using SE-HPLC and SDS-PAGE. Fractions showing acceptable purity were pooled and subjected to preparative gel filtration using a Superdex 200 prep-grade column (GE-Healthcare). The elution fractions containing the purified target molecule were pooled and buffer exchanged using a Sephadex G-25 column having 10 mM sodium acetate, 4.5% sorbitol pH 5.0 and concentrated to a typical concentration of approximately 1 mg / mL by ultrafiltration. The typical purity (measured by SE-HPLC) of these constructs ranged from 95.9% to 99.8% and the homogeneity (non-reducing SDS-PAGE) was between 72.2% and 99.0%.

[0341] 3) Antigen-binding protein molecules KiH-scFv-Fc and KiH-scDb-Fc containing a C-tag on the first polypeptide and a His-tag on the second polypeptide The target protein was purified from clarified CHO cell culture supernatant in a three-step procedure involving C-tag affinity chromatography, IMAC, and preparative SEC. In C-tag affinity chromatography, the clarified supernatant was loaded onto a CaptureSelect C-tag XL column (Thermo Scientific). After washing with phosphate-buffered saline pH 7.4, the protein was eluted with 20 mM sodium citrate pH 3.0. The purity of the fractions was analyzed using SE-HPLC and SDS-PAGE. Fractions showing acceptable purity were pooled and subjected to further IMAC purification. Thus, the sample was diluted (1:4) with equilibration buffer (50 mM Tris / HCL, 150 mM sodium chloride pH 7.5) and loaded onto a HisTrap FF column (GE Healthcare). After washing with equilibration buffer (50 mM Tris / HCL, 150 mM sodium chloride pH 7.5), the protein was eluted in a three-step gradient with 7% / 30% / 100% elution buffer (50 mM Tris / HCL, 0.4 M arginine, 500 mM imidazole pH 7.5). The purity of the fractions was analyzed using SE-HPLC and SDS-PAGE. Fractions showing acceptable purity were pooled and subjected to preparative gel filtration using a Superdex 200 prep-grade column (GE-Healthcare). The elution fractions containing the purified target molecule were pooled and buffer exchange was performed using a Sephadex G-25 column with 10 mM sodium acetate, 4.5% sorbitol pH 5.0, and concentrated to a typical concentration of approximately 1 mg / mL by ultrafiltration. The typical purity (measured by SE-HPLC) of these constructs ranged from 94.8% to 99.0%, and the homogeneity (non-reducing SDS-PAGE) was between 86.8% and 100%.

[0342] 4) scDb tribody The protein was purified from clarified CHO cell culture supernatant in a two-step procedure involving IMAC and preparative SEC. For IMAC, the clarified supernatant and 5 mM imidazole pH 7.0 were loaded onto a HisTrap FF column (GE Healthcare). After washing with equilibration buffer (50 mM Tris / HCL, 150 mM sodium chloride pH 7.5), the protein was eluted in a three-step gradient with 7% / 30% / 100% elution buffer (50 mM Tris / HCL, 0.4 M arginine, 500 mM imidazole pH 7.5). Fractions were analyzed for purity using SE-HPLC and SDS-PAGE. Fractions showing acceptable purity were pooled and subjected to preparative gel filtration using a Superdex 200 prep-grade column (GE-Healthcare). The elution fractions containing the purified target molecule were pooled and buffer exchanged using a Sephadex G-25 column with 10 mM sodium acetate, 4.5% sorbitol pH 5.0, and concentrated to a typical concentration of approximately 1 mg / mL by ultrafiltration. The typical purity (measured by SE-HPLC) of these constructs ranged from 95.1% to 100%, and the homogeneity (non-reducing SDS-PAGE) was above 85.8%.

[0343] a) scDb-Tribody_09-10 The protein was purified from clarified CHO cell culture supernatant in a two-step procedure involving Fab / lambda affinity chromatography and preparative SEC. In Fab / lambda affinity chromatography, the clarified supernatant was loaded onto a Fab Select Lambda column (GE Healthcare). After washing with phosphate buffered saline pH 7.4, the protein was eluted with 100 mM sodium acetate pH 3.5. Fractions were analyzed for purity using SE-HPLC and SDS-PAGE. Fractions showing acceptable purity were pooled and subjected to preparative gel filtration using a Superdex 200 prep-grade column (Ge-Healthcare). The elution fractions containing the purified target molecule were pooled and buffer exchanged using a Sephadex G-25 column with 10 mM sodium acetate, 4.5% sorbitol pH 5.0 and concentrated by ultrafiltration to a typical concentration of approximately 1 mg / mL. The typical purity (measured by SE-HPLC) of these constructs was greater than 94.8% and the homogeneity (non-reducing SDS-PAGE) was greater than 92.7%.

[0344] Protein analysis The homogeneity of the final samples was evaluated by SDS-PAGE under reducing and non-reducing conditions. Samples were mixed with either non-reducing 2X SDS PAGE sample buffer or reducing 2X SDS-PAGE sample buffer containing dithiothreitol (DTT) as the reducing agent. All samples were heated at 95 °C for 5 minutes before loading onto a 4-20% Criterion TGX Precast SDS Page Gel. 2 μg of purified protein sample was loaded. SDS-PAGE was run at 300 V for approximately 22 minutes in 1X Tris / glycine / SDS buffer to separate the proteins in the gel. Total protein was visualized in the gel using a Criterion Stain-free Molecular Imaging System (Biorad). Page Ruler Unstained Protein ladder was used as the molecular weight marker. The relative signal intensity of the product band in non-reducing SDS-PAGE was compared to possible high molecular weight or low molecular weight species.

[0345] The purity of the protein preparation was evaluated by analytical SE-HPLC using a Superdex 200 Increase 10 / 300GL column (GE-Healthcare).

[0346] The purified protein was stored as aliquots at -80 °C until further use.

[0347] Example 4: Binding of the CD16A antigen-binding protein to primary human NK cells at 37 °C, or binding to recombinant human CD16A soluble antigen by ELISA Method Isolation of PBMCs from buffy coats and enrichment of human NK cells PBMCs were isolated from buffy coats (German Red Cross, Mannheim, Germany) by density gradient centrifugation. The buffy coat samples were diluted with 2 - 3 volumes of PBS (Invitrogen, catalog number: 14190 - 169), layered onto a cushion of Lymphoprep (Stem Cell Technologies, catalog number: 07861), and centrifuged at 800 × g without interruption for 25 minutes at room temperature. The PBMCs located at the interface were collected, washed 3 times with PBS, and then cultured overnight in complete RPMI 1640 medium supplemented with 10% FCS without stimulation. For enrichment of NK cells, PBMCs were collected after overnight culture and subjected to negative selection once using the EasySep™ Human NK Cell Enrichment Kit (Stem Cell Technologies, catalog number: 19955) to isolate unstimulated human NK cells and the Big Easy EasySep™ Magnet (Stem Cell Technologies, catalog number: 18001) according to the manufacturer's instructions.

[0348] Cell binding assay and flow cytometry analysis An aliquot of enriched human NK cells was incubated at 37 °C for 45 min with 100 μL serial dilutions of the indicated CD16A AAF constructs in FACS buffer (PBS, Invitrogen, catalog number: 14190-169) containing 2% heat-inactivated FCS (Invitrogen, catalog number: 10270-106) and 0.1% sodium azide (Roth, Karlsruhe, Germany, catalog number: A1430.0100). After repeated washing with FACS buffer, cell-bound antibodies were detected with 15 μg / mL FITC-conjugated goat anti-human IgG (Dianova, catalog number: 109-095-098). After the final staining step, cells were washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg / mL propidium iodide (PI) (Sigma, catalog number: P4170) to remove dead cells. Fluorescence of live cells was measured using a Millipore Guava EasyCyte flow cytometer (Merck Millipore, Schwalbach, Germany). The mean fluorescence intensity of the cell samples was calculated using Incyte software (Merck Millipore, Schwalbach, Germany). After subtracting the fluorescence intensity values of cells stained with secondary and tertiary reagents alone, the values were used for non-linear regression analysis using GraphPad Prism software (GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla, CA, USA). For the calculation of K 3 fluorescence of live cells was measured. Fluorescence intensity values of cells stained with secondary and tertiary reagents alone were subtracted, and the resulting values were used for non-linear regression analysis using GraphPad Prism software (GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla, CA, USA). D The equation for one-site binding (hyperbola) was used for the calculation.

[0349] Analysis of soluble antigen binding of CD16A in ELISA A 96-well ELISA plate (Immuno MaxiSorp; Nunc) was coated overnight at 4°C with a recombinant NK cell inducer antibody format or a control antibody in 100 mM bicarbonate buffer. Antibodies were coated at a concentration of 2.0 - 5.0 μg / mL corresponding to an approximately 20 nM molar concentration, depending on the molecular weight. After a blocking step with 3% (w / v) non-fat dry milk (Merck) dissolved in PBS, serial dilutions of biotinylated recombinant human CD16A (48R - 158V) fused to the human IgG1 Fc portion in PBS containing 0.3% (w / v) non-fat dry milk were incubated on the plate for 1.5 hours at room temperature. After washing 3 times with 300 μL / well of PBS containing 0.1% (v / v) Tween 20, the plate was incubated with streptavidin-HRP (Roche) of the detection complex at a 1:10,000 dilution for 1 hour at room temperature. After washing 3 times with 300 μL / well of PBS containing 0.1% (v / v) Tween 20, the plate was incubated with a tetramethylbenzidine (TMB) substrate (Seramun) until color development was clearly visible. The reaction was stopped by adding 100 μL / well of 0.5 M H 2 SO 4 The absorbance was measured at 450 nm using a multi-label plate reader (Victor, Perkin Elmer). Absorbance values were plotted and analyzed using GraphPad Prism version 6.07 (GraphPad Software, La Jolla, CA, USA) with non-linear regression, sigmoid dose-response (variable slope), least squares (ordinary) fitting.

