VARIABLE REGIONS FOR NKp46 BINDING PROTEINS

Abstract

To provide NK cell specific antibodies applicable as bispecific antibodies, specifically NK cell specific antibodies with clearly defined new mechanisms of action capable of delivering benefits and suitable for use in addition to full length antibodies.SOLUTION: Provided are NKp46-binding immunoglobulin variable regions, and proteins, such as antibodies and multispecific proteins, that comprise the variable regions. The proteins can bind and specifically redirect NK cells to lyse a target cell of interest. The proteins have utility in the treatment of disease, notably cancer or infectious disease.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 271,474, filed December 28, 2015, the entire contents of which are incorporated by reference herein, including all figures and sequence listings.

[0002] Sequence Listing Reference This application is filed together with an electronic sequence listing, which is provided in a file entitled "NKp46-7 PCT_ST25 txt", created on December 19, 2016, and having a size of 376 KB. The information in the electronic format of this sequence listing is incorporated herein by reference in its entirety.

[0003] Immunoglobulin variable regions and proteins, such as antibodies and multispecific proteins, that include the variable regions are provided that can bind to and specifically redirect NK cells to lyse target cells of interest, and that are useful for the treatment of disease, particularly cancer or infectious disease. [Background technology]

[0004] Bispecific antibodies that bind to two different epitopes provide the opportunity to increase specificity, extend potency, and utilize novel mechanisms of action that cannot be achieved with conventional monoclonal antibodies. Various forms of bispecific antibodies that bind to two targets simultaneously have been reported. Also, using bispecific antibodies to crosslink two different receptors to inhibit signaling pathways has shown utility in various applications (see, for example, Non-Patent Document 1). Bispecific antibodies have been used to neutralize two different receptors. In another approach, bispecific antibodies are used to recruit immune effector cells, where activation of T cells is achieved in the vicinity of tumor cells by bispecific antibodies that simultaneously bind receptors on two different cell types (see, Non-Patent Document 2). Approaches developed to date have primarily involved bispecific antibodies that bind the CD3 complex on T cells to tumor-associated antigens. However, in another example, bispecific antibodies have been developed in which one arm binds CD16 (FcγRIIIa) and the other arm binds an antigen of interest, e.g., CD19 (see, Non-Patent Document 3).

[0005] Natural killer (NK) cells are a subpopulation of lymphocytes involved in non-conventional immunity. NK cells provide an efficient immunological surveillance mechanism that can eliminate unwanted cells, e.g., tumor or virus-infected cells. The characteristics and biological properties of NK cells include the expression of surface antigens including CD16, CD56, and / or CD57, the absence of α / β or γ / δ TCR complexes on the cell surface, the ability to bind to and kill cells that fail to express "self" MHC / HLA antigens by activation of specific cytolytic enzymes, the ability to kill tumor cells or other abnormal cells that express ligands for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or suppress immune responses.

[0006] NK cell activity is regulated by a complex mechanism involving both activating and inhibitory signals. Several different NK cell receptors have been identified that play important roles in NK cell-mediated recognition and killing of HLA class I-deficient target cells. One receptor is FcγR3a (CD16), which is not specific for NK cells but is involved in NK cell-mediated ADCC. Another NK cell receptor is NKp46, a member of the Ig superfamily. NKp46 is specific for NK cells and its cross-linking induced by certain mAbs increases the intracellular Ca ++ This results in strong NK cell activation that increases the levels of Fcγ-positive hematological malignancies, thereby inducing cytotoxicity and releasing lymphokines. (Patent Document 1) (Innate Pharma) discloses the use of a monospecific full-length IgG anti-NKp46 antibody that binds to Fcγ receptors in the treatment of Fcγ-positive hematological malignancies. It is believed that Fcγ receptors on tumor cells (e.g., B-cell malignancies) interact with the Fc domain of the anti-NKp46 antibody that binds to NK cells, thereby allowing the activated NK cells to access their target cells via the two reactive portions of the antibody (e.g., the antigen recognition domain and the Fc domain), enhancing the efficiency of the treatment. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2005 / 105858 Brochure [Non-patent literature]

[0008] [Non-Patent Document 1] Jackman,et al.,(2010)J.Biol.Chem.285:20850-20859 [Non-Patent Document 2] Baeuerle, PA, et al, (2009) Cancer Res 69(12):4941-4 [Non-Patent Document 3] Kellner et al.(2011)Cancer Lett.303(2):128-139 Summary of the Invention [Problem to be solved by the invention]

[0009] So far, no NK cell-specific bispecific antibody has been developed. The depleting agent that recruits NK cytotoxicity, such as antitumor antibodies, is typically full-length IgG1 that mediates ADCC via CD16. Despite the existence of various forms of bispecific antibodies, there is still a need in the art for proteins that have novel well-defined mechanisms of action that can provide benefits and can be used in addition to full-length antibodies. [Means for solving the problem]

[0010] In one aspect, the invention arises from the discovery of antibody hypervariable regions that cross-react with both human and non-human primate (e.g., Macaca fascicularis) NKp46 polypeptides. In another aspect, the invention arises from the discovery of antibody hypervariable regions that maintain function in single chain and multispecific proteins (e.g., polypeptides, single chain proteins, multi-chain proteins, including but not limited to antibody-based protein formats) that bind to NKp46 on NK cells.

[0011] Also provided is the epitope bound by the antigen-binding domain. The antigen-binding domain binds to an epitope on NKp46, providing a highly potent antigen-binding protein. The epitope is further shared by humans and non-human primates, including NKp46, which is expressed on the surface of NK cells.

[0012] The variable regions, when incorporated into a polypeptide (e.g., an antibody, Fc protein, scFv, etc.) that binds NKp46 monovalently, allow binding to NKp46 on isolated NK cells without inducing NKp46 activation (in the absence of target cells). The variable regions can bind NKp46 with high affinity as single-chain polypeptides, e.g., as tandem variable regions separated by a peptide linker, as in F(ab) format, and as monovalent binders, can bind to NKp46 epitopes that allow NK cells to lyse target cells. Such properties make them advantageous in a variety of applications, including in multispecific (e.g., bispecific) proteins that bind to NKp46 and an antigen of interest via heavy and light chain anti-NKp46 variable regions, and specifically bind to an antigen of interest on a target cell via hypervariable regions (e.g., heavy and light chain variable regions) that specifically bind to the antigen of interest. Such multispecific proteins can redirect NK cells to lyse target cells expressing the antigen of interest, e.g., cells involved in a disease. While the hypervariable regions and humanized variable regions disclosed herein maintain the monovalent binding of the single-chain form and can be advantageously used in configurations in which they are arranged on a single polypeptide chain, it will be understood that they may also be used in other applications, e.g., in other proteins in which the variable regions are on separate chains, e.g., bispecific antibodies and Fc proteins, or more generally, in monospecific and / or conventional anti-NKp46 antibodies, to bind to, modulate, and / or detect human and / or non-human primate NKp46 polypeptides in vivo or in vitro (e.g., in a biological sample).

[0013] In one aspect, an antigen binding domain (ABD) is provided that binds to both human and non-human primate NKp46 polypeptides (e.g., with similar affinity for NKp46-expressing cells as assessed by surface plasmon resonance and / or flow cytometry according to the methods herein). In one embodiment, the ABD is capable of binding to the NKp46 polypeptide as a single chain antigen binding domain (e.g., scFv). In one embodiment, the ABD comprises (or is comprised in) immunoglobulin heavy and light chain variable regions (e.g., the heavy and light chain variable domains are located on a single polypeptide chain). In another embodiment, the heavy and light chain variable domains are located on different polypeptide chains within a multimeric protein. The heavy chain variable region comprises a heavy chain framework region of human origin, and the light chain variable region comprises a light chain framework region of human origin, and optionally the heavy and / or light chain framework regions comprise one or more amino acid modifications (e.g., substitutions, back mutations where a residue is replaced by a residue present in the parent (e.g., murine) framework at that position), and optionally the amino acid modifications provide or increase binding to non-human primate NKp46. In one embodiment, a protein, Fc protein, antibody, or antibody fragment is provided that comprises such an ABD and / or variable region. In one aspect, an isolated and / or recombinant nucleic acid encoding such an antigen binding domain (ABD) is provided.

[0014] Examples of such proteins or polypeptides include, for example, single polypeptide chain NKp46 binding domains (ABDs that bind to human NKp46 located on a single polypeptide chain, e.g., a polypeptide comprising a VH and a VL separated by a (poly)peptide linker). In one embodiment, the single chain NKp46 binding domain comprises a VH and a VL domain as disclosed herein, separated by a peptide linker. The single polypeptide chain may be comprised in a multi-chain polypeptide comprising one or more additional polypeptide chains, or may be isolated as a single polypeptide chain. Another example of a protein or polypeptide comprising an anti-NKp46 ABD includes a multimeric protein comprising a first and a second polypeptide chain, where one chain comprises a VH domain of an anti-NKp46 ABD disclosed herein, and the other chain comprises a VL of an anti-NKp46 ABD disclosed herein, the chains being configured such that the VH and the VL form an antigen binding domain that binds to NKp46.

