Duplex Body

Abstract

The present invention relates to an antibody construct comprising: (i) at least four first binding domains (A), which can specifically bind to a first target (A'), which is an immunomodulatory antigen present on the surface of an innate immune effector cell, the immune effector cell being a natural killer cell or a macrophage; and (ii) a second binding domain (B), which can specifically bind to a second target (B'), which is an antigen present on the surface of a target cell. The present invention also relates to related nucleic acid molecules, vectors, host cells, methods for producing the antibody construct, pharmaceutical compositions, medical uses, and kits.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to an antibody construct comprising: (i) at least four first binding domains (A), which can specifically bind to a first target (A'), which is an immunomodulatory antigen present on the surface of an innate immune effector cell, the innate immune effector cell being a natural killer cell or a macrophage; and (ii) a second binding domain (B), which can specifically bind to a second target (B'), which is an antigen present on the surface of a target cell. The present invention also relates to related nucleic acid molecules, vectors, host cells, methods for producing the antibody construct, pharmaceutical compositions, medical uses, and kits. [Background technology]

[0002] background High expression of tumor antigens (e.g., EGFR, HER2) has been reported in various tumors, which has prompted the development of drugs (e.g., monoclonal antibodies) directed against these tumor targets. However, these tumor antigens are often heterogeneously expressed, either only in certain tumor subtypes or only in tumor tissues (Hoadley et al. 2007; Bedard et al. 2013; Passaro et al. 2020; Zhang et al. 2020). Diverse expression of markers on tumor cells and cancer stem cells reflects intratumoral heterogeneity. Low or reduced expression of tumor antigens (e.g., by downregulation or shedding) on ​​tumor cells and cancer stem cells can result in therapeutic resistance (Salih et al. 2002; Paczulla et al. 2019; Reim et al. 2009), because tumors may downregulate targeted tumor antigens during treatment as an evasion strategy. The general anti-tumor antibodies of the prior art are more effective in targeting tumor cells that express tumor antigens highly. However, it is believed that targeting only tumor cells that express tumor antigens highly will ultimately lead to the proliferation of tumor cells that express tumor antigens poorly and lack of clinical response to treatment. Tumor cells with low tumor antigens that are targeted may be the cause of relapse after initial complete remission. Therefore, targeted therapy for some tumor indications remains difficult due to tumor heterogeneity. Therefore, the object of the present invention is to provide means and methods for treating tumors even when tumor antigens are expressed heterogeneously. Summary of the Invention

[0003] overview The present invention is based on the surprising finding that bispecific antibody constructs, having at least four binding domains specific for immune-modulating antigens present on the surface of innate immune effector cells and at least one binding domain for an antigen present on the surface of a target cell, are capable of efficiently killing even target cells that have low or very low expression of the target antigen.

[0004] As shown in Example 10 herein, bispecific antibodies with one or two binding domains to EGFR and four binding domains to CD16A have surprisingly increased potency and efficacy against Daudi cells, which have very low expression of EGFR, compared to antibodies with only two binding domains to CD16A.

[0005] Therefore, the antibody construct of the present invention can be useful for tumor treatment because it can eliminate not only the cells in tumors that highly express the target antigen, but also the cells that express the target antigen at low or very low levels, including tumor stem cells.Therefore, the antibody construct can target the entire tumor.Therefore, the antibody construct of the present invention can be useful for targeting tumors that express the target antigen heterogeneously.

[0006] Thus, the present invention relates to a bispecific antibody construct comprising: (i) at least four first binding domains (A), which are capable of specifically binding to a first target (A'), which is an immunomodulatory antigen present on the surface of an innate immune effector cell, wherein the immune effector cell is a natural killer cell or a macrophage; and (ii) a second binding domain (B), which is capable of specifically binding to a second target (B'), which is an antigen present on the surface of a target cell.

[0007] The present invention also relates to a nucleic acid molecule comprising a sequence encoding an antibody construct of the invention.

[0008] The present invention also relates to a vector comprising the nucleic acid molecule of the present invention.

[0009] The present invention also relates to a host cell comprising a nucleic acid molecule of the invention or a vector of the invention.

[0010] The present invention also relates to a method for producing an antibody construct of the present invention, comprising culturing a host cell of the present invention under conditions allowing expression of the antibody construct of the present invention, and optionally recovering the produced antibody construct from the culture.

[0011] The present invention also relates to a pharmaceutical composition comprising an antibody construct of the invention or an antibody construct produced by a method of the invention.

[0012] The present invention also relates to an antibody construct of the invention for use in therapy.

[0013] The present invention also relates to a method of treating or ameliorating a proliferative disease, a neoplastic disease, a viral disease, or an immunological disorder, comprising administering to a subject in need thereof an antibody construct of the present invention or an antibody construct produced by the method of the present invention.

[0014] The present invention also relates to a method for simultaneously binding to a target cell and an immune effector cell, comprising administering to a subject an antibody construct of the present invention, wherein the target cell under-expresses a second target (B').

[0015] The present invention also relates to kits comprising an antibody construct of the invention or an antibody construct produced by a method of the invention, a nucleic acid molecule of the invention, a vector of the invention, and / or a host cell of the invention. [Brief description of the drawings]

[0016] [Figure 1-1] FIG. 1: Schematic diagram of antibody constructs (A–E) and reference antibody constructs (F–H) comprising at least four first binding domains. [Figure 1-2] See description of Figure 1-1. [Figure 1-3] See description of Figure 1-1. [Figure 2-1]FIG. 2: Biochemical characterization of antibody constructs. (A) Bi-scDb-IgAb_06 (SEQ ID NO: 162 and SEQ ID NO: 163), (B) Bi-scDb-Fc_01 (SEQ ID NO: 148), (C) Bi-scDb-Fc_02 (SEQ ID NO: 149), (D) aBi-scDb-Fc_01 (SEQ ID NO: 150 and SEQ ID NO: 151), (E) aBi-scDb-Fc_02 (SEQ ID NO: 152 and SEQ ID NO: 153), (F) aBi-scDb-Fc_03 (SEQ ID NO: 154 and SEQ ID NO: 155), (G) aBi-scDb-Fc_04 (SEQ ID NO: 156 and SEQ ID NO: (H) aBi-scDb-Fc_05 (SEQ ID NO: 158 and SEQ ID NO: 159), and (H) aBi-scDb-Fc_06 (SEQ ID NO: 160 and SEQ ID NO: 161). Left panel: Constructs purified by Protein A chromatography and preparative size-exclusion chromatography. Right panel: SDS-PAGE analysis under non-reducing (nR) or reducing (R) conditions. [Figure 2-2] See description of Figure 2-1. [Figure 2-3] See description of Figure 2-1. [Diagram 3] Concentration-dependent lysis of MCF-7 target cells by NK cells. Calcein-labeled MCF-7 cells were co-cultured with enriched primary human NK cells as effector cells at an E:T ratio of 5:1 in the presence of serial dilutions of the indicated antibodies. After 4 h of incubation, calcein fluorescence released into the supernatant from lysed target cells was quantified and used to calculate the percentage of specific lysis. Means and SD of duplicate values ​​are plotted. [Figure 4]Concentration-dependent lysis of Daudi target cells by NK cells. Calcein-labeled Daudi cells were co-cultured with enriched primary human NK cells as effector cells at an E:T ratio of 5:1 in the presence of serial dilutions of the indicated antibodies. After 4 h of incubation, calcein fluorescence released from lysed target cells into the supernatant was quantified and used to calculate the percentage of specific lysis. Means and SD of duplicate values ​​are plotted. [Diagram 5] Concentration-dependent induction of NK cell fratricide by different antibody constructs. Enriched primary human NK cells labeled with calcein were co-cultured with autologous NK cells as effector cells at a 1:1 E:T ratio in the presence of serial dilutions of the indicated antibodies. After 4 h of incubation, calcein fluorescence released from lysed target cells in the supernatant was quantified and used to calculate the % specific lysis. Anti-CD38 IgG1 (IgAb_51) was used as a positive control. Means and SD of duplicate values ​​are plotted. [Figure 6] Concentration-dependent phagocytosis of DK-MG target cells by macrophages. CMFDA-labeled DK-MG cells were co-cultured with human monocyte-derived macrophages as effector cells at an E:T ratio of 5:1 in the presence of serial dilutions of the indicated antibodies. After 4 h of incubation, cells were stained with anti-CD11b and the viability dye eF780. Phagocytosis of labeled target cells was quantified by analyzing the percentage of CMFDA+ / CD11b+ cells relative to live cells by flow cytometry. ADCP in the absence of antibodies was used for normalization. Means and SD of duplicate values ​​are plotted and represent one representative experiment. [Figure 7]Concentration-dependent phagocytosis of EGFR-low expressing MCF-7 target cells by macrophages. CMFDA-labeled MCF-7 cells were co-cultured with human monocyte-derived macrophages as effector cells at an E:T ratio of 5:1 in the presence of serial dilutions of the indicated antibodies. After 4 h of incubation, cells were stained with anti-CD11b and the viability dye eF780. Phagocytosis of labeled target cells was quantified by analyzing the percentage of CMFDA+ / CD11b+ cells relative to live cells by flow cytometry. ADCP in the absence of antibody was used for normalization. Means and SD of duplicate values ​​are plotted and represent one representative experiment. [Figure 8] Concentration-dependent lysis of A-431 target cells by NK cells. Calcein-labeled A-431 cells were co-cultured with enriched primary human NK cells as effector cells at an E:T ratio of 5:1 in the presence of serial dilutions of the indicated antibodies. After 4 h of incubation, calcein fluorescence released into the supernatant from lysed target cells was quantified and used to calculate normalized specific lysis. Means and SDs of duplicate values ​​are plotted. [Figure 9] Concentration-dependent lysis of BCMA+ MM.1S cells by NK cells. Calcein-labeled MM.1S cells were co-cultured with enriched primary human NK cells as effector cells at an E:T ratio of 5:1 in the presence of serial dilutions of the indicated antibodies. After 4 h of incubation, calcein fluorescence released into the supernatant from lysed target cells was quantified and used to calculate the percentage of specific lysis. Means and SD of duplicate values ​​are plotted. [Figure 10] Scoring of tumor cell lines for HER2 and EGFR expression based on specific antibody binding capacity (SABC). [Figure 11]Concentration-dependent phagocytosis of EGFR-expressing HCT-116 target cells by macrophages. CMFDA-labeled HCT-116 cells were co-cultured with human monocyte-derived macrophages as effector cells at an E:T ratio of 5:1 in the presence of serial dilutions of the indicated antibodies. After 4 h of incubation, cells were stained with anti-CD11b and the viability dye eF780. Phagocytosis of labeled target cells was quantified by analyzing the percentage of CMFDA+ / CD11b+ cells relative to live cells by flow cytometry. ADCP in the absence of antibodies was used for normalization. Means and SD of duplicate values ​​are plotted and represent one representative experiment. [Figure 12] Concentration-dependent lysis of CD19+ Daudi target cells by NK cells. Calcein-labeled Daudi cells were co-cultured with enriched primary human NK cells as effector cells at an E:T ratio of 5:1 in the presence of serial dilutions of the indicated antibodies. After 4 h of incubation, calcein fluorescence released from lysed target cells into the supernatant was quantified and used to calculate the percentage of specific lysis. Means and SD of duplicate values ​​are plotted. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] definition The term "binding domain" in the context of the present invention describes the feature of a domain that can specifically bind to / interact with / recognize a given target epitope or a given target site of a target molecule (antigen), for example an antigen present on the surface of an innate immune effector cell (such as CD16A or NKp46) and / or an antigen present on the surface of a target cell, respectively. The structure and / or function of the first binding domain (for example recognizing an antigen present on the surface of an innate immune effector cell), the structure and / or function of the second binding domain (for example recognizing an antigen present on the surface of a target cell), and also the structure and / or function of the third binding domain (recognizing an antigen present on the surface of a target cell) are preferably based on the structure and / or function of an antibody, for example a full-length or full-length immunoglobulin molecule, and / or are derived from the variable heavy chain (VH) and / or variable light chain (VL) domains of an antibody or a fragment thereof.

[0018] The term "specifically binds" as used herein means that the binding domain preferentially binds to or recognizes a target, even when its binding partner is present in a mixture of other molecules or other structures. Binding can be mediated by covalent or non-covalent interactions or a combination of both. In a preferred embodiment, "simultaneous binding to target cells and immune effector cells" includes the physical interaction of the binding domains with their targets on the cells, but also preferably includes the induction of an effect caused by the simultaneous binding to these two cells. Such an effect may be the immune effector function of the immune effector cells, such as a cytotoxic effect.

[0019] The term "antibody construct" refers to a molecule whose structure and / or function is based on the structure and / or function of an antibody, e.g., a full-length or full-length immunoglobulin molecule, and / or derived from the variable heavy (VH) and / or variable light (VL) domains of an antibody or a fragment thereof. Thus, the antibody construct is capable of specifically binding to its specific target or antigen. Furthermore, the binding region of the antibody construct defined in the present invention comprises the minimum structural requirements of an antibody, which allows target binding. This minimum requirement can be defined, for example, as the presence of at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region), preferably all six CDRs. Alternative approaches to define the minimal structural requirements of an antibody are to define the protein domains of the target protein that constitute the antibody epitopes, i.e. epitope regions (epitope clusters), within the structure of the specific target, respectively, or by reference to specific antibodies that compete with the epitopes for the defined antibody. Antibodies on which the constructs defined in the present invention are based include, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies, and human antibodies.

[0020] The binding region of the antibody construct defined in the present invention may, for example, comprise a group of CDRs as described above. Preferably, these CDRs are included in the framework of the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH); however, they do not have to be included in both. For example, the Fd fragment has two VH regions and often retains some of the antigen-binding function of the intact antigen-binding region. Further examples of antibody fragment, antibody variant, or binding domain formats include: (1) a Fab fragment, i.e., a monovalent fragment having the VL, VH, CL, and CH1 domains; (2) an F(ab')2 fragment, i.e., a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge domain; (3) an Fd fragment having two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of one arm of an antibody; (5) a dAb fragment having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) isolated complementarity determining regions (CDRs); and (7) single chain Fv (scFv), the latter being preferred (e.g., derived from an scFv library).

[0021] The antibody constructs defined in the present invention may comprise fragments of full-length antibodies, such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab')2, or "rIgG" ("half antibodies"). The antibody constructs defined in the present invention may also comprise modified antibody fragments, also called antibody variants, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv zippers, scFab, Fab2, Fab3, diabodies, single chain diabodies (scDb), tandem diabodies (TandAb's), tandem di-scFv, tandem tri-scFv, "multibodies" such as triabodies or tetrabodies, and single domain antibodies, such as nanobodies or single variable domain antibodies, which comprise only one variable domain, which may be VHH, VH, or VL, and specifically bind to an antigen or epitope independent of other V regions or V domains.

[0022] As used herein, the term "single chain Fv", "single chain antibody", or "scFv" refers to an antibody fragment of a single polypeptide chain that contains the variable regions from both the heavy and light chains but lacks a constant region. Generally, single chain antibodies further comprise a polypeptide linker between the VH and VL domains that allows them to form the desired structure that allows for antigen binding. A preferred linker for this purpose is a glycine serine linker, which preferably contains about 15 to about 30 amino acids. A preferred glycine serine linker may have one or more repeats of GGS, GGGS (SEQ ID NO: 1), or GGGGS (SEQ ID NO: 6). Such linkers preferably contain 5, 6, 7, 8, 9 and / or 10 repeats of GGS, preferably (GGS)6 (SEQ ID NO: 4) (preferably used for scFvs with the arrangement VH-VL) or preferably (GGS)7 (SEQ ID NO: 5) (preferably used for scFvs with the arrangement VL-VH). To stabilize "single chain antibodies", an interdomain disulfide bridge can be introduced using the H44-L100 mutation (Zhao et al., 2010). H44 represents amino acid number 44 in VH (Kabat numbering), which must be changed to a cysteine. Meanwhile, L100 represents amino acid number 100 in VL (Kabat numbering), which must be changed to a cysteine. Single chain antibodies are reviewed in detail by Plueckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).Various methods of making single chain antibodies are known, including those described in U.S. Patent Nos. 4,694,778 and 5,260,203; International Patent Publication WO 88 / 01649; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041. In certain embodiments, the single chain antibody may also be a bispecific antibody, a multispecific antibody, a human antibody, and / or a humanized antibody, and / or a synthetic antibody. As used herein, the term "bi-scFv" or "ta-scFv" (tandem scFv) refers to two scFvs fused together. Such bi-scFvs or ta-scFvs may include a linker between the two scFv moieties. Typically, the arrangement of the VH and VL domains in the polypeptide chains within each scFv may be in any order. This means that the "bi-scFv" of a "ta-scFv" may be arranged in the order VH(1)-VL(1)-VH(2)-VL(2), VL(1)-VH(1)-VH(2)-VL(2), VH(1)-VL(1)-VL(2)-VH(2), or VL(1)-VH(1)-VL(2)-VH(2), where (1) and (2) represent the first scFv and second scFv, respectively.

[0023] The term "double Fab" as used herein refers to two Fab fragments fused together, preferably in a staggered arrangement, where the first chain of the first Fab is fused N-terminally to the first chain of the second Fab, or the second chain of the first Fab is fused N-terminally to the second chain of the second Fab, or both, i.e., the first chain of the first Fab and the second chain of the first Fab are fused to the first and second chains of the second Fab, respectively. A linker may be present between the fused chains of the first and second Fab. The first and second chains of the first and second Fabs may be independently selected from a chain derived from the light chain of the Fab (VL-CL), a chain derived from the heavy chain of the Fab (VH-CH1), as long as each Fab comprises a VH, a VL, a CH1, and a CL. As an illustrative example, a chain derived from the light chain of the first Fab may be fused to a chain derived from the light chain of the second Fab. As another illustrative example, a chain derived from the heavy chain of a first Fab can be fused to a chain derived from the heavy chain of a second Fab. As yet another illustrative example, a chain derived from the heavy chain of a first Fab can be fused to a chain derived from the light chain of a second Fab. In some double Fabs, both chains of the two Fabs are fused together. For example, a chain derived from the light chain of a first Fab can be fused to a chain derived from the light chain of a second Fab, and at the same time, a chain derived from the heavy chain of a first Fab can be fused to a chain derived from the heavy chain of a second Fab. Alternatively, a chain derived from the light chain of a first Fab can be fused to a chain derived from the heavy chain of a second Fab, and at the same time, a chain derived from the heavy chain of a first Fab can be fused to a chain derived from the light chain of a second Fab. The fusion of two Fab chains may optionally include a linker. Suitable and preferred linkers include the upper hinge sequence (SEQ ID NO:11) or a glycine serine linker having up to about 20 amino acids, preferably up to 10 amino acids, or most preferably 10 amino acids, such as two repeats of GGGGS (SEQ ID NO:7).The glycine serine linker included in the dual Fab may have one or more repeats, for example 1, 2, 3, or 4 repeats, of GGS, GGGS (SEQ ID NO: 1), or GGGGS (SEQ ID NO: 6).

[0024] As used herein, "diabody" or "Db" refers to an antibody construct comprising two binding domains, which may be constructed using the heavy and light chains disclosed herein and by using the individual CDR regions disclosed herein. Typically, a diabody comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains of the same chain. Preferred linkers for this purpose include glycine-serine linkers having up to about 12 amino acids, preferably up to about 10 amino acids. A preferred glycine-serine linker may have one or more repeats of GGS, GGGS (SEQ ID NO: 1), or GGGGS (SEQ ID NO: 6). A preferred linker is (GGS)2 (SEQ ID NO: 2). Another preferred linker is (GGS)3 (SEQ ID NO: 3). Thus, the VH and VL domains of one fragment are forced to pair with the complementary VH and VL domains of another fragment, thereby forming two antigen-binding sites. A diabody may be formed by two separate polypeptide chains, each comprising a VH and a VL. Alternatively, all four variable domains may be contained in one single polypeptide chain, comprising two VH and two VL domains. In such a case, the diabody may also be referred to as a "single-chain diabody" or "scDb". Typically, an scDb comprises two chains of a non-single-chain diabody, fused together, preferably via a linker. A preferred linker for this purpose is a glycine serine linker, which preferably comprises about 15 to about 30 amino acids. A preferred glycine serine linker may have one or more repeats of GGS, GGGS (SEQ ID NO: 1), or GGGGS (SEQ ID NO: 6). Such linkers preferably contain 5, 6, 7, 8, 9 and / or 10 repeats of GGS, preferably (GGS)6 (SEQ ID NO: 4), or preferably (GGS)7 (SEQ ID NO: 5).In the polypeptide chain, the variable domains of the scDb can be arranged (from N-terminus to C-terminus) in the order VL-VH-VL-VH or VH-VL-VH-VL. Similarly, the spatial arrangement of the four domains in the tertiary / quaternary structure can be in the order VL-VH-VL-VH or VH-VL-VH-VL. The term diabody does not exclude that further binding domains are fused to the diabody. The "single-chain diabody" can also be stabilized by introducing an interdomain disulfide bridge using the H44-L100 mutation (Zhao et al., 2010). H44 represents amino acid number 44 in VH (Kabat numbering), which must be changed to a cysteine. Meanwhile, L100 represents amino acid number 100 in VL (Kabat numbering), which must be changed to a cysteine.

[0025] Furthermore, the definition of the term "antibody construct" also includes multivalent constructs, including bispecific constructs, which usually specifically bind only two antigenic structures, as well as multispecific / multispecific constructs that specifically bind more than two, e.g., three, four, or more, antigenic structures via separate binding domains. The antibody construct of the present invention is a multivalent (e.g., pentavalent or hexavalent) antibody construct that is at least bispecific (e.g., bispecific or trispecific). Furthermore, the definition of the term "antibody construct" also includes molecules that consist of only one polypeptide chain, as well as molecules that consist of multiple polypeptide chains, which may be identical (homodimers, homotrimers, or homooligomers) or different (heterodimers, heterotrimers, or heterooligomers). Examples of the above-identified antibodies and variants or derivatives thereof are described, inter alia, in Harlow and Lane, Antibodies a laboratory manual, CSHL Press (1988) and Using Antibodies: a laboratory manual, CSHL Press (1999), Kontermann and Dubel, Antibody Engineering, Springer, 2nd ed. 2010, and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0026] The term "valency" or "valency" means that a measured number of antigen-binding domains are present in an antigen-binding protein. Depending on the context, "valency" or "valency" may refer to the number of antigen-binding domains that are directed to a specific target, and does not exclude the presence of additional antigen-binding domains that are specific for other targets. As an illustrative example, natural IgG has two antigen-binding domains and is bivalent. As another illustrative example, the antibody construct defined in the present invention is at least tetravalent for a first target and comprises at least one additional binding domain that is specific for a second target.

[0027] The term "bispecific" as used herein means an antibody construct that is "at least bispecific", i.e., the antibody construct comprises at least a first binding domain and a second binding domain, where the first binding domain binds to one antigen or target (herein, an antigen present on the surface of a natural immune effector cell) and the second binding domain binds to another antigen or target (herein, an antigen present on the surface of a target cell). Thus, the antibody construct defined in the present invention comprises specificity for at least two different antigens or targets. For example, the first binding domain preferably binds to an extracellular epitope of an NK cell receptor of one or more species selected from humans, Macaca species, and rodent species.

[0028] The term "trispecific" as used herein means an antibody construct that is "at least trispecific", i.e., the antibody construct comprises at least a first binding domain, a second binding domain, and a third binding domain, where the first binding domain binds to a first antigen or target (herein, an antigen present on the surface of an innate immune effector cell), the second binding domain binds to a second antigen or target (herein, an antigen present on the surface of a target cell), and the third binding domain binds to a third antigen or target (herein, an antigen present on the surface of a target cell other than the second target). Thus, some antibody constructs defined in the present invention comprise specificity for at least three different antigens or targets. For example, the first binding domain preferably binds to an extracellular epitope of an NK cell receptor of one or more species selected from humans, macaque species, and rodent species.

[0029] "CD16A" or "CD16a" refers to the activating receptor CD16A, also known as FcγRIIIA, expressed on the cell surface of NK cells. CD16A is an activating receptor that triggers the cytotoxic activity of NK cells. The amino acid sequence of human CD16A is shown in UniProt entry P08637 (version 212 of August 12, 2020) and SEQ ID NO: 13. The affinity of antibodies to CD16A is directly related to their ability to trigger NK cell activation, and thus the higher the affinity to CD16A, the lower the dose of antibody required for activation. The antigen-binding site of the antigen-binding protein binds to CD16A, but preferably does not bind to CD16B. For example, an antigen-binding site comprising a heavy (VH) chain variable domain and a light (VL) chain variable domain that binds to CD16A but not to CD16B can be provided by an antigen-binding site that specifically binds to an epitope of CD16A comprising amino acid residues of the C-terminal sequence SFFPPGYQ ​​(positions 201-208 of SEQ ID NO:13) and / or residues G147 and / or Y158 of CD16A that are not present in CD16B.

[0030] "CD16B" refers to the receptor CD16B, also known as FcγRIIIB, expressed on neutrophils and eosinophils. This receptor is anchored by glycosylphosphatidylinositol (GPI) and is understood not to trigger any kind of cytotoxic activity of CD16B-positive immune cells.

[0031] The term "target cell" refers to a cell or group of cells that is the target of the mode of action exerted by the antibody construct of the present invention. This cell / group of cells includes, for example, pathological cells that are eliminated or inhibited by binding these cells to effector cells via the antibody construct of the present invention. A preferred target cell is a cancer cell.

[0032] The terms "target cell surface antigen" or "antigen present on the surface of a target cell", used synonymously, refer to an antigenic structure that is expressed by a cell and present on the cell surface so as to be accessible to the antibody constructs described herein. This may be a protein, preferably an extracellular portion of a protein, a peptide presented on the cell surface in association with MHC (including HLA-A2, HLA-A11, HLA-A24, HLA-B44, HLA-C4), or a carbohydrate structure, preferably a carbohydrate structure of a protein, such as a glycoprotein. This is preferably a tumor-associated or tumor-restricted antigen. It is envisaged that CD16A, CD56, NKG2A, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1, CD89, CD96, CD160, TIGIT, TIM-3, KIR2DL1-5, KIR3DL1-3, and KIR2DS1-5 are not target cell surface antigens according to the present invention.

[0033] The term "immunoregulatory antigen" as used herein refers to an antigen that is preferably a receptor. The antigen or preferably a receptor can receive and / or transmit signals, and its engagement is believed to affect the quality and strength of innate immune cell response. Such antigens include inhibitory receptors, activating receptors, adhesion molecules, and costimulatory molecules. Such "immunoregulatory antigens" include, but are not limited to, CD16A, CD56, NKG2A, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 (CD226), SLAMF7 (CD319), CD244 (2B4), OX40, CD47 / SIRPα, CD89, CD96, CD137, CD160, TIGIT, Nectin-4, PD-1, PD-L1, LAG-3, CTLA-4, TIM-3, KIR2DL1-5, KIR3DL1-3, KIR2DS1-5, KIR3DS1, and CD3. The term "immunoregulatory antigen" refers to a positive regulator of the immune response of immune cells (e.g., NK cells, macrophages), which can stimulate the function of the immune cells. The term "immunoregulatory antigen" also includes activating receptors, adhesion molecules, and costimulatory molecules. Activating receptors often detect self-molecules that are expressed under conditions of cellular stress. Some activating receptors signal, for example, through immunoreceptor tyrosine-based activation motifs (ITAMs), through immunoreceptor tyrosine-based switch motifs (ITSMs), or through other tyrosine-based signaling motifs that are usually included in the associated molecules. Such "immune activating antigens" include, but are not limited to, CD16A, CD56, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 (CD226), SLAMF7 (CD319), CD244 (2B4), OX40, CD137, CD89, CD160, and killer cell immunoglobulin-like receptors (KIR2DS1-5 and KIR3DS1). The term "immunosuppressive antigens" relates to negative regulators of the immune response of (innate) immune cells (e.g., NK cells, macrophages). "Immunosuppressive antigens" are preferably receptors.Inhibitory signals are usually transmitted through immunoreceptor tyrosine-based inhibitory motifs (ITIMs) located in the intracellular tails of the receptors. Such "immunosuppressive antigens" include, but are not limited to, NKG2A, TIGIT, PD-1, PD-L1, CD47, SIRPα, LAG-3, CTLA-4, CD96, TIM-3, CD137, KIR2DL1-5, and KIR3DL1-3.