[0350] Results The apparent binding affinity of the indicated CD16A antigen-binding protein was determined on primary human NK cells at 37°C in at least two independent experiments. Additionally, the soluble CD16A binding ability of the CD16A antigen-binding protein was analyzed by ELISA. Both methods demonstrate that the CD16A antigen-binding protein exhibits specific binding to CD16A. High affinity is obtained through bivalent CD16 binding. The absolute difference between the KD value measured by NK cell binding and the EC50 value measured by ELISA is due to differences in methodology, reagents, and assay setup. The results are shown in Table 2.

[0351] TIFF2025081307000004.tif252170TIFF2025081307000005.tif240170TIFF2025081307000006.tif249170TIFF2025081307000007.tif249170TIFF2025081307000008.tif245170

[0352] Example 5: Cytotoxic Activity of the CD16A Antigen-Binding Protein Against Tumor Target Cells Method: Culturing of Cell Lines A-431 (ATCC, catalog number: CRL-1555) was cultured under standard conditions in DMEM medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 100 IU / mL penicillin G sodium, and 100 μg / mL streptomycin sulfate (all components from Invitrogen). RPMI-8226 (DSMZ, catalog number: ACC402) and MM.1S (ATCC, catalog number: CRL2974) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 100 IU / mL penicillin G sodium, and 100 μg / mL streptomycin sulfate. SU-DHL-6 (DSMZ, catalog number: ACC572) was cultured in RPMI 1640 medium supplemented with 20% heat-inactivated FCS, 2 mM L-glutamine, and 100 IU / mL penicillin G sodium, and 100 μg / mL streptomycin sulfate. NCI-H929 (DSMZ, catalog number: ACC163) was cultured in RPMI 1640 medium supplemented with 20% heat-inactivated FCS, 2 mM L-glutamine, and 100 IU / mL penicillin G sodium, 100 μg / mL streptomycin sulfate, 1 mM sodium pyruvate, and 50 μM mercaptoethanol (all components from Invitrogen).

[0353] All cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO 2 2.

[0354] Isolation of PBMC from buffy coats and enrichment of human NK cells PBMCs were isolated from buffy coats (German Red Cross, Mannheim, Germany) by density gradient centrifugation. Buffy coat samples were diluted with 2 to 3 volumes of PBS (Invitrogen, catalog number: 14190-169), layered on a cushion of Lymphoprep (Stem Cell Technologies, catalog number: 07861), and centrifuged at 800 x g without interruption for 25 minutes at room temperature. PBMCs located at the interface were collected, washed 3 times with PBS, and then cultured overnight without stimulation in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 100 IU / mL penicillin G sodium, and 100 μg / mL streptomycin sulfate. For enrichment of NK cells, PBMCs were collected after overnight culture and subjected to negative selection once using the EasySep™ Human NK Cell Enrichment Kit (Stem Cell Technologies, catalog number: 19055) according to the manufacturer's instructions to immunomagnetically isolate unstimulated human NK cells and the Big Easy EasySep™ Magnet (Stem Cell Technologies, catalog number: 18001).

[0355] 4-hour Calcein Release Cytotoxicity Assay In the Calcein Release Cytotoxicity Assay, the indicated target cells were harvested from cultures, washed with RPMI 1640 medium without FCS, and labeled with 10 μM Calcein AM (Invitrogen / Molecular Probes, catalog number: C3100MP) in RPMI medium without FCS for 30 minutes at 37°C. After gentle washing, the labeled cells were resuspended in complete RPMI medium (RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 4 mM L-glutamine, 100 U / mL penicillin G sodium, 100 μg / mL streptomycin sulfate) to a density of 1x10 5 / mL. Then, 1x10 4Individual target cells were seeded in duplicate at a total volume of 200 μL / well into individual wells of a round-bottom 96-well microplate, together with primary human NK cells enriched at the indicated (effector to target) E:T ratio (usually 5:1 or 2:1) and the indicated antibody. Natural release, maximum release, and effector-mediated target killing in the absence of antibody were measured in quadruplicate on each plate.

[0356] After centrifugation at 200 g for 2 minutes, the assay was typically incubated for 4 hours (3 hours in some assays, as indicated) at 37 °C in a humidified atmosphere of 5% CO 2 2. Fifteen minutes before the end of incubation, 20 μL of 10% Triton X-100 in RPMI medium was added to the wells containing the target cells. 20 μL of RPMI medium was added to all other wells. 100 μL of cell culture supernatant was harvested from each well at 500 g after an additional 5 minutes of centrifugation, and the fluorescence of the released calcein was measured at 520 nm using a fluorescence plate reader (Victor 3, Perkin Elmer). Based on the measured counts, specific cell lysis was calculated according to the following formula: [fluorescence (sample) - fluorescence (spontaneous)] / [fluorescence (maximum) - fluorescence (spontaneous)] × 100%. Fluorescence (spontaneous) represents the fluorescence count from target cells in the absence of effector cells and antibody, and fluorescence (maximum) represents the total cell lysis induced by the addition of Triton X-100. The sigmoid dose-response curve and EC 50 values were calculated by non-linear regression / 4-parameter logistic fitting using GraphPad Prism software (GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla, California, USA).

[0357] The results of the cytotoxic activity of the antigen-binding protein against tumor target cells are shown in Table 3.

[0358] TIFF2025081307000009.tif255170TIFF2025081307000010.tif225170TIFF2025081307000011.tif231170

[0359] Example 6: Evaluation of NK-NK Lysis by CD16A Antigen-Binding Protein Method: Isolation of PBMCs from Buffy Coat and Enrichment of Human NK Cells PBMCs were isolated from buffy coat (German Red Cross, Mannheim, Germany) by density gradient centrifugation. Buffy coat samples were diluted with 2 - 3 volumes of PBS (Invitrogen, catalog number: 14190 - 169), layered on a cushion of Lymphoprep (Stem Cell Technologies, catalog number: 07861), and centrifuged at 800×g for 25 minutes without interruption at room temperature. PBMCs located at the interface were collected, washed 3 times with PBS, and then cultured overnight in complete RPMI 1640 medium supplemented with 10% FCS without stimulation. For enrichment of NK cells, PBMCs were collected after overnight culture and used once for negative selection using the EasySep™ Human NK Cell Enrichment Kit (Stem Cell Technologies, catalog number: 19055) to immunomagnetically isolate unstimulated human NK cells and Big Easy EasySep™ Magnet (Stem Cell Technologies, catalog number: 18001) according to the manufacturer's instructions.

[0360] 4-Hour Calcein Release Cytotoxicity Assay In the calcein release cytotoxicity assay to evaluate NK-NK cell lysis, half of the concentrated inactivated NK cells were washed with RPMI 1640 medium without FCS and labeled with 10 μM calcein AM (Invitrogen / Molecular Probes, catalog number: C3100MP) for 30 minutes at 37 °C in RPMI medium without FCS. After gentle washing, the labeled cells were resuspended in complete RPMI medium (RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 4 mM L-glutamine, 100 U / mL penicillin G sodium, 100 μg / mL streptomycin sulfate). Then, 5 x 10 4 target cells were seeded in duplicate at a total volume of 200 μL / well into individual wells of a round-bottom 96-well microplate together with NK cells derived from the same donor at an E:T ratio of 1:1 and the indicated antibodies. Natural release, maximum release, and target killing by effectors in the absence of antibody were measured in quadruplicate on each plate.

[0361] After centrifugation at 200 x g for 2 minutes, the assay was performed in 5% CO 2It was incubated at 37 °C for 4 hours in a humidified atmosphere. Fifteen minutes before the end of the incubation, 20 μL of 10% Triton X-100 in RPMI medium was added to the wells containing the target cells. 20 μL of RPMI medium was added to all other wells. 100 μL of the cell culture supernatant was collected from each well at 500 g after an additional 5-minute centrifugation, and the fluorescence of the released calcein was measured at 520 nm using a fluorescence plate reader (Victor 3, Perkin Elmer). Based on the measured counts, specific cell lysis was calculated according to the following formula: [Fluorescence (sample) - Fluorescence (spontaneous)] / [Fluorescence (maximum) - Fluorescence (spontaneous)] × 100%. Fluorescence (spontaneous) represents the fluorescence count from the target cells in the absence of effector cells and antibodies, and fluorescence (maximum) represents the total cell lysis induced by the addition of Triton X-100. The sigmoid dose-response curve was calculated by non-linear regression / 4-parameter logistic fitting using GraphPad Prism software (GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla, California, USA) to determine the EC 50 [pM] and E max [%] values were used.

[0362] The CD16A antigen-binding protein is considered negative for inducing NK-NK lysis if there is no or only a slight minimum lysis of less than 10% measurable at antibody concentrations up to 30 μg / mL in assays where daratumumab induced more than 50% NK lysis.