[0015] In one aspect, a protein or polypeptide (e.g., a monospecific, bispecific, or multispecific antibody or protein, scFv, F(ab) or F(ab)2, multispecific Fc protein) is provided that binds to a human NKp46 polypeptide, and optionally further binds to a non-human primate NKp46 polypeptide, the protein or polypeptide comprising: (a) a heavy chain variable region comprising human heavy chain framework regions (FR1, FR2, and FR3) derived from a human IGHV1-69 gene and heavy chain CDR1, 2, and 3 of the NKp46-1 antibody; and (b) a light chain variable region comprising human light chain framework regions (FR1, FR2, and FR3) derived from a human IGKV1-33 gene and light chain CDR1, 2, and 3 of the NKp46-1 antibody. Optionally, the CDRs are as defined by the Kabat numbering, Chotia numbering, or IMGT numbering. In one embodiment, the protein or polypeptide binds to a wild-type NKp46 polypeptide but has reduced binding to a mutant NKp46 polypeptide having a mutation (e.g., an alanine substitution) at any one or more of residues K41, E42, E119, Y121, and / or Y194 (see SEQ ID NO: 1) compared to binding to wild-type NKp46. In one embodiment, the heavy chain variable region comprises an amino acid sequence that shares at least 70%, 80%, 90%, 95%, or 98% identity with the amino acid sequence of SEQ ID NO:3, and / or the light chain variable region comprises an amino acid sequence that shares at least 70%, 80%, 90%, 95%, or 98% identity with the amino acid sequence of SEQ ID NO:4. In one embodiment, the heavy chain variable region comprises the amino acid sequence of the NKp46-1 H1 or H3 variable domain (or an amino acid sequence that shares at least 80, 90%, 95%, or 98% identity with this amino acid sequence), and the light chain variable region comprises the amino acid sequence of the NKp46-1 L1 variable domain (or an amino acid sequence that shares at least 80, 90%, 95%, or 98% identity with this amino acid sequence).

[0016] In one aspect, a protein or polypeptide (e.g., a monospecific, bispecific or multispecific antibody or protein, scFv, F(ab) or F(ab)2, a multispecific Fc protein) is provided that binds to a human NKp46 polypeptide, and optionally further binds to a non-human primate NKp46 polypeptide, the protein or polypeptide comprising: (a) a heavy chain variable region comprising human heavy chain framework regions (FR1, FR2, and FR3) derived from an IGHV4-30-4 gene and heavy chain CDR1, 2, and 3 of an NKp46-2 antibody; and (b) a light chain variable region comprising human light chain framework regions (FR1, FR2, and FR3) derived from an IGKV1-39 gene and light chain CDR1, 2, and 3 of an NKp46-2 antibody. Optionally, the CDRs are as defined by the Kabat numbering, Chotia numbering, or IMGT numbering. In one embodiment, the heavy chain variable region comprises an amino acid sequence that shares at least 70%, 80%, 90%, 95%, or 98% identity with the amino acid sequence of SEQ ID NO: 5, and / or the light chain variable region comprises an amino acid sequence that shares at least 70%, 80%, 90%, 95%, or 98% identity with the amino acid sequence of SEQ ID NO: 6. In one embodiment, the heavy chain variable region comprises an amino acid sequence of an NKp46-2 H1, H2, or H3 variable domain (or an amino acid sequence that shares at least 80, 90%, 95%, or 98% identity with this amino acid sequence), and the light chain variable region comprises an amino acid sequence of an NKp46-2 L1 variable domain (or an amino acid sequence that shares at least 80, 90%, 95%, or 98% identity with this amino acid sequence).

[0017] In one aspect, a protein or polypeptide (e.g., a monospecific, bispecific or multispecific antibody or protein, scFv, F(ab) or F(ab)2, a multispecific Fc protein) is provided that binds to a human NKp46 polypeptide, and optionally further binds to a non-human primate NKp46 polypeptide, the protein or polypeptide comprising: (a) a heavy chain variable region comprising human heavy chain framework regions (FR1, FR2, and FR3) derived from an IGHV1-69 gene and heavy chain CDR1, 2, and 3 of the NKp46-3 antibody; and (b) a light chain variable region comprising human light chain framework regions (FR1, FR2, and FR3) derived from an IGKV3-11 and / or IGKV3-15 gene (e.g., a mosaic variable region comprising both IGKV3-11 and IGKV3-15 sequences or segments) and light chain CDR1, 2, and 3 of the NKp46-3 antibody. Optionally, the CDRs are defined by Kabat numbering, Chotia numbering, or IMGT numbering. In one embodiment, the protein or polypeptide binds to a wild-type NKp46 polypeptide, but has reduced binding to a mutant NKp46 polypeptide having a mutation (e.g., an alanine substitution) at any one or more of residues P132, E133, I135, and / or S136 (see SEQ ID NO:1) compared to binding to wild-type NKp46. In one embodiment, the heavy chain variable region comprises an amino acid sequence that shares at least 70%, 80%, 90%, 95%, or 98% identity with the amino acid sequence of SEQ ID NO:7, and / or the light chain variable region comprises an amino acid sequence that shares at least 70%, 80%, 90%, 95%, or 98% identity with the amino acid sequence of SEQ ID NO:8. In one embodiment, the heavy chain variable region comprises the amino acid sequence of an NKp46-3 H1, H3, or H4 variable domain (or an amino acid sequence that shares at least 80, 90%, 95%, or 98% identity with this amino acid sequence), and the light chain variable region comprises the amino acid sequence of an NKp46-3 L1 variable domain (or an amino acid sequence that shares at least 80, 90%, 95%, or 98% identity with this amino acid sequence).

[0018] In one aspect, a protein or polypeptide (e.g., a monospecific, bispecific or multispecific antibody or protein, scFv, F(ab) or F(ab)2, a multispecific Fc protein) is provided that binds to a human NKp46 polypeptide, and optionally further binds to a non-human primate NKp46 polypeptide, the protein or polypeptide comprising: (a) a heavy chain variable region comprising human heavy chain framework regions (FR1, FR2, and FR3) derived from an IGHV1-46 and / or an IGHV1-69 gene (e.g., a mosaic variable region comprising both IGHV1-46 and IGHV1-69 sequences or segments) and heavy chain CDR1, 2, and 3 of the NKp46-4 antibody; and (b) a light chain variable region comprising human light chain framework regions (FR1, FR2, and FR3) derived from an IGKV1-NL1 gene and light chain CDR1, 2, and 3 of the NKp46-4 antibody. Optionally, the CDRs are defined by Kabat numbering, Chotia numbering, or IMGT numbering. In one embodiment, the protein or polypeptide binds to a wild-type NKp46 polypeptide, but has reduced binding to a mutant NKp46 polypeptide having a mutation (e.g., an alanine substitution) at any one or more of residues R101, V102, E104, and / or L105 (see SEQ ID NO: 1) compared to binding to wild-type NKp46. In one embodiment, the heavy chain variable region comprises an amino acid sequence that shares at least 70%, 80%, 90%, 95%, or 98% identity with the amino acid sequence of SEQ ID NO: 9, and / or the light chain variable region comprises an amino acid sequence that shares at least 70%, 80%, 90%, 95%, or 98% identity with the amino acid sequence of SEQ ID NO: 10. In one embodiment, the heavy chain variable region comprises the amino acid sequence of the NKp46-4 H1 variable domain (or an amino acid sequence that shares at least 80, 90%, 95%, or 98% identity with this amino acid sequence), and the light chain variable region comprises the amino acid sequence of the NKp46-4 L2 variable domain (or an amino acid sequence that shares at least 80, 90%, 95%, or 98% identity with this amino acid sequence).

[0019] In one aspect, a protein or polypeptide (e.g., a monospecific, bispecific or multispecific antibody or protein, scFv, F(ab) or F(ab)2, a multispecific Fc protein) is provided that binds to a human NKp46 polypeptide, and optionally further binds to a non-human primate NKp46 polypeptide, the protein or polypeptide comprising: (a) a heavy chain variable region comprising human heavy chain framework regions (FR1, FR2, FR3) derived from an IGHV4-30-4 gene and heavy chain CDRs 1, 2 and 3 of an NKp46-9 antibody; and (b) a light chain variable region comprising human light chain framework regions (FR1, FR2, FR3) derived from an IGKV1-39 gene and light chain CDRs 1, 2 and 3 of an NKp46-9 antibody. Optionally, the CDRs are as defined by the Kabat numbering, Chotia numbering, or IMGT numbering. In one embodiment, the heavy chain variable region comprises an amino acid sequence that shares at least 70%, 80%, 90%, 95%, or 98% identity with the amino acid sequence of SEQ ID NO: 13, and / or the light chain variable region comprises an amino acid sequence that shares at least 70%, 80%, 90%, 95%, or 98% identity with the amino acid sequence of SEQ ID NO: 14. In one embodiment, the heavy chain variable region comprises the amino acid sequence of the NKp46-9 H2 variable domain (or an amino acid sequence that shares at least 80, 90%, 95%, or 98% identity with this amino acid sequence), and the light chain variable region comprises the amino acid sequence of the NKp46-9 L1 or L2 variable domain (or an amino acid sequence that shares at least 80, 90%, 95%, or 98% identity with this amino acid sequence). In one embodiment, the heavy chain variable region comprises the amino acid sequence of the NKp46-9 H3 variable domain (or an amino acid sequence that shares at least 80, 90%, 95%, or 98% identity with this amino acid sequence), and the light chain variable region comprises the amino acid sequence of the NKp46-9 L1 or L2 variable domain (or an amino acid sequence that shares at least 80, 90%, 95%, or 98% identity with this amino acid sequence).