[0034] The antibody constructs of the present invention are at least bispecific, but may also contain additional specificities resulting in multispecific antibody constructs, such as trispecific antibody constructs, or constructs with more than three specificities (e.g., four, five, six, ...). However, even in these multispecific constructs, it is envisaged that only the first binding domain is specific for an antigen present on the surface of an innate immune effector cell. Examples of trispecific or multispecific antibody constructs are provided, for example, in WO 2015 / 158636, WO 2017 / 064221, WO / 2019 / 198051, and Ellwanger et al. (MAbs. 2019 Jul;11(5):899-918).

[0035] Considering that the antibody constructs defined in the present invention are (at least) bispecific, they do not exist in nature and they are significantly different from natural products. Thus, a "bispecific" antibody construct is an artificial hybrid antibody with at least two different binding sides with different specificities. Bispecific antibody constructs can be produced by various methods including fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315- 321 (1990).

[0036] The binding domain and the variable domain (VH / VL) of the antibody construct of the present invention may or may not include a peptide linker (spacer peptide). According to the present invention, the term "peptide linker" includes an amino acid sequence by which the amino acid sequence of one (variable and / or binding) domain and the amino acid sequence of another (variable and / or binding) domain of the antibody construct defined herein are linked to each other. A peptide linker may also be used to fuse one domain of the antibody construct defined herein to another domain. In such a case, it is preferably a short linker, preferably having a length of about 10 nm or less, preferably about 9 nm or less, preferably about 8 nm or less, preferably about 7 nm or less, preferably about 6 nm or less, preferably about 5 nm or less, preferably about 4 nm or less, or even shorter. The length of the linker is preferably determined as described by Rossmalen et al Biochemistry 2017, 56, 6565-6574, which also describes suitable linkers well known to those skilled in the art. An example of such a linker is a glycine-serine linker or a serine linker, preferably comprising about 75 amino acids or less, preferably about 50 amino acids or less. In an illustrative example, a suitable linker comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) GGGS sequences (SEQ ID NO: 6), such as (GGGGS)2 (SEQ ID NO: 7), (GGGGS)4 (SEQ ID NO: 8), or preferably (GGGGS)6 (SEQ ID NO: 9). Other illustrative examples of linkers are shown in SEQ ID NOs: 2-5. A preferred technical feature of such a peptide linker is that it does not contain any polymerization activity.

[0037] The antibody construct defined in the present invention is preferably an "in vitro generated antibody construct". This term refers to an antibody construct according to the above definition, in which all or part of the variable region (e.g., at least one CDR) is generated by a method other than immune cell selection, such as in vitro phage display, protein chip, or any other method that can test the binding ability of candidate sequences to antigen. Therefore, this term preferably does not include sequences generated only by using genome rearrangement in animal immune cells. It is also intended that the antibody construct of the present invention is a recombinant antibody or is based on a recombinant antibody. A "recombinant antibody" is an antibody generated by using recombinant DNA technology or genetic engineering.

[0038] The term "monoclonal antibody" (mAb) or monoclonal antibody construct as used herein refers to an antibody obtained from a population of substantially homogenous antibodies. That is, the individual antibodies that make up the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. In contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies against different determinants (or epitopes), monoclonal antibodies are highly specific and directed against a single antigenic side or determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and therefore are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies and should not be construed as requiring production of the antibody by any particular method.

[0039] To prepare monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used.For example, the monoclonal antibodies used can be produced by the hybridoma method first described by Koehler et al., Nature, 256: 495 (1975), or by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567).Examples of further techniques for producing human monoclonal antibodies include trioma technology, human B-cell hybridoma technology (Kozbor, Immunology Today 4 (1983), 72), and EBV hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).

[0040] In that case, standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE™) analysis can be used to screen the hybridomas to identify one or more hybridomas that produce antibodies that specifically bind to the designated antigen. Any form of the relevant antigen can be used as an immunogen, including recombinant antigens, naturally occurring forms, any variants or fragments thereof, and antigenic peptides thereof. Surface plasmon resonance used in the BIAcore system can be used to increase the efficiency of phage antibodies that bind to epitopes of target cell surface antigens (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). Another exemplary method of generating monoclonal antibodies includes screening protein expression libraries, such as phage display libraries or ribosome display libraries. Phage display methods are described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315-1317, Clackson et al., Nature, 352: 624-628 (1991), and Marks et al., J. Mol. Biol., 222: 581-597 (1991).

[0041] In addition to using display libraries, relevant antigens can be used to immunize non-human animals, such as rodents (such as mice, hamsters, rabbits, or rats). In one embodiment, the non-human animals contain at least a portion of a human immunoglobulin gene. For example, it is possible to artificially create mouse strains that are defective in mouse antibody production and have large fragments of the human Ig (immunoglobulin) locus. Using hybridoma technology, antigen-specific monoclonal antibodies derived from these genes and with desired specificity can be produced and selected. See, for example, XENOMOUSE™, Green et al. (1994) Nature Genetics 7:13-21, US 2003-0070185, WO 96 / 34096, and WO 96 / 33735.

[0042] Monoclonal antibodies can also be obtained from non-human animals and then modified, e.g., humanized, deimmunized, chimeric, etc., using recombinant DNA techniques known in the art. Examples of modified antibody constructs include humanized variants of non-human antibodies, "affinity matured" antibodies (see, e.g., Hawkins et al. J. Mol. Biol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837 (1991)), and antibody variants with altered effector functions (see, e.g., U.S. Patent No. 5,648,260; Kontermann and Dubel (2010), supra; and Little (2009), supra).

[0043] In immunology, affinity maturation is the process by which B cells produce antibodies with increasing affinity for antigens during an immune response. Repeated exposure to the same antigen induces the host to produce antibodies with increasingly higher affinities. Like the natural prototype, in vitro affinity maturation is based on the principle of mutation and selection. In vitro affinity maturation has been successfully used to optimize antibodies, antibody constructs, and antibody fragments. Random mutations within the CDRs are introduced using irradiation, chemical mutagens, or error-prone PCR. Additionally, genetic diversity can be increased by chain shuffling. Two or three rounds of mutation and selection using display methods such as phage display usually result in antibody fragments with affinities in the low nanomolar range.

[0044] A preferred type of amino acid substitution variant of an antibody construct involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further development have improved biological properties compared to the parent antibody from which they were generated. A convenient method for generating such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sides (e.g., 6-7 sides) are mutated to generate all possible amino acid substitutions on each side. The antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions to the gene III product of M13 packaged inside each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sides for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be useful to analyze the crystal structure of antigen-antibody complex to identify the contact points between binding domain and, for example, human target cell surface antigen.Such contact residues and adjacent residues are candidates for substitution by the techniques detailed herein.After making such variants, the panel of variants can be subjected to screening as described herein, and the antibody that has superior properties in one or more relevant assays can be selected for further development.

[0045] The monoclonal antibodies and antibody constructs of the present disclosure specifically include "chimeric" antibodies (immunoglobulins) and fragments of such antibodies, so long as they exhibit the desired biological activity, in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). As used herein, chimeric antibodies of interest include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and human constant region sequences. Various approaches to making chimeric antibodies have been described. See, e.g., Morrison et al., Proc. Natl. Acad. Sci USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., EP 0171496; EP 0173494; and GB 2177096.

[0046] Antibodies, antibody constructs, antibody fragments, or antibody variants can also be modified by specific deletion of human T cell epitopes (a method called "deimmunization"), for example by methods disclosed in WO 98 / 52976 or WO 00 / 34317. Briefly, the heavy and light chain variable domains of an antibody can be analyzed for peptides that bind to MHC class II; these peptides represent potential T cell epitopes (as defined in WO 98 / 52976 and WO 00 / 34317). As described in WO 98 / 52976 and WO 00 / 34317, a computer modeling approach called "peptide threading" can be applied to detect potential T cell epitopes, and further, a database of human MHC class II binding peptides can be searched for motifs present in VH and VL sequences. These motifs bind to any of the 18 major MHC class II DR allotypes, and therefore constitute potential T cell epitopes. Detected potential T cell epitopes can be eliminated by substituting a small number of amino acid residues in the variable domain, or preferably by single amino acid substitutions. Typically, conservative substitutions are made. In many, but not all cases, amino acids that occur frequently at certain positions in human germline antibody sequences can be used. Human germline sequences are disclosed, for example, in Tomlinson, et al. (1992) J. Mol. Biol. 227:776-798; Cook, GP et al. (1995) Immunol. Today Vol. 16 (5): 237-242; and Tomlinson et al. (1995) EMBO J. 14: 14:4628-4638. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, LA. et al. MRC Centre for Protein Engineering, Cambridge, UK).These sequences can be used as a source of human sequences, e.g., for the framework regions and CDRs. Consensus human framework regions, e.g., as described in U.S. Patent No. 6,300,064, can also be used.

[0047] "Humanized" antibodies, antibody constructs, such as antibody constructs of the invention, variants or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) are antibodies or immunoglobulins derived from mostly human sequences, with minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient's hypervariable region (as well as CDRs) are replaced by residues from a hypervariable region (donor antibody) of a non-human (e.g., rodent) species, such as mouse, rat, hamster, or rabbit, having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, as used herein, "humanized antibodies" may also include residues that are not present in either the recipient antibody or the donor antibody. These modifications are made to further refine and optimize antibody performance. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).

[0048] Humanized antibodies or fragments thereof can be produced by replacing the sequences of Fv variable domains that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for producing humanized antibodies or fragments thereof are provided by Morrison (1985) Science 229: 1202-1207; Oi et al. (1986) BioTechniques 4: 214; and US 5,585,089; US 5,693,761; US ​​5,693,762; US 5,859,205; and US 6,407,213. These methods include isolating, manipulating, and expressing nucleic acid sequences that code for all or part of the immunoglobulin Fv variable domains from at least one of the heavy or light chains. Such nucleic acids can be obtained from hybridomas that produce antibodies against a given target, as described above, and from other sources. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

[0049] Humanized antibodies may also be produced using transgenic animals such as mice that express human heavy and light chain genes but lack the ability to express endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR grafting method that may be used to prepare the humanized antibodies described herein (U.S. Pat. No. 5,225,539). All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace as many CDRs as are necessary for the binding of the humanized antibody to a given antigen.

[0050] Humanized antibodies can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions, and / or back mutations. Such altered immunoglobulin molecules can be generated by any of several techniques known in the art (e.g., Teng et al., Proc. Natl. Acad. Sci. USA, 80: 7308-7312, 1983; Kozbor et al., Immunology Today, 4: 7279, 1983; Olsson et al., Meth. Enzymol., 92: 3-16, 1982, and EP 239 400).

[0051] The terms "human antibody", "human antibody construct" and "human binding domain" include antibodies, antibody constructs, such as the antibody constructs of the present invention, and binding domains having antibody regions, e.g., variable and constant regions or domains, that substantially correspond to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (1991) supra. A human antibody, antibody construct, or binding domain as defined in the present invention may contain, for example, in the CDRs, and in particular in CDR3, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). A human antibody, antibody construct, or binding domain may have at least one, two, three, four, five, or more positions substituted with an amino acid residue not encoded by a human germline immunoglobulin sequence. However, the definitions of human antibody, antibody constructs, and binding domains used herein also contemplate "fully human antibodies" that include only non-artificial and / or genetically altered human sequences of antibodies, as may be obtained by using techniques or systems such as Xenomouse. Preferably, a "fully human antibody" does not include amino acid residues that are not encoded by human germline immunoglobulin sequences.

[0052] In some embodiments, the antibody construct as defined herein is an "isolated" or "substantially pure" antibody construct. When used to describe the antibody constructs disclosed herein, "isolated" or "substantially pure" means that the antibody construct has been identified, separated, and / or recovered from components present in its production environment. Preferably, the antibody construct is free or substantially free from all other components from its production environment. Contaminating components present in the production environment, e.g., from transfected recombinant cells, are substances that would typically interfere with diagnostic or therapeutic uses of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. The antibody construct may, for example, comprise at least about 5% or at least about 50% by weight of the total protein in a given sample. It is understood that an isolated protein may comprise 5% to 99.9% by weight of the total protein content, depending on the environment. The polypeptide may be produced at significantly higher concentrations using inducible promoters or high expression promoters to produce at high concentration levels. This definition includes the production of antibody constructs in a wide variety of organisms and / or host cells known in the art. In a preferred embodiment, the antibody construct is purified (1) to a sufficient extent to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequencer, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably silver staining. However, usually, isolated antibody constructs are prepared by at least one purification step.

[0053] According to the present invention, the binding domain is in the form of one or more polypeptides. Such polypeptides may contain proteinaceous and non-proteinaceous parts (e.g., chemical linkers or chemical cross-linking agents, such as glutaraldehyde). Proteins (including their fragments, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) contain two or more amino acids linked together via covalent peptide bonds (resulting in a chain of amino acids).

[0054] The term "polypeptide" or "polypeptide chain" as used herein refers to a group of molecules that usually consists of more than 30 amino acids. The terms "peptide", "polypeptide" and "protein" also refer to naturally modified peptides / polypeptides / proteins that are modified by post-translational modifications such as glycosylation, acetylation, and phosphorylation. As referred to herein, a "peptide", "polypeptide" or "protein" may also be chemically modified, for example pegylated. Such modifications are well known in the art and are described herein below. The above modifications (glycosylation, pegylation, etc.) also apply to the antibody constructs of the present invention.

[0055] Preferably, the binding domain that binds to the antigen present on the surface of the natural immune effector cell, the binding domain that binds to the antigen present on the surface of the target cell, and / or the binding domain that binds to another antigen present on the surface of the target cell are human binding domains.Antibody and antibody constructs that include at least one human binding domain avoid some of the problems associated with antibodies or antibody constructs that have variable and / or constant regions of non-human, e.g., rodents (e.g., mice, rats, hamsters, or rabbits).The presence of such rodent-derived proteins may cause rapid clearance of the antibody or antibody construct, or may result in the patient developing an immune response against the antibody or antibody construct.To avoid the use of rodent-derived antibodies or antibody constructs, human antibodies / antibody constructs or fully human antibodies / antibody constructs can be made by introducing human antibody functions into rodents so that the rodents produce fully human antibodies.

[0056] The ability to clone and reconstruct megabase-sized human loci in YACs and introduce them into the mouse germline represents a powerful approach for elucidating the functional components of very large or loosely mapped loci and for generating useful models of human disease. Furthermore, the use of such techniques to replace mouse loci with their human equivalents can provide unique insights into the expression and regulation of human gene products during development, their communication with other systems, and their involvement in the induction and progression of disease.

[0057] An important practical application of such a strategy is the "humanization" of the mouse humoral immune system. The introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated provides an opportunity to study the mechanisms underlying the programmed expression and assembly of antibodies and their role in B-cell development. Furthermore, such a strategy may provide an ideal source for the production of fully human monoclonal antibodies (mAbs), a key milestone to realizing the promise of antibody therapy in human diseases. Fully human antibodies or fully human antibody constructs are expected to minimize the immunogenic and allergic responses inherent to mouse or mouse-derivatized mAbs, and thus improve the efficacy and safety of the administered antibodies / antibody constructs. The use of fully human antibodies or fully human antibody constructs may be expected to provide substantial advantages in the treatment of chronic and recurrent human diseases that require repeated compound administration, such as inflammation, autoimmunity, and cancer.

[0058] One approach towards this goal has been to artificially create mouse strains deficient in mouse antibody production, carrying large fragments of the human Ig loci, in the hope that the mice will produce a large repertoire of human antibodies in the absence of mouse antibodies. The large human Ig fragments would maintain the large variable gene diversity and proper regulation of antibody production and expression. By taking advantage of the mechanisms used by mice for antibody diversification and selection and the lack of immune tolerance to human proteins, the human antibody repertoires recreated in these mouse strains should give rise to high affinity antibodies to any antigen of interest, including human antigens. Using hybridoma technology, antigen-specific human mAbs with the desired specificity could be easily produced and selected. This general strategy was demonstrated in conjunction with the creation of the first XenoMouse mouse strains (see Green et al. Nature Genetics 7:13- 21 (1994)). XenoMouse strains were engineered with yeast artificial chromosomes (YACs) containing 245 kb and 190 kb sized germline-configured fragments of the human heavy and kappa light chain loci, respectively, that contain the core sequences of the variable and constant regions. The human Ig-containing YACs proved to be compatible with the mouse system for both antibody rearrangement and expression, and had the ability to replace inactivated mouse Ig genes. This was demonstrated by their ability to induce B cell development, produce full human antibodies of the mature human repertoire, and generate antigen-specific human mAbs. These results also suggested that a substantially complete repertoire characteristic of the human humoral response to infection and immunization could be recapitulated by introducing larger portions of the human Ig loci, including a larger number of V genes, additional regulatory elements, and human Ig constant regions. The work of Green et al. has recently been extended to the introduction of more than 80% of the human antibody repertoire by introducing megabase-sized germline-configured YAC fragments of the human heavy and kappa light chain loci, respectively.See Mendez et al. Nature Genetics 15:146-156 (1997) and U.S. patent application Ser. No. 08 / 759,620.

[0059] The generation of XenoMouse mice is described in U.S. Patent Application Nos. 07 / 466,008, 07 / 610,515, 07 / 919,297, 07 / 922,649, 08 / 031,801, 08 / 112,848, 08 / 234,145, 08 / 376,279, 08 / 430,938, 08 / 464,584, 08 / 464,582, and 08 / 463,191. , 08 / 462,837, 08 / 486,853, 08 / 486,857, 08 / 486,859, 08 / 462,513, 08 / 724,752, and 08 / 759,620; as well as U.S. Patent Nos. 6,162,963; 6,150,584; 6,114,598; 6,075,181, and 5,939,598, and as further discussed and described in Japanese Patent Nos. 3068180 B2, 3068506 B2, and 3068507 B2. See also Mendez et al. Nature Genetics 15:146-156(1997) and Green and Jakobovits J. Exp. Med. 188:483-495(1998), EP0463151 B1, WO 94 / 02602, WO 96 / 34096, WO 98 / 24893, WO 00 / 76310, and WO 03 / 47336.

[0060] In an alternative approach, other companies, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked by including pieces (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a μ constant region, and a second constant region (preferably a γ constant region) form a construct for insertion into an animal. This approach is disclosed in U.S. Patent No. 5,545,807 to Surani et al. and U.S. Patent Nos. 5,545,806; 5,625,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; 5,814,318; 5,877,397; and 5,878,220, all of which are incorporated herein by reference. Nos. 5,874,299; and 6,255,458 to Krimpenfort and Berns, U.S. Pat. Nos. 5,591,669 and 6,023,010 to Berns et al., U.S. Pat. Nos. 5,612,205; 5,721,367; and 5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and U.S. Pat. and U.S. Patent Application Nos. 07 / 574,748, 07 / 575,962, 07 / 810,279, 07 / 853,408, 07 / 904,068, 07 / 990,860, 08 / 053,131, 08 / 096,762, 08 / 155,301, 08 / 161,739, 08 / 165,699, and 08 / 209,741 to International. See also EP 0546073 B1, WO 92 / 03918, WO 92 / 22645, WO 92 / 22647, WO 92 / 22670, WO 93 / 12227, WO 94 / 00569, WO 94 / 25585, WO 96 / 14436, WO 97 / 13852, and WO 98 / 24884, and U.S. Pat. No. 5,981,175.See also Taylor et al. (1992), Chen et al. (1993), Tuaillon et al. (1993), Choi et al. (1993), Lonberg et al. (1994), Taylor et al. (1994), and Tuaillon et al. (1995), Fishwild et al. (1996).

[0061] Kirin has also demonstrated the production of human antibodies from mice, where large chromosome fragments or entire chromosomes were introduced by microcell fusion. See European Patent Application Nos. 773288 and 843961. Xenerex Biosciences is developing a technology for potentially producing human antibodies. In this technology, SCID mice are reconstituted with human lymphocytes, such as B cells and / or T cells. The mice are then immunized with an antigen, and the mice can generate an immune response against the antigen. See U.S. Patent Nos. 5,476,996; 5,698,767; and 5,958,765.

[0062] The human anti-mouse antibody (HAMA) response has prompted industry to work on preparing chimeric or otherwise humanized antibodies. However, it is expected that some human anti-chimeric antibody (HACA) responses will be observed, especially during long-term or multiple use of antibodies. It would therefore be desirable to provide antibody constructs that include a human binding domain for an antigen present on the surface of an innate immune effector cell and / or a human binding domain for an antigen present on the surface of a target cell to eliminate the concerns and / or effects of a HAMA or HACA response.

[0063] The term "epitope" refers to the side on an antigen to which a binding domain, such as an antibody or immunoglobulin, or a derivative, fragment, or variant of an antibody or immunoglobulin, specifically binds. An "epitope" is antigenic, and thus the term epitope is sometimes referred to herein as an "antigenic structure" or "antigenic determinant." Thus, the binding domain is the "antigen interaction site." It is also understood that the binding / interaction defines the "specific recognition."

[0064] An "epitope" can be formed from both contiguous amino acids or non-contiguous amino acids adjacent to each other due to tertiary folding of a protein. A "linear epitope" is an epitope in which the primary amino acid sequence constitutes the recognized epitope. Typically, a linear epitope contains at least 3 or at least 4, and more usually at least 5 or at least 6 or at least 7, e.g., about 8 to about 10, amino acids in a unique sequence.

[0065] In contrast to linear epitopes, "conformational epitopes" are epitopes in which the primary sequence of amino acids that constitute the epitope is not the only defining element of the recognized epitope (e.g., the primary sequence of amino acids is not necessarily recognized by the binding domain). Typically, conformational epitopes contain more amino acids than linear epitopes. With respect to the recognition of conformational epitopes, the binding domain recognizes the three-dimensional structure of an antigen, preferably a peptide or protein or a fragment thereof (in the present invention, the antigenic structure for one of the binding domains is contained within the target cell surface antigen protein). For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbones that form a conformational epitope are in close proximity, thereby allowing the antibody to recognize the epitope. Methods for determining the conformation of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy and site-directed spin labeling, and electron paramagnetic resonance (EPR) spectroscopy.

[0066] The interaction of a binding domain with an epitope or an epitope-containing region implies that the binding domain exhibits appreciable affinity for the epitope / epitope-containing region on a particular protein or antigen (herein, e.g., an antigen present on the surface of an innate immune effector cell, e.g., CD16A, an antigen present on the surface of a target cell, and / or another antigen present on the surface of a target cell, respectively), and typically does not exhibit significant reactivity to proteins or antigens other than the surface of the innate immune effector cell, the antigen present on the surface of a target cell, and / or another antigen present on the surface of a target cell, etc. "Appreciable affinity" refers to a degree of affinity that is greater than or equal to about 10 -6 M(KD) or stronger. Preferably, the binding is with a binding affinity of about 10 -12 ~10 -8 M, 10 -12 ~10 -9 M, 10 -12 ~10 -10M, 10 -11 ~10 -8 M, preferably about 10 -11 ~10 -9 M. Whether a binding domain specifically reacts or binds to a target can be readily tested, inter alia, by comparing the reaction of the binding domain to a target protein or antigen with the reaction of the binding domain to other proteins or antigens, such as the surface of innate immune effector cells, the antigen present on the surface of target cells, and / or other antigens present on the surface of target cells.

[0067] The terms "essentially / substantially does not bind" or "cannot bind" mean that the binding domain of the invention does not bind to proteins or antigens other than, for example, antigens present on the surface of innate immune effector cells, antigens present on the surface of target cells, and / or other antigens present on the surface of target cells, i.e., when binding to antigens present on the surface of innate immune effector cells, antigens present on the surface of target cells, and / or other antigens present on the surface of target cells, respectively, is set to 100%, the binding domain does not show more than 30% reactivity to proteins or antigens other than antigens present on the surface of innate immune effector cells, antigens present on the surface of target cells, and / or other antigens present on the surface of target cells, preferably 20% or less, more preferably 10% or less, particularly preferably 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less.

[0068] Specific binding is believed to be influenced by specific motifs in the amino acid sequences of the binding domain and antigen. Thus, binding is achieved as a result of their primary, secondary, and / or tertiary structures, and as a result of secondary modifications of these structures. The specific interaction of an antigen interaction side with its specific antigen can result in the simple binding of said side to the antigen. Furthermore, the specific interaction of an antigen interaction side with its specific antigen can alternatively or additionally initiate a signal, for example, by inducing a conformational change of the antigen, oligomerization of the antigen, etc.

[0069] The term "variable" refers to that portion of an antibody or immunoglobulin domain (i.e., the "variable domain") that exhibits variability in sequence and is involved in determining the specificity and binding affinity of an individual antibody. The pairing of a variable heavy chain (VH) and a variable light chain (VL) together forms a single antigen-binding side.

[0070] The variability is not evenly distributed throughout the variable domain of an antibody, but is concentrated in subdomains of each of the heavy and light chain variable regions. These subdomains are called "hypervariable regions" or "complementarity determining regions" (CDRs). The more conserved (i.e., non-hypervariable) parts of the variable domains are called "framework" regions (FRMs or FRs), which provide a scaffold in three-dimensional space for the six CDRs to form the antigen-binding surface. Naturally occurring heavy and light chain variable domains each contain four FRM regions (FR1, FR2, FR3, and FR4), most often adopting a β-sheet configuration, connected by three hypervariable regions, which form loops that connect and in some cases form part of the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRMs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding side (Kabat et al., supra).

[0071] The term "CDR" and its plural "CDRs" refer to the complementarity determining regions, three of which make up the binding characteristics of the light chain variable region (CDR-L1, CDR-L2, and CDR-L3) and three of which make up the binding characteristics of the heavy chain variable region (CDR-H1, CDR-H2, and CDR-H3). The CDRs contain most of the residues responsible for the specific interactions of the antibody with the antigen and thus contribute to the functional activity of the antibody molecule: they are the main determinants of antigen specificity.

[0072] The precise boundaries and lengths of CDRs are varied by various classification and numbering systems. Thus, CDRs may be referred to using Kabat, Chothia, contact definitions, or any other boundary definitions, including the numbering systems described herein. Although the boundaries are different, each of these systems has some overlap in what constitutes the so-called "hypervariable regions" in variable sequences. Thus, the definitions of CDRs based on these systems may differ in terms of length and boundary regions with adjacent framework regions. See, for example, Kabat (an approach based on cross-species sequence variability), Chothia (an approach based on crystallographic studies of antigen-antibody complexes), and / or MacCallum (Kabat et al., supra; Chothia et al., J. Mol. Biol, 1987, 196: 901-917; and MacCallum et al., J. Mol. Biol, 1996, 262: 732). Yet another standard for characterizing antigen-binding side is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). To the extent that two residue identification techniques define overlapping but not identical regions, they can be combined to define hybrid CDRs. However, numbering according to the so-called Kabat system is preferred.

[0073] Typically, CDRs form loop structures that can be classified as canonical structures. The term "canonical structure" refers to the main chain conformation that an antigen-binding (CDR) loop adopts. Comparative structural studies have found that five of the six antigen-binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized based on the torsion angle of the polypeptide backbone. Thus, similar loops between antibodies can have very similar three-dimensional structures, despite the high degree of amino acid sequence variability found in most of the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196: 901; Chothia et al., Nature, 1989, 342: 877; Martin and Thornton, J. Mol. Biol, 1996, 263: 800). Furthermore, there is a relationship between the adopted loop structure and its surrounding amino acid sequence. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues present at key positions within the loop and within the conserved framework (i.e., outside the loop), and therefore assignment to a particular canonical class can be made based on the presence of these key amino acid residues.