[0363] Results: Each of the antigen-binding proteins was tested in at least two independent 4-hour calcein release cytotoxicity assays for NK cell-mediated NK lysis.

[0364] The results are summarized in Table 4 and Figures 17A - 17N, and the potency (EC 50 ) and efficacy (E max) has clearly demonstrated that it does not correlate with the recombinant CD16A antigen on NK cells or the apparent binding affinity of these proteins for CD16A (summarized in Table 2). Additionally, the efficacy (EC 50 ) of the CD16A antigen-binding protein on the lysis of antigen-positive tumor target cells also does not correlate with the ability to mediate NK-NK lysis. From these findings, it can be concluded that the tendency to mediate NK-NK lysis is not a characteristic of the anti-CD16A Fv domain itself, nor a function of their apparent affinity, but rather a characteristic property of the format, particularly the domain order of the variable heavy chain segment and variable light chain segment of a given CD16A antigen-binding protein.

[0365] TIFF2025081307000012.tif255170TIFF2025081307000013.tif229170TIFF2025081307000014.tif230170TIFF2025081307000015.tif225170TIFF2025081307000016.tif253170

[0366] Table 4 shows that antigen-binding proteins containing two CD16A antigen-binding moieties in the format of scDb, Db, or scFv fused to the Fc portion or Fab region do not induce NK-NK lysis when the variable regions are arranged from N-terminus to C-terminus in scDb V L -V H -V L -V H or scDb V L -V L -V H -V H or Db V L -V H or scFv V L -V H . Thereby, the results demonstrate that NK-NK lysis does not depend on the apparent binding affinity of these antigen-binding moieties for recombinant CD16A or CD16A on NK cells as defined by EC50 and Emax. Furthermore, NK-NK lysis is also independent of the target antigen-binding domain.

[0367] Example 7 - Re - induced Optimized Cell Killing (ROCK®): A Highly Versatile Multispecific and Qualified Antibody Platform for Involvement in Innate Immunity Overview Based on the ROCK® (Re - induced Optimized Cell Killing) platform, a portfolio of immunocyte inducers with unique properties was developed. This novel modular platform offers advantages in NK cell binding over the concepts of classical monoclonal antibodies and other inducers. Molecular design, binding affinity, activation of immune effector cells, and PK properties were considered. The ROCK® platform can be used to produce novel antibodies aimed at activating innate and adaptive immunity.

[0368] In this example, a novel and fully modular platform called the “Re - induced Optimized Cell Killing” (ROCK®) platform is presented. This platform contains a wide range of different formats with a unique anti - CD16A binding domain in order to generate a new class of NK cell - mobilizing antibodies aimed at activating innate and adaptive immunity.

[0369] Materials and Methods Generation of ROCK® Recombinant Antibodies, Controls, and Antigen Variants The gene sequences of different recombinant proteins (antigens, antibodies, controls) were synthesized by Thermo Fisher Scientific / Invitrogen GeneArt (Regensburg, Germany) or derived by PCR. Expression vectors encoding these recombinant proteins were generated by cloning each sequence element into the mammalian expression vector pcDNA5 / FRT (Life Technologies) or its modified version using standard molecular biology techniques. Soluble recombinant antigen variants were constructed as fusion proteins of the ECD sequence with monomeric Fc (Ying et al., "J Biol Chem" (2012) Vol. 287, pp. 19399-408). For the expression of cell surface-anchored antigen variants, the ECD sequence was fused to the transmembrane domain of human EGFR (Wang et al., "Blood" (2011) Vol. 118, pp. 1255-63), or anchored via GPI using the endogenous human CD16B sequence with the full-length propeptide sequence for post-translational processing and lipidation for GPI anchoring. In some expression constructs, the original vector was modified to contain two CMV promoter-controlled expression cassettes for co-expression of the two antibody chains. Further modification of the mammalian expression vector was to replace hygromycin resistance with the puromycin resistance gene. The expression constructs were further designed to contain the coding sequence of the N-terminal signal peptide to facilitate secretion. For recombinant fusion constructs encoding the fusion partner (e.g., antigen with IgG1 Fc portion) sequences, PCR amplification was performed using extended primers to construct the corresponding linker or connector sequences for gene fusion or restriction enzyme digestion. The resulting overlapping DNA fragments were inserted into the co-expression vector at the relevant positions using the Gibson assembly method to obtain the final construct (Gibson et al., "Nat Methods" (2010), Vol. 7, pp. 901-3; Gibson et al., "Nat Methods" (2009) Vol. 6, pp. 343-5). The sequences of all constructs were confirmed by the Sanger method at GATC (Cologne, Germany) using custom primers.

[0370] Soluble antibodies or antigens, or cell surface anchor antigens, were expressed in CHO as described above (Reusch et al., "Clin Cancer Res" (2016), Vol. 22, pp. 5829 - 38). The target protein was purified from the cell culture supernatant using one or two standard affinity chromatography methods (Protein A, Protein L, Capture Select C-tag, or IMAC), depending on the monomeric or multimeric (homo-dimeric, hetero-dimeric, or tetrameric) product form and the presence of a fusion affinity tag, κ light chain, or Fc portion. The total protein preparation was further polished by size exclusion chromatography and analyzed using SEC-MALS, SDS-PAGE, and UV-Vis spectroscopy.

[0371] Dot blot, SDS-PAGE, and Western blot The purified recombinant CD16 antigen variant was spotted onto a nitrocellulose membrane or mixed with sample buffer and separated on 4 - 20% Criterion TGX Precast Gels (Bio-Rad). The total protein was imaged using a Bio-Rad Molecular Imager Gel Documentation system. The protein was transferred by Western blotting to a PVDF Midi membrane using a Trans-Blot Turbo system (Bio-Rad). The membrane was blocked with 3% (w / v) non-fat dry milk (Merck) dissolved in TBS for 30 minutes, incubated with 2 - 4 μg / mL anti-CD16scFv antibody for 1 hour at room temperature, followed by washing with TBST (TBS containing 0.1% (v / v) Tween 20) and then washing twice with TBS. The membrane was incubated with anti-Penta-His-HRP (QIAGEN) diluted as a secondary detection conjugate 1:3000 for 1 hour at room temperature. After washing, a freshly prepared mixture of 0.66 mg / mL DAB, 0.02% CoCl 2 and 0.015% H 2 2 2 O was added to initiate colorimetric development and the membrane was washed with water to stop. The membrane was dried and photographed.

[0372] Analysis of CD16A Antigen Binding in ELISA A 96-well ELISA plate (Immuno MaxiSorp; Nunc) was coated overnight at 4°C in 100 mM carbonate buffer with either recombinant antigen or different NK cell inducer antibody formats. Depending on the molecular weight, 0, 5 - 3 μg / mL of antigen or 2 - 5 μg / mL of antibody format was coated. It was equivalent to approximately 30 nM for the coated antigen or 10 - 20 nM in terms of the number of moles for the coated antibody. After blocking with 3% (w / v) skim milk powder (Merck) dissolved in PBS, serial dilutions of His-tag scFv antibody or biotinylated CD16A-158V ECD - monomeric Fc fusion antigen in PBS containing 0.3% (w / v) skim milk powder were incubated for 1.5 h at room temperature on plates coated with either antigen or antibody, respectively. To assess binding competition, the scFv was titrated and incubated on the plate with 1 nM or 10 nM of 3G8 mAb. After washing three times with 300 μL / well of PBS containing 0.1% (v / v) Tween 20, the plate was incubated for 1 h at room temperature with a detection complex, either 1:3000 diluted Penta-His-HRP (Qiagen), Streptavidin-HRP (Roche), or for detecting competing 3G8, 1:10000 diluted Peroxidase AffiniPure Goat Anti-Mouse IgG(H+L) (Dianova). After washing, the plate was incubated with the tetramethylbenzidine (TMB) substrate (Seramun) for 1 - 2 min or until the color development was clearly visible. 0.5 M H 2 SO 4 (100 μL / well) was added to stop the reaction. Absorbance was measured at 450 nm (1 s) using a multi-well plate reader (Victor, Perkin Elmer). The absorbance values were plotted and a sigmoid dose-response curve was fitted with a non-linear regression model (4-parameter logistic fit) using GraphPad Prism version 6.07 (GraphPad Software, La Jolla, California, USA) to obtain the EC50 The value was determined.

[0373] Cell lines and cell culture NCI-H929 (DSMZ, catalog number: ACC-163), MC / CAR (ATCC, catalog number: CRL-8083), MM.1S (ATCC, catalog number: CRL-2974), RPMI-8226 (DSMZ, catalog number: ACC402), KARPAS-299 (DSMZ, catalog number: ACC31), and SW-982 (ATCC, catalog number: HTB-93) were purchased, and A-431 was obtained from Dr. G. Moldenhauer (DKFZ Heidelberg). All cells were cultured under standard conditions at 37 °C in a humidified 5% CO 2 atmosphere in DMEM (catalog number: 41965-039), IMDM (catalog number: 12440-053), or RPMI 1640 (catalog number: 21875-034) medium supplemented with 10% heat-inactivated fetal calf serum (FCS) (catalog number: 10270-106), 100 U / mL penicillin G / 100 mg / mL streptomycin (catalog number: 1540-122), and 2 mM L-glutamine (catalog number: 25030-024; all from Life Technologies).