[0020] In one aspect, there is provided a protein or polypeptide (such as, for example, a monoclonal antibody, scFv, multispecific polypeptide, bispecific antibody, DART®, or BiTe®, or a protein comprising such) that comprises an antigen binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) each comprising human FR1, 2, and 3 framework regions, or an ABD that binds to human and non-human primate NKp46 polypeptides, e.g., cell surface NKp46 polypeptides, the protein or polypeptide comprising (a) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% (or 100%) identical to the amino acid sequence of the NKp46-1 H1 or H3 variable domain set forth in SEQ ID NO: 199 or 200, respectively, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of the NKp46-1 L1 variable domain set forth in SEQ ID NO: 201; (b) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% (or 100%) identical to the amino acid sequence of the NKp46-2 H1, H2, or H3 variable domain set forth in SEQ ID NO:202, 203, or 203, respectively, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of the NKp46-2 L1 variable domain set forth in SEQ ID NO:205; (c) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% (or 100%) identical to the amino acid sequence of the NKp46-3 H1, H3, or H4 variable domain set forth in SEQ ID NO:206, 207, or 208, respectively, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of the NKp46-3 L1 variable domain set forth in SEQ ID NO:209; (d) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% (or 100%) identical to the amino acid sequence of the NKp46-4 H1, H2, or H3 variable domain set forth in SEQ ID NO:210, 211, or 212, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of the NKp46-4 L2 variable domain set forth in SEQ ID NO:213; (e) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% (or 100%) identical to the amino acid sequence of the NKp46-9 H2 variable domain set forth in SEQ ID NO: 215, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of the NKp46-9 L1 or L2 variable domain set forth in SEQ ID NO: 217 or 218, respectively; or (f) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% (or 100%) identical to the amino acid sequence of the NKp46-9 H3 variable domain set forth in SEQ ID NO: 215, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of the NKp46-9 L1 or L2 variable domain set forth in SEQ ID NO: 217 or 218, respectively. The VH and VL combinations are selected from the group consisting of:

[0021] In one aspect, there is provided a protein or polypeptide comprising an antigen binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) each comprising human FR1, 2, and 3 framework regions, or an ABD such that it binds to human and non-human primate NKp46 polypeptides, the protein or polypeptide comprising: (a) a VH comprising CDR1, 2, and 3 of a VH domain having the amino acid sequence set forth in SEQ ID NO:3 and FR1, 2, and 3 of a human IGHV1-69 gene segment, and a VL comprising CDR1, 2, and 3 of a VL domain having the amino acid sequence set forth in SEQ ID NO:4 and FR1, 2, and 3 of a human IGKV1-33 gene segment; (b) a VH comprising CDR1, 2, and 3 of a VH domain having the amino acid sequence set forth in SEQ ID NO:5 and FR1, 2, and 3 of a human IGHV4-30-4 gene segment, and a VL comprising CDR1, 2, and 3 of a VL domain having the amino acid sequence set forth in SEQ ID NO:6 and FR1, 2, and 3 of a human IGKV1-39 gene segment; (c) a VH comprising CDR1, 2, and 3 of a VH domain having the amino acid sequence set forth in SEQ ID NO: 7 and FR1, 2, and 3 of a human IGHV1-69 gene segment, and a VL comprising CDR1, 2, and 3 of a VL domain having the amino acid sequence set forth in SEQ ID NO: 8 and FR1, 2, and 3 of a human IGKV3-11 and / or IGKV3-15 gene segment; (d) a VH comprising CDR1, 2, and 3 of a VH domain having the amino acid sequence set forth in SEQ ID NO: 9 and FR1, 2, and 3 of a human IGHV1-46 and / or IGHV1-69 gene segment, and a VL comprising CDR1, 2, and 3 of a VL domain having the amino acid sequence set forth in SEQ ID NO: 10 and FR1, 2, and 3 of a human IGKV1-NL1 gene segment; (e) a VH domain comprising CDR1, 2, and 3 of a VH domain having the amino acid sequence set forth in SEQ ID NO: 13 and FR1, 2, and 3 of a human IGHV4-30-4 gene segment; and a VL domain comprising CDR1, 2, and 3 of a VL domain having the amino acid sequence set forth in SEQ ID NO: 14 and FR1, 2, and 3 of a human IGKV1-39 gene segment. The VH and VL combinations are selected from the group consisting of:

[0022] In any aspect, the antigen binding domain may comprise a VH and a VL that comprise amino acid sequences that are at least 80%, 90%, 95%, or 98% (or 100%) identical to the VH and VL, respectively, of any one of the following antibodies: NKp46-1 H1L1, NKp46-1 H3L1, NKp46-2 H1L1, NKp46-2 H2L1, NKp46-2 H3L1, NKp46-3 H1L1, NKp46-3 H3L1, NKp46-3 H4L1, NKp46-4 H1L2, NKp46-4 H2L2, NKp46-4 H3L2, NKp46-9 H2L1, NKp46-9 H2L2, NKp46-9 H3L1, or NKp46-9 H3L2.

[0023] In one aspect, a protein or polypeptide (e.g., an antibody, a multispecific protein) is provided that comprises an antigen binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) each comprising human FR1, 2, and 3 framework regions, or an ABD such that it binds to an NKp46 polypeptide, the protein or polypeptide being an antibody such as: NKp46-1 H1L1, NKp46-1 H3L1, NKp46-2 H1L1, NKp46-2 H2L1, NKp46-2 H3L1, NKp46-3 H1L1, NKp46-3 H3L1, NKp46-3 H4L1, NKp46-4 H1L2, NKp46-4 H2L2, NKp46-4 H3L2, NKp46-9 H2L1, NKp46-9 H2L2, NKp46-9 H3L1, or NKp46-9 H3L2, H3L3, and H3L2. The VH and VL sequences of these antibodies are listed in Table C and in Example 1, part B.

[0024] In one embodiment, NKp46-1 VL may comprise an amino acid substitution at Kabat position 87, optionally where the residue at position 87 is a phenylalanine. NKp46-1 VH may comprise an amino acid substitution at Kabat residues 27, 66, and / or 67, and optionally further comprise substitutions at Kabat residues 37, 48, and / or 91. Optionally, the residue present at a particular position is a residue substituted at that position herein, e.g., as shown in Example 1.

[0025] In one embodiment, NKp46-2 VL may comprise an amino acid substitution at Kabat position 48, optionally where the residue at position 48 is a valine. NKp46-2 VH may comprise an amino acid substitution at Kabat residues 27 and / or 71, optionally further comprising a substitution at Kabat residues 48 and / or 67, and optionally further comprising a substitution at Kabat residue 31. Optionally, the residue present at a particular position is a residue substituted at that position herein, e.g., as shown in Example 1.

[0026] In one embodiment, NKp46-3 VL may comprise an amino acid substitution at Kabat position 49, optionally where the residue at position 49 is a lysine. NKp46-3 VH may comprise an amino acid substitution at Kabat residue 27, and optionally further comprise a substitution at Kabat residue 48 and / or 67, and optionally further comprise a substitution at Kabat residue 69. Optionally, the residue present at a particular position is a residue substituted at that position herein, e.g., as shown in Example 1.

[0027] In one embodiment, NKp46-4 VL may comprise amino acid substitutions at Kabat positions 36 and / or 48, optionally where the residue at position 36 is a phenylalanine and optionally where the residue at position 48 is a valine. NKp46-4 VH may comprise amino acid substitutions at Kabat residues 30, 48 and / or 93, optionally further comprising a substitution at Kabat residue 67, and optionally further comprising a substitution at Kabat residue 69. Optionally, the residue present at a particular position is a residue substituted at that position herein, e.g., as shown in Example 1.

[0028] In one embodiment, NKp46-9 VL may comprise an amino acid substitution at Kabat position 36, optionally where the residue at position 36 is a cysteine, and optionally further comprises a substitution at Kabat residue 48, and optionally where the residue at position 48 is a valine. NKp46-9 VH may comprise an amino acid substitution at Kabat residue 71, and optionally further comprises a substitution at Kabat residue 27, and optionally further comprises a substitution at Kabat residues 48 and / or 67. Optionally, the residue present at a particular position is a residue substituted at that position herein, e.g., as shown in Example 1.

[0029] In one aspect of any embodiment, the protein or polypeptide binds monovalently to NKp46. In an aspect of any embodiment, the protein or polypeptide comprises or includes no more than one antigen binding domain that binds to NKp46.