[0074] The term "canonical structure" may also include considerations of the linear sequence of an antibody, for example as listed by Kabat (Kabat et al., supra). The Kabat numbering scheme is a widely adopted standard for numbering the amino acid residues of an antibody variable domain in a consistent manner, and is the preferred scheme applied in the present invention as noted elsewhere herein. Additional structural considerations may also be used to define the canonical structure of an antibody. For example, differences not fully reflected by the Kabat numbering can be accounted for by the Chothia et al. numbering system and / or can be revealed by other techniques, such as crystallography and two- or three-dimensional computer modeling. Thus, a given antibody sequence may be placed into a canonical class that allows, among other things, the identification of an appropriate chassis sequence (e.g., based on a desire to include various canonical structures in a library). The Kabat numbering for the amino acid sequence of an antibody and structural considerations as described by Chothia et al., supra, and their correlation to interpret the canonical appearance of antibody structures are described in the literature. The subunit structures and three-dimensional configurations of various classes of immunoglobulins are well known in the art. For a review of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988. A comprehensive reference in immunoinformatics is the IMGT (International ImMunoGenetics Information System) three-dimensional (3D) structure database (Ehrenmann et al., 2010, Nucleic Acids Res., 38, D301-307). IMGT / 3D Structure DB structural data is obtained from the Protein Data Bank (PDB) and annotated based on the IMGT classification concepts using internal tools.Therefore, the IMGT / 3D structure DB provides the closest genes and alleles expressed in the amino acid sequences of the 3D structures by aligning them with the IMGT domain reference directory. In the case of antigen receptors, this directory includes the amino acid sequences of the domains encoded by translation of the constant genes and germline variable and binding genes. The CDR regions of the amino acid sequences of the present inventors are preferably revealed by using the IMGT / 3D structure database.

[0075] The CDR3 of the light chain and especially the CDR3 of the heavy chain may constitute the most important determinant for antigen binding within the variable region of the light and heavy chains. In some antibody constructs, the heavy chain CDR3 is likely to constitute the main contact area between the antigen and the antibody. An in vitro selection scheme that changes only the CDR3 can be used to change the binding properties of the antibody or to determine which residues contribute to antigen binding. Thus, the CDR3 is typically the largest source of molecular diversity within the antibody binding side. For example, H3 can be as short as just 2 amino acid residues or more than 26 amino acids.

[0076] In classical full-length antibodies or immunoglobulins, each light (L) chain is linked to a heavy (H) chain by one covalent disulfide bond, and 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 an antibody is contained within the heavy chain constant domain and can interact with Fc receptors located on the cell surface, for example.

[0077] After assembly and somatic mutation, antibody gene sequences show high diversity, and these diversified genes span 10 10It has been estimated that each of the nucleotide sequences encodes a number of different antibody molecules (Immunoglobulin Genes, 2nd ed., eds. Jonio et al., Academic Press, San Diego, CA, 1995). Thus, the immune system provides a repertoire of immunoglobulins. The term "repertoire" refers to at least one nucleotide sequence derived in whole or in part from at least one sequence encoding at least one immunoglobulin. These sequences can result from in vivo rearrangement of V, D, and J segments of heavy chains and V and J segments of light chains. Alternatively, these sequences can be generated from cells in which rearrangement occurs, e.g., in response to in vitro stimulation. Alternatively, some or all of these sequences can be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods, see, e.g., U.S. Pat. No. 5,565,332. A repertoire can include only one sequence or can include multiple sequences, including sequences in a genetically diverse collection.

[0078] The antibody constructs defined in the present invention may also comprise additional domains, for example, useful for the isolation of the molecule or related to the adapted pharmacokinetic profile of the molecule. The domains useful for the isolation of the antibody constructs may be selected from peptide motifs or secondarily introduced moieties that can be captured in an isolation method, for example, an isolation column. Non-limiting embodiments of such additional domains include peptide motifs known as Myc tags, HAT tags, HA tags, TAP tags, GST tags, chitin-binding domains (CBD tags), maltose-binding protein (MBP tags), Flag tags, Strep tags and variants thereof (e.g., StrepII tags), and His tags. All of the antibody constructs disclosed herein that feature identified CDRs may comprise a His tag domain, commonly known as a repeat of consecutive His residues, preferably 5 and more preferably 6 His residues (hexahistidine), in the amino acid sequence of the molecule. The His tag may, for example, be located at the N-terminus or C-terminus of the antibody construct, preferably at the C-terminus. Most preferably, the hexahistidine tag is linked to the C-terminus of the antibody construct according to the invention by a peptide bond. Furthermore, the conjugate system PLGA-PEG-PLGA may be combined with the polyhistidine tag for sustained release applications and improved pharmacokinetic profiles.

[0079] Amino acid sequence modifications of the antibody constructs 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. The amino acid sequence variants of the antibody constructs are prepared by introducing appropriate nucleotide changes into the antibody construct nucleic acid or by peptide synthesis. Any of the amino acid sequence modifications described below should result in an antibody construct that still retains the desired biological activity of the unmodified parent molecule (e.g., against an antigen present on the surface of an innate immune effector cell, an antigen present on the surface of a target cell, and / or against other antigens present on the surface of a target cell).

[0080] The term "amino acid" or "amino acid residue" typically refers to an amino acid having its art-recognized definition, e.g., an amino acid selected from the group consisting of alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may also be used, if desired. Generally, amino acids can be grouped as having nonpolar side chains (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); negatively charged side chains (e.g., Asp, Glu); positively charged side chains (e.g., Arg, His, Lys); or uncharged polar side chains (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0081] Amino acid modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody construct. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes may also alter post-translational processing of the antibody construct, for example, changing the number or location of glycosylation sites.

[0082] For example, 1, 2, 3, 4, 5, or 6 amino acids may be inserted, substituted, or deleted in each CDR (depending, of course, 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 FR. Preferably, the amino acid sequence insertion into the antibody construct includes amino- and / or carboxyl-terminal fusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Corresponding modifications may also be performed within the third binding domain of the antibody construct defined in the present invention. Insertional variants of the antibody construct defined in the present invention include the fusion of an enzyme or a polypeptide to the N- or C-terminus of the antibody construct.

[0083] The most interesting sites for substitution mutagenesis include (but are not limited to) the CDRs of heavy and / or light chains, especially the hypervariable regions, but also contemplate changing the FRs of heavy and / or light chains.These substitutions are preferably conservative substitutions as described herein.Preferably, depending on the length of CDR or FR, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids can be substituted in CDR, 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 can be substituted in framework regions (FR).For example, if a CDR sequence contains 6 amino acids, it is envisaged that 1, 2 or 3 of these amino acids are substituted. Similarly, if a CDR sequence comprises 15 amino acids, it is envisaged that 1, 2, 3, 4, 5 or 6 of these amino acids may be substituted.

[0084] A useful method for identifying specific residues or regions of an antibody construct that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" and is described by Cunningham and Wells in Science, 244: 1081-1085 (1989). Herein, a residue or target residues within the antibody construct are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and substituted with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the epitope.

[0085] The amino acid positions that show functional sensitivity to these substitutions are then more precisely defined by introducing another or other variant at or to the substitution site. Thus, the site or region for introducing the amino acid sequence variant is predetermined, but the nature of the mutation itself does not have to be predetermined. For example, to analyze or optimize the action of the mutation at a given site, alanine scanning or random mutagenesis can be performed on the target codon or region, and the expressed antibody construct variants are screened for the optimal combination of desired activity. Techniques for making substitution mutations at a predetermined site in DNA with a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Screening of mutants is performed using an assay for antigen binding activity, such as binding to an antigen present on the surface of an innate immune effector cell, an antigen present on the surface of a target cell, and / or another antigen present on the surface of a target cell.

[0086] In general, when amino acids are replaced in one or more or all of the CDRs of the heavy and / or light chain, the resulting "replaced" sequence is preferably at least 60% or at least 65% identical to the "original" CDR sequence, more preferably at least 70% or at least 75%, even more preferably at least 80% or at least 85%, and particularly preferably at least 90% or at least 95% identical. This means that the degree to which the original sequence is identical to the "replaced" sequence depends on the length of the CDR. For example, a CDR with 5 amino acids is preferably at least 80% identical to its replaced amino acid sequence in order for at least one amino acid to be replaced. Thus, the CDRs of an antibody construct may have different degrees of identity to their replaced sequences, for example, CDRL1 may have at least 80% identity and CDRL3 may have at least 90% identity.

[0087] Preferred substitutions (or replacements) are conservative substitutions. However, any substitution (including non-conservative substitutions or one or more of the "exemplary substitutions" listed in Table 3 below) is envisaged, as long as the antibody construct retains the ability to bind, for example, via the first binding domain (A) to an antigen present on the surface of an innate immune effector cell, via the second binding domain (B) to an antigen present on the surface of a target cell, and / or via an optional third binding domain (C) to another antigen present on the surface of a target cell, and / or its CDR has identity to the sequence replaced at that time (at least 60% or at least 65%, more preferably at least 70% or at least 75%, even more preferably at least 80% or at least 85%, and particularly preferably at least 90% or at least 95% identical to the "original" CDR sequence).

[0088] Conservative substitutions are shown under the heading of "preferred substitutions" in Table 1. If such substitutions result in altered biological activity, then more extensive changes, referred to as "exemplary substitutions" in Table 1 or further described below for amino acid classes, can be introduced and the products screened for the desired characteristics.

[0089] (Table 1) Amino acid substitutions TIFF2024529381000001.tif102128

[0090] Substantial modification of the biological properties of the antibody constructs of the invention is achieved by selecting substitutions that differ significantly in their impact on (a) the structure of the polypeptide backbone in the substituted region, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Naturally occurring residues are divided into the following groups based on common side chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr, asn, gln; (3) acidic: asp, glu; (4) basic: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

[0091] Non-conservative substitutions involve exchanging a member of one of these classes for another. Any cysteine ​​residue not involved in maintaining the proper conformation of the antibody construct may be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bond(s) may be added to the antibody to improve stability (particularly where the antibody is an antibody fragment, such as an Fv fragment).

[0092] For amino acid sequences, sequence identity and / or similarity may be determined by inspection or using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395, preferably using the default settings. Preferably, the percent identity is calculated by FastDB using the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis", Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

[0093] An example of a useful algorithm is PILEUP. PILEUP uses progressive pairwise alignments to generate a multiple sequence alignment from a group of related sequences. PILEUP can also plot a tree showing the clustering relationships used to generate the alignment. PILEUP uses a simplified version of the progressive alignment method of Feng & Doolittle, 1987, J. Mol. Evol. 35:351-360; the method is similar to that described by Higgins and Sharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0094] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. USA 90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program, taken from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to default values. Adjustable parameters are set with the following values: overlap span=1, overlap ratio=0.125, word threshold (T)=11. The HSP S and HSP S2 parameters are dynamic values, determined by the program itself depending on the composition of the particular sequence and the composition of the particular database in which the sequence of interest is searched, however these values ​​can be adjusted to increase sensitivity.

[0095] Another useful algorithm is Gapped BLAST, reported by Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses the BLOSUM-62 substitution score; the threshold T parameter is set to 9; the two-hit method for triggering ungapped extensions imposes a cost of 10+k on the gap length k; Xu is set to 16, and Xg is set to 40 in the database search stage and 67 in the output stage of the algorithm. Gapped alignments are triggered by a score equivalent to approximately 22 bits.

[0096] Generally, the amino acid homology, similarity or identity between each variant CDR or VH / VL sequence is at least 60% with respect to the sequence shown herein, and more typically, the homology or identity is preferably increased to at least 65% or 70%, more preferably at least 75% or 80%, and even more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and approximately 100%. In a similar manner, the "nucleic acid sequence identity percentage (%)" for the nucleic acid sequence of the binding protein specified herein is defined as the percentage of nucleotide residues in the candidate sequence that are identical to the nucleotide residues in the coding sequence of the antibody construct. In one specific method, the BLASTN module of WU-BLAST-2 is used with default parameters, and the overlap span and overlap ratio are set to 1 and 0.125, respectively.

[0097] Generally, the nucleic acid sequence homology, similarity or identity between the nucleotide sequences encoding the individual variant CDRs or VH / VL sequences and the nucleotide sequences set forth herein will be at least 60%, more typically the homology or identity will be at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and preferably close to 100%. Thus, a "variant CDR" or "variant VH / VL region" is one that has a designated homology, similarity or identity to a parent CDR / VH / VL as defined in the present invention and has a biological function, including but not limited to, at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the specificity and / or activity of the parent CDR or VH / VL.

[0098] In one embodiment, the identity percentage of the antibody construct according to the present invention to human germline is 70% or more or 75% or more, more preferably 80% or more or 85% or more, even more preferably 90% or more, and most preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, or even 96% or more. Identity to human antibody germline gene products is considered an important feature to reduce the risk that the therapeutic protein will cause an immune response to the drug in the patient being treated. Hwang & Foote ("Immunogenicity of engineered antibodies"; Methods 36 (2005) 3-10) demonstrate that reducing the non-human portion of the drug-antibody construct reduces the risk of generating anti-drug antibodies in the patient being treated. A comparison of a comprehensive number of clinically evaluated antibody drugs and their respective immunogenicity data shows that humanization of the V region of an antibody tends to make the protein less immunogenic (5.1% of patients on average) compared to antibodies with unmodified non-human V regions (23.59% of patients on average). Therefore, for V region-based protein therapeutics in the form of antibody constructs, a high degree of identity to human sequences is desirable. For this purpose of measuring germline identity, the V region of the VL can be aligned with the amino acid sequences of human germline V and J segments (http: / / vbase.mrc-cpe.cam.ac.uk / ) using Vector NTI software, and the percentage of amino acid sequences can be calculated by dividing the identical amino acid residues by the total number of amino acid residues in the VL. The same can be done for the VH segment (http: / / vbase.mrc-cpe.cam.ac.uk / ), except that the VH CDR3 may be omitted due to its high diversity and lack of existing human germline VH CDR3s to align with. Recombinant techniques can then be used to increase sequence identity to human antibody germline genes.

[0099] The term "EGFR" refers to the epidermal growth factor receptor (EGFR in humans; ErbB-1; HER1), including all isoforms or variants that have been described with activation, mutations, and implicated in pathophysiological processes. The EGFR antigen-binding site recognizes an epitope in the extracellular domain of EGFR. In certain embodiments, the antigen-binding site specifically binds human EGFR and cynomolgus monkey EGFR. The epidermal growth factor receptor (EGFR) is a member of the HER family of receptor tyrosine kinases, which consists of four members: EGFR (ErbB1 / HER1), HER2 / neu (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Stimulation of the receptor by ligand binding (e.g., EGF, TGFa, HB-EGF, neuregulin, betacellulin, amphiregulin) activates the intrinsic receptor tyrosine kinase within the intracellular domain via tyrosine phosphorylation, promoting receptor homo- or heterodimerization with HER family members. These intracellular phospho-tyrosines serve as docking sites for various adaptor proteins or enzymes, including SHC, GRB2, PLCg, and PI(3)K / Akt, which simultaneously initiate many signaling cascades that affect cell proliferation, angiogenesis, apoptosis resistance, invasion, and metastasis.

[0100] The term "immune effector cell" as used herein may refer to any white blood cell or precursor that is involved in protecting the body from, for example, cancer, disease caused by infectious agents, foreign substances, or autoimmune reactions. For example, immune effector cells include B lymphocytes (B cells), T lymphocytes (T cells, including CD4+T cells and CD8+T cells), NK cells, NKT cells, monocytes, macrophages, dendritic cells, mast cells, granulocytes, such as neutrophils, basophils, and eosinophils, innate lymphoid cells (ILCs, including ILC-1, ILC-2, and ILC-3), γδT cells, or any combination thereof. The term "innate immune effector cell" refers to immune effector cells that belong to the innate immune system. For example, immune effector cells include natural, modified, or engineered NK cells, monocytes, macrophages, dendritic cells, mast cells, granulocytes, such as neutrophils, basophils, and eosinophils, innate lymphoid cells (ILCs, including ILC-1, ILC-2, and ILC-3), innate immune cells derived from induced pluripotent stem cells (iPSCs) (e.g., iPSC-NK cells, iPSC-macrophages), engineered immune effector cells (e.g., T cells) expressing immune regulatory receptors (e.g., CD16A, NKp46), or any combination thereof. Preferably, the term innate immune effector cells refers to NK cells and / or macrophages.

[0101] Natural killer (NK) cells are CD56+CD3- large granular lymphocytes that can kill virus-infected and transformed cells and constitute an essential cell subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676). Unlike cytotoxic CD8+ T lymphocytes, NK cells can generate cytotoxicity against tumor cells without the need for prior sensitization and can also eradicate MHC-I negative cells (Narni-Mancinelli E, et al. Int Immunol 2011 23:427-431). NK cells are safer effector cells because they avoid potentially lethal complications such as cytokine storm (Morgan RA, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011 365:725-733), and on-target and off-tumor effects.

[0102] Monocytes are produced by the bone marrow from hematopoietic stem cell precursors called monoblasts. Monocytes circulate in the bloodstream for approximately 1-3 days and then typically migrate into tissues throughout the body. Monocytes constitute 3-8% of the white blood cells in the blood. Within tissues, monocytes mature into various types of macrophages in various anatomical locations. Monocytes have two main functions in the immune system: (1) recruiting resident macrophages and dendritic cells under normal conditions, and (2) in response to inflammatory signals, monocytes can rapidly migrate (approximately 8-12 hours) to sites of infection in tissues and divide / differentiate into macrophages and dendritic cells to elicit an immune response. Monocytes are usually identified in stained smears based on their large bilobed nuclei.

[0103] Macrophages are potent effectors of the innate immune system and can perform at least three distinct antitumor functions: phagocytosis, cell-mediated cytotoxicity, and antigen presentation to orchestrate adaptive immune responses. Whereas T cells require antigen-dependent activation via the T cell receptor or chimeric immune receptors, macrophages can be activated in a variety of ways. Direct macrophage activation is antigen-independent and relies on mechanisms such as pathogen-associated molecular pattern recognition by Toll-like receptors (TLRs). Immune complex-mediated activation is antigen-dependent but requires the presence of antigen-specific antibodies and the absence of inhibitory CD47-SIRPa interactions.

[0104] T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), based on the presence of the T cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus gland (although some also mature in the tonsils). There are several subsets of T cells, each with distinct functions.

[0105] T helper cells (TH cells) assist other white blood cells in immunological processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, helper T cells divide rapidly and secrete small proteins called cytokines that regulate or assist active immune responses. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, TH9, or TFH, which secrete different cytokines to promote different types of immune responses.

[0106] Cytotoxic T cells (TC cells or CTLs) destroy virus-infected and tumor cells and are also implicated in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize targets by binding to antigens bound to MHC class I molecules, which are present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated into an anergic state, thereby preventing autoimmune diseases.

[0107] Memory T cells are a subset of antigen-specific T cells that persist for long periods after an infection has cleared. Upon re-exposure to the cognate antigen, memory T cells rapidly proliferate into large numbers of effector T cells, thus providing the immune system with a "memory" of past infections. Memory cells can be either CD4+ or CD8+. Typically, memory T cells express the cell surface protein CD45RO.

[0108] Regulatory T cells (Treg cells), previously known as suppressor T cells, are crucial for the maintenance of immune tolerance. Their main role is to shut down T cell-mediated immunity towards the end of the immune response and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus. Two main classes of CD4+ Treg cells have been described: natural Treg cells and adaptive Treg cells.

[0109] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) act as a bridge between the adaptive and innate immune systems. Unlike conventional T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d.

[0110] As used herein, the term "engineered immune effector cells" refers to immune cells that are genetically modified by methods or tools that allow gene editing. Examples of methods for immune cell engineering include, but are not limited to, viral transduction, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR / Cas9. Engineered immune cells can also include adaptive immune cells (e.g., T cells) that acquire innate immune functions, for example, by expressing immune regulatory receptors (e.g., CD16A, NKp46) that are normally expressed by innate immune cells (Source: (CD16A CAR T cells D'Aloia et al Chimeric Antigen Receptor T Cells, 18(2):278-290).

[0111] As used herein, the term "half-life prolonging domain" refers to a portion that increases the serum half-life of an antibody construct. The half-life prolonging domain may comprise a portion of an antibody, such as the Fc portion of an immunoglobulin, the hinge domain, the CH2 domain, the CH3 domain, and / or the CH4 domain. Although not optimal, the half-life prolonging domain may also comprise elements that are not contained in an antibody, such as an albumin-binding peptide, an albumin-binding protein, or transferrin, to name just a few. It is preferred that the half-life prolonging domain does not have an immunomodulatory function. When the half-life prolonging domain comprises the hinge domain, the CH2 domain, and / or the CH3 domain, it is preferred that the half-life prolonging domain does not essentially bind to Fc receptors. This can be achieved, for example, by "silencing" the Fcγ receptor binding domain.

[0112] As used herein, "silencing" of an Fc receptor binding domain or an Fcγ receptor binding domain refers to any modification that reduces the binding of the CH2 domain to an Fc receptor, particularly an Fcγ receptor. Such modifications can be made by substitution and / or deletion of one or more amino acids involved in Fc(γ) receptor binding. Such mutations are well known in the art and are described, for example, by Saunders (2019, Front. Immunol. 10:1296). For example, the mutation can be located at any one of positions 233, 234, 235, 236, 237, 239, 263, 265, 267, 273, 297, 329, and 331. Examples of such mutations are: deletion of Glu233→Pro, Glu233, Leu234→Phe, Leu234→Ala, Leu234→Gly, Leu234→Glu, Leu234→Val, deletion of Leu234, Leu235→Glu, Leu235→Ala, Leu235→Arg, Leu235→Phe, deletion of Leu235, deletion of Gly236, Gly237→Ala, Ser239→Lys, Val263→Leu, Asp265→Ala, Ser267→Lys, Val273→Glu, Asn297→Gly, Asn297→Ala, Lys332→Ala, Pro329→Gly, Pro331→Ser, and combinations thereof. Preferably, such modifications include one or both of Leu234→Ala and Leu235→Ala (also known as "LALA" mutations). Preferably, such modifications further include Pro329→Gly mutations, also known as "LALA-PG" mutations (Leu234→Ala, Leu235→Ala, and Pro329→Gly). Preferably, such modifications include one, two, or three of the mutations Leu234→Phe, Leu235→Glu, and Asp265→Ala, more preferably all three of these mutations. The combination of Leu234→Phe, Leu235→Glu, and Asp265→Ala is a preferred modification in the present invention, also known as "FEA" mutations. Preferably, such modifications further include Asn297→Gly.Such preferred modifications include the mutations Leu234→Phe, Leu235→Glu, Asp265→Ala, and Asn297→Gly.

[0113] The term "fratricide" in the present invention refers to the reduction of effector cells by cytotoxic killing, and thus the reduction of available effector cell populations / compartments. Fratricide can be caused by the cross-linking of two immune cells. As an illustrative example, cross-linking of NK cells can cause the killing of either or both of the NK cells. In the case where an antibody construct recruits two different types of effector cells, such as NK cells and macrophages or NK cells and T cells, the elimination of one type of effector cell by the other type of effector cell is also understood as fratricide. Fratricide can be measured, for example, by an assay essentially as described in Example 7.

[0114] Detailed Description The present invention is based on the finding that tumor mass is highly heterogeneous with respect to the expression level (high, medium, low) of a given tumor antigen. In the course of antitumor treatment, tumors may downregulate targeted tumor antigens during treatment as an evasive strategy. Tumor cells with low targeted tumor antigens may be the cause of relapse after the first CR. This is considered to be a common challenge for all strategies that target tumor antigens.

[0115] When applying bispecific or multispecific immune effector cell engagers, downregulation of targeted tumor antigens reduces the number of engaged signaling-competent tumor antigen / immune cell antigen complexes below the threshold required for efficient killing by innate immune receptor cells such as NK cells. However, the inventors of the present application have surprisingly found that by increasing the number of immune effector cell binding domains for immune modulatory antigens per individual tumor antigen engaged, better killing of tumor cells with fewer targeted tumor antigens may be achieved.

[0116] Thus, the present invention provides a bispecific or multispecific immune effector cell engager format with tetravalency for immune modulatory antigens of innate immune effector cells to increase the activation threshold and affinity / cell surface retention of innate immune effector cells. By increasing the number of binding domains for innate immune effector cell antigens, one tumor antigen can induce activation via up to four innate immune effector cell antigens, such as CD16A.

[0117] The use of engagers comprising at least four binding domains for immune-modulating antigens of innate immune cells may result in long-lasting maximum efficacy of innate immune cells such as NK cells and / or macrophages. Furthermore, the killing kinetics of such engagers may be faster when compared to conventional engagers having less than four (e.g., one or two) binding domains for immune-modulating antigens of innate immune cells. Bispecific or multispecific engagers having at least four binding domains for immune-modulating antigens of innate immune cells may also show greater responses and thus result in complete eradication of target positive tumor cells. This is particularly interesting for combination with a dual targeting approach in which the engager is specific for at least two antigens present on the surface of the target cell. Thus, the bispecific and multispecific engagers of the present invention may be a preferred format for targeting tumor antigens (including peptide / MHC-I complexes) that are very low in abundance.

[0118] Therefore, the antibody construct of the present invention can effectively target tumor cells with downregulated expression of target antigen or tumor cells that naturally express low levels of target antigen.This makes the antibody construct particularly useful for treating natural tumors that express tumor antigens heterogeneously, since the antibody construct can target the entire tumor.In addition, the antibody construct can also prevent tumor recurrence by killing low-expressing cells, such as tumor stem cells.

[0119] Therefore, the present invention contemplates an antibody construct comprising: (i) at least four first binding domains (A), said first binding domains (A) being capable of specifically binding to a first target (A') that is an antigen present on the surface of an innate immune effector cell; and (ii) a second binding domain (B), said second binding domain (B) being capable of specifically binding to a second target (B') that is an antigen present on the surface of a target cell. The antigen present on the surface of an innate immune cell is preferably an immunoregulatory antigen. The innate immune cell is preferably a natural killer cell or a macrophage.

[0120] With respect to the first binding domain (A), the term "at least 4" includes 5, 6, 7, 8, or even higher numbers. However, 4, 5, and 6 are preferred, with 4 being most preferred. Similarly, with respect to the second binding domain (B), the antibody construct of the present disclosure may include multiple second binding domains, for example, 2, 3, 4, or even more second binding domains. However, the antibody construct of the present invention preferably includes one or two second binding domains (B), with two being most preferred. It is understood that the second target (B') is present on the surface of a target cell that is not the same cell as the innate immune effector cell expressing the first target (A').

[0121] The antibody construct of the present invention may be capable of binding to target cells and (natural) immune effector cells simultaneously. Binding to (natural) immune effector cells may be via at least one of at least four first binding domains (A). Binding to target cells may be via at least one second binding domain (B) and / or at least one optional third binding domain (C). The antibody construct of the present disclosure may be bispecific. The antibody construct of the present disclosure may also be trispecific. However, bispecific antibody constructs are preferred.

[0122] Binding of one or more of the at least four first binding domains (A) to a first target (A') can enhance the functionality of immune effector cells by inducing activating signals or blocking inhibitory signals, said first target (A') being referred to herein as an "immunoregulatory antigen", particularly on NK cells and / or macrophages. Such "immunomodulatory antigens" include, but are not limited to, CD16A, CD56, NKG2A, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 (CD226), SLAMF7 (CD319), CD244 (2B4), OX40, CD47, SIRPα, CD89, CD96, CD137, CD160, TIGIT, Nectin-4, PD-1, PD-L1, LAG-3, CTLA-4, TIM-3, KIR2DL1-5, KIR3DL1-3, KIR2DS1-5, KIR3DS1, and CD3. Furthermore, the first target (A') is an immunomodulatory antigen that can be grouped into various categories according to the mechanism of action: (1) antigens that induce the activation of immune effector cells. (2) Inhibitory antigens on effector cells, including but not limited to CD16A, CD56, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 (CD226), SLAMF7 (CD319), CD244 (2B4), OX40, CD137, CD89, CD160, and killer cell immunoglobulin-like receptors (KIR2DS1-5 and KIR3DS1), herein referred to as "immunostimulatory antigens." (3) Inhibitory antigens on effector cells, including but not limited to NKG2A, TIGIT, PD-1, PD-L1, CD47, SIRPα, LAG-3, CTLA-4, CD96, TIM-3, CD137, KIR2DL1-5, and KIR3DL1-3, herein referred to as "immunosuppressive antigens," may be disrupted to counteract inhibition and / or functional exhaustion. The first binding partner (A) to the "immunostimulatory antigen" is preferably an agonist. The first binding partner (A) to the "immunosuppressive antigen" is preferably an antagonist.

[0123] Antigens that induce effector cell activation can be further classified into groups based on the signaling cascade: (1) CD3ζ-dependent / CD16A-associated signaling, e.g., CD16A, NKp46, NKp30, and (2) CD3ζ-independent signaling, e.g., but not limited to, NKG2D, NKp44, NKp80, DNAM-1 (CD226), SLAMF7 (CD319), CD244 (2B4), and killer cell immunoglobulin-like receptor (e.g., KIR2DS1).