[0374] Isolation of human NK cells Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy volunteers (German Red Cross, Mannheim, Germany) by density gradient centrifugation using Lymphoprep (StemCell Technologies, catalog number: 07861) as described above (Fuss et al., "Curr Protoc Immunol" (2009), Chapter 7, Section VII, page 1). PBMCs were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 100 U / mL penicillin G sodium / 100 μg / mL streptomycin in a humidified atmosphere at 37 °C and 5% CO 2Cultured under O / N conditions, and then NK cells were enriched by negative bead selection using EasySEP™ Negative NK Cell Enrichment Kit (StemCell Technologies, catalog number: 17955) according to the manufacturer's instructions. The purity of NK cell isolation was measured by flow cytometry, and usually, CD56 + cells were demonstrated to be less than 80% (data not shown).

[0375] Cell binding assay and flow cytometry analysis 0.2 - 1x10 6An aliquot of the enriched human NK cells was incubated for 45 min at 37 °C with 100 μL serial dilutions of the indicated constructs in FACS buffer (PBS, Invitrogen, catalog number: 14190-169) containing 2% heat-inactivated FCS (Invitrogen, catalog number: 10270-106), 0.1% sodium azide (Roth, Karlsruhe, Germany, catalog number: A1430.0100) in the absence of 10 mg / mL polyclonal human IgG (Gammanorm, Octapharma), or in the presence of 10 mg / mL polyclonal human IgG when so indicated. After repeated washing with FACS buffer, cell-bound His-tagged scFv, TandAb, or aTriFlex antibodies were detected with 10 μg / mL anti-His mAb 13 / 45 / 31-2 (Dianova, Hamburg, Germany, catalog number: DIA910-1MG), followed by detection with 15 μg / mL FITC-conjugated goat anti-mouse IgG (Dianova, catalog number: 115-095-062). The bispecific BCMA / CD16A antibody construct was detected with soluble His-tagged BCMA, followed by 10 μg / mL anti-His mAb 13 / 45 / 31-2, followed by 15 μg / mL FITC-conjugated goat anti-mouse IgG, and the bispecific Fc-containing EGFR / CD16A construct with 15 μg / mL FITC-conjugated goat anti-human IgG (Dianova, catalog number: 109-095-098). After the final staining step, the cells were washed again and resuspended in 0.2 mL FACS buffer containing 2 μg / mL propidium iodide (PI) (Sigma, catalog number: P4170) to remove dead cells. The fluorescence of 2 - 5×10 3 live cells was measured using a Millipore Guava EasyCyte flow cytometer (Merck Millipore, Schwalbach, Germany) or a CytoFlex cytometer (Beckman Coulter, Krefeld, Germany), and the median fluorescence intensity of the cell sample was determined. After subtracting the fluorescence intensity values of cells stained with secondary and / or tertiary reagents alone, the values were used for non-linear regression analysis. The equilibrium dissociation constant (K D) was calculated using one-site binding (hyperbola) fit and GraphPad Prism software V6 or V7 (GraphPad Software, La Jolla, CA, USA).

[0376] Cytotoxicity assay For the calcein release assay, target cells were labeled with 10 mM calcein AM (Life Technologies, catalog number: C3100MP) for 30 min at 37°C in RPMI medium, washed, and then 1 × 10 4 tumor target cells were seeded into individual wells of a 96-well microplate with 200 μL of effector cells at the indicated effector:target (E:T) ratios in the presence of increasing antibody concentrations. For the evaluation of NK fratricide, 5 × 10 4 calcein-labeled primary human NK cells were co-incubated with autologous NK cells at a 1:1 E:T ratio in the presence of increasing antibody concentrations. Unless otherwise indicated, after incubation at 37°C for 4 h in a humidified 5% CO 2 atmosphere, the fluorescence (F) of calcein released into the supernatant was measured at 520 nm using a plate reader (Victor 3 or EnSight, Perkin Elmer, Turku, Finland). Specific cell lysis was calculated as follows: [F(sample) - F(spontaneous)] / [F(max) - F(spontaneous)] x 100%. F(spontaneous) represents the fluorescence released from target cells in the absence of effector cells and antibody, and F(max) represents the fluorescence released after total cell lysis induced to a final concentration of 1% by the addition of Triton X-100 (Roth, catalog number: 3051.2). The mean values of specific target cell lysis (%) and standard deviation (SD) were plotted, and the in vitro potency (EC 50 ) was determined by fitting a non-linear regression model to a sigmoid dose-response curve (variable slope) using GraphPad Prism (v6 and v7; GraphPad Software, La Jolla, CA, USA).

[0377] Surface plasmon resonance Kinetic binding analysis of monovalent interactions with CD16A and qualitative dissociation phase comparison of monovalent and bivalent anti-CD16A binding analytes were performed on a Biacore T200 Instrument (GE Healthcare) at 25°C for dilutions using HBS-P+ (10 mM HEPES, 150 mM NaCl, 0.05% (w / v) polysorbate 20, pH 7.4) as the running buffer. The CAP sensor chip (Biotin Capture Kit, GE Healthcare) was preconditioned overnight using HBS-P+ buffer. In the pre-capture step, the chip surface was treated with Biotin CAPTure reagent (GE Healthcare) at 5 μL / min for 100 seconds in flow cells 1-4.

[0378] For kinetic binding analysis of various anti-CD16A scFvs, biotinylated mono-Fc (silencing)-Avi-tagged receptor was prepared in HBS-P+ and captured in flow cells Fc2 and Fc4 (approximately 40 RU for interaction measurements with human CD16A-158V, human CD16A-158F, scFv, and approximately 10 RU for interaction measurements with IgG). IgG and scFv were prepared in HBS-P+ buffer at the indicated series of concentrations and injected at a flow rate of 40 μL / min (association time 180 seconds, dissociation time 300 seconds) onto the reference surfaces (Fc1, Fc3) and receptor capture surfaces (Fc2, Fc4). Interactions were measured using the Multi Cycle Kinetic mode (all antibodies were prepared in a 4-fold dilution series: 150 nM - 0.586 nM scFv-Ab16 中 , 50 nM - 0.195 nM scFv-Ab16 高 , 500 nM - 1.953 nM scFv-AB16 低, scFv-3G8 and human IgG1 Fc enhanced from 200 nM to 0.781 nM, human IgG1 and human IgG1 Fc silenced from 2000 nM to 7.813 nM), and then, a regeneration cycle with 6 M guanidine-HCl / 250 mM NaOH was performed at 10 mL / min for 120 seconds. The data was referenced by subtracting the signal from the reference surface (Fc2~1, Fc4~3) and zero concentration (buffer control) signals. The kinetic evaluation was performed by fitting the data to a 1:1 binding model (locally fitting Rmax and RI) using Biacore T200 Evaluation Software (v3.1). For qualitative comparison of the sensorgrams, single concentrations (312.5 nM) of scFv and IgG were injected onto the reference and receptor capture surfaces at increasing capture levels (human CD16A-158V 180 RU and human CD16A-158F 90 RU). The reference curves were normalized to the maximum value of each curve and overlaid. For the study of receptor retention by qualitative dissociation phase comparison of monovalent and bivalent CD16A-binding antibodies or ligands, biotinylated mono-Fc (silencing)-Avi-tagged receptor was prepared in HBS-P+ and captured in flow cells Fc2 and Fc3 (human CD16A-158V approximately 160 RU, cynomolgus monkey CD16 approximately 320 RU). Ab16 高 Antibodies (scFv-IgAb, Db-Fc, KiH-scDb-Fc, TandAb) incorporating the binding domain and comparator, enhanced human IgG1 Fc (S239D / I332E), and monovalent binding scFv-Ab16 高 were diluted to a concentration of 50 nM in HBS-P+ buffer and injected onto the reference surface (Fc1) and receptor capture surfaces (Fc2, Fc3) at a flow rate of 30 μL / min (association time 180 seconds, dissociation time 3 hours). The surface was regenerated with 6 M guanidine-HCl / 250 mM NaOH at 10 mL / min for 120 seconds. The data was referenced by subtracting the signal from the reference surface (Fc2~1, Fc4~3) and zero concentration (buffer control) signals. The reference curves were normalized to the maximum value of each curve and overlaid.

[0379] Pharmacokinetic study of ROCK inducer The basic pharmacokinetic parameters of different ROCK (registered trademark) platform formats were evaluated in CD-1 Swiss mice. The in-life study was conducted by Heidelberg Pharma GmbH. CD-1 Swiss female mice (for example, n = 24) were administered a single intravenous dose of 0.3 mg (approximately 10 mg / kg body weight) of the test substance. Blood was collected at different time points for 1 to 3 weeks. Serum was collected from the blood and analyzed using established assays based on ELISA or MSD technology. A representative blood sampling schedule ...

Claims

1. 1. A multispecific antigen-binding protein comprising: (a) at least a first target antigen binding portion; (b) at least two CD16A antigen binding portions, wherein said at least two CD16A antigen binding portions are fused to a Fab fragment comprising at least one constant domain, or to an Fc portion.

2. 2. The multispecific antigen-binding protein of claim 1, wherein at least one of the two CD16A antigen-binding moieties is an antigen-binding molecule selected from the group consisting of a single-chain Fv (scFv), a single-chain diabody (scDb), and a diabody Db.