[0030] In one embodiment, the protein or polypeptide comprises a human Fc domain or a portion thereof, which Fc domain or a portion thereof is capable of binding to the human neonatal Fc receptor (FcRn) and a human CD16 polypeptide. Because FcγRIIIa (CD16) is not present on all NK cells, conventional therapeutic antibodies (e.g., human isotype IgG1) designed to confer antibody-dependent cellular cytotoxicity (ADCC) via FcγRIIIa may not recruit all NK cells; in contrast, the present protein can engage all NK cells via NKp46, and thus the present protein can engage all NK cells via NKp46. + CD16 - NK cells and NKp46 + CD16 + It is useful to activate or increase the cytolytic activity of NK cells.

[0031] In one embodiment, the protein or polypeptide comprises a human Fc domain or a portion thereof, which Fc domain or portion thereof is capable of binding to a human neonatal Fc receptor (FcRn) and does not bind to a human CD16 polypeptide.

[0032] In one embodiment, the protein or polypeptide is a single chain polypeptide. In one embodiment, the protein or polypeptide is a multimeric polypeptide, optionally a dimeric, trimeric, or tetrameric protein.

[0033] Advantageously, in one embodiment, in the presence of NK cells and target cells, the multispecific protein is capable of binding to (i) an antigen of interest on the target cell and (ii) NKp46 on the NK cell, and upon binding to both the antigen of interest and NKp46 on the target cell, is capable of inducing signaling in the NK cell by NKp46 and / or activating the NK cell (the protein acts as an NKp46 agonist), thereby promoting NK cell activation and / or lysis of the target cell, in particular by the activation signal delivered by NKp46. In a particular advantageous embodiment, the multispecific comprises a single antigen-binding domain that binds to NKp46 (the protein binds NKp46 monovalently). In one embodiment, the protein is capable of inducing signaling in the NK cell by NKp46, upon binding to both the antigen of interest on the target cell and NKp46 on the NK cell. In one embodiment, the protein comprises a first antigen-binding domain and a second antigen-binding domain, one of the first or second antigen-binding domain binds to a human NKp46 polypeptide, and the other of the first or second antigen-binding domain binds to an antigen of interest expressed on a target cell.

[0034] In one embodiment, the multispecific protein is a monomer. In one embodiment, the multispecific protein is a dimer, e.g., a heterodimer, trimer, or tetramer. In one embodiment, the protein is a heterodimer, heterotrimer, or tetramer comprising a first polypeptide chain comprising VH and VL domains of an ABD that binds NKp46 (VH and VL as disclosed herein) separated by a linker peptide, and an optional Fc domain, and a second polypeptide chain comprising one or more variable regions or antigen binding domains that bind an antigen of interest, and an optional Fc domain. In one embodiment, both the first and second polypeptide chains comprise an Fc domain, and the protein comprises a dimeric Fc domain, and optionally, the Fc domain is capable of binding to human CD16. In one embodiment, the monomeric or dimeric protein comprises a protein having a domain structure with an Fc domain between the antigen binding domain (ABD) that binds NKp46 and the antigen binding domain that binds the antigen of interest. In one embodiment, the multispecific Fc-derived polypeptide is a bispecific antibody.

[0035] In one embodiment of any of the proteins herein, the antigen-binding domain of the multispecific protein that binds to an antigen of interest binds to an antigen (e.g., a polypeptide) expressed by a target cell to be lysed by an NK cell. Optionally, such an antigen is expressed by a cancer cell, a tumor-associated immune cell (e.g., an immunosuppressant or regulatory cell), a virally infected cell, or a cell that contributes to an autoimmune or inflammatory disease. Optionally, the antigen of interest is a cancer antigen that is known to be capable of being internalized into a cell when contacted with a full-length human IgG1 antibody that specifically binds to it.

[0036] In one aspect of any of the embodiments herein, there is provided a recombinant nucleic acid encoding the VH and / or VL, antibody heavy and / or light chains, single chain antigen binding domains, first, second, third or further polypeptide chains of any of the proteins of the disclosure. In one aspect of any of the embodiments herein, there is provided a recombinant host cell comprising a nucleic acid of the disclosure.

[0037] Any method may be further characterized as including any step disclosed in the present application, particularly including the "Description of Embodiments". The present invention further relates to methods of identifying, testing, and / or producing the proteins disclosed herein. The present invention further relates to multispecific proteins obtainable by any of the present methods. The present disclosure further relates to pharmaceutical or diagnostic agents of the multispecific proteins disclosed herein. The present disclosure further relates to methods of using the multispecific proteins in methods of treatment or diagnosis.

[0038] These and additional advantageous aspects and features of the present invention may be further described elsewhere herein. [Brief description of the drawings]

[0039] [Figure 1] FIG. 1 shows that anti-CD19-F1-anti-CD3 does not induce T / B cell aggregation in the presence of B221 (CD19) or JURKAT (CD3) cell lines when separated, but induces cell aggregation when both B221 and JURKAT cells are co-cultured. [Figure 2-1] 2A-F show the different domain arrangements of the bispecific proteins produced. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above. [Figure 2-6] Same as above. [Diagram 3]Figures 3A-B show that bispecific F1 and F2 antibodies with NKp46-binding regions based on NKp46-1, NKp46-2, NKp46-3, or NKp46-4, respectively, were able to direct resting NK cells to their CD19-positive Daudi tumor target cells, whereas an isotype control antibody did not result in the elimination of the Daudi cells. Rituximab (RTX) served as a positive control for ADCC, and the maximum response obtained with RTX (10 μg / ml in this assay) was 21.6% specific lysis. [Figure 4-1] FIG. 4A (upper panel CD107, lower panel CD69) shows that a bispecific antibody with NKp46 and CD19 binding regions in the F2 form protein did not activate resting NK cells in the absence of target cells, whereas a full-length anti-NKp46 antibody and the positive control alemtuzumab activated NK cells. [Figure 4-2] Figure 4B shows that in the presence of Daudi target cells, bispecific anti-NKp46xanti-CD19 antibodies (containing NKp46-1, NKp46-2, NKp46-3, or NKp46-4 binding domains, respectively) activated resting NK cells (upper panel CD107, lower panel CD69), whereas full-length anti-CD19 at best showed only very low activation of NK cells. Neither full-length anti-NKp46 antibodies nor alemtuzumab showed a substantial increase in activation beyond that observed in the presence of NK cells alone. [Figure 4-3] Figure 4C (upper panel CD107, lower panel CD69) shows that in the presence of CD19-negative HUT78 cells, none of the bispecific anti-NKp46xanti-CD19 antibodies (containing each of the NKp46-1, NKp46-2, NKp46-3, or NKp46-4 variable regions) activated NK cells. However, full-length anti-NKp46 antibodies and alemtuzumab caused detectable activation of NK cells at levels similar to that observed in the presence of NK cells alone. An isotype control antibody did not induce activation. [Diagram 5]Figures 5A-B show that at a low effector:target ratio of 1:1, each of the bispecific anti-NKp46xanti-CD19 antibodies activated NK cells in the presence of Daudi cells, and that the bispecific anti-NKp46xanti-CD19 was much more potent than the anti-CD19 antibody, as was the ADCC-inducing antibody full-length human IgG1. The upper panel shows CD107 (Figure 5A) and the lower panel shows CD69 (Figure 5B). [Figure 6] 6A-B show that each NKp46xCD19 bispecific protein (single chain form F3, and multimeric forms F5 and F6), but not rituximab and human IgG1 isotype control (IC) antibodies, induced specific lysis of Daudi (FIG. 6A) or B221 (FIG. 6B) cells by the human KHYG-1 CD16-negative hNKp46-positive NK cell line. [Figure 7] We show that the F5 format of the NKp46xCD19 bispecific protein, in which the Fc domain binds to CD16, is much more potent in mediating Daudi target cell lysis than a full-length IgG1 anti-CD19 antibody or the F6 format of the bispecific protein. The F6 format of the bispecific anti-CD19, in which the Fc domain does not bind to CD16, is as potent in mediating NK cell lysis of Daudi target cells as the full-length IgG1 anti-CD19 antibody, which is noteworthy given that the control IgG1 anti-CD19 antibody binds CD19 bivalently. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] As used herein, "a" or "an" may mean one or more. When used in the claims with the word "comprising," the words "a" or "an" may mean one or more than one.

[0041] When "comprising" is used, this can optionally be replaced with "consisting essentially of" and further optionally with "consisting of."

[0042] The term "antigen-binding domain" as used herein refers to a domain comprising a three-dimensional structure capable of immunospecifically binding to an epitope. Thus, in one embodiment, said domain may comprise a hypervariable region, optionally a VH and / or VL domain of an antibody chain, optionally at least one VH domain. In another embodiment, the binding domain may comprise at least one complementarity determining region (CDR) of an antibody chain. In another embodiment, the binding domain may comprise a polypeptide domain from a non-immunoglobulin scaffold.