[0124] Depending on the selection of antigens for the at least four first binding domains (A), various cell types may be targeted / activated, including but not limited to CD16A, CD56, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 (CD226), SLAMF7 (CD319), CD244 (2B4), OX40, CD137, CD160, KIR2DS1-5, NKG2A, TIGIT, PD NK cells may be targeted / activated with an antigen selected from the group including CD16A, CD89, SLAMF7, SIRPα, and / or CD47. Furthermore, depending on the antigen, various subpopulations (e.g., CD56, CD89, CD16A, CD89, SLAMF7, SIRPα, and / or CD47) may be targeted / activated. 弱陽性 CD16 強陽性 NK cells, CD56 強陽性 CD16 陰性 NK cells, peripheral or tissue-resident NK cells, M1 or M2 macrophages, tumor-associated macrophages) can also be addressed.

[0125] In some embodiments, the at least four first binding domains (A) are specific for a (CD) antigen, preferably selected from the group consisting of CD16A, CD56, NKG2A, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 (CD226), SLAMF7 (CD319), CD244 (2B4), OX40, CD47, SIRPα, CD89, CD96, CD137, CD160, TIGIT, Nectin-4, PD-1, PD-L1, LAG-3, CTLA-4, TIM-3, KIR2DL1-5, KIR3DL1-3, KIR2DS1-5, KIR3DS1, and CD3.

[0126] According to the present disclosure, the first target (A') is preferably selected from the group consisting of CD16A, CD56, NKG2A, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 (CD226), SLAMF7 (CD319), CD244 (2B4), OX40, CD47, SIRPα, CD89, CD96, CD137, CD160, TIGIT, Nectin-4, PD-1, PD-L1, LAG-3, CTLA-4, TIM-3, KIR2DL1-5, KIR3DL1-3, KIR2DS1-5, KIR3DS1, and CD3, with CD16A and NKp46 being preferred and CD16A being most preferred. In this context, the preferred first target (A') is a target present on NK cells and / or macrophages. At least four first binding domains (A) are capable of binding to the same epitope of a first target (A').

[0127] The antibody construct of the present disclosure may comprise a third binding domain (C) capable of specifically binding to a third target (C'). The third target (C') is an antigen present on the surface of a target cell other than the second target (B'). However, the antibody construct of the present disclosure may comprise multiple third binding domains (C), for example, two, three, four or even more third binding domains (C). However, it is preferred that the antibody construct of the present disclosure comprises one or two third binding domains (B). Thus, a preferred antibody construct of the present disclosure may comprise one second binding domain (B) and one tertiary binding domain (C), two second binding domains (B) and one tertiary binding domain (C), one second binding domain (B) and two tertiary binding domains (C), or two second binding domains (B) and two tertiary binding domains (C), with one second binding domain (B) and one tertiary binding domain (C) being most preferred.

[0128] The first binding domain (A) is preferably derived from an antibody. The first binding domain (A) preferably comprises the VH domain and the VL domain of an antibody. The exemplary structure of the first binding domain (A) comprises the pair of VL and VH that can be comprised in Fv, scFv, Fab, or diabody (Db), scDb, bi-scFv, or double Fab, where the pair of VL and VH that is comprised in scDb or bi-scFv is preferred.

[0129] The second binding domain (B) is also preferably derived from an antibody. The second binding domain (B) preferably comprises the VH and VL domains of an antibody. Exemplary structures of the second binding domain (B) include the VL and VH pairs that can be comprised in Fv, scFv, Fab, or diabody (Db), scDb, or double Fab, where scFv or Fab are preferred.

[0130] The third binding domain (C) is also preferably derived from an antibody. The third binding domain (C) preferably comprises the VH and VL domains of an antibody. Exemplary structures of the third binding domain (C) include the VL and VH pairs that can be comprised in Fv, scFv, Fab, or diabody (Db), scDb, or double Fab, where scFv or Fab are preferred.

[0131] The antibody construct of the present disclosure may comprise a fourth domain (D), which comprises a half-life prolonging domain as described herein. The half-life prolonging domain may comprise a CH2 domain, the Fcγ receptor binding domain of the CH2 domain being silenced. The half-life prolonging domain may comprise two such CH2 domains. Whenever the half-life prolonging domain comprises a CH2 domain, the Fcγ receptor binding domain of the CH2 domain is silenced. The half-life prolonging domain may comprise one CH3 domain. The half-life prolonging domain may comprise two CH3 domains. The half-life prolonging domain may comprise one hinge domain. The half-life prolonging domain may comprise two hinge domains. The half-life prolonging domain may comprise one CH2 domain and one CH3 domain. In such a case, the CH2 domain and the CH3 domain are preferably fused to each other, preferably in the order (amino to carboxyl) of CH2 domain-CH3 domain. Non-limiting examples of such fusions are shown in SEQ ID NOs: 39-58. The half-life prolonging domain may comprise one hinge domain and one CH2 domain. In such cases, the hinge domain and the CH2 domain are preferably fused to each other, preferably in the order (amino to carboxyl) as hinge domain-CH2 domain. The half-life prolonging domain may comprise one hinge domain, one CH2 domain, and one CH3 domain. In such cases, the hinge domain, the CH2 domain, and the CH3 domain are preferably fused to each other, preferably in the order (amino to carboxyl) as hinge domain-CH2 domain-CH3 domain. The half-life prolonging domain may comprise two hinge domain-CH2 domain elements, two CH2 domain-CH3 domain elements, or two hinge domain-CH2 domain-CH3 domain elements. In such cases, the two fusions may be located on two different polypeptide chains. Alternatively, the fusions may be located on the same polypeptide chain.An illustrative example where two hinge domain-CH2 domain-CH3 domain elements are arranged on the same polypeptide chain is the "single chain Fc" or "scFc" format. In this case, both hinge-CH2-CH3 subunits are fused together via a linker that allows assembly of the Fc domain. A preferred linker for this purpose is a glycine serine linker, which preferably contains about 20 to about 40 amino acids. A preferred glycine serine linker may have one or more repeats of GGS, GGGS (SEQ ID NO: 1), or GGGGS (SEQ ID NO: 6). Such linkers preferably contain 4 to 8 repeats of GGGGS (e.g., 4, 5, 6, 7, or 8 repeats). Such linkers are preferably (GGGGS)6 (SEQ ID NO 9). Illustrative examples of such scFc domains are shown in SEQ ID NOs: 2 to 5. Further scFc constant domains are known in the art and are described, inter alia, in WO 2017 / 134140.

[0132] Typically, with respect to the second target (B') and / or optionally the third target (C'), the antibody construct of the present disclosure can be monovalent, bivalent, trivalent, or even higher valency to any one of the second target (B') and / or optionally the third target (C'). With respect to the first target (A'), the antibody construct of the present disclosure can be tetravalent, or even higher valency. Thus, the antibody construct of the present disclosure may comprise 4, 5, 6, or even more of any one of the first binding domains (A). The antibody construct of the present disclosure may comprise 1, 2, 3, or even more of the second binding domain (B). Optionally, the antibody construct of the present disclosure may comprise 1, 2, 3, or even more of the third binding domain (C). For the antibody construct of the present disclosure, it is preferred that the antibody construct is at least tetravalent for the first target (A') and at least monovalent or at least bivalent for the second target (B'). For the antibody construct of the present disclosure, it is even more preferred that the antibody construct is at least tetravalent for the first target (A'), at least monovalent for the second target (B'), and at least monovalent for the third target (C'). More preferably, the antibody construct of the present disclosure is tetravalent for the first target (A') and monovalent for the second target (B'). More preferably, the antibody construct of the present disclosure is tetravalent for the first target (A'), monovalent for the second target (B'), and monovalent for the third target (C'). Even more preferably, the antibody construct of the present disclosure is tetravalent for the first target (A') and bivalent for the second target (B'). For the antibody construct of the present disclosure, it is preferred that the antibody construct comprises at least two or four first binding domains (A) and at least one or at least two second binding domains (B). For the antibody construct of the present disclosure, it is even more preferred that the antibody construct comprises at least four first binding domains (A), at least one second binding domain (B), and at least one third binding domain (C). More preferably, the antibody construct of the present disclosure comprises four first binding domains (A) and one second binding domain (B).More preferably, the antibody construct of the present invention comprises four first binding domains (A), one second binding domain (B), and one third binding domain (C). Even more preferably, the antibody construct of the present disclosure comprises four first binding domains (A) and two second binding domains (B).

[0133] It is also envisaged that in order to reduce immune effector cell fratricide, the four first binding domains (A) should be positioned relative to each other in such a way that simultaneous binding to two immune effector cells is reduced or preferably prevented. This can be achieved, for example, by shortening the distance between the first binding domains (A). For example, the first first binding domain (A1) and the second first binding domain (A2) can be fused to each other to form a pair (A1A2), also referred to herein as a dimer. Such a dimer (A1A2) can be in the form of a bi-scFv, double fab, Db or scDb. However, such a dimer (A1A2) is preferably in the form of a bi-scFv or scDb. The spatial arrangement of the variable domains of the bi-scFv can be in any suitable order, with the order VH-VL-VH-VL being preferred. The spatial arrangement of the variable domains of the scDb can be in any suitable order, with the order VL-VH-VL-VH being preferred. The most preferred form of the dimer (A1A2) consisting of two first binding domains is the scDb format. In such a scDb, the domains of the polypeptide on the polypeptide chain are preferably arranged (N to C) in the order VL-VH-VL-VH.

[0134] Similarly, the third first binding domain (A3) and the fourth binding domain (A4) can be fused together to form a pair (A3A4) also called a dimer. Again, such a dimer (A3A4) can be in the form of a bi-scFv, double Fab, Db, or scDb. However, such a dimer (A3A4) is preferably in the form of a bi-scFv or scDb. The spatial arrangement of the variable domains of a bi-scFv can be in any suitable order, with the order VH-VL-VH-VL being preferred. The spatial arrangement of the variable domains of a scDb can be in any suitable order, with the order VL-VH-VL-VH being preferred. The most preferred form of the dimer (A3A4) consisting of two first binding domains is the scDb format. In such a scDb, the domains of the polypeptide on the polypeptide chain are preferably arranged (from N to C) in the order VL-VH-VL-VH.

[0135] At least four first binding domains (A) are preferably fused to the fourth domain. The first binding domains can be arranged as monomers, but it is preferred that the first binding domains are in the form of at least two dimers ((A1A2) and (A3A4)). The preferred fourth binding domain comprises two hinge domain-CH2 domain-CH3 domain elements, which can be in the form of an scFc or preferably in the form of an Fc. Usually, the four first binding domains (A) can be fused to any N-terminus or C-terminus of the scFc or preferably of the Fc. In a preferred arrangement, the first binding domain and the second first binding domain (A1A2) that are fused to each other are fused to the C-terminus of the CH3 domain of the fourth domain (D). In a preferred arrangement, the first binding domain and the second first binding domain (A1A2) that are fused to each other are fused to the N-terminus of the hinge of the fourth domain (D). In a preferred configuration, the third binding domain and the fourth first binding domain (A3A4) that are fused to each other are fused to the C-terminus of the CH3 domain of the fourth domain (D). In a preferred configuration, the third binding domain and the fourth first binding domain (A3A4) that are fused to each other are fused to the N-terminus of the hinge of the fourth domain (D).

[0136] In the antibody construct of the present disclosure, the first and second first binding domains (A1A2) fused to each other may be fused to the C-terminus of the CH3 domain of the fourth domain (D), while the third and fourth first binding domains (A3A4) fused to each other are fused to the N-terminus of the hinge of the fourth domain (D). However, to make the distance between the first binding domains shorter, it is preferred that both dimers of the first binding domains, i.e. (A1A2) and (A3A4), are fused to either the two N-terminus of the Fc of the fourth domain (D), or more preferably, the two C-terminus of the Fc of the fourth domain (D). Thus, in a preferred antibody construct of the present disclosure, the first and second first binding domains (A1A2) fused to each other are fused to the N-terminus of the first hinge domain of the fourth domain (D), while the third and fourth first binding domains (A3A4) fused to each other are fused to the N-terminus of the second hinge domain of the fourth domain (D). In an even more preferred antibody construct of the present disclosure, the first and second first binding domains (A1A2) fused to each other are fused to the C-terminus of the first CH3 domain of the fourth domain (D), while the third and fourth first binding domains (A3A4) fused to each other are fused to the C-terminus of the second CH3 domain of the fourth domain (D).

[0137] In the antibody construct of the present disclosure, the second binding domain (B) can be fused to the fourth domain (D). Usually, the second binding domain (B) can be fused at any position suitable for such fusion, in particular at any N-terminus or C-terminus of the fourth domain (D). Thus, the second binding domain (B) can be fused to the N-terminus of the hinge domain of the fourth domain (D). The second binding domain can also be fused to the C-terminus of the CH3 domain of the fourth domain (D).

[0138] When the antibody construct comprises at least two second binding domains (B), the at least two second binding domains can be fused individually at any position suitable for such fusion, in particular at any N-terminus or C-terminus of the fourth domain (D). For example, one second binding domain (B) can be fused to the N-terminus of the hinge domain of the fourth domain (D), while another second binding domain (B) is fused to the C-terminus of the CH3 domain of the fourth domain (D). However, the arrangement in which the two second binding domains are both fused to the N-terminus of the fourth domain (D) or both fused to the C-terminus of the fourth domain (D) is preferred. Thus, it is preferred that the second binding domain (B) is fused to the N-terminus of the hinge of the fourth domain (D), while another second binding domain (B) is fused to the N-terminus of another hinge of the fourth domain (D). It is also preferred that a second binding domain (B) is fused to the C-terminus of the CH3 of the fourth domain (D) while another second binding domain (B) is fused to the C-terminus of another CH3 of the fourth domain (D).

[0139] In the antibody construct comprising the third binding domain (C), the third binding domain (C) can be fused to the fourth domain (D). Usually, the third binding domain (C) can be fused at any position suitable for such fusion, in particular at any N-terminus or C-terminus of the fourth domain (D). Thus, the third binding domain (C) can be fused to the N-terminus of the hinge domain of the fourth domain (D). The third binding domain can also be fused to the C-terminus of the CH3 domain of the fourth domain (D).

[0140] In the antibody construct of the present disclosure, the second binding domain (B) can be fused to the N-terminus of the hinge domain of the fourth domain (D), while the third binding domain (C) is fused to the C-terminus of the CH3 domain of the fourth domain (D), or vice versa. However, the arrangement in which the second binding domain (B) and the third binding domain (C) are both fused to the N-terminus of the fourth domain (D) or both fused to the C-terminus of the fourth domain (D) is preferred. Thus, it is preferred that the second binding domain (B) is fused to the N-terminus of the hinge of the fourth domain (D), while the third binding domain (C) is fused to the N-terminus of another hinge of the fourth domain (D). It is also preferred that the second binding domain (B) is fused to the C-terminus of the CH3 of the fourth domain (D) while the third binding domain (C) is fused to the C-terminus of another CH3 of the fourth domain (D).

[0141] In a preferred antibody construct, a first dimer (A1A2) consisting of two first binding domains and a second dimer (A3A4) consisting of two first binding domains are fused to the two C-termini of the Fc region. Such a fusion format is exemplarily shown in Figure 1A-C. In the first dimer (A1A2), the two first binding domains (A1 and A2) are preferably fused together in the form of a diabody or a single chain diabody, preferably via the VL domain of the first first binding domain (A1). Similarly, in the second dimer (A3A4), the two first binding domains (A3 and A4) are preferably fused together in the form of a diabody or a single chain diabody, preferably via the VL domain of the third first binding domain (A3). The first dimer (A1A2) and / or the second dimer (A3A4) may be fused to the constant domain of an antibody via a linker. Such linkers are preferably short linkers, preferably having a length of about 10 nm or less, preferably about 9 nm or less, preferably about 8 nm or less, preferably about 7 nm or less, preferably about 6 nm or less, preferably about 5 nm or less, preferably about 4 nm or less, or preferably even shorter. The length of the linker is preferably determined as described by Rossmalen et al Biochemistry 2017, 56, 6565-6574, which also describes suitable linkers well known to those skilled in the art. An example of a suitable linker is a glycine-serine linker or a serine linker, preferably comprising about 75 amino acids or less, preferably about 50 amino acids or less. In an illustrative example, a suitable linker comprises one or more GGGGS sequences (SEQ ID NO:6), such as (GGGGS)2 (SEQ ID NO:7), (GGGGS)4 (SEQ ID NO:8), or preferably (GGGGS)6 (SEQ ID NO:9). Other illustrative examples of linkers are shown in SEQ ID NOs:2-5.The first dimer (A1A2) and / or the second dimer (A3A4) are preferably scDb fragments fused to the two C-terminus of the Fc domain, preferably via the VL domain of the scDb. Thus, the arrangement of the polypeptide chains (from N to C) is preferably ...-CH2-CH3-VL-VH-VL-VH, optionally with a linker between the Fc and the scDb. The one or two second binding domains (B) can be located at any suitable position of the antibody construct. Similarly, the third binding domain (C) can also be located at any suitable position of the antibody construct. If the antibody construct comprises an Fc region, the second binding domain (B) and / or the third binding domain (C) can be located at the N-terminus of the Fc region, either directly or by being linked via at least one part of the hinge domain. Other linkers disclosed herein can also be used to link the second binding domain and / or the third binding domain to the Fc domain. However, for this purpose, a hinge domain is preferred. The second binding domain (B) can be any suitable structure disclosed herein, including Fab and scFv, but is preferably an scFv structure. Similarly, the third binding domain (C) can be any suitable structure disclosed herein, including Fab and scFv, but is preferably an scFv structure. In the case of scFv, the scFv is preferably fused to the Fc domain via the VL region contained in the scFv.

[0142] A preferred antibody construct of the present invention is preferably in the format essentially as shown in Figure 1A. Such an antibody construct comprises an immunoglobulin having two scDb fragments fused to the C-terminus of the heavy chain, optionally via a linker, preferably a glycine-serine linker or a serine linker, preferably a glycine-serine linker, preferably comprising about 75 amino acids or less, preferably about 50 amino acids or less. In an illustrative example, a suitable linker comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) GGGS sequences (SEQ ID NO: 6), such as (GGGGS)2 (SEQ ID NO: 7), (GGGGS)4 (SEQ ID NO: 8), or preferably (GGGGS)6 (SEQ ID NO: 9). Other illustrative examples of linkers are shown in SEQ ID NOs: 2-5. One of these two scDbs contains two first binding domains (A1 and A2), and the other scDb also contains two first binding domains (A3 and A4). One second binding domain (B) is fused to one N-terminus of one Fc region. Such an antibody construct consists of two polypeptide chains, i.e., in the configuration VH(B)-VL(B)-hinge-CH2-CH3-VL(A2)-VH(A1)-VL(A1)-VH(A2) (or less optimally VH(B)-VL(B)-hinge-CH2-CH3-VH(A2)-VL(A1)-VH(A1)-VL(A2), VL(B)-VH(B)-hinge-CH2-CH3-VL(A2)-VH(A1)-VL(A1)-VH ... H(A2), or VL(B)-VH(B)-hinge-CH2-CH3-VH(A2)-VL(A1)-VH(A1)-VL(A2)), and another polypeptide chain of the arrangement hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4) (or less optimally hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4)). Illustrative examples of such antibody constructs are shown in SEQ ID NOs: 152-153 and SEQ ID NOs: 158-159.

[0143] A preferred antibody construct of the present invention is preferably in the format essentially as shown in FIG. 1B. Such an antibody construct comprises an immunoglobulin having two scDb fragments fused to the C-terminus of the heavy chain, optionally via a linker, preferably a glycine-serine linker or a serine linker, preferably a glycine-serine linker, preferably comprising about 75 amino acids or less, preferably about 50 amino acids or less. In an illustrative example, a suitable linker comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) GGGS sequences (SEQ ID NO: 6), such as (GGGGS)2 (SEQ ID NO: 7), (GGGGS)4 (SEQ ID NO: 8), or preferably (GGGGS)6 (SEQ ID NO: 9). Other illustrative examples of linkers are shown in SEQ ID NOs: 2-5. One of these two scDbs contains two first binding domains (A1 and A2), the other scDb also contains two first binding domains (A3 and A4), and two second binding domains (B) are fused to the N-terminus of the Fc region.Such antibody constructs may comprise one or more of two polypeptide chains, i.e., one or more of the following configurations: VH(B)-VL(B)-hinge-CH2-CH3-VL(A2)-VH(A1)-VL(A1)-VH(A2) (or less optimally VH(B)-VL(B)-hinge-CH2-CH3-VH(A2)-VL(A1)-VH(A1)-VL(A2); VL(B)-VH(B)-hinge-CH2-CH3-VL(A2)-VH(A1)-VL(A1)-VH(A2); or VL(B)-VH(B)-hinge-CH2-CH3-VH(A2)-VL(A1)-VH(A1)-VL(A2)). and another polypeptide chain of the configuration VH(B)-VL(B)-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4) (or less optimally VH(B)-VL(B)-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4), VL(B)-VH(B)-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4), or VL(B)-VH(B)-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4)). Illustrative examples of such antibody constructs are shown in SEQ ID NOs: 148-149.

[0144] A preferred antibody construct of the invention is preferably in the format defined below. Such an antibody construct comprises an immunoglobulin with two scDb fragments fused to the C-terminus of the heavy chain, optionally via a linker, preferably a glycine-serine linker or a serine linker, preferably a glycine-serine linker, preferably comprising about 75 amino acids or less, preferably about 50 amino acids or less. In an illustrative example, a suitable linker comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) GGGS sequences (SEQ ID NO: 6), such as (GGGGS)2 (SEQ ID NO: 7), (GGGGS)4 (SEQ ID NO: 8), or preferably (GGGGS)6 (SEQ ID NO: 9). Other illustrative examples of linkers are shown in SEQ ID NOs: 2-5. One of these two scDbs contains two first binding domains (A1 and A2), the other scDb also contains two first binding domains (A3 and A4), and the second binding domain (B) and the third binding domain (C) are fused to the N-terminus of the Fc region.Such antibody constructs may comprise one or more of two polypeptide chains, i.e., one or more of the following configurations: VH(B)-VL(B)-hinge-CH2-CH3-VL(A2)-VH(A1)-VL(A1)-VH(A2) (or less optimally VH(B)-VL(B)-hinge-CH2-CH3-VH(A2)-VL(A1)-VH(A1)-VL(A2); VL(B)-VH(B)-hinge-CH2-CH3-VL(A2)-VH(A1)-VL(A1)-VH(A2); or VL(B)-VH(B)-hinge-CH2-CH3-VH(A2)-VL(A1)-VH(A1)-VL(A2)). and another polypeptide chain of the configuration VH(C)-VL(C)-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4) (or less optimally VH(C)-VL(C)-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4), VL(C)-VH(C)-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4), or VL(C)-VH(C)-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4)).

[0145] A preferred antibody construct of the present invention is preferably in the format essentially as shown in Figure 1C. Such an antibody construct comprises an immunoglobulin having two scDb fragments fused to the C-terminus of the heavy chain, optionally via a linker, preferably a glycine-serine linker or a serine linker, preferably a glycine-serine linker, preferably comprising about 75 amino acids or less, preferably about 50 amino acids or less. In an illustrative example, a suitable linker comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) GGGS sequences (SEQ ID NO: 6), such as (GGGGS)2 (SEQ ID NO: 7), (GGGGS)4 (SEQ ID NO: 8), or preferably (GGGGS)6 (SEQ ID NO: 9). Other illustrative examples of linkers are shown in SEQ ID NOs: 2-5. One of these two scDbs contains two first binding domains (A1 and A2), the other scDb also contains two first binding domains (A3 and A4), and the two second binding domains (B) are formed by immunoglobulin binding sites.Such antibody constructs comprise four polypeptide chains, i.e., two light chains of the configuration VL(B)-CL and VL(B)-CL, two light chains of the configuration VH(B)-CH1-hinge-CH2-CH3-VL(A2)-VH(A1)-VL(A1)-VH(A2) (or, less optimally or not, VH(B)-CH1-hinge-CH2-CH3-VH(A2)-VL(A1)-VH(A1)-VL(A2), VH(B)-CH1-hinge-CH2-CH3-VL(A2)-VH(A1)-VL(A1)-VH(A2), or VH(B)-CH1-hinge-CH2-CH3-VH(A2)-VL(A1)-VH(A2) ( The scDb may comprise one heavy chain fused to an scDb of the configuration VH(B)-CH1-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4) (or, less optimally, VH(B)-CH1-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4), VH(B)-CH1-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4), or VH(B)-CH1-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4)). Illustrative examples of such antibody constructs are shown in SEQ ID NOs: 162-163.

[0146] A preferred antibody construct of the invention is preferably in the format defined below. Such an antibody construct comprises an immunoglobulin with two scDb fragments fused to the C-terminus of the heavy chain, optionally via a linker, preferably a glycine-serine linker or a serine linker, preferably a glycine-serine linker, preferably comprising about 75 amino acids or less, preferably about 50 amino acids or less. In an illustrative example, a suitable linker comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) GGGS sequences (SEQ ID NO: 6), such as (GGGGS)2 (SEQ ID NO: 7), (GGGGS)4 (SEQ ID NO: 8), or preferably (GGGGS)6 (SEQ ID NO: 9). Other illustrative examples of linkers are shown in SEQ ID NOs: 2-5. One of these two scDbs contains two first binding domains (A1 and A2), the other scDb also contains two first binding domains (A3 and A4), one second binding domain (B) and one third binding domain (C) are formed by immunoglobulin binding sites.Such antibody constructs comprise four polypeptide chains, i.e., two light chains of the configuration VL(B)-CL and VL(C)-CL, two light chains of the configuration VH(B)-CH1-hinge-CH2-CH3-VL(A2)-VH(A1)-VL(A1)-VH(A2) (or, less optimally or not, VH(B)-CH1-hinge-CH2-CH3-VH(A2)-VL(A1)-VH(A1)-VL(A2), VH(B)-CH1-hinge-CH2-CH3-VL(A2)-VH(A1)-VL(A1)-VH(A2), or VH(B)-CH1-hinge-CH2-CH3-VH(A2)-VL(A1)-VH( The scDb may comprise one heavy chain fused to an scDb of the configuration VH(C)-CH1-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4) (or, less optimally, VH(C)-CH1-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4), VH(C)-CH1-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4), or VH(C)-CH1-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4)).

[0147] In the antibody construct of the present disclosure, the first dimer (A1A2) consisting of two first binding domains and the second dimer (A3A4) consisting of two first binding domains can also be fused to the N-terminus of a pair of two constant domains (e.g., dimers) of an antibody, such as a pair of two CH3 domains, a pair of two CH2 domains, or a pair of CH1 domain and CL domain. In a preferred embodiment, the first dimer (A1A2) is fused to the N-terminus of one CH2 domain, and the second dimer (A3A4) is fused to the N-terminus of another CH2 domain. In a preferred embodiment, the first dimer (A1A2) and the second dimer (A3A4) are fused to the two N-terminus of the Fc region. For the antibody construct of the present disclosure, it is preferred that the first dimer (A1A2) is fused to the N-terminus of the first hinge domain, and the second dimer (A3A4) is fused to the N-terminus of the second hinge domain. Such a fusion format is exemplarily shown in Figure 1D. The first dimer (A1A2) and / or the second dimer (A3A4) may be fused to the constant domain of the antibody via a linker (e.g., a glycine-serine linker or a serine linker, preferably a glycine-serine linker, preferably comprising about 75 amino acids or less, preferably about 50 amino acids or less) disclosed herein. In illustrative examples, suitable linkers include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) GGGS sequences (SEQ ID NO:6), such as (GGGGS)2 (SEQ ID NO:7), (GGGGS)4 (SEQ ID NO:8), or preferably (GGGGS)6 (SEQ ID NO:9). Other illustrative examples for linkers are shown in SEQ ID NOs:2-5 or are hinge domains, with hinge domains being preferred.