3. 3. The multispecific antigen-binding protein of claim 1 or 2, wherein each of the at least two CD16A antigen-binding portions is an scFv.

4. The multispecific antigen-binding protein of claim 1 or 2, wherein the at least two CD16A antigen-binding portions are in scDb format.

5. Each of the at least two CD16A antigen-binding moieties comprises a light chain variable region (V Ｌ ) and the heavy chain variable region (V Ｈ ) wherein the variable region at the N-terminus of the polypeptide chain is Ｌ 5. The multispecific antigen-binding protein of claim 1, wherein

6. The variable regions of the at least two CD16A antigen binding moieties in the polypeptide chain are Ｌ -V Ｈ , V Ｌ -V Ｌ -V Ｈ -V Ｈ , or V Ｌ -V Ｈ -V Ｌ -V Ｈ 6. The multispecific antigen-binding protein of claim 5 , wherein the amino acids are arranged in the order:

7. The variable regions of the at least two CD16A antigen binding moieties in the polypeptide chain are Ｌ -V Ｈ 7. The multispecific antigen-binding protein of claim 5 or 6, wherein the amino acids are located in the order: N-terminus to C-terminus.

8. The variable regions of the at least two CD16A antigen binding moieties in the polypeptide chain are Ｌ -V Ｈ -V Ｌ -V Ｈ 7. The multispecific antigen-binding protein of claim 5 or 6, wherein the amino acids are located in the order: N-terminus to C-terminus.

9. (i) the C-terminus of the polypeptide chain is fused to the N-terminus of the CH2 domain of the Fc portion; or (ii) the C-terminus of the polypeptide chain is fused to the N-terminus of the hinge of the Fc portion; (iii) the N-terminus of the polypeptide chain is fused to the C-terminus of the CH3 domain of the Fc portion; or (iv) The multispecific antigen-binding protein of any one of claims 5 to 8, wherein the N-terminus of said polypeptide chain is fused to the C-terminus of a Fab fragment.

10. 10. The multispecific antigen-binding protein of any one of claims 5 to 9, wherein the N-terminus of said polypeptide chain is fused to the C-terminus of the CH3 domain of said Fc portion.

11. 11. The multispecific antigen-binding protein of any one of claims 1 to 10, wherein the Fc portion is selected from the group consisting of a monomeric CH2-CH3 fragment, a heterodimeric Fc region, and a homodimeric Fc region.

12. The multispecific antigen-binding protein of any one of claims 1 to 11, wherein said Fc portion does not bind to Fcγ receptors but maintains binding to neonatal Fc receptors.

13. 13. The multispecific antigen-binding protein of claim 12, wherein a scDb or Db comprising two CD16 antigen-binding moieties is fused to the C-terminus of one chain of the Fab fragment and the first target antigen-binding moiety is fused to the C-terminus of the other chain of the Fab fragment.

14. 14. The multispecific antigen-binding protein of claim 13, wherein the Fab fragment comprises an HSA antigen-binding Fv at the N-terminus.

15. 15. The multispecific antigen-binding protein of any one of claims 1 to 14, wherein the antigen-binding protein is tetravalent.

16. 16. The multispecific antigen-binding protein of claim 15, comprising at least two target antigen-binding portions.

17. 17. The multispecific antigen-binding protein of claim 16, comprising a first target antigen-binding portion, a second target antigen-binding portion, and at least two CD16A antigen-binding portions fused to a dimeric Fc portion.

18. 2. The multispecific antigen binding protein of claim 1, wherein a CD16A antigen binding portion is fused to the C-terminus of each heavy chain of an IgG, said IgG comprising a first target antigen binding Fv portion in each of said two Fabs at the N-terminus, and a second target antigen binding portion is fused C-terminally to each of the CL domains.

19. the CD16A antigen binding portion comprises (i) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 50; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 51; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 52; and / or 19. The multispecific antigen-binding protein of any one of claims 1 to 18, comprising: (i) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO:53; a CDR2 having the amino acid sequence set forth in SEQ ID NO:54; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

55.

20. the CD16A antigen binding portion comprises (i) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75; and / or (ii) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

78.

21. 21. The multispecific antigen-binding protein of any one of claims 1 to 20, wherein said CD16A antigen-binding portion comprises (i) a heavy chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:3, and / or (ii) a light chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:

2.

22. 22. The multispecific antigen-binding protein of any one of claims 1 to 21, wherein the CD16A antigen-binding portion comprises (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:3, and / or (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

2.

23. The multispecific antigen-binding protein of any one of claims 1 to 15, wherein the first target antigen is selected from BCMA and EGFR.

24. 24. The multispecific antigen-binding protein of claim 23, wherein the first target antigen is BCMA.

25. 25. The multispecific antigen-binding protein of claim 24, wherein the protein is a tetramer comprising a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 61 or 63 and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 62 or 64.

26. The protein is (i) a first polypeptide having an amino acid sequence as set forth in SEQ ID NO:61 and a second polypeptide having an amino acid sequence as set forth in SEQ ID NO:62; and (ii) a first polypeptide having an amino acid sequence as set forth in SEQ ID NO:61 and a second polypeptide having an amino acid sequence as set forth in SEQ ID NO:64; and (iii) a first polypeptide having an amino acid sequence as set forth in SEQ ID NO:63 and a second polypeptide having an amino acid sequence as set forth in SEQ ID NO:62; and (iv) a first polypeptide having the amino acid sequence set forth in SEQ ID NO:63 and a second polypeptide having the amino acid sequence set forth in SEQ ID NO:

64.

27. the BCMA antigen binding portion (i) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 67; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 68; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 69; and / or (ii) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 70; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 71; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

72.

28. the BCMA antigen binding portion (i) a heavy chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 65; and / or 25. The multispecific antigen-binding protein of claim 24, comprising: (ii) a light chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:

66.

29. the BCMA antigen binding portion (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65, and / or 25. The multispecific antigen-binding protein of claim 24, comprising: (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

66.

30. the antigen binding protein comprising: (i) a CD16A antigen-binding portion comprising: (a) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75; and (b) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

78. (ii) a BCMA antigen-binding portion comprising: (a) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO:67; a CDR2 having the amino acid sequence set forth in SEQ ID NO:68; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:69; and (ii) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 70; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 71; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

72.

31. 31. A pharmaceutical composition comprising the multispecific antigen-binding protein of any one of claims 1 to 30 and a pharma- ceutically acceptable carrier.

32. A multispecific antigen-binding protein according to any one of claims 1 to 30 for use as a medicament.

33. 32. A pharmaceutical composition according to claim 31 for use as a medicament.

34. 32. A method for treating or ameliorating a disease, comprising administering to a subject in need thereof a multispecific antigen-binding protein according to any one of claims 1 to 30 or a pharmaceutical composition according to claim 31.

35. 35. The method of claim 34, wherein the disease is cancer.

36. 36. The method of claim 34 or 35, wherein the disease is a blood cancer.

37. The method according to any one of claims 34 to 36, wherein the disease is multiple myeloma.

38. 32. A method for treating and / or preventing a disease in a subject, comprising administering to a subject in need thereof a bispecific antigen-binding protein according to any one of claims 1 to 30 or a pharmaceutical composition according to claim 31.

39. 39. The method of claim 38, comprising administering the bispecific antigen binding protein intravenously.

40. 39. The method of claim 38, comprising administering the bispecific antigen-binding protein subcutaneously.

41. 41. The method of any one of claims 38 to 40, comprising administering a second therapy.

42. 42. The method of claim 41, wherein the second therapy is selected from the group consisting of a therapy comprising an anti-PD-1 antibody, a therapy comprising an anti-PD-L1 antibody, a therapy comprising a CD3 bispecific antibody, a therapy comprising an anti-TIGIT antibody, a therapy comprising an anti-VEGF antibody, and a therapy comprising an anti-FcRH5 antibody.

43. 42. The method of claim 41, wherein the second therapy is a cytokine therapy.

44. 44. The method of claim 43, wherein the cytokine therapy comprises administration of a cytokine selected from the group consisting of IL-15, IL-2, IL-12, IL-21, and IL-6.

45. 45. The method of claim 44, wherein the cytokine therapy comprises administration of IL-2.

46. The method of claim 44, wherein the cytokine therapy comprises administration of IL-15.

47. The method according to any one of claims 34 to 46, wherein the subject is a cancer patient.

48. The method of any one of claims 34 to 46, wherein the subject is a blood cancer patient.

49. The method of any one of claims 34 to 48, wherein the subject is a multiple myeloma patient.

50. The method of any one of claims 34 to 49, wherein the subject is a relapsed / refractory multiple myeloma patient.

51. The method of any one of claims 34 to 50, wherein the subject has undergone anti-CD38 therapy.

52. The method of any one of claims 34-51, wherein the subject has received daratumumab.

53. 53. The method of any one of claims 34 to 52, wherein the subject is daratumumab naive, daratumumab resistant, daratumumab refractory, or daratumumab relapsed.

54. 54. The method of claim 53, wherein the subject is daratumumab-resistant, daratumumab-refractory, or daratumumab-relapsed.

55. The method of any one of claims 34 to 54, wherein the subject expresses a CD16A polymorphism.

56. 56. The method of claim 55, wherein the CD16A polymorphism is the CD16A-158V / F polymorphism.