[0043] The term "antibody" is used in a broad sense herein and specifically includes full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), as well as antibody fragments and derivatives, so long as they exhibit the desired biological activity. Various techniques suitable for producing antibodies are set forth, for example, in Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). An "antibody fragment" includes a full-length antibody, e.g., a portion of its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)3, Fv (typically the VL and VH domains of a single arm of an antibody), single chain Fv (scFv), dsFv, Fd fragments (typically the VH and CH1 domains), and dAb (typically the VH domain) fragments; VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappabodies (see, e.g., Ill et al., Protein Eng 1997;10:949-57); camelid IgG; IgNAR; and multispecific antibody fragments formed from antibody fragments and one or more isolated CDRs or functional paratopes, which isolated CDRs or antigen-binding residues or polypeptides may be joined or linked together to form a functional antibody fragment. Various types of antibody fragments are described and reviewed in, for example, Holliger and Hudson, Nat Biotechnol 2005;23,1126-1136; WO2005040219, and US Patent Publication Nos. 20050238646 and 20020161201.

[0044] The term "antibody derivatives" as used herein includes full-length antibodies, or fragments of antibodies, e.g., including at least the antigen binding or variable region thereof, in which one or more amino acids have been chemically modified, e.g., by alkylation, pegylation, acylation, ester formation, or amide formation, etc. This includes, but is not limited to, pegylated antibodies, cysteine-pegylated antibodies, and variants thereof.

[0045] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al. 1991), and / or amino acid residues from the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol 1987;196:901-917). Typically, the numbering of amino acid residues in this region is done according to the method described in Kabat et al., supra. Phrases such as "Kabat position", "variable domain residue numbering as in Kabat" and "according to Kabat" herein refer to this numbering system for the heavy chain variable domain or the light chain variable domain. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or an insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of CDR H2 (residue 52a according to Kabat) and an inserted residue after heavy chain FR residue 82 (such as residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of homologous regions of the antibody's sequence with a "reference" Kabat numbered sequence.

[0046] As used herein, "framework" or "FR" residues refer to the regions of an antibody variable domain excluding those portions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions separated by the CDRs (FR1, FR2, FR3, and FR4).

[0047] As defined herein, a "constant region" refers to the constant region from an antibody encoded by one of the light or heavy chain immunoglobulin constant region genes. As used herein, a "constant light chain" or "light chain constant region" refers to the region of an antibody encoded by the kappa (Cκ) or lambda (Cλ) light chain. The constant light chain typically comprises a single domain and refers to positions 108-214 of Cκ, or Cλ, as defined herein, numbering according to the EU index (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda). As used herein, "constant heavy chain" or "heavy chain constant region" refers to the region of an antibody that is encoded by the μ, δ, γ, α, or ε genes, respectively, and that defines the antibody's isotype as IgM, IgD, IgG, IgA, or IgE. In full-length IgG antibodies, the constant heavy chain as defined herein refers to the region from the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, thus comprising positions 118-447, numbering according to the EU index.

[0048] As used herein, "Fab" or "Fab region" refers to a polypeptide comprising the VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to this region in isolation, or to this region in the context of a polypeptide, a multispecific polypeptide, or an ABD, or other embodiments outlined herein.

[0049] As used herein, a "single-chain Fv" or "scFv" refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. Methods for making scFvs are well known in the art. For a review of methods for making scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0050] As used herein, "Fv" or "Fv fragment" or "Fv region" refers to a polypeptide comprising the VL and VH domains of a single antibody.

[0051] As used herein, "Fc" or "Fc region" refers to a polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. In IgA and IgM, Fc may include the J chain. In IgG, Fc includes immunoglobulin domains Cγ2 (CH2) and Cγ3 (CH3), and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include at least residues C226, P230, or A231 to its carboxy terminus, as numbered according to the EU index. Fc may refer to this region in isolation, as described below, or this region in the context of an Fc polypeptide. As used herein, an "Fc polypeptide" or "Fc-derived polypeptide" refers to a polypeptide that comprises all or a portion of an Fc region. Fc polypeptides include, but are not limited to, antibodies, Fc fusions, and Fc fragments.

[0052] As used herein, a "variable region" refers to a region of an antibody that includes one or more Ig domains substantially encoded by either the VL (including Vκ (VK) and Vλ) genes and / or the VH genes that constitute the light (including κ and λ) and heavy chain immunoglobulin loci, respectively. A light or heavy chain variable region (VL or VH) consists of a "framework" or "FR" region interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". In the context of framework regions and CDRs, they are precisely defined, for example, as in Kabat (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., USDepartment of Health and Human Services, (1983)) and as in Chothia. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serve to position and align the CDRs, which are primarily responsible for binding to the antigen.

[0053] The term "specifically binds" means that an antibody or polypeptide can bind to a binding partner, e.g., NKp46, preferably in a competitive binding assay, assessed using either a recombinant form of the protein, an epitope therein, or the native protein present on the surface of an isolated target cell. Competitive binding assays and other methods of determining specific binding are described further below and are well known in the art.

[0054] When an antibody or polypeptide is said to "compete" with a particular monoclonal antibody (e.g., NKp46-1, -2, -4, -6, or -9 in the context of an anti-NKp46 mono- or bispecific antibody), this "competes" means that the antibody or polypeptide competes with the monoclonal antibody in a binding assay with either a recombinant target (e.g., NKp46) molecule or a surface-expressed target (e.g., NKp46) molecule. For example, if a test antibody reduces the binding of NKp46-1, -2, -4, -6, or -9 to an NKp46 polypeptide or an NKp46-expressing cell in a binding assay, the antibody is said to "compete" with NKp46-1, -2, -4, -6, or -9, respectively.

[0055] The term "affinity" as used herein refers to the strength of binding of an antibody or polypeptide to an epitope. The affinity of an antibody is determined by the dissociation constant K, defined as [Ab] x [Ag] / [Ab-Ag]. D where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant K A is 1 / K D The affinity of a mAb is defined by the following: Preferred methods for determining the affinity of a mAb are described in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), the entire contents of which are incorporated herein by reference. One preferred standard method known in the art for determining the affinity of a mAb uses surface plasmon resonance (SPR) screening (e.g., by analysis on a BIAcore™ SPR analyzer).

[0056] In the context of this invention, a "determinant" designates a site of interaction or binding on a polypeptide.

[0057] The term "epitope" refers to an antigenic determinant, which is an area or region of an antigen to which an antibody or polypeptide binds. A protein epitope may include amino acid residues directly involved in binding as well as those that are effectively blocked by a particular antigen-binding antibody or peptide, i.e., those within the "footprint" of an antibody. A protein epitope is the simplest form or smallest area on a complex antigen molecule that can bind, for example, to an antibody or receptor. Epitopes can be linear or conformational / structured. The term "linear epitope" is defined as an epitope that is composed of contiguous amino acid residues on a linear sequence of amino acids (primary structure). The term "conformation or structural epitope" is defined as an epitope that is composed of amino acid residues that are not all contiguous and therefore represents a discrete portion of a linear sequence of amino acids that are adjacent to each other due to folding of the molecule (secondary, tertiary, and / or quaternary structure). A conformational epitope is determined by a three-dimensional structure. Thus, the term "conformational" is frequently used synonymously with the term "structural."

[0058] An "amino acid modification" herein refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. An example of an amino acid modification herein is a substitution. An "amino acid modification" herein refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. An "amino acid substitution" or "substitution" herein refers to the replacement of an amino acid at a given position in a protein sequence with another amino acid. For example, the substitution Y50W refers to a variant of a parent peptide in which the tyrosine at position 50 is replaced with a tryptophan. A "variant" of a polypeptide refers to a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide, typically a native or "parent" polypeptide. A polypeptide variant may have one or more amino acid substitutions, deletions, and / or insertions at specific positions in the native amino acid sequence.

[0059] "Conservative" amino acid substitution is an amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties.Families of amino acid residues with similar side chains are known in the art, and include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0060] The term "identity" or "identical" as used in the context of two or more polypeptide sequences refers to the degree of sequence relatedness between the polypeptides, as determined by the number of matches between two or more strings of amino acid residues. "Identity" refers to the percent of exact matches between two or more similar sequences, with gap alignments (if any) performed by a particular computational model or computer program (i.e., "algorithm"). Identity between related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0061] Preferred methods for determining identity are designed to obtain the largest match between the sequences tested. Methods for determining identity are described in published computer programs. Preferred computer program methods for determining identity between two sequences include GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)), including the GCG program package. The BLASTX program is published by the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith Waterman algorithm can also be used to determine identity.

[0062] An "isolated" molecule is one that is the predominant species in a composition in which it is found relative to the class of molecules to which it belongs (i.e., an isolated molecule constitutes at least about 50% of the type of molecule in the composition, and typically constitutes at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% or more of the species of molecules, e.g., peptides, in the composition). Typically, the composition of the polypeptide exhibits 98%, 98%, or 99% homogeneity for the polypeptide relative to all peptide species present in the composition, or at least for substantially active peptide species in the context of the proposed use.