[0148] Typically, the hinge domain included in the antibody construct of the present disclosure may comprise a full-length hinge domain, for example the hinge domain shown in SEQ ID NO: 26. The hinge domain may also comprise a shortened and / or modified hinge domain. The shortened hinge domain may comprise the upper hinge domain, for example as shown in SEQ ID NO: 27, or the middle hinge domain, for example as shown in SEQ ID NO: 28, but not the entire hinge domain, the latter being preferred. The hinge domain preferred in the present invention exhibits modulated flexibility compared to antibody constructs with wild-type hinge domains as described in Dall'Acqua et al (J Immunol. 2006 Jul 15;177(2):1129-38) or WO 2009 / 006520. For some antibody constructs of the present disclosure, hinge domains exhibiting low flexibility are preferred, especially when the dimer (A1A2) and / or the second dimer (A3A4) are fused to the hinge domain. Furthermore, preferred hinge domains are characterized in that they consist of less than 25 amino acid residues. More preferably, the length of the hinge is between 10 and 20 amino acid residues. The hinge domain comprised in the antibody construct of the present disclosure may also comprise or consist of the IgG2 subtype hinge sequence ERKCCVECPPCP (SEQ ID NO: 23), the IgG3 subtype hinge sequence ELKTPLDTTHTCPRCP (SEQ ID NO: 30) or ELKTPLGDTTHTCPRCP (SEQ ID NO: 131), and / or the IgG4 subtype hinge sequence ESKYGPPCPSCP (SEQ ID NO: 132). Further hinge domains that can be used in the present invention are known to those skilled in the art and are described, for example, in WO 2017 / 134140.

[0149] When the first dimer (A1A2) is fused to the N-terminus of one CH2 domain and the second dimer (A3A4) is fused to the N-terminus of another CH2 domain, for example, when the first dimer (A1A2) and the second dimer (A3A4) are fused to the two N-terminus of Fc region, one second binding domain (B) can be fused to the C-terminus of Fc region, optionally via a linker as disclosed herein.The second binding domain is preferably in the form of scFv.The scFv is preferably fused to the Fc region via its VH domain. Such antibody constructs comprise two polypeptide chains, i.e., in the configuration VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3-VH(B)-VL(B) (or less optimally VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3-VH(B)-VL(B), VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3-VL(B)- VH(B), or VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3-VL(B)-VH(B)), and another polypeptide chain of the configuration VL(A4)-VH(A3)-VL(A3)-VH(A4)-hinge-CH2-CH3 (or less optimally VH(A4)-VL(A3)-VH(A3)-VL(A4)-hinge-CH2-CH3). Illustrative examples of such antibody constructs are shown in SEQ ID NOs: 150-151 and SEQ ID NOs: 156-157.

[0150] When the first dimer (A1A2) is fused to the N-terminus of one CH2 domain and the second dimer (A3A4) is fused to the N-terminus of another CH2 domain, for example, when the first dimer (A1A2) and the second dimer (A3A4) are fused to the two N-terminus of Fc region, two second binding domains (B) can be fused to the C-terminus of Fc region, optionally via a linker as disclosed herein. These second binding domains are preferably in the form of scFv. The scFv is preferably fused to the Fc region via its VH domain. Such antibody constructs may comprise one or more of two polypeptide chains, i.e., one or more of the following configurations: VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3-VH(B)-VL(B) (or less optimally VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3-VH(B)-VL(B); VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3-VL(B)-VH(B); or VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3-VL(B)-VH(B)). and another polypeptide chain of the configuration VL(A4)-VH(A3)-VL(A3)-VH(A4)-hinge-CH2-CH3-VH(B)-VL(B) (or less optimally VH(A4)-VL(A3)-VH(A3)-VL(A4)-hinge-CH2-CH3-VH(B)-VL(B), VL(A4)-VH(A3)-VL(A3)-VH(A4)-hinge-CH2-CH3-VL(B)-VH(B), or VH(A4)-VL(A3)-VH(A3)-VL(A4)-hinge-CH2-CH3-VL(B)-VH(B).

[0151] When the first dimer (A1A2) is fused to the N-terminus of one CH2 domain and the second dimer (A3A4) is fused to the N-terminus of another CH2 domain, for example, when the first dimer (A1A2) and the second dimer (A3A4) are fused to the two N-terminus of Fc region, the second binding domain (B) and the third binding domain (C) can be fused to the C-terminus of Fc region, optionally via a linker as disclosed herein.The second binding domain and / or the third binding domain are preferably in the form of scFv.The scFv is preferably fused to Fc region via its VH domain. Such antibody constructs may comprise one or more of two polypeptide chains, i.e., one or more of the following configurations: VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3-VH(B)-VL(B) (or less optimally VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3-VH(B)-VL(B); VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3-VL(B)-VH(B); or VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3-VL(B)-VH(B)). and another polypeptide chain of the configuration VL(A4)-VH(A3)-VL(A3)-VH(A4)-hinge-CH2-CH3-VH(C)-VL(C) (or less optimally VH(A4)-VL(A3)-VH(A3)-VL(A4)-hinge-CH2-CH3-VH(C)-VL(C), VL(A4)-VH(A3)-VL(A3)-VH(A4)-hinge-CH2-CH3-VL(C)-VH(C), or VH(A4)-VL(A3)-VH(A3)-VL(A4)-hinge-CH2-CH3-VL(C)-VH(C).

[0152] In the antibody construct of the present disclosure, the first dimer (A1A2) consisting of two first binding domains may be fused to the N-terminus of the first hinge-CH2-CH3 element of the fourth domain (D), while the second dimer (A3A4) consisting of two first binding domains may be fused to the C-terminus of the second hinge-CH2-CH3 element of the fourth domain (D). In such a case, the second binding domain (B) may be fused to the C-terminus of the first hinge-CH2-CH3 element of the fourth domain (D). The first and second dimers (A1A2) and (A3A4) are preferably in the form of scDb. The second binding domain (B) is preferably in the form of scFv. Such a fusion format is exemplarily shown in FIG. 1E. Such antibody constructs comprise two polypeptide chains, i.e., in the configuration VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3-VH(B)-VL(B) (or less optimally VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3-VH(B)-VL(B), VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3-VL(B)- VH(B), or VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3-VL(B)-VH(B)), and another polypeptide chain of the arrangement hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4) (or less optimally hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4)). Illustrative examples of such antibody constructs are shown in SEQ ID NOs: 154-155 and SEQ ID NOs: 160-161.

[0153] In the antibody construct of the present disclosure, the first dimer (A1A2) consisting of two first binding domains may be fused to the N-terminus of the first hinge-CH2-CH3 element of the fourth domain (D), while the second dimer (A3A4) consisting of two first binding domains may be fused to the C-terminus of the second hinge-CH2-CH3 element of the fourth domain (D). In such a case, the second binding domain (B) may be fused to the N-terminus of the second hinge-CH2-CH3 element of the fourth domain (D). The first dimer and the second dimer (A1A2) and (A3A4) are preferably in the form of a scDb. The second binding domain (B) is preferably in the form of a scFv. Such antibody constructs may comprise two polypeptide chains, i.e., one polypeptide chain of the configuration VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3 (or, less optimally, VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3), and one polypeptide chain of the configuration VH(B)-VL(B)-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4) ( or less optimally, the additional polypeptide chain may be VH(B)-VL(B)-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4); VL(B)-VH(B)-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4); or VL(B)-VH(B)-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4)).

[0154] In the antibody construct of the present disclosure, the first dimer (A1A2) consisting of two first binding domains may be fused to the N-terminus of the first hinge-CH2-CH3 element of the fourth domain (D), while the second dimer (A3A4) consisting of two first binding domains may be fused to the C-terminus of the second hinge-CH2-CH3 element of the fourth domain (D). In such a case, one second binding domain (B) may be fused to the C-terminus of the first hinge-CH2-CH3 element of the fourth domain (D), and another second binding domain (B) may be fused to the N-terminus of the second hinge-CH2-CH3 element of the fourth domain (D). The first and second dimers (A1A2) and (A3A4) are preferably in the form of scDbs. These second binding domains (B) are preferably in the form of scFvs. Such antibody constructs can be constructed as a single antibody consisting of two polypeptide chains, i.e. in the configuration VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3-VH(B)-VL(B) (or less optimally VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3-VH(B)-VL(B), VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3-VL(B)-VH(B), or VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3-VL(B)-VH(B). and another polypeptide chain of the configuration VH(B)-VL(B)-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4) (or less optimally VH(B)-VL(B)-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4), VL(B)-VH(B)-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4), or VL(B)-VH(B)-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4)).

[0155] In the antibody construct of the present disclosure, the first dimer (A1A2) consisting of two first binding domains may be fused to the N-terminus of the first hinge-CH2-CH3 element of the fourth domain (D), while the second dimer (A3A4) consisting of two first binding domains may be fused to the C-terminus of the second hinge-CH2-CH3 element of the fourth domain (D). In such a case, the second binding domain (B) may be fused to the C-terminus of the first hinge-CH2-CH3 element of the fourth domain (D), and the third binding domain (C) may be fused to the N-terminus of the second hinge-CH2-CH3 element of the fourth domain (D). The first and second dimers (A1A2) and (A3A4) are preferably in the form of scDbs. The second binding domain (B) and the third binding domain (C) are preferably in the form of an scFv. Such antibody constructs may comprise one or more of two polypeptide chains, i.e., one or more of the following configurations: VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3-VH(B)-VL(B) (or less optimally VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3-VH(B)-VL(B); VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3-VL(B)-VH(B); or VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3-VL(B)-VH(B)). and another polypeptide chain of the configuration VH(C)-VL(C)-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4) (or less optimally VH(C)-VL(C)-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4), VL(C)-VH(C)-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4), or VL(C)-VH(C)-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4)).

[0156] In the antibody construct of the present disclosure, the first dimer (A1A2) consisting of two first binding domains may be fused to the N-terminus of the first hinge-CH2-CH3 element of the fourth domain (D), while the second dimer (A3A4) consisting of two first binding domains may be fused to the C-terminus of the second hinge-CH2-CH3 element of the fourth domain (D). In such a case, the third binding domain (C) may be fused to the C-terminus of the first hinge-CH2-CH3 element of the fourth domain (D), and the second binding domain (B) may be fused to the N-terminus of the second hinge-CH2-CH3 element of the fourth domain (D). The first and second dimers (A1A2) and (A3A4) are preferably in the form of scDbs. The second binding domain (B) and the third binding domain (C) are preferably in the form of an scFv. Such antibody constructs may comprise one or more of two polypeptide chains, i.e., one or more of the following configurations: VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3-VH(C)-VL(C) (or less optimally VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3-VH(C)-VL(C); VL(A2)-VH(A1)-VL(A1)-VH(A2)-hinge-CH2-CH3-VL(C)-VH(C); or VH(A2)-VL(A1)-VH(A1)-VL(A2)-hinge-CH2-CH3-VL(C)-VH(C)). and another polypeptide chain of the configuration VH(B)-VL(B)-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4) (or less optimally VH(B)-VL(B)-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4), VL(B)-VH(B)-hinge-CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4), or VL(B)-VH(B)-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4)).

[0157] Usually, the antibody constructs described herein having two scDb fragments containing four first binding domains (A1-A4) fused to the C-terminus of the two heavy chains are preferred. Among these antibody constructs, those containing one or two second binding domains (B) in the form of scFvs fused to one or two N-terminus of the Fc region are most preferred. The antibody constructs described herein having one scDb fragment containing two first binding sites (A1 and A2 or A3 and A4) fused to the C-terminus of the heavy chain and another scDb fragment containing two first binding sites (A3 and A4 or A1 and A2) fused to the N-terminus of the Fc region are not optimal. The antibody constructs described herein having two scDb fragments containing four first binding domains (A1-A4) fused to the two N-terminus of the Fc region are not preferred.

[0158] Ideally, the distance between the binding sites of the first binding domain (A1, A2, A3, A4) is short. Therefore, it is preferred that the two binding domains are within a distance of about 30 or less, preferably about 25 nm or less, more preferably about 22 nm or less, more preferably about 20 nm or less, more preferably about 19 nm or less, more preferably about 18 nm or less, more preferably about 17 nm or less, more preferably about 16 nm or less, more preferably about 15 nm or less, more preferably about 14 nm or less, more preferably about 13 nm or less, more preferably about 12 nm or less, more preferably about 11 nm or less, more preferably about 10 nm or less, more preferably about 9 nm or less, more preferably about 8 nm or less, more preferably about 7 nm or less, more preferably about 6 nm or less, more preferably about 5 nm or less. Preferably, the distance is measured as the distance from the center of the binding site. The distance between the domains is preferably measured as the distance between the two first binding domains (A1, A2, A3, A4, ...) that are the longest distance from each other. To measure the distance between the two binding domains, a crystal structure is preferred. If a crystal structure is not available, structural considerations according to Rossmalen et al Biochemistry 2017, 56, 6565-6574 are preferably applied, especially with regard to the linker.

[0159] When the antibody construct of the present invention comprises a CH3 region, modifications can be introduced into the CH3 region to enhance the heterodimer pairing of polypeptides comprising the CH3 region. The CH3 region can be modified by the "knob-into-hole" technique, which is described in detail with some examples in, for example, WO96 / 027011; Ridgway, J., B., et al., Protein Eng 9 (1996) 617-621; and Merchant, AM, et al., Nat Biotechnol 16 (1998) 677-681. In this method, the interaction surface of the two CH3 domains is modified to increase the heterodimerization of both heavy chains comprising these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be a "knob" and the other is a "hole". Introduction of disulfide bridges stabilizes the heterodimers (Merchant, AM, et al., Nature Biotech 16 (1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

[0160] Thus, the antibody constructs of the present disclosure may be further characterized in that the CH3 domain of one polypeptide chain meets the CH3 domain of another polypeptide chain at an interface that comprises the original interface between the antibody CH3 domains, where the interface has been modified to facilitate formation of the antibody construct. The modifications may be characterized as follows: a) the CH3 domain of one polypeptide chain is modified so that, within the original interface of the CH3 domain of one polypeptide chain that meets the original interface of the CH3 domain of the other polypeptide chain in the antibody construct, certain amino acid residues are replaced with amino acid residues with a larger side chain volume, thereby creating a protuberance within the interface of the CH3 domain of one polypeptide chain that can be positioned in a recess within the interface of the CH3 domain of the other polypeptide chain, and b) the CH3 domain of the other polypeptide chain is modified so that, within the original interface of the second CH3 domain that meets the original interface of the first CH3 domain in the antibody construct, certain amino acid residues are replaced with amino acid residues with a smaller side chain volume, thereby creating a recess within the interface of the second CH3 domain into which the protuberance within the interface of the first CH3 domain can be positioned.

[0161] Preferably, the amino acid residues with larger side chain volumes are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues with smaller side chain volumes are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0162] Both CH3 domains are further modified by introducing a cysteine ​​(C) as the amino acid at the corresponding position of each CH3 domain so that a disulfide bridge between both CH3 domains can be formed.

[0163] In a preferred embodiment, the antibody construct comprises a T366W mutation in the CH3 domain of the "knob chain" and a T366S, L368A, and Y407V mutation in the CH3 domain of the "hole chain". Additional interchain disulfide bridges between the CH3 domains can also be used, for example by introducing a Y349C mutation in the CH3 domain of the "knob chain" and an E356C or S354C mutation in the CH3 domain of the "hole chain" (Merchant, AM, et al., Nature Biotech 16 (1998) 677-681). Alternatively, the antibody construct can comprise a T366Y mutation in the CH3 domain of the "knob chain" and a Y407T mutation in the "hole chain". Other knob-in-hole technologies that can be used similarly are described in Labrijn AF, Janmaat ML, Reichert JM, Parren P. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov 2019; 18:585-608. Preferred types of knob chain CH2-CH3 heavy chain constant domains are shown in SEQ ID NOs: 44, 46, 48, 50, 52, 54, 56, and 58. Preferred types of hole chain CH2-CH3 heavy chain constant domains are shown in SEQ ID NOs: 43, 45, 47, 49, 51, 53, 55, and 57.

[0164] In the preferred antibody construct, at least four first binding domains (A) can specifically bind to CD16A, and preferably comprise the ability to distinguish between CD16A and CD16B. In other words, at least four first binding domains (A) preferably bind to CD16A with higher affinity than CD16B, and the affinity may be at least about 10 times higher, at least about 100 times higher, or at least about 1000 times higher. More preferably, at least four first binding domains (A) do not essentially bind to CD16B. Therefore, it is understood that the first binding domain is preferably not a non-silenced CH2 domain, i.e., a CH2 domain that can bind to both CD16A and CD16B.

[0165] Thus, it is preferred that the at least four first binding domains (A) bind to an epitope of CD16A that includes the C-terminal sequence SFFPPGYQ ​​(positions 201-209 of SEQ ID NO: 13) of CD16A and / or the amino acid residues of residues G147 and / or Y158, which are not present in CD16B. It is preferred in the present invention that the first binding domains that bind to CD16A on the surface of effector cells bind to an epitope on CD16A that is closer to the membrane than the physiological Fcγ receptor binding domain of CD16A. Binding domains that specifically bind to an epitope that includes Y158 are preferred, since this epitope is closer to the cell membrane and thus further contributes to reducing the possibility of simultaneous binding to a second immune effector cell. Exemplary each binding domain is characterized, for example, by the following group of CDRs: CDR-H1 as set forth in SEQ ID NO: 77, CDR-H2 as set forth in SEQ ID NO: 78, CDR-H3 as set forth in SEQ ID NO: 79, CDR-L1 as set forth in SEQ ID NO: 80, CDR-L2 as set forth in SEQ ID NO: 81, CDR-L3 as set forth in SEQ ID NO: 82, and a binding domain that binds the same epitope; CDR-H1 as set forth in SEQ ID NO: 83, CDR-H2 as set forth in SEQ ID NO: 84, CDR-H3 as set forth in SEQ ID NO: 85, CDR-L1 as set forth in SEQ ID NO: 86, CDR-L2 as set forth in SEQ ID NO: 87, CDR-L3 as set forth in SEQ ID NO: 88, and a binding domain that binds the same epitope; and CDR-H1 as depicted in SEQ ID NO: 77, CDR-H2 as depicted in SEQ ID NO: 89, CDR-H3 as depicted in SEQ ID NO: 79, CDR-L1 as depicted in SEQ ID NO: 80, CDR-L2 as depicted in SEQ ID NO: 81, CDR-L3 as depicted in SEQ ID NO: 82, and a binding domain that binds the same epitope. A preferred CD16A binding domain is characterized by the following group of CDRs: CDR-H1 as depicted in SEQ ID NO: 83, CDR-H2 as depicted in SEQ ID NO: 84, CDR-H3 as depicted in SEQ ID NO: 85, CDR-L1 as depicted in SEQ ID NO: 86, CDR-L2 as depicted in SEQ ID NO: 87, CDR-L3 as depicted in SEQ ID NO: 88, and a binding domain binding to the same epitope. Examples of such CD16A binders are also described in WO2020043670.

[0166] In some embodiments, the at least four first binding domains (A) comprise the same CDR sequences. In some embodiments, one or more, preferably all, of the at least four first binding domains (A) comprise a VH region comprising CDR-H1, CDR-H2, and CDR-H3, and a VL region comprising CDR-L1, CDR-L2, and CDR-L3 selected from the following: CDR-H1 as set forth in SEQ ID NO: 77, CDR-H2 as set forth in SEQ ID NO: 78, CDR-H3 as set forth in SEQ ID NO: 79, CDR-L1 as set forth in SEQ ID NO: 80, CDR-L2 as set forth in SEQ ID NO: 81, CDR-L3 as set forth in SEQ ID NO: 82, and a binding domain that binds the same epitope; CDR-H1 as set forth in SEQ ID NO: 83, CDR-H2 as set forth in SEQ ID NO: 84, CDR-H3 as set forth in SEQ ID NO: 85, CDR-L1 as set forth in SEQ ID NO: 86, CDR-L2 as set forth in SEQ ID NO: 87, CDR-L3 as set forth in SEQ ID NO: 88, and a binding domain that binds the same epitope (which is preferred); and CDR-H1 as depicted in SEQ ID NO: 77, CDR-H2 as depicted in SEQ ID NO: 89, CDR-H3 as depicted in SEQ ID NO: 79, CDR-L1 as depicted in SEQ ID NO: 80, CDR-L2 as depicted in SEQ ID NO: 81, CDR-L3 as depicted in SEQ ID NO: 82, and a binding domain that binds the same epitope.

[0167] In some embodiments, the at least four first binding domains (A) comprise the same VL and VH sequences. In some preferred embodiments, one or more, preferably all, of the at least four first binding domains (A) comprise a pair of VH and VL chains having sequences as shown in a sequence pair selected from the group consisting of SEQ ID NO: 59 and SEQ ID NO: 68, SEQ ID NO: 60 and SEQ ID NO: 69, and SEQ ID NO: 61 and SEQ ID NO: 70, with SEQ ID NO: 60 and SEQ ID NO: 69 being preferred. In some embodiments, the at least four first binding domains (A) comprise the same amino acid sequence.

[0168] In some embodiments, one or more, preferably all, of the at least four first binding domains (A) comprise a VH domain comprising the following three heavy chain CDRs and a VL domain comprising the following three light chain CDRs: CDR-H1 as depicted in SEQ ID NO: 83, CDR-H2 as depicted in SEQ ID NO: 84, CDR-H3 as depicted in SEQ ID NO: 85, CDR-L1 as depicted in SEQ ID NO: 86, CDR-L2 as depicted in SEQ ID NO: 87, and CDR-L3 as depicted in SEQ ID NO: 88.

[0169] In some preferred embodiments, one or more, preferably all, of the at least four first binding domains (A) comprise a pair of VH and VL chains having sequences as set forth in a sequence pair selected from the group consisting of SEQ ID NO: 60 and SEQ ID NO: 69.

[0170] Antibodies against the first target (A') of the present disclosure are well known in the art.Antibodies against CD16A are described, for example, in WO2020043670.Antibodies against CD56 are described, for example, in WO2012138537 and WO2017023780.Antibodies against NKG2A are described, for example, in WO2008009545, WO2009092805, WO2016032334, WO2020094071, WO2020102501.Antibodies against NKG2D are described, for example, in WO2009077483, WO2018148447, WO2019157366.Antibodies against NKp30 are described, for example, in WO2020172605. Antibodies against NKp46 are described, for example, in WO2011086179 and WO2016209021. Antibodies against DNAM-1 are described, for example, in WO2013140787. Antibodies against SLAMF7 are described, for example, in US2018208653. Antibodies against OX40 are described, for example, in WO2007062245, US2010136030, US2019100596, WO2013008171, WO2013028231. Antibodies against CD47, SIRPα are described, for example, in WO9727873, WO2005044857, US2014161799. Antibodies against CD89 are described, for example, in WO02064634, WO2020084056. Antibodies against CD96 are described, for example, in WO2019091449. Antibodies against CD137 are described, for example, in WO2005035584, WO2006088464, US2006188439. Antibodies against CD160 are described, for example, in US2012003224, US2013122006. Antibodies against TIGIT are described, for example, in US2020040082 and WO2019062832. Antibodies against Nectin-4 are described, for example, in WO2018158398. Antibodies against PD-1 are described, for example, in WO2009014708, US2012237522, US2013095098, and US2011229461.Antibodies against PD-L1 are described, for example, in US2012237522, WO2014022758, WO2014055897, and WO2014195852. Antibodies against LAG-3 are described, for example, in WO2008132601, US2016176965, and WO2010019570. Antibodies against CTLA-4 are described, for example, in WO2005092380, US2009252741, and WO2006066568. Antibodies against TIM-3 are described, for example, in US2014134639, WO2011155607, and WO2015117002. Antibodies against KIR2DS1-5 are described, for example, in WO2016031936. Antibodies against CD3 are described, for example, in US6750325, WO9304187, and WO9516037.

[0171] In some preferred embodiments, the at least four first binding domains (A) are specific for NKG2D. One or more, preferably all, of the at least four first binding domains (A) preferably comprise the following three heavy chain CDRs and three light chain CDRs: CDR-H1 as shown in SEQ ID NO: 96, CDR-H2 as shown in SEQ ID NO: 97, CDR-H3 as shown in SEQ ID NO: 98, CDR-L1 as shown in SEQ ID NO: 99, CDR-L2 as shown in SEQ ID NO: 100, CDR-L3 as shown in SEQ ID NO: 101.

[0172] In some preferred embodiments, one or more, preferably all, of the at least four first binding domains (A) comprise a pair of VH and VL chains having the sequences shown in the sequence pair SEQ ID NO: 63 and SEQ ID NO: 72.

[0173] In some preferred embodiments, the at least four first binding domains (A) are specific for NKp46. One or more, preferably all, of the at least four first binding domains (A) preferably comprise the following three heavy chain CDRs and three light chain CDRs: CDR-H1 as shown in SEQ ID NO: 90, CDR-H2 as shown in SEQ ID NO: 91, CDR-H3 as shown in SEQ ID NO: 92, CDR-L1 as shown in SEQ ID NO: 93, CDR-L2 as shown in SEQ ID NO: 94, and CDR-L3 as shown in SEQ ID NO: 95.

[0174] In some preferred embodiments, one or more, preferably all, of the at least four first binding domains (A) comprise a pair of VH and VL chains having the sequences shown in the sequence pair SEQ ID NO: 62 and SEQ ID NO: 71.

[0175] In some embodiments, the second binding domain (B) is specific for a second target (B') that is a tumor-associated antigen. The second target (B') is preferably selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD52, CD70, CD74, CD79b, CD123, CLL1, BCMA, FCRH5, EGFR, EGFRvlll, HER2, GD2.

[0176] In some embodiments, the third binding domain (B) is specific for a third target (B') that is a tumor-associated antigen. The third target (B') is preferably selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD52, CD70, CD74, CD79b, CD123, CLL1, BCMA, FCRH5, EGFR, EGFRvlll, HER2, GD2.

[0177] These cell surface antigens, present on the surface of target cells, have been implicated in specific disease entities. CD30 is a cell surface antigen characteristic of malignant cells in Hodgkin's lymphoma. CD19, CD20, CD22, CD70, CD74, and CD79b are cell surface antigens characteristic of malignant cells in non-Hodgkin's lymphomas (diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), T-cell lymphomas (both peripheral and cutaneous, including transformed mycosis fungoides / Sézary syndrome TMF / SS and anaplastic large cell lymphoma (ALCL)). CD52, CD33, CD123, and CLL1 are cell surface antigens characteristic of malignant cells in leukemias (chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML)). BCMA, FCRH5 are cell surface antigens characteristic of malignant cells of multiple myeloma. EGFR, HER2, GD2 are cell surface antigens characteristic of solid tumors (triple negative breast cancer (TNBC), breast cancer BC, colorectal cancer (CRC), non-small cell lung cancer (NSCLC), small cell carcinoma (SCLC, also known as "small cell lung cancer" or "oat cell carcinoma"), prostate cancer (PC), glioblastoma (also known as glioblastoma multiforme (GBM)).

[0178] Antibodies against such targets are well known in the art.Antibodies against CD19 are described, for example, in WO2018002031, WO2015157286, and WO2016112855.Antibodies against CD20 are described, for example, in WO2017185949, US2009197330, and WO2019164821.Antibodies against CD22 are described, for example, in WO2020014482, WO2013163519, and US10590197.Antibodies against CD30 are described, for example, in WO2007044616, WO2014164067, and WO2020135426. Antibodies against CD33 are described, for example, in WO2019006280, WO2018200562, and WO2016201389. Antibodies against CD52 are described, for example, in WO2005042581, WO2011109662, and US2003124127. Antibodies against CD70 are described, for example, in US2012294863, WO2014158821, and WO2006113909. Antibodies against CD74 are described, for example, in WO03074567, US2014030273, and WO2017132617. Antibodies against CD79b are described, for example, in US2009028856, US2010215669, and WO2020088587. Antibodies against CD123 are described, for example, in US2017183413, WO2016116626, and US10100118. Antibodies against CLL1 are described, for example, in WO2020083406. Antibodies against BCMA are described, for example, in WO02066516, US10745486, and US2019112382. Antibodies against FCRH5 are described, for example, in US2013089497. Antibodies against EGFR are described, for example, in WO9520045, WO9525167, and WO02066058. Antibodies against EGFRvlll are described, for example, in WO2017125831. Antibodies against HER2 are described, for example, in US2011189168, WO0105425, and US2002076695.Antibodies against GD2 are described, for example, in WO8600909, WO8802006, and US5977316.