57. 1. A bispecific antigen-binding protein comprising: (a) at least one BCMA binding moiety; (b) at least two CD16A antigen-binding portions, wherein said at least two CD16A antigen-binding portions are fused to a Fab fragment comprising at least one constant domain, or to an Fc portion.

58. 58. The bispecific antigen-binding protein of claim 57, wherein at least one of the two CD16A antigen-binding moieties is an antigen-binding molecule selected from the group consisting of a single chain Fv (scFv), a single chain diabody (scDb), and a diabody Db.

59. 59. The bispecific antigen-binding protein of claim 57 or 58, wherein each of the at least two CD16A antigen-binding portions is an scFv.

60. 60. The bispecific antigen-binding protein of any one of claims 57 to 59, wherein the at least two CD16A antigen-binding portions are in scDb format.

61. Each of the at least two CD16A antigen-binding moieties comprises a light chain variable region (V Ｌ ) and the heavy chain variable region (V Ｈ ) wherein the variable region at the N-terminus of the polypeptide chain is Ｌ 60. The bispecific antigen-binding protein of any one of claims 57 to 59,

62. The variable regions of the at least two CD16A antigen binding moieties in the polypeptide chain are Ｌ -V Ｈ , V Ｌ -V Ｌ -V Ｈ -V Ｈ , or V Ｌ -V Ｈ -V Ｌ -V Ｈ 62. The bispecific antigen-binding protein of claim 61 , wherein the amino acids are arranged in the order N-terminus to C-terminus:

63. The variable regions of the at least two CD16A antigen binding moieties in the polypeptide chain are Ｌ -V Ｈ 63. The bispecific antigen-binding protein of claim 61 or 62, wherein the amino acids are located in the order N-terminus to C-terminus:

64. The variable regions of the at least two CD16A antigen binding moieties in the polypeptide chain are Ｌ -V Ｈ -V Ｌ -V Ｈ 63. The bispecific antigen-binding protein of claim 61 or 62, wherein the amino acids are located in the order N-terminus to C-terminus:

65. (i) the C-terminus of the polypeptide chain is fused to the N-terminus of the CH2 domain of the Fc portion; or (ii) the C-terminus of the polypeptide chain is fused to the N-terminus of the hinge of the Fc portion; (iii) the N-terminus of the polypeptide chain is fused to the C-terminus of the CH3 domain of the Fc portion; or (iv) The bispecific antigen-binding protein of any one of claims 61 to 64, wherein the N-terminus of said polypeptide chain is fused to the C-terminus of a Fab fragment.

66. 65. The bispecific antigen-binding protein of any one of claims 61 to 64, wherein the N-terminus of said polypeptide chain is fused to the C-terminus of the CH3 domain of said Fc portion.

67. 67. The bispecific antigen-binding protein of any one of claims 57 to 66, wherein the Fc portion is selected from the group consisting of a monomeric CH2-CH3 fragment, a heterodimeric Fc region, and a homodimeric Fc region.

68. 68. The bispecific antigen-binding protein of any one of claims 57 to 67, wherein said Fc portion does not bind to an Fcγ receptor but retains binding to a neonatal Fc receptor.

69. 68. The bispecific antigen-binding protein of any one of claims 57 to 67, wherein the Fc portion comprises at least one effector-less mutation.

70. 70. The bispecific antigen binding protein of claim 69, wherein the at least one effectorless mutation is selected from the group consisting of C220S, C229S, E233P, L234A, L234V, L234F, L235A, L235E, P238S, D265A, N297A, N297Q, and P331S.

71. 71. The bispecific antigen-binding protein of claim 69 or 70, wherein the at least one effectorless mutation is selected from the group consisting of L234A, L234V, L234F, L235A, L235E, P238S, and D265A.

72. 72. The bispecific antigen-binding protein of any one of claims 69 to 71, wherein said at least one effectorless mutation is selected from the group consisting of L234F, L235E, and D265A.

73. 73. The bispecific antigen-binding protein of any one of claims 69 to 72, wherein the Fc portion has two effectorless mutations.

74. 74. The bispecific antigen-binding protein of claim 73, wherein the two effectorless mutations are L234F and L235E.

75. 73. The bispecific antigen-binding protein of any one of claims 69 to 72, wherein the Fc portion has three effectorless mutations.

76. 76. The bispecific antigen-binding protein of claim 75, wherein the three effectorless mutations are L234F, L235E, and D265A.

77. 78. The bispecific antigen-binding protein of any one of claims 57 to 77, wherein the at least two CD16A antigen-binding portions are fused to an Fc portion.

78. 78. The bispecific antigen-binding protein of any one of claims 57 to 77, comprising two BCMA targeting moieties.

79. 79. A bispecific antigen-binding protein according to any one of claims 57 to 78, comprising two CD16A antigen-binding moieties and two BCMA targeting moieties.

80. 80. The bispecific antigen binding protein of claim 79, wherein each of said two CD16A antigen binding moieties is fused to the pre-C-terminus of a respective heavy chain of an IgG and each of said two BCMA targeting moieties is fused to the N-terminus of said IgG.

81. 81. The bispecific antigen-binding protein of any one of claims 77 to 80, having the structure shown in Figure 13.

82. 78. The bispecific antigen-binding protein of any one of claims 57 to 77, comprising one BCMA targeting moiety.

83. 83. The bispecific antigen-binding protein of claim 82, wherein the BCMA targeting portion is fused to the N-terminus of the Fc portion to which the at least two CD16A antigen-binding portions are fused.

84. 84. A bispecific antigen-binding protein according to claim 82 or 83, having the structure shown in Figure 5.

85. the CD16A antigen binding portion comprises (i) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 50; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 51 or 56; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 52; and / or (ii) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO:53; a CDR2 having the amino acid sequence set forth in SEQ ID NO:54; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

55.

86. 85. The bispecific antigen-binding protein of claim 84, wherein the heavy chain variable region CDR2 has the amino acid sequence set forth in SEQ ID NO:

51.

87. 86. The bispecific antigen-binding protein of claim 85, wherein the heavy chain variable region CDR2 has the amino acid sequence set forth in SEQ ID NO:

56.

88. the CD16A antigen binding portion comprises (i) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75; and / or (ii) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

78.

89. 89. The bispecific antigen-binding protein of any one of claims 57 to 88, wherein the CD16A antigen-binding portion comprises (i) a heavy chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:3, and / or (ii) a light chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:

2.

90. 90. The bispecific antigen-binding protein of any one of claims 57 to 89, wherein the CD16A antigen-binding portion comprises (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:3, and / or (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

2.

91. the BCMA antigen binding portion (i) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 67; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 68; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 69; and / or (ii) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 70; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 71; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

72.

92. 92. The bispecific antigen-binding protein of any one of claims 57-91, wherein the BCMA antigen-binding portion comprises (i) a heavy chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:65, and / or (ii) a light chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:

66.

93. 93. The bispecific antigen-binding protein of any one of claims 57-92, wherein the BCMA antigen-binding portion comprises (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:65, and / or (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

66.

94. two CD16A antigen-binding moieties and two BCMA targeting moieties, (a) each of the CD16A antigen-binding portions has an amino acid sequence as set forth in SEQ ID NO:73; Ｈ CDR1, V having the amino acid sequence shown in SEQ ID NO:74 Ｈ CDR2, V having the amino acid sequence set forth in SEQ ID NO:75 Ｈ CDR3, V having the amino acid sequence set forth in SEQ ID NO:76 Ｌ CDR1, V having the amino acid sequence set forth in SEQ ID NO:77 Ｌ CDR2, and V having the amino acid sequence shown in SEQ ID NO:78 Ｌ comprises CDR3; (b) each of the BCMA targeting moieties has an amino acid sequence as set forth in SEQ ID NO:67; Ｈ CDR1, V having the amino acid sequence shown in SEQ ID NO:68 Ｈ CDR2 having the amino acid sequence shown in SEQ ID NO:69, CDR3 having the amino acid sequence shown in SEQ ID NO:70 Ｌ CDR1, V having the amino acid sequence shown in SEQ ID NO:71 Ｌ CDR2, and V having the amino acid sequence shown in SEQ ID NO:72 Ｌ 94. A bispecific antigen-binding protein according to any one of claims 57 to 74, 77 to 81 and 85 to 93, comprising a CDR3.

95. two CD16A antigen-binding moieties and two BCMA targeting moieties, (a) each of the CD16A antigen-binding portions comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2; 95. The bispecific antigen-binding protein of any one of claims 57-74, 77-81 and 85-94, wherein each of said BCMA targeting moieties comprises (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:65, and (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

66.

96. 96. The bispecific antigen-binding protein of any one of claims 57-74, 77-81 and 85-95, which is a tetramer comprising a first polypeptide having the amino acid sequence set forth in SEQ ID NO:61 and a second polypeptide having the amino acid sequence set forth in SEQ ID NO:

62.

97. 94. The bispecific antigen-binding protein of any one of claims 57 to 93, which is a tetramer comprising a first polypeptide having the amino acid sequence set forth in SEQ ID NO:63 and a second polypeptide having the amino acid sequence set forth in SEQ ID NO:

64.

98. (i) the CD16A antigen-binding portion is, respectively: (a) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75; (b) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 78; (ii) the BCMA antigen binding portion is, respectively: (a) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO:67; a CDR2 having the amino acid sequence set forth in SEQ ID NO:68; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:69; (ii) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 70; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 71; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

72.