[0063] In the context of this specification, "treatment" or "treating" refers to preventing, alleviating, managing, curing, or ameliorating one or more symptoms or clinically relevant signs of a disease or disorder, unless stated to the contrary. For example, "treatment" of a patient who has no identified symptoms or clinically relevant signs of a disease or disorder is preventative or prophylactic therapy, while "treatment" of a patient who has identified symptoms or clinically relevant signs of a disease or disorder generally is not preventative or prophylactic therapy.

[0064] As used herein, "NK cells" refers to a subpopulation of lymphocytes involved in non-conventional immunity. NK cells can be identified by certain characteristics and biological properties, such as expression of certain surface antigens, including CD56 and / or NKp46 on human NK cells, absence of α / β or γ / δ TCR complexes on the cell surface, ability to bind to and kill cells that fail to express "self" MHC / HLA antigens by activation of specific cytolytic machinery, ability to kill tumor cells or other abnormal cells that express ligands for NK activating receptors, and ability to release protein molecules called cytokines that stimulate or suppress immune responses. Any of these characteristics and activities can be used to identify NK cells by methods known in the art. Any subpopulation of NK cells is encompassed by the term NK cells. In the context of this specification, "active" NK cells refer to biologically active NK cells, including NK cells that have the ability to lyse target cells or promote the immune function of other cells. NK cells can be obtained by various techniques known in the art, such as isolation from blood samples, cytoadsorption apheresis, tissue or cell collection, etc. Useful protocols for assays involving NK cells are described in Natural Killer Cells Protocols (edited by Campbell KS and Colonna M). Human Press. pp. 219-238 (2000).

[0065] The term "internalization" is used interchangeably with "internalization into a cell" and refers to molecular, biochemical, and cellular events related to the process of transferring a molecule from the extracellular surface of a cell to the intracellular surface of a cell. The processes involved in the internalization of molecules into a cell are well known and can include, among others, the internalization of extracellular molecules (e.g., hormones, antibodies, and small organic molecules); membrane-bound molecules (e.g., cell surface receptors); and complexes of membrane-bound molecules bound to extracellular molecules (e.g., ligands bound to transmembrane receptors or antibodies bound to membrane-bound molecules). Thus, "induction and / or increase of internalization" includes events that initiate internalization into a cell and / or increase the rate and / or extent of internalization into a cell.

[0066] As used herein, an agent having "agonist" activity at Nkp46 is an agent that can cause or increase "NKp46 signaling". "NKp46 signaling" refers to the ability of an NKp46 polypeptide to activate or transduce an intracellular signaling pathway. Changes in NKp46 signaling activity can be measured, for example, in assays designed to measure changes in the NKp46 signaling pathway, such as by monitoring phosphorylation of signaling elements, assays measuring the binding of specific signaling elements to other proteins or intracellular structures, or in biochemical activity of components such as kinases, or indirectly by assays designed to measure expression of a reporter gene under the control of an NKp46-sensitive promoter or enhancer, or by downstream effects mediated by NKp46 polypeptides (e.g., activation of a specific cytolytic apparatus in NK cells). The reporter gene can be a naturally occurring gene (e.g., monitoring cytokine production) or can be a gene artificially introduced into the cell. Other genes can be placed under the control of such regulatory elements and thus serve to report the level of NKp46 signaling.

[0067] "NKp46" refers to a protein or polypeptide encoded by the Ncr1 gene or a cDNA prepared from such a gene. Any naturally occurring isoforms, alleles, or variants are encompassed by the term NKp46 polypeptide (e.g., an NKp46 polypeptide that is 90%, 95%, 98%, or 99% identical to SEQ ID NO: 1, or a contiguous sequence of at least 20, 30, 50, 100, or 200 amino acid residues thereof). The sequence of 304 amino acid residues of human NKp46 (isoform a) is shown below: [ka]

[0068] SEQ ID NO:1 corresponds to NCBI Accession No. NP_004820, the disclosure of which is incorporated herein by reference. Human NKp46 mRNA sequence is set forth in NCBI Accession No. NM_004829, the disclosure of which is incorporated herein by reference. The amino acid residue sequence of the extracellular domain of the macaca fascicularis (cynomolgus monkey) NKp46 polypeptide is as follows: [ka]

[0069] Preparation of anti-NKp46 antibody The anti-NKp46 antigen binding domain binds to the extracellular portion of a human NKp46 polypeptide. In one aspect, the VH and VL comprising protein of the present disclosure is or comprises a humanized antibody. In one aspect, the antibody comprises a constant domain selected from human IgG1, IgG2, IgG3, and IgG4 constant domains, and optionally comprises one or more amino acid modifications. In one aspect, the anti-NKp46 antibody is a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a F(ab)2 fragment, a F(ab')2 fragment, an Fv fragment, a heavy chain Ig (llama Ig or camel Ig), a V fragment, ... HHThe antibody fragments include those selected from the group consisting of scFV, dsFV, minibody, diabody, triabody, kappabody, IgNAR, and multispecific antibodies. The agent may optionally further comprise an Fc domain. In one embodiment, the antibody is in at least partially purified form. In one embodiment, the antibody is in essentially isolated form.

[0070] Antibodies can be produced by various techniques known in the art. For example, as described in PCT Application No. PCT / EP2016 / 064537 (Innate Pharma), filed June 23, 2016, the disclosure of which is incorporated herein by reference, immunological screening assays that can evaluate antibody competition can be used to select antibodies that bind to the same epitope on NKp46, as they can evaluate the antigen-binding domain of the present invention.

[0071] Typically, the anti-NKp46 antibodies or NKp46 binding proteins provided herein have a binding affinity of about 10 to an NKp46 polypeptide (e.g., an NKp46 polypeptide produced in the Examples herein). 4 ~about 10 11 M -1 (For example, about 10 8 ~about 10 10 M -1 For example, in certain aspects, the present disclosure provides antibodies that have an affinity in the range of 1×10 for NKp46, as determined, for example, by surface plasmon resonance (SPR) screening (e.g., by analysis on a BIAcore™ SPR analyzer). -9 The average dissociation constant (K D In a more specific exemplary embodiment, the present disclosure provides an anti-NKp46 antibody or NKp46 binding protein having a 1×10 IgG antibody binding domain for human or cynomolgus monkey NKp46 protein. -8 M ~ approx. 1×10 -10 M, or approximately 1 x 10 -9 M ~ approx. 1×10-11 The present invention provides an anti-NKp46 antibody or NKp46 binding protein having a KD of 0.01 M. In one embodiment, the NKp46 antibody or NKp46 binding protein has a dissociation constant (KD) for human and cynomolgus monkey NKp46 proteins that differ by 1-log or 2-log. D ).

[0072] The antibody or NKp46 binding protein can be characterized by an average KD (i.e., affinity higher than) of, for example, about 100 nanomolar, about 60 nanomolar, about 10 nanomolar, about 5 nanomolar, or 1 nanomolar or less, preferably sub-nanomolar or less, or optionally about 500 picomolar, about 200 picomolar, about 100 picomolar, or about 10 picomolar or less. The KD can be determined, for example, by immobilizing recombinantly produced human NKp46 protein on a chip surface, followed by adding the antibody to be tested in solution.

[0073] DNA encoding an antibody or other NKp46 binding protein that binds to an epitope present on the NKp46 polypeptide is isolated and introduced into a suitable expression vector for transfection into a suitable host. The host is then used for recombinant production of the antibody or other NKp46 binding protein. For example, DNA can be introduced into an expression vector and transfected into a host cell that does not normally produce immunoglobulin proteins, such as Escherichia coli (E. coli) cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to synthesize the desired protein in the recombinant host cell.

[0074] In one embodiment, the proteins and antibodies herein bind to the D1 domain of NKp46, the D2 domain of NKp46, or a region spanning both the D1 and D2 domains of the NKp46 polypeptide of SEQ ID NO: 1 (at the boundary of the D1 and D2 domains, the D1 / D2 junction). -8 Less than M, 10 -9 Less than M or 10 -10 K under M DIt has an affinity for human NKp46, characterized by:

[0075] In another embodiment, the protein or antibody binds to NKp46 at substantially the same epitope on NKp46 as antibody NKp46-1, NKp46-2, NKp46-3, or NKp46-4. In another embodiment, the antibody at least partially overlaps with or includes at least one residue of the segment bound by NKp46-1, NKp46-2, NKp46-3, or NKp46-4. In one embodiment, all the key residues of the epitope are in the segment corresponding to domain D1 or D2. In one embodiment, the antibody binds to residues present in the D1 domain and residues present in the D2 domain. In one embodiment, the antibody or protein binds to an epitope that includes one, two, three, four, five, six, or more residues in the segment corresponding to domain D1 or D2 of the NKp46 polypeptide of SEQ ID NO:1. In one embodiment, the antibody or protein binds to domain D1 and binds to an epitope comprising one, two, three, or four of residues R101, V102, E104, and / or L105. In one embodiment, the antibody or protein binds to the domain D1 / D2 junction and binds to an epitope comprising one, two, three, four, or five of residues K41, E42, E119, Y121, and / or Y194. In one embodiment, the antibody binds to domain D2 and binds to an epitope comprising one, two, three, or four of residues P132, E133, I135, and / or S136.