[0179] In some preferred embodiments, the second binding domain (B) and / or the third binding domain (C) are specific for EGFR and preferably comprise a VH domain comprising the following three heavy chain CDRs and a VL domain comprising the following three light chain CDRs: CDR-H1 as depicted in SEQ ID NO: 114, CDR-H2 as depicted in SEQ ID NO: 115, CDR-H3 as depicted in SEQ ID NO: 116, CDR-L1 as depicted in SEQ ID NO: 117, CDR-L2 as depicted in SEQ ID NO: 118, CDR-L3 as depicted in SEQ ID NO: 119.

[0180] In some preferred embodiments, the second binding domain (B) and / or the third binding domain (C) comprises a pair of VH and VL chains having the sequences shown in the sequence pair SEQ ID NO: 66 and SEQ ID NO: 75.

[0181] In some preferred embodiments, the second binding domain (B) and / or the third binding domain (C) are specific for BCMA and preferably comprise a VH domain comprising the following three heavy chain CDRs and a VH domain comprising the following three light chain CDRs: CDR-H1 as depicted in SEQ ID NO: 102, CDR-H2 as depicted in SEQ ID NO: 103, CDR-H3 as depicted in SEQ ID NO: 104, CDR-L1 as depicted in SEQ ID NO: 105, CDR-L2 as depicted in SEQ ID NO: 106, CDR-L3 as depicted in SEQ ID NO: 107.

[0182] In some preferred embodiments, the second binding domain (B) and / or the third binding domain (C) comprises a pair of VH and VL chains having the sequences shown in SEQ ID NO:64 and SEQ ID NO:73.

[0183] In some preferred embodiments, the second binding domain (B) and / or the third binding domain (C) are specific for CD19 and preferably comprise a VH domain comprising the following three heavy chain CDRs and a VH domain comprising the following three light chain CDRs: CDR-H1 as depicted in SEQ ID NO: 108, CDR-H2 as depicted in SEQ ID NO: 109, CDR-H3 as depicted in SEQ ID NO: 110, CDR-L1 as depicted in SEQ ID NO: 111, CDR-L2 as depicted in SEQ ID NO: 112, CDR-L3 as depicted in SEQ ID NO: 113.

[0184] In some preferred embodiments, the second binding domain (B) and / or the third binding domain (C) comprises a pair of VH and VL chains having the sequences shown in SEQ ID NO:65 and SEQ ID NO:74.

[0185] In some preferred embodiments, the second binding domain (B) and / or the third binding domain (C) are specific for HER2 and preferably comprise a VH domain comprising the following three heavy chain CDRs and a VH domain comprising the following three light chain CDRs: CDR-H1 as depicted in SEQ ID NO: 120, CDR-H2 as depicted in SEQ ID NO: 121, CDR-H3 as depicted in SEQ ID NO: 122, CDR-L1 as depicted in SEQ ID NO: 123, CDR-L2 as depicted in SEQ ID NO: 124, and CDR-L3 as depicted in SEQ ID NO: 125.

[0186] In some preferred embodiments, the second binding domain (B) and / or the third binding domain (C) comprises a pair of VH and VL chains having the sequences shown in SEQ ID NO:67 and SEQ ID NO:76.

[0187] The antibody construct of the present invention is preferably an antibody construct selected from the group consisting of SEQ ID NOs: 148, 149, 150 and 151, 152 and 153, 154 and 155, 156 and 157, 158 and 159, 160 and 161, 162 and 163, 180 to 183, 190, and 191 and 192.

[0188] The antibody construct of the present invention is preferably a variant of an antibody construct selected from the group consisting of SEQ ID NOs: 148, 149, 150 and 151, 152 and 153, 154 and 155, 156 and 157, 158 and 159, 160 and 161, 162 and 163, 180 to 183, 190, and 191 and 192, having at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99% sequence identity to any one of these aforementioned antibody constructs, preferably with the proviso that the CDR sequences contained in these antibody constructs are not modified.

[0189] The antibody constructs of the present invention are characterized by inducing a low degree of fratricide, also referred to as a "reduced" degree of fratricide. The degree of fratricide can be measured in a cytotoxicity assay, for example an assay essentially as described in Example 7. Such an assay is preferably performed as follows: For the calcein release NK cell fratricide assay, enriched primary human NK cells are labeled with 10 μM of the fluorescent dye calcein AM for 30 minutes and then incubated with 5×10 4Aliquots of labeled cells are seeded, preferably in duplicate, into individual wells of a round-bottom 96-well microplate at a 1:1 effector:target (E:T) ratio with unlabeled enriched autologous NK cells in the presence of 10 1:5 dilutions starting at 100 μg / mL of the designated test or control antibody construct. Anti-CD38 IgG1 with Fab domain derived from daratumumab (IgAb_51, SEQ ID NO: 166 and SEQ ID NO: 167) is preferably used as a positive control. Control samples for measuring spontaneous release, maximum release, and antibody-independent lysis by effector cells are tested, preferably in 4 replicates. After 4 hours of incubation, 100 μL of cell-free culture supernatant is collected from each well and the fluorescent calcein released from lysed target cells is quantified using a multiplate fluorescence reader. After the fluorescence of spontaneously lysed cells is subtracted from all samples, the fluorescence of each sample should be normalized to that of completely lysed cells to reveal specific lysis for each sample. The mean and standard deviation (SD) of specific target cell lysis (%) can be plotted, and in vitro potency (EC 50 ) and efficacy (E max ) can be revealed.

[0190] In some embodiments, "low fratricide" means that the degree of fratricide of the test molecule, such as the antibody construct of the present invention, is about 40% or less. The degree of fratricide of the antibody construct of the present invention is preferably about 35% or less, more preferably about 30% or less, more preferably about 25% or less, more preferably about 22% or less, more preferably about 20% or less, more preferably about 19% or less, more preferably about 18% or less, more preferably about 17% or less, more preferably about 16% or less, more preferably about 15% or less, more preferably about 14% or less, more preferably about 13% or less, more preferably about 12% or less, more preferably about 11% or less, more preferably about 10% or less, and this measurement is preferably performed at a concentration of 100 μg / mL.

[0191] In some embodiments, the antibody constructs of the present invention induce a lower degree of fratricide compared to the anti-CD38 antibodies shown in SEQ ID NO: 167 and SEQ ID NO: 168, preferably measured using a test antibody and control at a concentration of 100 μg / mL.

[0192] In some embodiments, the antibody construct of the present invention has a higher potency (lower EC50) in a cytotoxicity assay compared to a reference antibody having only two or only one first binding domain (A). A preferred reference antibody is preferably bivalent for a first target (A') and bivalent for a second target (B'). A preferred reference antibody consists of a full-length immunoglobulin specific for a first target (A') to which an scFv specific for the second target (B') is fused to the C-terminus of each heavy chain. As an illustrative example, such a reference antibody may have the heavy and light chain sequences shown in SEQ ID NO: 170 and SEQ ID NO: 171. Alternatively, a preferred reference antibody consists of a full-length immunoglobulin specific for a second target (B') to which an scFv specific for the first target (A') is fused to the C-terminus of each heavy chain. As an illustrative example, such a reference antibody may have the heavy and light chain sequences shown in SEQ ID NO: 176 and SEQ ID NO: 177. At least four first binding domains of the antibody of the present invention preferably comprise the same CDR sequences as the binding domain specific for the first target (A') of the reference antibody. Even more preferably, at least four first binding domains of the antibody of the present invention preferably comprise the same VL and VH sequences as the binding domain specific for the first target (A') of the reference antibody. The potency (EC50) is preferably measured in a cytotoxicity assay essentially as described in Example 6. In some embodiments, the EC50 of the antibody construct of the present invention has a value that is about 0.5 times or less, preferably about 0.4 times or less, preferably about 0.3 times or less, preferably about 0.2 times or less, preferably about 0.1 times, compared to the EC50 of the reference antibody. In principle, the potency can be measured using any target cell expressing the second target (B'). However, the cell is preferably a tumor or cancer cell line. The target cell may highly express the second target (B'). In such a case, the EC50 of the antibody construct of the present invention may have a value that is about 0.5 times or less than that of the reference antibody.However, the increase in efficacy becomes more pronounced when target cells that express low or even very low levels of the second target (B') are used. In such cases, the EC50 of the antibody construct of the present invention may have a value that is about 0.5 times or less than the EC50 of the reference antibody, preferably about 0.4 times or less, preferably about 0.3 times or less, preferably about 0.2 times or less, and more preferably about 0.1 times or less than the EC50 of the reference antibody.

[0193] There are several methods in the art for measuring the expression level of a second target (B') on a cell line. A preferred method according to the present disclosure is the measurement of specific antibody binding capacity (SABC). SACB assays are known in the art (Serke et al., 1998, Cytometry, 33(2):179-87). Such assays can be performed essentially as described in Example 5. Specifically, the density of antigen present on the surface of one or more cell lines can be measured using a QIFIKIT (Dako) and a suitable antibody, such as anti-HER2 mAb MAB 1129 (RnD Systems) or anti-EGFR mAb H11 (Dianova), according to the manufacturer's instructions. Briefly, 1×10 6 An aliquot of cells can be stained with an appropriate antibody (e.g., mAb MAB 1129 or mAb H11) followed by F(ab')2 fragment of FITC-conjugated goat anti-mouse IgG. As a negative control, 1 x 10 6An aliquot of cells can be stained with a negative control antibody (e.g., mAb 9E10 (Acris)) followed by F(ab')2 fragment of FITC-conjugated goat anti-mouse IgG. To calculate the specific antibody binding capacity, calibration beads, including five bead populations with different numbers of mAb molecules, can be stained with F(ab')2 fragment of FITC-conjugated goat anti-mouse IgG. A calibration curve can be generated from the resulting median fluorescence intensity. This calibration curve can be used to calculate the specific antibody binding capacity (SABC) of each antibody (e.g., mAb MAB 1129 or mAb H11) for each cell line. High-expressing cell lines for various second targets (B') have been described in the art. For EGFR, the high-expressing cell line is A-431. For CD19, the high-expressing cell line is JOK-1 (Reusch et al., 2015 MAbs, 7(3): 584-604). For CD20, the high expressing cell line is DHL-10 (Watanabe et al., J Immunol February 1, 2015, 194 (3) 911-920). For CD22, the high expressing cell line is JOK-1. For CD30, the high expressing cell line is HDLM-2 (Zhao et al, 2015, ASCO abstract 3050). For CD33, the high expressing cell line is MOLM-13 (Friedrich et al., 2014, Mol Cancer Ther 13(6):1549-1557). For CD52, the high expressing cell line is U-698 (human protein atlas). For CD70, the high expressing cell line is U-266 (human protein atlas). For CD74, the high expressing cell line is HDLM-2 (human protein atlas). For CD79b, the high expressing cell line is Daudi (Engelberts et al, 2020, EBioMedicine, vol. 52, 102625). For CD123, the high expressing cell line is MOLM-13. For CLL1, the high expressing cell line is EOL-1. For BCMA, the high expressing cell line is NCI-H929.For FCRH5, the high expressing cell line is U-698 (human protein atlas). For EGFRvIII, the high expressing cell line is DK-MG. For HER2, the high expressing cell line is SK-BR-3. For GD2, the high expressing cell line is T98G (Golinelli et al, 2020 Cancer Gene Therapy 27:558-570). Any one of the aforementioned cell lines is a preferred reference cell line for the high expressing cell line for each secondary target. A cell line is preferably classified as a high expressing cell line if it has an SABC score of at least 50% for each secondary target (B') compared to the reference high expressing cell line. A cell line is preferably classified as a low expressing cell line if it has an SABC score of 15% or less for each secondary target (B') compared to the reference high expressing cell line. A cell line is preferably classified as very low expressing cell line if it has an SABC score of 5% or less for each secondary target (B') compared to a reference high expressing cell line. Very low expressing cell line should be understood as a subgroup of low expressing cell line. It is understood that low expressing cell line and very low expressing cell line preferably still show detectable expression of each secondary target (B') in SABC assay method.

[0194] In some embodiments, the antibody construct of the present invention has a higher efficacy (higher E) in a cytotoxicity assay compared to a reference antibody having only two or only one first binding domain (A). max). A preferred reference antibody is preferably bivalent for a first target (A') and bivalent for a second target (B'). A preferred reference antibody consists of a full-length immunoglobulin specific for a first target (A') to which an scFv specific for the second target (B') is fused to the C-terminus of each heavy chain. As an illustrative example, such a reference antibody may have the heavy and light chain sequences shown in SEQ ID NO: 170 and SEQ ID NO: 171. Alternatively, a preferred reference antibody consists of a full-length immunoglobulin specific for a second target (B') to which an scFv specific for the first target (A') is fused to the C-terminus of each heavy chain. As an illustrative example, such a reference antibody may have the heavy and light chain sequences shown in SEQ ID NO: 176 and SEQ ID NO: 177. At least four first binding domains of the antibody of the present invention preferably comprise the same CDR sequences as the binding domain specific for the first target (A') of the reference antibody. Even more preferably, at least four first binding domains of the antibody of the present invention preferably comprise the same VL and VH sequences as the binding domain specific for the first target (A') of the reference antibody. Efficacy (E max ) is preferably measured in a cytotoxicity assay essentially as described in Example 6.

[0195] The present invention also relates to nucleic acid molecules (DNA and RNA) that comprise nucleotide sequences encoding the antibody constructs disclosed herein. The present disclosure also encompasses vectors that comprise the nucleic acid molecules of the present invention. The present invention also encompasses host cells that comprise said nucleic acid molecules or said vectors. Because the degeneracy of the genetic code allows some codes to be replaced by other codons that designate the same amino acid, the present disclosure is not limited to a particular nucleic acid molecule that encodes the antibody constructs described herein, but encompasses any nucleic acid molecule that comprises a nucleotide sequence that encodes a functional polypeptide. In this regard, the present disclosure also relates to nucleotide sequences that encode the antibody constructs of the present disclosure.

[0196] The nucleic acid molecules disclosed in the present application may be "operably linked" to a regulatory sequence (or regulatory sequences) to allow expression of the nucleic acid molecule.

[0197] A nucleic acid molecule, such as DNA, is said to be "capable of expressing a nucleic acid molecule" or "allowing for expression of a nucleotide sequence" if it contains sequence elements that contain information for transcriptional and / or translational regulation and such sequences are "operably linked" to a nucleotide sequence that encodes a polypeptide. An operable linkage is one in which the regulatory sequence elements and the sequence to be expressed are joined in a manner that allows gene expression. The exact nature of the regulatory regions necessary for gene expression can vary from species to species, but generally these regions include promoters, which in prokaryotes include both the promoter itself, i.e., the DNA elements that direct transcription initiation and the DNA elements that, when transcribed into RNA, signal translation initiation. Usually, such promoter regions include 5' non-coding sequences involved in the initiation of transcription and translation, such as the -35 / -10 box and Shine-Dalgarno elements in prokaryotes, or the TATA box, CAAT sequence, and 5' capping element in eukaryotes. These regions may also contain enhancer or repressor elements, as well as translated signal or leader sequences for targeting the native polypeptide to a particular compartment of the host cell.

[0198] In addition, the 3' non-coding sequences may also contain regulatory elements involved in transcription termination, polyadenylation, etc. However, if these termination sequences are not fully functional in a particular host cell, the termination sequences may be replaced with signals functional in that cell.

[0199] Therefore, the nucleic acid molecule of the present disclosure can include regulatory sequences such as promoter sequences.In some embodiments, the nucleic acid molecule of the present disclosure includes promoter sequences and transcription termination sequences.The example of the promoter that is useful for expression in eukaryotic cells is SV40 promoter or CMV promoter.

[0200] The nucleic acid molecules of the disclosure can also be part of a vector or any other type of cloning vehicle, such as a plasmid, phagemid, phage, baculovirus, cosmid, or artificial chromosome.

[0201] Such cloning vehicles can contain, apart from the aforementioned regulatory sequences and nucleic acid sequences encoding the antibody constructs described herein, replication and control sequences derived from a species compatible with the host cell used for expression, as well as a selection marker which confers a selectable phenotype on transformed or transfected cells. Many suitable cloning vectors are known in the art and are commercially available.

[0202] The present disclosure also relates to a method for producing the antibody construct of the present disclosure, which is produced starting from a nucleic acid encoding the antibody construct or any subunit thereof. This method can be carried out in vivo, for example, the polypeptide can be produced in a bacterial or eukaryotic host organism and then isolated from the host organism or a culture thereof. It is also possible to produce the antibody construct of the present disclosure in vitro, for example, using an in vitro translation system.

[0203] When producing antibody constructs in vivo, the nucleic acid encoding such polypeptides is introduced into suitable bacterial or eukaryotic host organisms using recombinant DNA technology.For this purpose, host cells can be transformed with cloning vectors that contain the nucleic acid molecules encoding the antibody constructs described herein using established standard methods.The host cells can then be cultured under conditions that allow the expression of heterologous DNA and thus the synthesis of the corresponding polypeptide or antibody construct.Subsequently, the polypeptide or antibody construct is recovered from either the cells or the culture medium.

[0204] Suitable host cells can be eukaryotic, for example, an immortalized mammalian cell line (eg, HeLa cells or CHO cells) or primary mammalian cells.

[0205] The antibody constructs of the present disclosure described herein do not necessarily have to be made or produced solely by genetic engineering. Rather, such polypeptides can also be obtained by chemical synthesis, such as Merrifield solid-phase polypeptide synthesis, or by in vitro transcription and translation. Methods for solid-phase and / or solution-phase synthesis of proteins are well known in the art (see, for example, Bruckdorfer, T. et al. (2004) Curr. Pharm. Biotechnol. 5, 29-43).

[0206] Antibody constructs of the present disclosure may be produced by in vitro transcription / translation using well-established methods known to those skilled in the art.

[0207] The present invention also provides a composition, preferably a pharmaceutical composition, comprising the antibody construct of the present invention.

[0208] Certain embodiments provide pharmaceutical compositions comprising an antibody construct as defined in the present invention and one or more other excipients, such as those exemplarily described in this section and elsewhere herein.In this regard, excipients can be used in the present invention for a variety of purposes, such as for adjusting the physical, chemical, or biological properties of the formulation, for example adjusting the viscosity, and / or for the process of one aspect of the present invention to enhance the efficacy and / or stabilize such formulation, as well as for the process of combating degradation and damage due to pressures that occur during manufacture, transportation, storage, preparation before use, administration, and thereafter.

[0209] In certain embodiments, pharmaceutical compositions may include formulation materials to alter, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition (see REMINGTON'S PHARMACEUTICAL SCIENCES, 18" Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company). In such embodiments, suitable formulation materials include, but are not limited to, the following: Charged amino acids, preferably lysine, lysine acetate, arginine, glutamic acid, and / or histidine, including amino acids such as glycine, alanine, glutamine, asparagine, threonine, proline, 2-phenylalanine Antimicrobial agents, such as antibacterial and antifungal agents ·Antioxidants such as ascorbic acid, methionine, sodium sulfite, or sodium bisulfite; Buffers, buffer systems, and buffering agents used to maintain compositions at physiological pH or slightly lower pH; examples of buffering agents are borates, bicarbonates Tris-HCI, citrate, phosphate, or other organic acids, succinate, phosphate, and histidine; for example, Tris buffer at a pH of about 7.0 to 8.5; · Non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate; Aqueous carriers, including water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media; · Biodegradable polymers such as polyester; · Bulking agents such as mannitol or glycine; · Chelating agents such as ethylenediaminetetraacetic acid (EDTA); ·Tonicity and absorption retarding agents; Complexing agents such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin Fillers; Monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrins); the carbohydrates may be non-reducing sugars, preferably trehalose, sucrose, octasulfate, sorbitol, or xylitol; · a (low molecular weight) protein, polypeptide or proteinaceous carrier, preferably of human origin, such as human or bovine serum albumin, gelatin or immunoglobulins; Colouring and flavouring agents; Sulfur-containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [α]-monothioglycerol, and sodium thiosulfate; · Diluents; ·emulsifier; · Hydrophilic polymers such as polyvinylpyrrolidone; · A salt-forming counterion, such as sodium; · preservatives such as antimicrobials, antioxidants, chelating agents, and inert gases; examples are benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide; · Metal complexes such as Zn-protein complexes; · Solvents and co-solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugars and sugar alcohols, such as trehalose, sucrose, octasulfate, mannitol, sorbitol, or xylitol stachyose, mannose, sorbose, xylose, ribose, myo-inositose, galactose, lactitol, ribitol, myo-inositol, galactitol, glycerol, cyclitols (e.g. inositol), polyethylene glycol, and polyhydric sugar alcohols; ·Suspending agents; Surfactants or wetting agents, e.g., pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal; surfactants may be surfactants, preferably with a molecular weight greater than 1.2 KD, and / or polyethers, preferably with a molecular weight greater than 3 KD; non-limiting examples of preferred surfactants are Tween 20, Tween 40, Tween 60, Tween 80, and Tween 85; non-limiting examples of preferred polyethers are PEG 3000, PEG 3350, PEG 4000, and PEG 5000; ·Stability enhancers such as sucrose or sorbitol; · an alkali metal halide, preferably sodium chloride or potassium chloride, an osmolality increasing agent such as mannitol sorbitol; Parenteral delivery vehicles, including sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils; Intravenous delivery vehicles, including fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose).

[0210] It will be apparent to one of skill in the art that various components of a pharmaceutical composition (e.g., those listed above) may have different effects, e.g., amino acids may act as buffers, stabilizers, and / or antioxidants; mannitol may act as a bulking agent and / or osmolality increasing agent; sodium chloride may act as a delivery vehicle and / or osmolality increasing agent, etc.

[0211] In certain embodiments, the optimal pharmaceutical composition is determined by those skilled in the art, for example, depending on the intended administration route, delivery mode, and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra.For example, suitable vehicle or carrier can be water for injection, physiological saline, or artificial cerebrospinal fluid, optionally supplemented with other substances that are common in compositions for parenteral administration.Neutral buffered saline or saline mixed with serum albumin is yet another exemplary vehicle.

[0212] In one embodiment of the pharmaceutical composition according to one aspect of the present invention, the composition is administered to the patient intravenously.

[0213] Methods and protocols for intravenous (iv) administration of the pharmaceutical compositions described herein are well known in the art.

[0214] The antibody constructs and / or pharmaceutical compositions of the present invention are preferably used in the prevention, treatment or amelioration of a disease, preferably selected from a proliferative disease, a neoplastic disease, a viral disease and / or an immunological disorder. Preferably, the neoplastic disease is a malignant disease, preferably a cancer.

[0215] In one embodiment of the antibody construct and / or pharmaceutical composition of the invention, the identified malignant disease is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, and solid tumors.

[0216] According to the present disclosure, the antibody construct and / or pharmaceutical composition of the present invention is preferably for use in treating tumors that contain cells expressing a second target (B'). The expression of the second target (B') in the tumor may be heterogeneous. For example, the second target (B') may be more highly expressed in certain tumor subtypes or in tumor tissue. However, despite heterogeneous intratumoral expression, tumors can be classified into high or low expressers for a particular antigen based on the overall expression in the tumor or tumor tissue. The preferred method of such classification is by immunohistochemistry.

[0217] The antibody construct of the present invention can be used to treat any cancer tumor that expresses the second target (B'), including cancer or tumor that expresses the second target highly or that expresses the second target low or very low, with the latter two being preferred. The antibody construct of the present invention is considered to be particularly advantageous for treating cancer or tumor that expresses the second target low or very low, thanks to at least four first binding domains that can activate natural immune effector cells even at low or very low expression of the second target (B'). The antibody construct of the present invention is further preferably for use in treating diseases that include malignant cells that express the second target (B') low or very low. Thus, the antibody construct of the present invention may be for use in treating cells that show reduced expression of the second target (B') on tumor cells and / or cancer stem cells (e.g., by downregulation or shedding), which may otherwise result in treatment resistance.

[0218] The antibody construct of the present invention is not only effective for treating cells that express the second target (B') at high levels, but also effective for treating cells that express the second target at low levels or at very low levels, so the antibody construct can also be useful for preventing disease recurrence. The antibody construct of the present invention can also be effective against low or very low expressing cells for the second target (B'), so these cells can be effectively removed by treatment with the antibody construct of the present invention. In another situation, for example, when using other therapies such as other antibody constructs, such low expressing cells may avoid treatment with other therapies and cause disease recurrence. Therefore, the antibody construct of the present invention can be for use in the treatment of disease and / or the prevention of disease recurrence. In particular, the use in the treatment of disease can include the prevention of disease recurrence.

[0219] The present invention also provides a method for treating or ameliorating a disease, comprising administering to a subject in need thereof an antibody construct according to the present invention.

[0220] In one embodiment of the method for treating or ameliorating a disease, the subject suffers from a proliferative disease, a neoplastic disease, an infectious disease, such as a viral disease, or an immunological disorder.Preferably, the neoplastic disease is a malignant disease, preferably a cancer.

[0221] In one embodiment of said method for treating or ameliorating a disease, said malignant disease is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, and solid tumors.

[0222] The present invention also relates to a method of simultaneously binding to target cells and immune effector cells, comprising administering to a subject an antibody construct of the present invention, said target cells preferably expressing low or very low expression of a second target (B'). Such a method is preferably for treating or ameliorating a disease as defined herein. The simultaneous binding to target cells and immune effector cells preferably includes target cell-specific activation of immune effector cells.

[0223] The present invention also relates to kits comprising the antibody constructs of the invention, the nucleic acid molecules of the invention, the vectors of the invention, or the host cells of the invention. Typically, kits of the invention include a container containing the antibody constructs of the invention, the nucleic acid molecules of the invention, the vectors of the invention, or the host cells of the invention, as well as one or more other containers containing materials desirable from a commercial and user standpoint, including, optionally, buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0224] In some embodiments, the antibody constructs of the present invention mediate (preferably concentration-dependent) lysis of target cells expressing low levels of the target antigen. For example, all EGFR / CD16A bispecific antibodies tested in the examples mediated concentration-dependent lysis of target cells expressing low levels of EGFR (mean SABC measured for MCF-7: 4546) and very low levels of EGFR (mean SABC measured for Daudi: 868). Furthermore, all EGFR / NKp46 bispecific antibodies tested in the examples mediated concentration-dependent lysis by NK cells of A431 target cells expressing low levels of HER2.

[0225] In some embodiments, the antibody constructs of the present invention having four first binding domains (A) show higher potency and / or efficacy than constructs having two or one first binding domain (A). For example, all three constructs with four anti-Fv domains (Bi-scDb-Fc_02, aBi-scDb-Fc_05, and Bi-scDb-IgAb_06) tested in the Examples showed higher potency and efficacy than constructs having two or one anti-CD16A Fv domain. Among these constructs, aBi-scDb-Fc_05, which has only one anti-EGFR domain, showed lower potency compared to constructs having two anti-EGFR Fv domains. The IgG-based construct Bi-scDb-IgAb_06 showed slightly but reproducibly lower efficacy than the Fc-based constructs, suggesting that the long distance between the effector and target binding domains has a negative impact on efficacy. Furthermore, a higher potency and efficacy was demonstrated for the construct with four anti-NKp46 domains (AIG-2scDb_06) than for the construct with two anti-NKp46 domains (AIG-2scFv_27).

[0226] In some embodiments, the antibody construct of the present invention having four first binding domains (A) mediates concentration-dependent phagocytosis by macrophages more effectively than the antibody construct having two first binding domains (A) against target cells expressing a second target (B'). The target cells may highly express the second target (B'). Alternatively, the target cells may lowly express the second target (B'). Against target cells expressing high levels of EGFR, all of the tested EGFR / CD16A bispecific antibodies mediated concentration-dependent phagocytosis by macrophages, with the constructs containing four anti-Fv domains (Bi-scDb-Fc_02, aBi-scDb-Fc_05, and Bi-scDb-IgAb_06) showing the highest efficacy. However, against target cells expressing low levels of EGFR, only constructs with four anti-Fv domains (Bi-scDb-Fc_02, aBi-scDb-Fc_05, and Bi-scDb-IgAb_06) induced phagocytosis by macrophages to similar levels.

[0227] The present invention is further characterized by the following items.