99. 99. A pharmaceutical composition comprising the bispecific antigen-binding protein of any one of claims 57 to 98 and a pharma- ceutically acceptable carrier.

100. 99. A bispecific antigen-binding protein according to any one of claims 57 to 98 for use as a medicament.

101. 100. A pharmaceutical composition according to claim 99 for use as a medicament.

102. 100. A method for treating and / or preventing a disease comprising administering a bispecific antigen-binding protein according to any one of claims 57 to 98 or a pharmaceutical composition according to claim 99 to a subject in need thereof.

103. 103. The method of claim 102, wherein the disease is cancer.

104. 104. The method of claim 102 or 103, wherein the disease is a blood cancer.

105. The method according to any one of claims 102 to 104, wherein the disease is multiple myeloma.

106. 106. The method of any one of claims 102 to 105, comprising administering the bispecific antigen-binding protein intravenously.

107. 106. The method of any one of claims 102 to 105, comprising administering the bispecific antigen-binding protein subcutaneously.

108. 108. The method of any one of claims 102 to 107, comprising administering a second therapy.

109. 109. The method of claim 108, wherein the second therapy is selected from the group consisting of a therapy comprising an anti-PD-1 antibody, a therapy comprising an anti-PD-L1 antibody, a therapy comprising a CD3 bispecific antibody, a therapy comprising an anti-TIGIT antibody, a therapy comprising an anti-VEGF antibody, and a therapy comprising an anti-FcRH5 antibody.

110. 109. The method of claim 108, wherein the second therapy is a cytokine therapy.

111. 111. The method of claim 110, wherein the cytokine therapy is administration of a cytokine selected from the group consisting of IL-15, IL-2, IL-12, IL-21, IL-17, IL-18, IL-23, IL-27, and IL-6.

112. The method of claim 111, wherein the cytokine is IL-15.

113. The method of claim 111, wherein the cytokine is IL-2.

114. The method of any one of claims 102 to 113, wherein the subject is a cancer patient.

115. The method of any one of claims 102 to 114, wherein the subject is a blood cancer patient.

116. The method of any one of claims 102 to 115, wherein the subject is a multiple myeloma patient.

117. The method of any one of claims 102-116, wherein the subject is a relapsed / refractory multiple myeloma patient.

118. The method of any one of claims 102 to 117, wherein the subject has undergone anti-CD38 therapy.

119. The method of any one of claims 102-118, wherein the subject has received daratumumab.

120. The method of any one of claims 102-119, wherein the subject is daratumumab naive, daratumumab resistant, daratumumab refractory, or daratumumab relapsed.

121. 121. The method of claim 120, wherein the subject is daratumumab-resistant, daratumumab-refractory, or daratumumab-relapsed.

122. The method of any one of claims 102 to 121, wherein the subject expresses a CD16A polymorphism.

123. The method of claim 122, wherein the CD16A polymorphism is the CD16A-158V / F polymorphism.

124. 99. The multispecific antigen-binding protein of any one of claims 1 to 30 or the bispecific antigen-binding protein of any one of claims 57 to 98, wherein the multispecific antigen-binding protein or the bispecific antigen-binding protein, when administered to a subject, does not substantially deplete or reduce the Natural Killer (NK) cell population in said subject.

125. 103. A method of treating a subject having a depleted or reduced NK cell population comprising administering a multispecific antigen binding protein according to any one of claims 1 to 30 or a bispecific antigen binding protein according to any one of claims 57 to 98.

126. The method of claim 125, wherein the subject has previously been treated with an anti-CD38 therapy.

127. 127. The method of claim 126, wherein the anti-CD38 therapy is daratumumab therapy.

128. 1. A bispecific antigen binding protein comprising two CD16A antigen binding moieties and two BCMA targeting moieties, each of said two CD16A antigen binding moieties fused to the C-terminus of a respective heavy chain of an IgG, and each of said two BCMA targeting moieties fused to the N-terminus of each of said heavy chains of said IgG, wherein: (i) the CD16A antigen-binding portion is, respectively: (a) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75; (b) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 78; (ii) the BCMA antigen binding portion is, respectively: (a) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO:67; a CDR2 having the amino acid sequence set forth in SEQ ID NO:68; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:69; (ii) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 70; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 71; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

72.

129. 1. A method of treating cancer in a subject, comprising administering to the subject a bispecific antigen binding protein comprising two CD16A antigen binding moieties and two BCMA targeting moieties, wherein each of the two CD16A antigen binding moieties is fused to the C-terminus of a respective heavy chain of an IgG, and each of the two BCMA targeting moieties is fused to the N-terminus of each of the heavy chains of the IgG; (i) the CD16A antigen-binding portion is, respectively: (a) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75; (b) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 78; (ii) the BCMA antigen binding portion is, respectively: (a) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO:67; a CDR2 having the amino acid sequence set forth in SEQ ID NO:68; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:69; (ii) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 70; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 71; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

72.

130. 130. The method of claim 129, wherein the cancer is multiple myeloma.

131. An antibody or antigen binding protein that binds to or is capable of binding to CD16A and BCMA, (i) a first heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75; (ii) a first light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 78; (iii) a second heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO:67; a CDR2 having the amino acid sequence set forth in SEQ ID NO:68; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:69; (iv) a second light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO:70; a CDR2 having the amino acid sequence set forth in SEQ ID NO:71; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

72.

132. (i) the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:3; (ii) the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:2; (iii) the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:65; (iv) the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:66; (v) the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:3, and the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:2; (vi) the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:65, and the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:66; or (vii) the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:3, and the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:2; and the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:65, and the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

66.

132. An antibody or antigen-binding protein as described in claim 131.

133. 133. The antibody or antigen-binding protein of claim 131 or 132, which does not bind to CD16B.

134. 134. The antibody or antigen binding protein of any one of claims 131 to 133, which does not substantially deplete or reduce a subject's natural killer (NK) cell population when administered to said subject.

135. 135. The antibody or antigen binding protein of any one of claims 131 to 134, wherein the CD16A is human CD16A.

136. 136. The antibody or antigen binding protein of any one of claims 131 to 135, wherein the BCMA is human BCMA.

137. 137. A pharmaceutical composition comprising the antibody or antigen-binding protein of any one of claims 131 to 136 and a pharma- ceutically acceptable carrier.

138. 137. An antibody or antigen-binding protein according to any one of claims 131 to 136 for use as a medicament.

139. 138. A pharmaceutical composition according to claim 137 for use as a medicament.

140. 137. A method for treating and / or preventing a disease, comprising administering a bispecific antigen-binding protein according to any one of claims 131 to 136 or a pharmaceutical composition according to claim 137 to a subject in need thereof.

141. 141. The method of claim 140, wherein the disease is cancer.

142. 142. The method of claim 140 or 141, wherein the disease is a blood cancer.

143. The method of any one of claims 140 to 142, wherein the disease is multiple myeloma.

144. 144. The method of any one of claims 140 to 143, comprising administering the antibody or antigen binding protein intravenously or subcutaneously.

145. 145. The method of any one of claims 140 to 144, comprising administering a second therapeutic agent.

146. 146. The method of claim 145, wherein the second therapy is selected from the group consisting of a therapy comprising an anti-PD-1 antibody, a therapy comprising an anti-PD-L1 antibody, a therapy comprising a CD3 bispecific antibody, a therapy comprising an anti-TIGIT antibody, a therapy comprising an anti-VEGF antibody, and a therapy comprising an anti-FcRH5 antibody.

147. 146. The method of claim 145, wherein the second therapy is a cytokine therapy.

148. 148. The method of claim 147, wherein said cytokine therapy is administration of a cytokine selected from the group consisting of IL-15, IL-2, IL-12, IL-21, IL-17, IL-18, IL-23, IL-27, and IL-6.

149. The method of claim 148, wherein the cytokine is IL-15.

150. The method of claim 148, wherein the cytokine is IL-2.

151. The method of any one of claims 140 to 150, wherein the subject is a cancer patient.

152. The method of any one of claims 140 to 151, wherein the subject is a blood cancer patient.

153. The method of any one of claims 140 to 152, wherein the subject is a multiple myeloma patient.

154. The method of any one of claims 140 to 153, wherein the subject is a relapsed / refractory multiple myeloma patient.

155. The method of any one of claims 140 to 154, wherein the subject has undergone anti-CD38 therapy.

156. The method of any one of claims 140-155, wherein the subject has received daratumumab.

157. The method of any one of claims 140-156, wherein the subject is daratumumab naive, daratumumab resistant, daratumumab refractory, or daratumumab relapsed.

158. The method of claim 157, wherein the subject is daratumumab-resistant, daratumumab-refractory, or daratumumab-relapsed.

159. The method of any one of claims 140 to 158, wherein the subject expresses a CD16A polymorphism.

160. 160. The method of claim 159, wherein the CD16A polymorphism is the CD16A-158V / F polymorphism.

161. 137. A method of treating a subject having a depleted or reduced NK cell population comprising administering an antibody or antigen binding protein according to any one of claims 131 to 136, or a pharmaceutical composition according to claim 137.

162. The method of claim 161, wherein the subject has previously been treated with an anti-CD38 therapy.

163. 163. The method of claim 162, wherein the anti-CD38 therapy is daratumumab therapy.