[0076] The Examples section herein describes the structure of a series of mutant human NKp46 polypeptides. The binding of anti-NKp46 antibodies or proteins to cells transfected with NKp46 mutants was measured and compared to the ability of anti-NKp46 antibodies or proteins to bind to wild-type NKp46 polypeptide (SEQ ID NO: 1). As used herein, reduced binding between an anti-NKp46 antibody or protein and a mutant NKp46 polypeptide means that there is a reduced binding affinity (e.g., measured by known methods, e.g., FACS testing of cells expressing a particular mutant or by Biacore testing of binding to the mutant polypeptide) and / or a reduced overall binding capacity of the anti-NKp46 antibody or protein (e.g., evidenced by a reduced Bmax in a plot of anti-NKp46 antibody concentration versus polypeptide concentration). A significant reduction in binding indicates that the mutant residue is directly involved in binding to the anti-NKp46 antibody or protein or is in close proximity to the binding protein when the anti-NKp46 antibody or protein binds to NKp46. Thus, the epitope preferably includes such residues and may include additional residues adjacent to such residues.

[0077] In some embodiments, a significant decrease in binding means that the binding affinity and / or binding capacity between an anti-NKp46 antibody or protein and a mutant NKp46 polypeptide is decreased by more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% relative to the binding between the antibody or protein and a wild-type NKp46 polypeptide (e.g., the polypeptide set forth in SEQ ID NO:1). In certain embodiments, the binding is reduced below the detectable limit. In some embodiments, a significant decrease in binding is evidenced when the binding of an anti-NKp46 antibody to a mutant NKp46 polypeptide is less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%) of the binding observed between an anti-NKp46 antibody and a wild-type NKp46 polypeptide (e.g., the polypeptide set forth in SEQ ID NO:1 (or the extracellular domain thereof)). Such binding measurements can be carried out using a variety of binding assays known in the art, a specific example of one such assay is described in the Examples section.

[0078] In some embodiments, anti-NKp46 antibodies or proteins are provided that exhibit significantly reduced binding to mutant NKp46 polypeptides in which residues in a wild-type NKp46 polypeptide (e.g., SEQ ID NO:1) have been substituted, in the abbreviation notation used herein: wild-type residue: position in polypeptide: mutant residue numbered as shown in SEQ ID NO:1.

[0079] In some embodiments, the anti-NKp46 antibody or protein binds to a wild-type NKp46 polypeptide, but has reduced binding to a mutant NKp46 polypeptide having a mutation (e.g., an alanine substitution) at any one or more of residues R101, V102, E104, and / or L105 (see SEQ ID NO:1) compared to binding to wild-type NKp46.

[0080] In some embodiments, the anti-NKp46 antibody or protein binds to a wild-type NKp46 polypeptide, but has reduced binding to a mutant NKp46 polypeptide having a mutation (e.g., an alanine substitution) at any one or more of residues K41, E42, E119, Y121, and / or Y194 (see SEQ ID NO:1) compared to binding to wild-type NKp46.

[0081] In some embodiments, the anti-NKp46 antibody or protein binds to a wild-type NKp46 polypeptide, but has reduced binding to a mutant NKp46 polypeptide having a mutation (e.g., an alanine substitution) at any one or more of residues P132, E133, I135, and / or S136 (see SEQ ID NO:1) compared to binding to wild-type NKp46.

[0082] The amino acid sequences of the heavy and light chain variable regions of parent antibodies NKp46-1, NKp46-2, NKp46-3, and NKp46-4 are set forth herein in Table B. The amino acid sequences of the heavy and light chain variable regions of humanized antibodies NKp46-1, NKp46-2, NKp46-3, NKp46-4, and NKp46-9, which demonstrated high binding affinity to both human and non-human primate NKp46, are set forth herein in Table C.

[0083] The heavy chain variable region of the NKp46-1, NKp46-2, NKp46-3, NKp-46-4, NKp46-6, or NKp46-9 antibody comprises, for each antibody, a human heavy chain FR1 framework region; an HCDR1 region comprising an amino acid sequence set forth in Table A or a sequence of at least 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids thereof, wherein one or more of these amino acids may be substituted with a different amino acid; a human heavy chain FR2 framework region; an amino acid sequence set forth in Table A or a human heavy chain FR3 framework region; and an HCDR3 region comprising an amino acid sequence set forth in Table A or a sequence of at least 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids thereof, wherein one or more of these amino acids may be substituted with a different amino acid. Optionally, the variable region further comprises a human heavy chain FR4 framework region.

[0084] The light chain variable region of the NKp46-1, NKp46-2, NKp46-3, NKp-46-4, NKp46-6, or NKp46-9 antibody comprises, for each antibody, a human light chain FR1 framework region; a LCDR1 region comprising an amino acid sequence set forth in Table A or a sequence of at least 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids thereof, wherein one or more of these amino acids are optionally substituted with a different amino acid; a human light chain FR2 framework region; an amino acid sequence set forth in Table A or at least a sequence of at least 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids thereof, wherein one or more of these amino acids are optionally substituted with a different amino acid; a human light chain FR3 framework region; and an LCDR3 region comprising an amino acid sequence set forth in Table A or a sequence of at least 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids thereof, wherein one or more of these amino acids may be deleted or substituted with a different amino acid. Optionally, the variable region further comprises a human light chain FR4 framework region.

[0085] Examples of VH and VL combinations include: (a) a VH comprising CDR1, 2, and 3 of SEQ ID NO: 3 and FR1, 2, and 3 of a human IGHV1-69 gene segment, and a VL comprising CDR1, 2, and 3 of SEQ ID NO: 4 and FR1, 2, and 3 of a human IGKV1-33 gene segment; (b) a VH comprising CDR1, 2, and 3 of SEQ ID NO:5 and FR1, 2, and 3 of a human IGHV4-30-4 gene segment, and a VL comprising CDR1, 2, and 3 of SEQ ID NO:6 and FR1, 2, and 3 of a human IGKV1-39 gene segment; (c) a VH comprising CDR1, 2, and 3 of SEQ ID NO: 7 and FR1, 2, and 3 of a human IGHV1-69 gene segment, and a VL comprising CDR1, 2, and 3 of SEQ ID NO: 8 and FR1, 2, and 3 of a human IGKV3-11 and / or IGKV3-15 gene segment; (d) a VH comprising CDR1, 2, and 3 of SEQ ID NO: 9 and FR1, 2, and 3 of a human IGHV1-46 and / or IGHV1-69 gene segment, and a VL comprising CDR1, 2, and 3 of SEQ ID NO: 10 and FR1, 2, and 3 of a human IGKV1-NL1 gene segment; or (e) a VH comprising CDR1, 2, and 3 of SEQ ID NO: 13 and FR1, 2, and 3 of a human IGHV4-30-4 gene segment, and a VL comprising CDR1, 2, and 3 of SEQ ID NO: 14 and FR1, 2, and 3 of a human IGKV1-39 gene segment; Examples include:

[0086] In another aspect, exemplary VH and VL combinations include: (a) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to an amino acid sequence of an NKp46-1 H1 or H3 variable domain, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to an amino acid sequence of an NKp46-1 L1 variable domain; (b) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to an amino acid sequence of an NKp46-2 H1, H2, or H3 variable domain, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to an amino acid sequence of an NKp46-2 L1 variable domain; (c) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to an amino acid sequence of an NKp46-3 H1, H3, or H4 variable domain, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to an amino acid sequence of an NKp46-3 L1 variable domain; (d) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of an NKp46-4 H1 variable domain, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of an NKp46-4 L2 variable domain; (e) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of an NKp46-9 H2 variable domain, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of an NKp46-9 L1 or L2 variable domain; or (f) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of an NKp46-9 H3 variable domain, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of an NKp46-9 L1 or L2 variable domain. Examples include:

[0087] In one embodiment, said CDRs are according to Kabat numbering, e.g., as shown in Table A. In one embodiment, said CDRs are according to Chotia numbering, e.g., as shown in Table A. In one embodiment, said CDRs are according to IMGT numbering, e.g., as shown in Table A.

[0088] In one embodiment, a particular VH or VL comprises amino acid substitutions (e.g., backmutations) in the human framework at the Kabat positions set forth in Example 1; optionally, the amino acid residue substituted in Example 1 at the corresponding Kabat position can be identified as being present at a particular position.

[0089] In another aspect of any of the embodiments herein, any of the heavy and light chain CDR1, 2, and 3 may be characterized by the sequence of at least 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids thereof and / or as having an amino acid sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90%, or 95% sequence identity with the corresponding SEQ ID NO or specific CDR or set of CDRs set forth in Table A.

[0090] The sequences of the CDRs according to the IMGT, Kabat and Chothia definition systems are summarized below in Table A. The sequences of the variable chains of the antibodies according to the invention are set forth below in Table B. In any embodiment herein, the VL or VH sequence may be designated or numbered to include or lack a signal peptide or any portion thereof.