[0228] Item 1. Below: (i) at least four first binding domains (A), which are capable of specifically binding to a first target (A'), which is an immune modulatory antigen present on the surface of an innate immune effector cell, wherein the immune effector cell is a natural killer cell or a macrophage; and (ii) a second binding domain (B) capable of specifically binding to a second target (B'), which is an antigen present on the surface of a target cell; An antibody construct comprising:

[0229] Item 2. The antibody construct of item 1, which simultaneously binds to one target cell and one immune effector cell.

[0230] Item 3. The antibody of the construct of item 1 or 2, wherein the first target (A') is an immunostimulatory or immunosuppressive antigen.

[0231] Item 4. The antibody construct of any one of the preceding items, wherein the first target (A') is selected from the group consisting of CD16A, CD56, NKG2A, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 (CD226), SLAMF7 (CD319), CD244 (2B4), OX40, CD47, SIRPα, CD89, CD96, CD137, CD160, TIGIT, Nectin-4, PD-1, PD-L1, LAG-3, CTLA-4, TIM-3, KIR2DL1-5, KIR3DL1-3, KIR2DS1-5, KIR3DS1, and CD3.

[0232] Item 5. The antibody construct of any one of the preceding items, which is bispecific.

[0233] Item 6. A third binding domain (C) capable of specifically binding to a third target (C') other than the second target (B'), which is an antigen present on the surface of a target cell. 5. The antibody construct of any one of items 1 to 4, comprising:

[0234] Item 7. The antibody construct of any one of the preceding items, further comprising a fourth domain (D) comprising a half-life prolonging domain.

[0235] Item 8. The antibody construct of item 7, wherein the half-life extending domain comprises a CH2 domain and the Fcγ receptor binding domain is silenced.

[0236] Item 9. The antibody construct of item 7 or 8, wherein the half-life extending domain comprises a CH3 domain.

[0237] Item 10. The antibody construct of any one of items 7 to 9, comprising at least one hinge domain and a CH3 domain fused to a CH2 domain in the format hinge-CH2 domain-CH3 domain in amino to carboxyl order.

[0238] Item 11. The antibody construct of any one of items 7 to 10, comprising at least two hinge-CH2 domain-CH3 domain elements.

[0239] Item 12. The antibody construct of any one of the preceding items, wherein the second binding domain (B) comprises an antibody VH domain and a VL domain.

[0240] Item 13. The antibody construct of any one of the preceding items, wherein the second binding domain (B) is a Fab or scFv.

[0241] Item 14. The antibody construct of any one of the preceding items, wherein the second target (B') is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD52, CD70, CD74, CD79b, CD123, CLL1, BCMA, FCRH5, EGFR, EGFRvlll, HER2, and GD2.

[0242] Item 15. The antibody construct of any one of items 6 to 14, wherein the third binding domain (C) comprises an antibody VH domain and a VL domain.

[0243] Item 16. The antibody construct of any one of items 6 to 15, wherein the third binding domain (C) is a Fab or scFv.

[0244] Item 17. The antibody construct of any one of items 6 to 16, wherein the third target (C') is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD52, CD70, CD74, CD79b, CD123, CLL1, BCMA, FCRH5, EGFR, EGFRvlll, HER2, and GD2.

[0245] Item 18. The antibody construct of any one of the preceding items, wherein the first binding domain (A) comprises an antibody VH domain and a VL domain.

[0246] Item 19. The antibody construct of any one of the preceding items, wherein the first target (A') is CD16A.

[0247] Item 20. The antibody construct of any one of the preceding items, wherein the first binding domain (A) binds to an epitope on CD16A that is C-terminal to the physiological Fcγ receptor binding domain, said epitope preferably comprising Y158 of SEQ ID NO: 13.

[0248] Item 21. The antibody construct of any one of the preceding items, wherein the four binding domains (A) are positioned relative to one another in such a way that simultaneous binding to two immune effector cells is reduced or preferably prevented.

[0249] Item 22. At least four first binding domains (A) (a) contain the same CDR sequences, (b) comprise the same VL and VH sequences; and / or (c) containing the same amino acid sequence; The antibody construct of any one of the preceding items.

[0250] Item 23. The first binding domain (A1) and the second binding domain (A2) of the four first binding domains (A) are fused to each other (A1A2) in the form of a bi-scFv, double Fab, Db or scDb, preferably in the form of a bi-scFv or scDb, preferably in the form of a scDb, and the variable domain of the scDb is preferably V L -V H -V L -V H The antibody construct of any one of the preceding items, wherein the antibody construct is arranged in the order of:

[0251] Item 24. The third first binding domain (A3) and the fourth first binding domain (A4) of the four first binding domains (A) are fused to each other (A3A4) in the form of a bi-scFv, double Fab, Db or scDb, preferably in the form of a bi-scFv or scDb, preferably in the form of a scDb, and the variable domain of the scDb is preferably V L -V H -V L -V H The antibody construct of any one of the preceding items, wherein the antibody construct is arranged in the order of:

[0252] Item 25. The antibody construct of any one of items 7 to 24, wherein the first first binding domain and the second first binding domain (A1A2), which are fused to each other, are fused to the C-terminus of the CH3 domain of the fourth domain (D).

[0253] Item 26. The antibody construct of any one of items 7 to 24, wherein the first first binding domain and the second first binding domain (A1A2), which are fused to each other, are fused to the N-terminus of the hinge of the fourth domain (D).

[0254] Item 27. The antibody construct of any one of items 7 to 24, wherein the third first binding domain and the fourth first binding domain (A3A4), which are fused to each other, are fused to the C-terminus of the CH3 domain of the fourth domain (D).

[0255] Item 28. The antibody construct of any one of items 7 to 24, wherein the third first binding domain and the fourth first binding domain (A3A4), which are fused to each other, are fused to the N-terminus of the hinge of the fourth domain (D).

[0256] Item 29. The antibody construct of any one of items 7 to 24, wherein the first first binding domain and the second first binding domain (A1A2), which are fused to each other, are fused to the C-terminus of the first CH3 domain of the fourth domain (D), and the third first binding domain and the fourth first binding domain (A3A4), which are fused to each other, are fused to the C-terminus of the second CH3 domain of the fourth domain (D).

[0257] Item 30. The antibody construct of any one of items 7 to 24, wherein the first first binding domain and the second first binding domain (A1A2), which are fused to each other, are fused to the N-terminus of the first hinge of the fourth domain (D), and the third first binding domain and the fourth first binding domain (A3A4), which are fused to each other, are fused to the N-terminus of the second hinge of the fourth domain (D).

[0258] Item 31. The antibody construct of any one of items 7 to 24, wherein the first first binding domain and the second first binding domain (A1A2), which are fused to each other, are fused to the C-terminus of the CH3 domain of the fourth domain (D), and the third first binding domain and the fourth first binding domain (A3A4), which are fused to each other, are fused to the N-terminus of the hinge of the fourth domain (D).

[0259] Item 32. The antibody construct of any one of items 7 to 29 and 31, wherein the second binding domain (B) is fused to the N-terminus of the hinge of the fourth domain (D).

[0260] Item 33. The antibody construct of any one of items 7 to 28 and 30 to 31, wherein the second binding domain (B) is fused to the C-terminus of the CH3 domain of the fourth domain (D).

[0261] Item 34. The antibody construct of any one of items 7 to 25, 27, and 29, wherein a second binding domain (B) is fused to the N-terminus of the hinge of the fourth domain (D) and another second binding domain (B) is fused to the N-terminus of another hinge of the fourth domain (D).

[0262] Item 35. The antibody construct of any one of items 7 to 24, 26, 28, and 30, wherein a second binding domain (B) is fused to the C-terminus of the CH3 domain of the fourth domain (D), and another second binding domain (B) is fused to the C-terminus of another CH3 domain of the fourth domain (D).

[0263] Item 36. The antibody construct of any one of items 7 to 28 and 31, wherein the second binding domain (B) is fused to the N-terminus of the hinge of the fourth domain (D) and another second binding domain (B) is fused to the C-terminus of the CH3 domain of the fourth domain (D).

[0264] Item 37. The antibody construct of any one of items 7 to 28 and 30 to 33, wherein the third binding domain (C) is fused to the C-terminus of the CH3 domain of the fourth domain (D).

[0265] Item 38. The antibody construct of any one of items 7 to 29 and 31 to 33, wherein the third binding domain (C) is fused to the N-terminus of the hinge of the fourth domain (D).

[0266] Item 39. The antibody construct of any one of items 7 to 25, 27, and 29, wherein the second binding domain (B) is fused to the N-terminus of a hinge of the fourth domain (D) and the third binding domain (C) is fused to the N-terminus of another hinge of the fourth domain (D).

[0267] Item 40. The antibody construct of any one of items 7 to 24, 26, 28, and 30, wherein the second binding domain (B) is fused to the C-terminus of a CH3 domain of the fourth domain (D) and the third binding domain (C) is fused to the C-terminus of another CH3 domain of the fourth domain (D).

[0268] Item 41. The antibody construct of any one of items 7 to 28 and 31, wherein the second binding domain (B) is fused to the N-terminus of the hinge of the fourth domain (D) and the third binding domain (C) is fused to the C-terminus of the CH3 domain of the fourth domain (D).

[0269] Item 42. The antibody construct of any one of items 7 to 28 and 31, wherein the second binding domain (B) is fused to the C-terminus of the CH3 domain of the fourth domain (D) and the third binding domain (C) is fused to the N-terminus of the hinge of the fourth domain (D).

[0270] Item 43. The antibody construct of any one of items 7 to 24, wherein the first first binding domain and the second first binding domain (A1A2), which are fused to each other, are fused to the C-terminus of the first CH3 domain of the fourth domain (D), and the third first binding domain and the fourth first binding domain (A3A4), which are fused to each other, are fused to the C-terminus of the second CH3 domain of the fourth domain (D), and the second binding domain (B) is fused to the N-terminus of the hinge of the fourth domain (D).

[0271] Item 44. The antibody construct of item 43, wherein a separate second binding domain (B) is fused to the N-terminus of a separate hinge of the fourth domain (D).

[0272] Item 45. The antibody construct of item 43, wherein the third binding domain (C) is fused to the N-terminus of another hinge of the fourth domain (D).

[0273] Item 46. The antibody construct of any one of items 7 to 24, wherein the first first binding domain and the second first binding domain (A1A2), which are fused to each other, are fused to the N-terminus of the first hinge of the fourth domain (D), and the third first binding domain and the fourth first binding domain (A3A4), which are fused to each other, are fused to the N-terminus of the second hinge of the fourth domain (D), and the second binding domain (B) is fused to the C-terminus of the CH3 domain of the fourth domain (D).

[0274] Item 47. The antibody construct of item 46, wherein a further second binding domain (B) is fused to the C-terminus of a further CH3 domain of the fourth domain (D).

[0275] Item 48. The antibody construct of item 47, wherein the third binding domain (C) is fused to the C-terminus of another CH3 domain of the fourth domain (D).

[0276] Item 49. The antibody construct of any one of items 7 to 24, wherein the first first binding domain and the second first binding domain (A1A2), which are fused to each other, are fused to the N-terminus of the first hinge-CH2-CH3 element of the fourth domain (D), and the third first binding domain and the fourth first binding domain (A3A4), which are fused to each other, are fused to the C-terminus of the second hinge-CH2-CH3 element of the fourth domain (D).

[0277] Item 50. The antibody construct of item 49, wherein the second binding domain (B) is fused to the C-terminus of the first hinge-CH2-CH3 element of the fourth domain (D).

[0278] Item 51. The antibody construct of item 50, wherein a separate second binding domain (B) is fused to the N-terminus of the second hinge-CH2-CH3 element of the fourth domain (D).

[0279] Item 52. The antibody construct of item 50, wherein the third binding domain (C) is fused to the N-terminus of the second hinge-CH2-CH3 element of the fourth domain (D).

[0280] Item 53. The antibody construct of item 49, wherein the second binding domain (B) is fused to the N-terminus of the second hinge-CH2-CH3 element of the fourth domain (D).

[0281] Item 54. The antibody construct of item 53, wherein the third binding domain (C) is fused to the C-terminus of the first hinge-CH2-CH3 element of the fourth domain (D).

[0282] Item 55. The first binding domain (A) is below: (a) CDR-H1 as depicted in SEQ ID NO: 77, CDR-H2 as depicted in SEQ ID NO: 78, CDR-H3 as depicted in SEQ ID NO: 79, CDR-L1 as depicted in SEQ ID NO: 80, CDR-L2 as depicted in SEQ ID NO: 81, and CDR-L3 as depicted in SEQ ID NO: 82; (b) CDR-H1 as set forth in SEQ ID NO: 83, CDR-H2 as set forth in SEQ ID NO: 84, CDR-H3 as set forth in SEQ ID NO: 85, CDR-L1 as set forth in SEQ ID NO: 86, CDR-L2 as set forth in SEQ ID NO: 87, and CDR-L3 as set forth in SEQ ID NO: 88; and (c) CDR-H1 as depicted in SEQ ID NO: 77, CDR-H2 as depicted in SEQ ID NO: 89, CDR-H3 as depicted in SEQ ID NO: 79, CDR-L1 as depicted in SEQ ID NO: 80, CDR-L2 as depicted in SEQ ID NO: 81, and CDR-L3 as depicted in SEQ ID NO: 82. A VH region comprising CDR-H1, CDR-H2, and CDR-H3 and a VL region comprising CDR-L1, CDR-L2, and CDR-L3 selected from 4. The antibody construct of any one of the preceding items.

[0283] Item 56. The antibody construct of any one of the preceding items, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 148, 149, 150 and 151, 152 and 153, 154 and 155, 156 and 157, 158 and 159, 160 and 161, 162 and 163, 180 to 183, 190, and 191 and 192.

[0284] Item 57. The antibody construct of any one of the preceding items, which induces a lower degree of NK cell fratricide in a cytotoxicity assay compared to a reference antibody having the heavy and light chain sequences of SEQ ID NO: 166 and SEQ ID NO: 167.

[0285] Item 58. The antibody construct of any one of the preceding items, which induces NK cell fratricide of 40% or less.

[0286] Item 59. The antibody construct of any one of the preceding items, having higher potency (lower EC50) in a cytotoxicity assay compared to a reference antibody having only two or only one first binding domain (A).

[0287] Item 60. The antibody construct of item 59, wherein the EC50 of the antibody construct is preferably measured using a target cell that highly expresses the second target (B') and has a value that is about 0.5 times or less than the EC50 of the reference antibody.

[0288] Item 61. The antibody construct of item 59, wherein the EC50 of the antibody construct is preferably measured using a target cell that low-expresses the second target (B') and has a value that is about 0.1 times or less than the EC50 of the reference antibody.

[0289] Item 62. Higher efficacy (higher E) in a cytotoxicity assay compared to a reference antibody having only two or only one first binding domain (A). max5. The antibody construct of any one of the preceding claims, comprising

[0290] Item 63. A nucleic acid molecule comprising a sequence encoding the antibody construct of any one of items 1 to 62.

[0291] Item 64. A vector comprising the nucleic acid molecule of Item 63.

[0292] Item 65. A host cell comprising the nucleic acid molecule of Item 63 or the vector of Item 64.

[0293] Item 66. A method for producing an antibody construct according to any one of items 1 to 62, comprising culturing a host cell according to item 65 under conditions allowing expression of the antibody construct according to any one of items 1 to 62, and optionally recovering the produced antibody construct from the culture.

[0294] Item 67. A pharmaceutical composition comprising the antibody construct of any one of items 1 to 62 or the antibody construct produced by the method of item 66.

[0295] Item 68. The antibody construct of any one of items 1 to 62 for use in therapy.

[0296] Item 69. The antibody construct of any one of items 1 to 62 or the antibody construct produced by the method of item 65 for use in the prevention, treatment, or amelioration of a disease selected from a proliferative disease, a tumor disease, a viral disease, or an immunological disorder.

[0297] Item 70. The antibody construct for use according to item 69, wherein the disease is a cancer or tumor, preferably a cancer or tumor that underexpresses the second target (B').

[0298] Item 71. The antibody construct for use according to item 69 or 70, wherein the disease comprises malignant cells that under-express the second target (B').

[0299] Item 72. The antibody construct for use according to any one of items 69 to 71, wherein the use prevents recurrence of the disease.

[0300] Item 73. A method for treating or ameliorating a proliferative disease, a neoplastic disease, a viral disease, or an immunological disorder, comprising administering to a subject in need thereof an antibody construct according to any one of items 1 to 62 or an antibody construct produced by the method of item 66.

[0301] Item 74. A method for simultaneously binding one target cell and one immune effector cell, comprising administering to a subject an antibody construct according to any one of items 1 to 62, wherein the target cell under-expresses a second target (B').

[0302] Item 75. A kit comprising the antibody construct of any one of items 1 to 62 or the antibody construct produced by the method of item 66, the nucleic acid molecule of item 63, the vector of item 64, and / or the host cell of item 65.

[0303] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context specifically dictates otherwise. Thus, for example, reference to "a reagent" includes one or more of such various reagents, and reference to "the method" includes reference to equivalent steps and methods known to those of skill in the art that may be modified or used in place of the methods described herein.

[0304] Unless otherwise specified, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0305] The term "and / or" whenever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by that term".

[0306] As used herein, the term "about" or "approximately" means within 10% (plus (+) or minus (-)), preferably within 5%, more preferably within 2%, and even more preferably within 1% of a given value or range. However, the term also includes the actual number, for example, about 20 includes 20.

[0307] The terms "less than" or "greater than" are inclusive of the actual number. For example, less than 20 means "less than" or "equal to." Similarly, "greater than" or "greater than" means "greater than" or "equal to," or "greater than" or "equal to," respectively.

[0308] Throughout this specification and the claims which follow, unless the context dictates otherwise, the word "comprise," and variations such as "comprises" and "comprising," are understood to mean the inclusion of a recited integer or step, or group of integers or steps, but not the exclusion of any other integers and steps or groups of integers and steps. As used herein, the term "comprising" can be replaced by the terms "containing" or "including," or, as sometimes used herein, the term "having."

[0309] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0310] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. For example, disclosure of the term "comprising" includes disclosure of the term "consisting essentially of" as well as disclosure of the term "consisting of."

[0311] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc. described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0312] All publications and patents cited throughout the text of this specification, whether supra or infra, including all patents, patent applications, scientific publications, manufacturer's specifications, instruction manuals, and the like, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention. To the extent that material incorporated by reference contradicts or is inconsistent with this specification, the present specification shall take precedence over any such material.

[0313] A further understanding of the present invention and its advantages will be gained from the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. EXAMPLES

[0314] Example 1: Expression and purification of antibody constructs Stable expression of antibody constructs was performed as described by Ellwanger et al. (MAbs. 2019 Jul;11(5):899-918). Duplexbody constructs were purified from clarified CHO cell culture supernatants in a two- or three-step procedure involving either Protein A combined with IMAC, Protein L, or C-tag combined with IMAC followed by preparative SEC, respectively. For Protein A, the clarified supernatant was loaded onto a HiTrap MabSelectSuRe column. 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 with 10 mM sodium acetate (pH 3.5) and 10 mM glycine / HCL (pH 2.0). For Protein L, the clarified supernatant was loaded onto a 5 mL HiTrap Protein L chromatography column. 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 with 10 mM glycine / HCL (pH 3.0) and 10 mM glycine / HCL (pH 2.0). In the case of C-tag, the clarified supernatant was loaded onto a CaptureSelect C-tag XL column. After washing with phosphate-buffered saline (pH 7.4), the protein was eluted with 20 mM sodium citrate (pH 3.0). In the case of IMAC, the fractions containing the target protein were loaded onto a HisTrap FF chromatography column. After washing with IMAC A buffer, the his-tagged target protein was eluted by washing sequentially with 25% IMAC B buffer and 100% IMAC B buffer. The purity of the fractions was analyzed using SE-HPLC and SDS-PAGE. Fractions showing acceptable purity were collected and subjected to preparative gel filtration using a Superdex 200 preparative grade column. Eluate fractions containing purified duplexbody constructs were collected, subjected to buffer exchange with 10 mM sodium acetate, 4.5% sorbitol (pH 5.0) using a Sephadex G-25 column, and concentrated by ultrafiltration.The final samples were evaluated by SDS-PAGE under reducing and non-reducing conditions (see Figure 2). Samples were mixed with non-reducing 2x SDS-PAGE sample buffer or with reducing 2x SDS-PAGE sample buffer containing dithiothreitol (DTT) as a reducing agent. All samples were heated at 95°C for 5 min and then loaded onto 4-20% Criterion TGX precast SDS Page gels. 2 μg of purified protein sample was used per lane. To separate these proteins in the gel, SDS-PAGE was run at 300 V for approximately 22 min in 1x Tris / glycine / SDS buffer. Total proteins were visualized in the gel using a Criterion unstained molecular imaging system (Biorad Bio-Rad). Page Ruler unstained protein ladder was used as a molecular weight marker. Purity (Bi-scDb-IgAb_06 (SEQ ID NO: 162 and SEQ ID NO: 163) approx. 93%, Bi-scDb-Fc_01 (SEQ ID NO: 148) approx. 92%, Bi-scDb-Fc_02 (SEQ ID NO: 149) approx. 94%, aBi-scDb-Fc_01 (SEQ ID NO: 150 and SEQ ID NO: 151) approx. 98%, aBi-scDb-Fc_02 (SEQ ID NO: 152 and SEQ ID NO: 153) approx. 98%, aBi-scDb-Fc_03 (SEQ ID NO: 154 and SEQ ID NO: 155) approx. 98%, aBi-scDb-Fc_04 (SEQ ID NO: 156 and SEQ ID NO: The purity of aBi-scDb-Fc_05 (SEQ ID NO: 157) approx. 97%, aBi-scDb-Fc_05 (SEQ ID NO: 158 and SEQ ID NO: 159) approx. 96%, and aBi-scDb-Fc_06 (SEQ ID NO: 160 and SEQ ID NO: 161) approx. 96%) was assessed by analytical SE-HPLC using a Superdex 200 Increase 10 / 300GL column. The purified proteins were stored in aliquots at -80°C until further use.

[0315] Example 2: Isolation of peripheral blood mononuclear cells (PBMCs) from buffy coat PBMCs were isolated from the buffy coat by density gradient centrifugation. Buffy coat samples were diluted with 2-3 volumes of PBS, layered on top of a layer of Lymphoprep (Stem Cell Technologies, Cat. No.: 7861), and centrifuged at 800×g without brake at room temperature for 25 min. PBMCs located at the interface were harvested and washed three times with PBS before being used for enrichment of PBMC subsets or flow cytometric analysis. In most cases, PBMCs were cultured overnight in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U / mL sodium penicillin G, and 100 μg / mL streptomycin sulfate (referred to herein as complete RPMI 1640 medium) at 37°C and 5% CO2 in a humidified atmosphere before being used for enrichment of NK cells.

[0316] Example 3: Enrichment of NK cells from human PBMCs and differentiation into macrophages NK cells were enriched from PBMCs using the EasySep™ NK Enrichment Kit for Immunomagnetic Isolation of Intact Human NK Cells (Stem Cell Technologies, Cat. No. 17955) according to the manufacturer's instructions. The purity of the NK cell isolation was measured by flow cytometry. For macrophage differentiation, PBMCs were discarded after overnight culture, whereas adherent mononuclear cells were used for the subsequent differentiation protocol. Complete RPMI1640 medium supplemented with human M-CSF (50 ng / mL final) was added to the monocytes and replenished every 5-6 days. Adherent macrophages were harvested for subsequent analysis using Accutase treatment after 1-4 weeks, depending on cell morphology, density, and proliferation.

[0317] Example 4: Cultivation of tumor cell lines The MCF-7 cell line was purchased from DSMZ (cat. no. ACC115) and cultured under standard conditions at 37°C and 5% CO2 in a humidified atmosphere in RPMI1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U / mL sodium penicillin G, 100 μg / mL streptomycin sulfate, and 1 mM sodium pyruvate as recommended by the supplier.

[0318] The Daudi cell line was purchased from DSMZ (catalog number ACC78) and cultured under standard conditions at 37°C and 5% CO2 in a humidified atmosphere in RPMI1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U / mL sodium penicillin G, and 100 μg / mL streptomycin sulfate as recommended by the supplier.

[0319] The MM.1S cell line was purchased from ATCC (catalog number CRL-2974) and cultured under standard conditions at 37°C and 5% CO2 in a humidified atmosphere in RPMI1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U / mL sodium penicillin G, and 100 μg / mL streptomycin sulfate as recommended by the supplier.

[0320] The DK-MG cell line was purchased from DSMZ (catalog number ACC277) and cultured under standard conditions at 37°C and 5% CO2 in a humidified atmosphere in RPMI1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U / mL sodium penicillin G, and 100 μg / mL streptomycin sulfate as recommended by the supplier.

[0321] The HCT-116 cell line was purchased from DSMZ (cat. no. ACC581) and cultured under standard conditions at 37°C and 5% CO2 in a humidified atmosphere in RPMI1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U / mL sodium penicillin G, 100 μg / mL streptomycin sulfate as recommended by the supplier.

[0322] Example 5: Quantification of specific antibody binding capacity as a measure of antigen expression levels in tumor cell lines The densities of HER2 and EGFR present on the surface of various cell lines were measured using the QIFIKIT (Dako) and anti-HER2 mAb MAB 1129 (RnD Systems) or anti-EGFR mAb H11 (Dianova) according to the manufacturer's instructions. 6 Aliquots of cells were stained with mAb MAB 1129 or mAb H11, followed by F(ab')2 fragments of FITC-conjugated goat anti-mouse IgG. As a negative control, 1 × 10 6 Aliquots of cells were stained with mAb 9E10 (Acris) followed by F(ab')2 fragment of FITC-conjugated goat anti-mouse IgG. To calculate the specific antibody binding capacity, calibration beads containing five bead populations with different numbers of mAb molecules were stained with F(ab')2 fragment of FITC-conjugated goat anti-mouse IgG. A standard curve was generated from the resulting median fluorescence intensity. This standard curve was used to calculate the specific antibody binding capacity (SABC) of mAbs MAB 1129 and mAb H11 for the various cell lines. The HER2 density and EGFR density (SABC) in Table 1 represent the average value of at least two independent experiments. The SABC value measured with anti-HER2 mAb MAB 1129 and the HER2 IHC score were used to generate an artificial EGFR score based on the SABC value measured with anti-EGFR mAb H11 (Table 1). Scoring of tumor cell lines for HER2 and EGFR expression based on specific antibody binding capacity (SABC) is shown in FIG.

[0323] Table 1. Specific antibody binding ability (SABC) measured using anti-HER2 mAb MAB 1129 and anti-EGFR mAb H11 against various tumor cell lines, and scoring of expression levels. nd: not measured. TIFF2024529381000002.tif56153

[0324] Example 6: Calcein release cytotoxicity assay against tumor target cells using NK cells as effector cells (E:T=5:1) in the presence of increasing concentrations of different antibody constructs For calcein release cytotoxicity assay, target cells were labeled with 10 μM of the fluorescent dye calcein AM (Invitrogen, Cat. No.: C3100MP) for 30 min and then incubated with 1 × 10 4 Aliquots of labeled target cells were seeded in duplicate, unless otherwise indicated, into individual wells of round-bottom 96-well microplates at a 5:1 effector:target (E:T) ratio with freshly isolated and enriched primary human NK cells in the presence of 12 1:5 dilutions of the indicated antibodies, usually starting at 30 μg / mL. Control samples for measuring spontaneous release, maximum release, and antibody-independent lysis by effector cells were tested in four replicates. After 4 h of incubation, 100 μL of cell-free culture supernatant was collected from each well and fluorescent calcein released from lysed target cells was quantified using a multiplate fluorescence reader. After the fluorescence of spontaneously lysed cells was subtracted from all samples, the fluorescence of each sample was normalized to the fluorescence of completely lysed cells to account for specific lysis for each sample. The mean and standard deviation (SD) of specific target cell lysis (%) were plotted and in vitro potency (EC 50 ) and efficacy (E max ) was revealed.