164. 1. An antibody or antigen binding protein comprising at least one arm that binds or is capable of binding to CD16A and at least one arm that binds or is capable of binding to BCMA, (i) said at least one arm that binds to or is capable of binding to CD16A, (a) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75; (b) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 78; (ii) said at least one arm that binds to or is capable of binding to BCMA is (a) a heavy chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO:67; a CDR2 having the amino acid sequence set forth in SEQ ID NO:68; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:69; (ii) a light chain variable region comprising: a CDR1 having the amino acid sequence set forth in SEQ ID NO:70; a CDR2 having the amino acid sequence set forth in SEQ ID NO:71; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

72.

165. The antibody or antigen binding protein of claim 164, wherein the at least one arm that binds or is capable of binding to CD16A is different from the at least one arm that binds or is capable of binding to BCMA.

166. (i) the at least one arm that binds to or is capable of binding to CD16A comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:3; (ii) the at least one arm that binds to or is capable of binding to CD16A comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2; (iii) the at least one arm that binds to or is capable of binding to BCMA comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:65; (iv) the at least one arm that binds to or is capable of binding to BCMA comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:66; (v) the at least one arm that binds to or is capable of binding to CD16A comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2; (vi) the at least one arm that binds to or is capable of binding to BCMA comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:65 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:66; or (vii) the antibody or antigen binding protein of claim 164 or 165, wherein the at least one arm that binds to or is capable of binding to CD16A comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; and the at least one arm that binds to or is capable of binding to BCMA comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

66.

167. 167. The antibody or antigen-binding protein of any one of claims 164 to 166, which does not bind to CD16B.

168. 168. The antibody or antigen binding protein of any one of claims 164 to 167, which does not substantially deplete or reduce a subject's natural killer (NK) cell population when administered to said subject.

169. 169. The antibody or antigen binding protein of any one of claims 164 to 168, wherein the CD16A is human CD16A.

170. 170. The antibody or antigen binding protein of any one of claims 164 to 169, wherein the BCMA is human BCMA.

171. 171. A pharmaceutical composition comprising the antibody or antigen-binding protein of any one of claims 164 to 170 and a pharma- ceutically acceptable carrier.

172. 171. An antibody or antigen-binding protein according to any one of claims 164 to 170 for use as a medicament.

173. 173. A pharmaceutical composition according to claim 172 for use as a medicament.

174. 172. A method for treating and / or preventing a disease, comprising administering to a subject in need thereof an antibody or antigen binding protein according to any one of claims 164 to 170, or a pharmaceutical composition according to claim 171.

175. 175. The method of claim 174, wherein the disease is cancer.

176. 176. The method of claim 174 or 175, wherein the disease is a blood cancer.

177. The method of any one of claims 174 to 176, wherein the disease is multiple myeloma.

178. 178. The method of any one of claims 174 to 177, comprising administering the antibody or antigen binding protein intravenously or subcutaneously.

179. 179. The method of any one of claims 174 to 178, comprising administering a second therapeutic agent.

180. 180. The method of claim 179, wherein the second therapy is selected from the group consisting of a therapy comprising an anti-PD-1 antibody, a therapy comprising an anti-PD-L1 antibody, a therapy comprising a CD3 bispecific antibody, a therapy comprising an anti-TIGIT antibody, a therapy comprising an anti-VEGF antibody, and a therapy comprising an anti-FcRH5 antibody.

181. 180. The method of claim 179, wherein the second therapy is a cytokine therapy.

182. 182. The method of claim 181, wherein said cytokine therapy is administration of a cytokine selected from the group consisting of IL-15, IL-2, IL-12, IL-21, IL-17, IL-18, IL-23, IL-27, and IL-6.

183. The method of claim 182, wherein the cytokine is IL-15.

184. The method of claim 182, wherein the cytokine is IL-2.

185. The method of any one of claims 174 to 184, wherein the subject is a cancer patient.

186. The method of any one of claims 174 to 185, wherein the subject is a blood cancer patient.

187. The method of any one of claims 174 to 186, wherein the subject is a multiple myeloma patient.

188. The method of any one of claims 174-187, wherein the subject is a relapsed / refractory multiple myeloma patient.

189. The method of any one of claims 174 to 188, wherein the subject has undergone anti-CD38 therapy.

190. The method of any one of claims 174-189, wherein the subject has received daratumumab.

191. The method of any one of claims 174 to 190, wherein the subject is daratumumab naive, daratumumab resistant, daratumumab refractory, or daratumumab relapsed.

192. The method of claim 191, wherein the subject is daratumumab-resistant, daratumumab-refractory, or daratumumab-relapsed.

193. The method of any one of claims 174 to 192, wherein the subject expresses a CD16A polymorphism.

194. 194. The method of claim 193, wherein the CD16A polymorphism is the CD16A-158V / F polymorphism.

195. 172. A method of treating a subject having a depleted or reduced NK cell population comprising administering an antibody or antigen binding protein according to any one of claims 164 to 170, or a composition according to claim 171.

196. The method of claim 195, wherein the subject has previously been treated with an anti-CD38 therapy.

197. 197. The method of claim 196, wherein the anti-CD38 therapy is daratumumab therapy.

198. 1. An antibody or antigen binding protein that binds to or is capable of binding to BCMA, comprising: (i) a heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO:67; a CDR2 having the amino acid sequence set forth in SEQ ID NO:68; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:69; and (ii) a light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO:70; a CDR2 having the amino acid sequence set forth in SEQ ID NO:71; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

72.

199. The antibody or antigen binding protein of claim 198, wherein the BCMA is human BCMA.

200. 200. The antibody or antigen binding protein of claim 198 or 199, further comprising at least one arm that binds to or is capable of binding to CD16A.

201. The antibody or antigen binding protein of claim 200, wherein the CD16A is human CD16A.

202. The antibody or antigen binding protein of claim 200 or 201, wherein the at least one arm that binds to or is capable of binding to CD16A comprises: (i) a heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75; and (ii) a light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77; and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

78.

203. (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2; (iii) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:65; (iv) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:66; (v) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:3, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2; (vi) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66; or (vii) the antibody or antigen binding protein of any one of claims 198-202, comprising a first heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:3, a first light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2; and a second heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:65, and a second light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

66.

204. 204. A pharmaceutical composition comprising the antibody or antigen-binding protein of any one of claims 198 to 203 and a pharma- ceutically acceptable carrier.

205. 204. An antibody or antigen-binding protein according to any one of claims 198 to 203 for use as a medicament.

206. 208. A pharmaceutical composition according to claim 207 for use as a medicament.

207. 206. A method for treating and / or preventing a disease, comprising administering a bispecific antigen binding protein according to any one of claims 198 to 203 or a pharmaceutical composition according to claim 204 to a subject in need thereof.

208. The method of claim 207, wherein the disease is cancer.

209. The method of claim 207 or 208, wherein the disease is a blood cancer.

210. The method of any one of claims 207 to 209, wherein the disease is multiple myeloma.

211. 211. The method of any one of claims 207 to 210, comprising administering the antibody or antigen binding protein intravenously or subcutaneously.

212. 212. The method of any one of claims 207 to 211, comprising administering a second therapeutic agent.

213. 213. The method of claim 212, wherein the second therapy is selected from the group consisting of a therapy comprising an anti-PD-1 antibody, a therapy comprising an anti-PD-L1 antibody, a therapy comprising a CD3 bispecific antibody, a therapy comprising an anti-TIGIT antibody, a therapy comprising an anti-VEGF antibody, and a therapy comprising an anti-FcRH5 antibody.

214. 213. The method of claim 212, wherein the second therapy is a cytokine therapy.

215. 215. The method of claim 214, wherein said cytokine therapy is administration of a cytokine selected from the group consisting of IL-15, IL-2, IL-12, IL-21, IL-17, IL-18, IL-23, IL-27, and IL-6.

216. The method of claim 215, wherein the cytokine is IL-15.

217. The method of claim 215, wherein the cytokine is IL-2.

218. The method of any one of claims 207 to 217, wherein the subject is a cancer patient.

219. The method of any one of claims 207 to 218, wherein the subject is a blood cancer patient.

220. The method of any one of claims 207 to 219, wherein the subject is a multiple myeloma patient.

221. The method of any one of claims 207-220, wherein the subject is a relapsed / refractory multiple myeloma patient.

222. The method of any one of claims 207 to 221, wherein the subject has undergone anti-CD38 therapy.

223. The method of any one of claims 207-222, wherein the subject has received daratumumab.

224. The method of any one of claims 207 to 223, wherein the subject is daratumumab naive, daratumumab resistant, daratumumab refractory, or daratumumab relapsed.

225. The method of claim 224, wherein the subject is daratumumab-resistant, daratumumab-refractory, or daratumumab-relapsed.

226. The method of any one of claims 207 to 225, wherein the subject expresses a CD16A polymorphism.

227. The method of claim 226, wherein the CD16A polymorphism is the CD16A-158V / F polymorphism.

228. 205. A method of treating a subject having a depleted or reduced NK cell population comprising administering an antibody or antigen binding protein according to any one of claims 198-203, or a composition according to claim 204.

229. The method of claim 228, wherein the subject has previously been treated with an anti-CD38 therapy.

230. 230. The method of claim 229, wherein the anti-CD38 therapy is daratumumab therapy.

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