[0325] Example 7: A 4-hour calcein release assay to assess NK cell fratricide induced by increasing concentrations of various antibody constructs For the calcein-release NK cell fratricide assay, enriched primary human NK cells were labeled with 10 μM of the fluorescent dye calcein AM for 30 min and then incubated with 5 × 10 4Aliquots of labeled cells were seeded in duplicate into individual wells of round-bottom 96-well microplates at a 1:1 effector:target (E:T) ratio with unlabeled enriched autologous NK cells in the presence of ten 1:5 dilutions starting at 100 μg / mL of the indicated antibodies. Anti-CD38 IgG1 with Fab domain derived from daratumumab (IgAb_51, SEQ ID NO: 166 and SEQ ID NO: 167) was used as a positive control. Control samples for measuring spontaneous release, maximum release, and antibody-independent lysis by effector cells were tested in four replicates. After 4 hours of incubation, 100 μL of cell-free culture supernatant was collected from each well and fluorescent calcein released from lysed target cells was quantified using a multiplate fluorescence reader. After subtracting the fluorescence of spontaneously lysed cells from all samples, the fluorescence of each sample was normalized to the fluorescence of completely lysed cells to determine specific lysis for each sample. Means and standard deviations (SD) of specific target cell lysis (%) were plotted and in vitro potency (EC 50 ) and efficacy (E max ) was revealed.

[0326] Example 8: Phagocytosis assay against tumor target cells using macrophages as effector cells (E:T=5:1) in the presence of increasing concentrations of different antibody constructs For phagocytosis assays, macrophages were seeded in 96-well UpCell plates and cultured overnight. Target cells were labeled with 0.5 μM CellTracker™ Green CMFDA dye for 30 min at 37°C, washed, and cultured overnight. Target cells were overlaid on top of macrophages (E:T ratio of 5:1), and the indicated antibodies were added in duplicate at graded concentrations (0.3 pg / mL to 30 μg / mL). After 4 h of incubation, cells were detached from the culture plate by incubation on ice and stained with A700-labeled anti-CD11b and fixable viability dye eF780 for 30 min at 4°C. Phagocytosis of labeled target cells was assessed by CMFDA staining of live cells. + / CD11b + The percentage of cells was quantified by flow cytometry analysis. ADCP in the absence of antibody was used for normalization. Furthermore, the reduction of labeled target cells was measured by the ratio of CMFDA to live cells. + / CD11b - The percentage of cells was quantified by analysis by flow cytometry. The reduction of target cells in the absence of antibody was used for normalization.

[0327] Example 9: Potency and efficacy of EGFR-targeted natural cell engagers in four cytotoxicity assays against MCF-7 target cells expressing low levels of EGFR The in vitro ADCC activity of multivalent anti-CD16A natural cell engagers targeting EGFR (Bi-scDb-Fc_02 (SEQ ID NO: 149), aBi-scDb-Fc_05 (SEQ ID NO: 158 and SEQ ID NO: 159), Bi-scDb-IgAb_06 (SEQ ID NO: 162 and SEQ ID NO: 163)) was compared with bivalent anti-EGFR engagers (scFv-IgAb_43 (SEQ ID NO: 174 and SEQ ID NO: 175), scFv-IgAb_167 (SEQ ID NO: 178 and SEQ ID NO: 179)), and Fc-enhanced (S239D / I332E) anti-EGFR IgG1 (IgAb_53 (SEQ ID NO: 168 and SEQ ID NO: 169)). To compare the results of the EGFR-specific IgG1-specific IgG1-specific IgG1-specific IgG2 ... In summary, these results indicate that multivalent CD16A engagement is advantageous not only in terms of the potency of natural cell engagers to induce ADCC, but also in terms of efficacy in NK cell-mediated target cell lysis.

[0328] Table 2. Potency and efficacy of anti-EGFR antibody constructs as measured in a cytotoxicity assay against MCF-7 target cells. Efficacy (EC 50 ) and efficacy (E max) was measured in a calcein release cytotoxicity assay against MCF-7 target cells using NK cells as effector cells at an E:T ratio of 5:1. Mean values ​​of three independent experiments are shown. TIFF2024529381000003.tif92153

[0329] Example 10: Potency and efficacy of EGFR-targeted natural cell engagers in a 4-hour cytotoxicity assay against Daudi target cells expressing very low levels of EGFR To assess the impact of multivalent CD16A engagement on the ADCC activity of EGFR-targeting natural cell engagers, a 4-hour calcein release cytotoxicity assay was performed on Daudi target cells expressing very low levels of EGFR (mean SABC:868) at a 5:1 E:T ratio and enriched primary human NK cells as effector cells. Using assays performed as described in Example 6, multivalent anti-CD16A constructs (Bi-scDb-Fc_02 (SEQ ID NO: 149), aBi-scDb-Fc_05 (SEQ ID NO: 158 and SEQ ID NO: 159), Bi-scDb-IgAb_06 (SEQ ID NO: 162 and SEQ ID NO: 163)) were compared with constructs containing two anti-CD16A domains (scFv-IgAb_43 (SEQ ID NO: 174 and SEQ ID NO: 175) and scFv-IgAb_167 (SEQ ID NO: 178 and SEQ ID NO: 179)) and with Fc-enhanced (S239D / I332E) anti-EGFR in three independent assays with NK cells from different blood donors. IgG1 (IgAb_53 (SEQ ID NO: 168 and SEQ ID NO: 169)). The results summarized in Table 3 and the exemplary graph in FIG. 4 demonstrate the superior potency and efficacy of the multivalent anti-CD16A constructs compared to bivalent anti-CD16A scFv-IgAb constructs or Fc-enhanced anti-EGFR IgG1. Notably, aBi-scDb-Fc_05, which has only one Fv domain against EGFR, shows approximately half the potency compared to other constructs containing four anti-CD16A domains and two anti-EGFR domains. These results clearly demonstrate the advantages of multivalent anti-CD16A engagement and bivalent tumor targeting for natural cell engagers that mediate ADCC.

[0330] Table 3. Potency and efficacy of anti-EGFR antibody constructs measured in a cytotoxicity assay against Daudi target cells. Efficacy (EC 50 ) and efficacy (Emax ) was measured in a calcein release cytotoxicity assay against Daudi target cells using NK cells as effector cells at an E:T ratio of 5:1. Mean values ​​of three independent experiments are shown. TIFF2024529381000004.tif91153

[0331] Example 11: Evaluation of NK cell fratricide mediated by multivalent anti-CD16A engagers in a 4-hour calcein release cytotoxicity assay To assess whether the multivalent anti-CD16A natural cell engager targeting EGFR has the ability to crosslink NK cells with other NK cells, thereby inducing NK cell fratricide, a 4-hour calcein release assay was performed using enriched primary human NK cells as target cells and autologous NK cells as effector cells, as described in Example 7. The independent assay summary in Table 4 and the exemplary graph in Figure 5 show the potent and effective NK cell fratricide induced by anti-CD38 IgG1 with a Fab domain derived from daratumumab (IgAb_51 (SEQ ID NO: 166 and SEQ ID NO: 167)), which was used as a positive control. Fc-enhanced anti-EGFR IgG1 (IgAb_53 (SEQ ID NO: 168 and SEQ ID NO: 169)) and scFv-IgAbs (scFv-IgAb_43 (SEQ ID NO: 174 and SEQ ID NO: 175) and scFv-IgAb_167 (SEQ ID NO: 178 and SEQ ID NO: 179)) did not induce NK-NK cell lysis or induced only slight NK-NK cell lysis (mean E max Multivalent natural cell engagers containing four anti-CD16A Fv domains induced NK cell fratricide with slightly higher efficacy. However, compared to anti-CD38 IgG1, multivalent anti-CD16A engagers induced a lower degree of NK cell fratricide (mean E max :<30%) was induced only at high antibody concentrations (>100 pM).

[0332] Table 4. Potency and efficacy of anti-EGFR antibody constructs as measured in the NK cell fratricide assay. Efficacy (EC 50 ) and efficacy (E max ) was measured in a 4-h calcein release NK cell fratricide assay using enriched primary human NK cells as target and effector cells at an E:T ratio of 1:1. Mean values ​​of three independent experiments are shown. na, not applicable; § was determined in two independent assays. TIFF2024529381000005.tif105153

[0333] Example 12: 4-hour phagocytosis assay on DK-MG cells To assess the effect of multivalent CD16A engagement on the ADCP activity of EGFR-targeting natural cell engagers, a 4-h phagocytosis assay was performed on DK-MG target cells expressing high levels of EGFR (mean SABC:222648) and monocyte-derived human macrophages as effector cells at an E:T ratio of 5:1. Multivalent anti-CD16A constructs (Bi-scDb-Fc_02 (SEQ ID NO: 149), aBi-scDb-Fc_05 (SEQ ID NO: 158 and SEQ ID NO: 159), Bi-scDb-IgAb_06 (SEQ ID NO: 162 and SEQ ID NO: 163)) were compared with a construct containing two anti-CD16A domains (scFv-IgAb_43 (SEQ ID NO: 174 and SEQ ID NO: 175)) and with Fc wild type anti-EGFR IgG1 (IgAb_49 (SEQ ID NO: 164 and SEQ ID NO: 166)) in three independent assays with macrophages generated from monocytes of different blood donors using the assay performed as described in Example 8. 165)) and Fc-enhanced (S239D / I332E) anti-EGFR IgG1 (IgAb_53 (SEQ ID NO: 168 and SEQ ID NO: 169)). The representative exemplary graphs in FIG. 6 demonstrate that the multivalent anti-CD16A constructs induced higher phagocytosis compared to bivalent anti-CD16A scFv-IgAb constructs, Fc wild-type anti-EGFR IgG1 antibodies, or Fc-enhanced anti-EGFR IgG1 antibodies. The lowest degree of phagocytosis was induced by IgAb_49 and Fc-enhanced anti-EGFR IgG (IgAb_53). These results clearly demonstrate the advantage of multivalent anti-CD16A engagement for natural cell engagers that mediate ADCP.

[0334] Example 13: 4-hour phagocytosis assay on MCF-7 cells To assess the effect of multivalent CD16A engagement on the ADCP activity of EGFR-targeting natural cell engagers, a 4-h phagocytosis assay was performed on MCF-7 target cells expressing low levels of EGFR (mean SABC: 4,546) and monocyte-derived human macrophages as effector cells at a 5:1 E:T ratio. Multivalent anti-CD16A constructs (Bi-scDb-Fc_02 (SEQ ID NO: 149), aBi-scDb-Fc_05 (SEQ ID NO: 158 and SEQ ID NO: 159), Bi-scDb-IgAb_06 (SEQ ID NO: 162 and SEQ ID NO: 163)) were compared with a construct containing two anti-CD16A domains (scFv-IgAb_43 (SEQ ID NO: 174 and SEQ ID NO: 175)) and with Fc wild type anti-EGFR IgG1 (IgAb_49 (SEQ ID NO: 164 and SEQ ID NO: 166)) in three independent assays with macrophages generated from monocytes of different blood donors using the assay performed as described in Example 8. The multivalent anti-CD16A constructs were compared to the multivalent anti-CD16A construct IgAb_43 (SEQ ID NO: 165)) and to the Fc-enhanced (S239D / I332E) anti-EGFR IgG1 (IgAb_53 (SEQ ID NO: 168 and SEQ ID NO: 169)). The representative exemplary graphs in FIG. 7 demonstrate that the multivalent anti-CD16A constructs induce phagocytosis. In contrast, scFv-IgAb_43, IgAb_49, and IgAb_53 did not induce phagocytosis of MCF-7 target cells by macrophages. These results clearly demonstrate the advantage of multivalent anti-CD16A engagement over ADCP-mediated natural cell engagers.

[0335] Example 14: 4-hour calcein release assay for A-431 To compare the in vitro ADCC activity of the EGFR-targeting multivalent anti-NKp46 natural cell engager (AIG-2scDb_06 (SEQ ID NOs: 180-183)) with the bivalent anti-HER2 engager (AIG-2scFv_27 (SEQ ID NOs: 184-187)), a 4-hour calcein release cytotoxicity assay was performed against A-431 target cells expressing low levels of HER2 (average SABC: 6,150) using primary enriched human NK cells as effector cells at an E:T ratio of 5:1 as described in Example 6. The representative graph in FIG. 8 clearly demonstrates the superior potency and efficacy of the multivalent natural cell engager with four anti-NKp46 Fv domains compared to the bivalent anti-NKp46 construct. In summary, these results indicate that multivalent NKp46 engagement is advantageous not only in terms of the potency of natural cell engagers to induce ADCC, but also in terms of efficacy in NK cell-mediated target cell lysis.

[0336] Example 15: ELISA studies of binding of EGFR / CD16A engagers or BCMA / CD16A engagers to CD16A To assess the binding of EGFR / CD16 or BCMA / CD16 antibodies to the coated antigen in an ELISA, 96-well ELISA plates (Immuno Maxisorp, Nunc) were coated overnight at 4° C. with recombinant CD16 antigen variants fused to monomeric human Fc at a concentration of 1.5 μg / mL in 100 mM carbonate-bicarbonate buffer. After blocking with 3% (w / v) nonfat dry milk in phosphate-buffered saline (PBS), serial dilutions of bispecific antibody constructs were incubated on the antigen-coated plates for 1.5 hours at room temperature. After washing three times with 300 μL / well of PBS containing 0.1% (v / v) Tween 20, the plates were incubated with 5 μg / mL of anti-AFM24 mAb 62-1-1 for 1 h, followed by washing and detection with peroxidase-conjugated goat anti-mouse IgG (H+L)-HRPO (MinX Hu,Bo,Ho) diluted 1:10,000 in PBS containing 0.3% (w / v) nonfat dry milk. After washing, the plates were incubated with tetramethylbenzidine substrate (Seramun) for 1–2 min. The reaction was stopped by adding 0.5 M H2SO4 (100 μL / well). Absorbance at 450 nm was measured using a multiwell plate reader, plotted, and EC was calculated by fitting a nonlinear regression model to a sigmoidal dose-response curve (four-parameter logistic fit) using GraphPad Prism software. 50 The value was calculated.

[0337] To assess binding of soluble CD16A antigen to coated or antigen-captured EGFR / CD16 or BCMA / CD16 antibodies, 96-well ELISA plates (Immuno Maxisorp, Nunc) were coated overnight at 4° C. with various multivalent bispecific engagers at concentrations of 3.5-5 μg / mL (equivalent to 24-27 nM) in 100 mM carbonate-bicarbonate buffer. For the capture approach, 96-well ELISA plates (Immuno Maxisorp, Nunc) were coated overnight at 4° C. with His-tagged human EGFR extracellular domain or His-tagged BCMA extracellular domain at concentrations of 3.0 μg / mL or 0.4 μg / mL, respectively. After blocking with 3% (w / v) nonfat dry milk dissolved in phosphate-buffered saline (PBS), the antigen-coated plates were used to capture various multivalent bispecific engagers at concentrations of 3–5 μg / mL mixed in PBS containing 0.3% (w / v) nonfat dry milk. After washing three times with 300 μL / well of PBS containing 0.1% (v / v) Tween 20, the plates were incubated for 1.5 h at room temperature with serial dilutions of biotinylated dimeric or monomeric CD16A antigen diluted in PBS containing 0.3% (w / v) nonfat dry milk. After washing, the plates were incubated for 1 h with the detection conjugate streptavidin-HRP diluted 1:10,000 in PBS containing 0.3% (w / v) nonfat dry milk, followed by washing and incubation with tetramethylbenzidine substrate (Seramun) for 1–2 min. The reaction was stopped by adding 0.5 M H2SO4 (100 μL / well). The absorbance at 450 nm was measured using a multiwell plate reader, plotted, and the EC was calculated by fitting a nonlinear regression model to a sigmoidal dose-response curve (four-parameter logistic fit) using GraphPad Prism software. 50 The value was calculated.

[0338] The ELISA results summarized in Table 5 show overall comparable binding strengths of bivalent and tetravalent CD16 binding engager constructs to the CD16A antigen in ELISA using various assay configurations.

[0339] Table 5. Half-maximal binding values ​​for CD16 binding of tetravalent or bivalent CD16 binding constructs targeting EGFR or BCMA were analyzed in ELISA. Concentration-dependent binding of antibody constructs to coated recombinant CD16A antigen or soluble monomeric or dimeric CD16 antigen variants to coated or target antigen-captured antibody constructs was analyzed in ELISA. Half-maximal binding concentrations (EC) were calculated by fitting a nonlinear regression model to a sigmoidal dose-response curve (four-parameter logistic fit) using GraphPad Prism software. 50 ) was sought. TIFF2024529381000006.tif131152TIFF2024529381000007.tif154152

[0340] Example 16: BCMA using bispecific NK cell engagers with multivalent CD16A binding capacity + Cytotoxicity Assay for Target Cells To assess the potency and efficacy of multivalent BCMA-directed NK cell engagers, and concomitantly, the specificity of EGFR-targeted NK cell engagers, calcein-labeled BCMA at a 5:1 E:T ratio was administered in the presence of increasing concentrations of the indicated constructs essentially as described in Example 6. + / EGFR - A 4-hour calcein release cytotoxicity assay was performed on MM.1S target cells and enriched NK cells as effector cells. The results of the assay, summarized in Table 6 and shown in the exemplary graph in Figure 9, demonstrate potent (EC50 range of 1.0 pM to 2.4 pM) inhibition of MM.1S target cells mediated by BCMA-specific engagers in a concentration-dependent manner. 50value) and valid (E in the range of 46.4% to 62.8%) max Despite multivalent binding to CD16A NK cells, EGFR-targeted engagers do not express EGFR - did not induce lysis of MM.1S target cells, demonstrating the specificity of the multivalent anti-CD16A natural cell engager in mediating lysis of target cells by NK cells only if the target antigen was expressed on the target cell.

[0341] (Table 6) BCMA + Potency and efficacy of multivalent antibody constructs measured in a 4-hour cytotoxicity assay against MM.1S target cells Efficacy (EC 50 ) and efficacy (E max ) was measured in a calcein release cytotoxicity assay against calcein-labeled MM.1S target cells using enriched primary human NK cells as target and effector cells at an E:T ratio of 5:1. Mean values ​​of three independent experiments are shown. na, not applicable. TIFF2024529381000008.tif116152

[0342] Example 17: Efficacy of EGFR-targeted natural cell engagers in a 4-hour cytotoxicity assay against Daudi target cells expressing very low levels of EGFR The evaluation of ADCC activity by multivalent CD16A engagement was extended to a comparison of two different sequences targeting CD16A using a 4-hour calcein release cytotoxicity assay against Daudi target cells as described in Example 10, in six independent assays with NK cells from different blood donors, using multivalent anti-CD16A constructs (Bi-scDb-Fc_02 (SEQ ID NO: 149), scFv-Fc-scDb_04 (SEQ ID NO: 190) together with a construct containing two anti-CD16A domains (scFv-IgAb_43 (SEQ ID NO: 174 and SEQ ID NO: 175)). The results summarized in Table 7 confirm the superior efficacy of multivalent anti-CD16A constructs compared to bivalent anti-CD16A scFv-IgAb constructs.

[0343] Table 7. Efficacy of anti-EGFR antibody constructs measured in a cytotoxicity assay against Daudi target cells Effectiveness (E max ) was measured in a calcein release cytotoxicity assay against Daudi target cells using NK cells as effector cells at an E:T ratio of 5:1. Mean values ​​of three independent experiments are shown. TIFF2024529381000009.tif50128

[0344] Example 18: 4-hour phagocytosis assay on HCT-116 cells To further demonstrate the effect of multivalent CD16A engagement on the ADCP activity of EGFR-targeting natural cell engagers, a 4-hour phagocytosis assay was performed on HCT-116 target cells (mean SABC: 33,822), which express intermediate levels of EGFR. As described in Example 8, the multivalent anti-CD16A constructs (Bi-scDb-Fc_02 (SEQ ID NO: 149), aBi-scDb-Fc_05 (SEQ ID NO: 158 and SEQ ID NO: 159), Bi-scDb-IgAb_06 (SEQ ID NO: 162 and SEQ ID NO: 163)) were compared with a construct containing two anti-CD16A domains (scFv-IgAb_43 (SEQ ID NO: 174 and SEQ ID NO: 175)), Fc wild-type anti-EGFR IgG1 (IgAb_49 (SEQ ID NO: 164 and SEQ ID NO: 165)), and Fc-enhanced (S239D / I332E) anti-EGFR IgG1 (IgAb_49 (SEQ ID NO: 164 and SEQ ID NO: 165)) in three independent assays using macrophages generated from monocytes of different blood donors. (IgAb_53 (SEQ ID NO: 168 and SEQ ID NO: 169)). The representative exemplary graphs in FIG. 11A demonstrate phagocytosis induction by multivalent anti-CD16A constructs. In contrast, scFv-IgAb_43, IgAb_49, and IgAb_53 showed only low levels of phagocytosis of HCT-116 target cells by macrophages. Furthermore, the representative exemplary graphs in FIG. 11B show a concentration-dependent reduction of HCT-116 target cells mediated by multivalent anti-CD16A constructs measured at the end of a 4-hour co-culture with macrophages. In contrast, there was no concentration-dependent reduction of HCT-116 target cells in the presence of scFv-IgAb_43, IgAb_49, and IgAb_53. These results clearly demonstrate the advantage of multivalent anti-CD16A engagement over ADCP-mediated natural cell engagers.

[0345] Example 19: Efficacy of CD19-targeted natural cell engagers in a 4-hour cytotoxicity assay against Daudi target cells To assess the effect of multivalent CD16A engagement on the ADCC activity of CD19-targeting natural cell engagers, a 4-hour calcein release cytotoxicity assay was performed on Daudi target cells expressing high levels of CD19 and enriched primary human NK cells as effector cells at an E:T ratio of 5:1. Using the assay performed as described in Example 6, a multivalent anti-CD16A construct (IG-scDb_10 (SEQ ID NO: 191 and SEQ ID NO: 192)) was compared to a construct containing two anti-CD16A domains (scFv-IgAb_398 (SEQ ID NO: 193 and SEQ ID NO: 194)) in two independent assays with NK cells from different blood donors. Representative exemplary graphs in Figure 12 demonstrate the superior potency and efficacy of multivalent anti-CD16A constructs compared to bivalent anti-CD16A scFv-IgAb constructs. The results clearly demonstrate the advantages of multivalent anti-CD16A binding and bivalent tumor targeting over natural cell engagers that mediate ADCC.

[0346] Array Table TIFF2024529381000010.tif224153TIFF2024529381000011.tif236153TIFF2024529381000012.tif225153TIFF2024529381000013.tif239153TIFF2024529381000014.tif237153TIFF2024529381000015.tif231153TIFF2024529381000016.tif239153TIFF2024529381000017.tif231153TIFF2024529381000018.tif206153TIFF2024529381000019.tif206153TIFF2024529381000020.tif234153TIFF2024529381000021.tif228153TIFF2024529381000022.tif190153TIFF2024529381000023.tif203153TIFF2024529381000024.tif234153TIFF2024529381000025.tif73153

[0347] The embodiments illustratively described herein can be suitably implemented in the absence of any element or elements, or any limitation not specifically disclosed herein. Thus, the terms and expressions used herein are used as descriptive terms, not limiting, and when using such terms and expressions, there is no intention to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, although the embodiments of the invention are specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention. Also, each of the subspecies and subgeneric groups falling within the scope of the general disclosure also forms part of the invention. This includes the general description of the invention with a condition or negative limitation that excludes any subject matter from the genus, whether or not the subject matter to be removed is specifically recited herein. Furthermore, when a feature is described in terms of a Markush group, the disclosure will also be recognized by those skilled in the art that it is also described in terms of any individual member or subgroup of members of the Markush group.

[0348] Equivalents: Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims. Further embodiments will be apparent from the appended claims.

Claims

1. below: (i) at least four first binding domains (A), which are capable of specifically binding to a first target (A'), which is an immunomodulatory antigen present on the surface of an innate immune effector cell, wherein the immune effector cell is a natural killer cell or a macrophage; (ii) a second binding domain (B), which is capable of specifically binding to a second target (B'), which is an antigen present on the surface of a target cell; and (iii) a fourth domain (D) containing a half-life extending domain containing two CH3 domains; An antibody construct comprising: The first and second first binding domains (A1A2), which are fused to each other, are fused to the C-terminus of the first CH3 domain of the fourth domain (D), while the third and fourth first binding domains (A3A4), which are fused to each other, are fused to the C-terminus of the second CH3 domain of the fourth domain (D); Antibody construct.

2. 2. The antibody construct of claim 1, wherein the first target (A') is selected from the group consisting of CD16A, CD56, NKG2A, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 (CD226), SLAMF7 (CD319), CD244 (2B4), OX40, CD47, SIRPα, CD89, CD96, CD137, CD160, TIGIT, Nectin-4, PD-1, PD-L1, LAG-3, CTLA-4, TIM-3, KIR2DL1-5, KIR3DL1-3, KIR2DS1-5, KIR3DS1, and CD3.

3. a third binding domain (C) capable of specifically binding to a third target (C') other than the second target (B'), which is an antigen present on the surface of a target cell; 2. The antibody construct of claim 1, comprising:

4. The antibody construct of claim 1, wherein the second binding domain (B) comprises the VH and VL domains of an antibody.

5. 2. The antibody construct of claim 1, wherein the second target (B') is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD52, CD70, CD74, CD79b, CD123, CLL1, BCMA, FCRH5, EGFR, EGFRvIIIl, HER2, and GD2.

6. 2. The antibody construct of claim 1, wherein the first binding domain (A) comprises the VH and VL domains of an antibody.

7. 2. The antibody construct of claim 1, wherein the first target (A') is CD16A.

8. 2. The antibody construct of claim 1, wherein the first binding domain (A) binds to an epitope on CD16A that is C-terminal to the physiological Fcγ receptor binding domain, said epitope preferably comprising Y158 of SEQ ID NO:

13.

9. 2. The antibody construct of claim 1, wherein the second binding domain (B) is fused to the N-terminus of the hinge of the fourth domain (D).

10. 10. The antibody construct of claim 9, wherein a separate second binding domain (B) is fused to the N-terminus of a separate hinge of the fourth domain (D).

11. The first binding domain (A) comprises: below: (a) CDR-H1 as set forth in SEQ ID NO: 77, CDR-H2 as set forth in SEQ ID NO: 78, CDR-H3 as set forth in SEQ ID NO: 79, CDR-L1 as set forth in SEQ ID NO: 80, CDR-L2 as set forth in SEQ ID NO: 81, and CDR-L3 as set forth in SEQ ID NO: 82; (b) CDR-H1 as set forth in SEQ ID NO: 83, CDR-H2 as set forth in SEQ ID NO: 84, CDR-H3 as set forth in SEQ ID NO: 85, CDR-L1 as set forth in SEQ ID NO: 86, CDR-L2 as set forth in SEQ ID NO: 87, and CDR-L3 as set forth in SEQ ID NO: 88; and (c) CDR-H1 as set forth in SEQ ID NO: 77, CDR-H2 as set forth in SEQ ID NO: 89, CDR-H3 as set forth in SEQ ID NO: 79, CDR-L1 as set forth in SEQ ID NO: 80, CDR-L2 as set forth in SEQ ID NO: 81, and CDR-L3 as set forth in SEQ ID NO:

82. a VH region comprising CDR-H1, CDR-H2, and CDR-H3 and a VL region comprising CDR-L1, CDR-L2, and CDR-L3 selected from 2. The antibody construct of claim 1, comprising:

12. 2. The antibody construct of claim 1, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 148, 149, 150 and 151, 152 and 153, 154 and 155, 156 and 157, 158 and 159, 160 and 161, 162 and 163, and 180 to 183, 190, and 191 and 192.

13. A nucleic acid molecule comprising a sequence encoding the antibody construct of any one of claims 1 to 12, or a vector comprising said nucleic acid molecule.

14. A host cell comprising the nucleic acid molecule or vector of claim 13.

15. 13. A method for producing an antibody construct according to any one of claims 1 to 12, comprising culturing a host cell comprising a nucleic acid molecule comprising a sequence encoding said antibody construct, or a vector comprising said nucleic acid molecule, under conditions allowing expression of said antibody construct, and optionally recovering the produced antibody construct from the culture.

16. A pharmaceutical composition comprising the antibody construct of any one of claims 1 to 12.

17. A pharmaceutical composition comprising the antibody construct of any one of claims 1 to 12 for the prevention, treatment or amelioration of a disease selected from a proliferative disease, a neoplastic disease, a viral disease or an immunological disorder.

18. A kit comprising the antibody construct of any one of claims 1 to 12, a nucleic acid molecule comprising a sequence encoding said antibody construct, a vector comprising said nucleic acid molecule, and / or a host cell comprising said nucleic acid molecule or said vector.

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