[0091] [Table 1-1]

[0092] [Table 1-2]

[0093] [Table 2]

[0094] [Table 3]

[0095] Multispecific antibodies and polypeptides An antigen binding domain (ABD) that binds to NKp46 can be derived from the anti-NKp46 CDR, VH, and VL sequences provided herein (see the "Anti-NKp46 Variable Domains" section). The antigen binding domains can be arranged in the same or separate polypeptides to form an antigen binding domain that can bind to NKp46 (e.g., human NKp46 expressed on the cell surface). The ABD can be made, for example, as an scFv, tandem scFv, Bite, DART, Fab, F(ab)2, antibody, bispecific antibody, or monomeric or multimeric Fc protein.

[0096] For the construction of multispecific proteins (e.g., antibodies, tandem scFvs, BiTe, DARTs, Fc proteins), antigen binding domains that bind antigens of interest (other than NKp46) can be readily derived from a variety of immunoglobulin or non-immunoglobulin scaffolds, such as the Z domain of Staphylococcus protein A, affibodies based on engineered Kunitz domains, monobodies or adnectins based on the 10th extracellular domain of human fibronectin III, anticalins derived from lipocalins, DARPins (designed ankyrin repeat domains), multimerized LDLR-A modules, avimers, or cysteine-rich knottin peptides. See, e.g., Gebauer and Skerra (2009) Current Opinion in Chemical Biology 13:245-255, the disclosure of which is incorporated herein by reference.

[0097] The variable domain that binds an antigen of interest (other than NKp46) may be in the form of an antibody, e.g., the related VL and VH domains found on two polypeptide chains, or a single chain antigen-binding domain, e.g., scFv, V H Domain, V L Domain, dAb, V-NAR domain, or V H The antigen-binding domains (e.g., ABD1 and ABD2) can also be easily obtained from the antibody as Fab.

[0098] Typically, antibodies are first obtained by immunization of a non-human animal, e.g., a mouse, with an immunogen that includes a polypeptide for which it is desired to obtain an antibody (e.g., a human polypeptide), or a fragment or derivative thereof, typically an immunogenic fragment. Immunizing a non-human mammal with an antigen can be performed in any manner known in the art for stimulating the production of antibodies in mice (see, e.g., E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the entire disclosure of which is incorporated herein by reference).

[0099] Antigen binding domains (ABDs) that bind antigens of interest (other than NKp46) for use in multispecific polypeptides can be selected based on the desired cellular target and can include, for example, cancer antigens, bacterial antigens, or viral antigens. As used herein, the term "bacterial antigen" includes, but is not limited to, intact, attenuated, or killed bacteria, any structural or functional bacterial protein or carbohydrate, or any peptide portion of a bacterial protein of sufficient length (typically about 8 amino acids or more) to be antigenic. Examples include gram-positive bacterial antigens and gram-negative bacterial antigens. In some embodiments, the bacterial antigen is selected from the group consisting of Helicobacter spp., particularly Helicobacter pyloris; Borelia spp., particularly Borelia burgdorferi; Legionella spp., particularly Legionella pneumophilia; Mycobacteria spp., particularly M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae; Staphylococcus spp., particularly Staphylococcus aureus. aureus; Neisseria species, especially N. gonorrhoeae and N. meningitidis; Listeria species, especially Listeria monocytogenes; Streptococcus species, especially S. pyogenes, S. agalactiae and S. faecalis; S. bovis, Streptococcus pneumoniae (S.pneumonias; anaerobic Streptococcus spp; pathogenic Campylobacter spp; Enterococcus spp; Haemophilus spp, especially Haemophilus influenzae; Bacillus spp, especially Bacillus anthracis; Corynebacterium spp, especially Corynebacterium diphtheriae; Erysipelothrix spp, especially Erysipelothrix rhusiopathiae; Clostridium spp, especially C. perfringens and C. tetani.tetani; Enterobacter spp., especially Enterobacter aerogenes; Klebsiella spp., especially Klebsiella 1S pneumoniae; Pasteurella spp., especially Pasteurella multocida; Pasteurella spp., especially Pasteurella multocida; Bacteroides spp.; Fusobacterium spp., especially Fusobacterium nucleatum; Streptobacillus spp., especially Streptobacillus moriformis; moniliformis; Treponema species, particularly Treponema pertenue; Leptospira; pathogenic Escherichia species; and Actinomyces species, particularly Actinomyces israelli.

[0100] The term "viral antigen" as used herein includes, but is not limited to, whole intact, attenuated, or killed viruses, any structural or functional viral protein, or any peptide portion of a viral protein of sufficient length (typically about 8 amino acids or more) to be antigenic.Sources of viral antigens include, but are not limited to, Retroviridae (e.g., human immunodeficiency viruses, e.g., HIV-1 (also called HTLV-III, LAV, or HTLV-III / LAV, or HIV-III; and other isolates, e.g., HIV-LP); Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains causing gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps, measles virus, respiratory syncytial virus, Orthomyxoviridae (e.g., influenza viruses); Bunyaviridae (e.g., hantaviruses, bunyaviruses, phleboviruses, and nairoviruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); Bornabilidae; Hepadnaviridae (hepatitis B viruses); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviruses (most adenoviruses); Herpesviridae (herpes simplex viruses (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpes viruses); Poxviridae (variolaviruses, vaccinia viruses, pox viruses); and Iridaviridae (e.g., African swine fever viruses); and unclassified viruses (e.g., the agent of delta hepatitis (defective satellite of hepatitis B virus)). Examples of viruses include those from the following families: Hepatitis C; Norwalk and related viruses; and Astrovirus. Alternatively, viral antigens may be made recombinantly.

[0101] As used herein, the terms "cancer antigen" and "tumor antigen" are used interchangeably and refer to antigens (other than NKp46) that are differentially expressed by cancer cells or that are expressed by non-tumor cells (e.g., immune cells) that have a pro-tumor effect (e.g., immunosuppressive effect) and can therefore be used to target cancer cells. Cancer antigens are clearly antigens that can potentially stimulate tumor-specific immune responses. Some of these antigens are encoded by normal cells but are not necessarily expressed or are expressed at a lower level or less frequently. These antigens can be characterized as antigens that are normally silent (i.e., not expressed) in normal cells, antigens that are expressed only at certain stages of differentiation, and antigens that are transiently expressed, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutated cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutated p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes, such as those carried by RNA and DNA tumor viruses. Still other cancer antigens can be expressed on immune cells capable of mediating tumor-promoting effects, such as monocytes or macrophages, and optionally suppressor T cells, regulatory T cells, or myeloid-derived suppressor cells.

[0102] Cancer antigens are usually surface antigens of normal cells that are overexpressed or expressed at abnormal times or by the target cell population. Ideally, the target antigen is expressed only on proliferating cells (e.g., tumor cells) or tumor-promoting cells (e.g., immune cells with immunosuppressive effects), but this is only rarely observed in practice. As a result, target antigens are often selected based on differential expression between proliferating diseased tissues and healthy tissues. Examples of cancer antigens include receptor tyrosine kinase-like orphan receptor 1 (ROR1), Cripto, CD4, CD20, CD30, CD19, CD38, CD47, glycoprotein NMB, CanAg, Her2 (ErbB2 / Neu), Siglec family members such as CD22 (Siglec2) or CD33 (Siglec3), CD79, CD138, CD171, PSCA, L1-CAM, PSMA (prostate specific membrane antigen), BCMA, CD52, CD56, CD80, CD70, E-selectin, EphB2, melanotransferrin, Mud6, and TMEFF2. Examples of cancer antigens also include immunoglobulin immunoglobulin superfamily (IgSF) antigens, such as cytokine receptors, Killer-Ig-like receptors, CD28 family proteins, such as Killer-Ig-like receptor 3DL2 (KIR3DL2), B7-H3, B7-H4, B7-H6, PD-L1, and IL-6 receptor. Examples include, but are not limited to, MAGE, MART-1 / Melan-A, gp100, major histocompatibility complex class I-related chain A and B polypeptides (MICA and MICB), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal-related antigen (CRC)-C017-1A / GA733, protein tyrosine kinase 7 (PTK7), receptor protein tyrosine kinase 3 (TYRO-3), nectins (e.g., nectin-4), major histocompatibility complex class I-related chain A and B polypeptides (MICA and MICB), the UL16 binding protein (ULBP) family of proteins, the retinoic acid early transcript-1 (RAET1) family of proteins, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA),T cell receptor / CD3 zeta chain, MAGE family of tumor antigens, GAGE ​​family of tumor antigens, anti-Mullerian hormone type II receptor, delta-like ligand 4 (DLL4), DR5, ROR1 (also known as receptor tyrosine kinase-like orphan receptor 1 or NTRKR1 (EC 2.7.10.1)), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC family, VEGF, VEGF receptor, angiopoietin-2, PDGF, TGF-α, EGF, EGF receptor, members of the human EGF-like receptor family, e.g., HER-2 / neu, HER-3, HER-4, or heterodimeric receptors consisting of at least one HER subunit, gastrin releasing peptide receptor antigen, Muc-1, CA125, integrin receptors, αvβ3 integrin, α5β1 integrin, Tegrin, αIIbβ3-integrin, PDGFβ receptor, SVE-cadherin, IL-8 receptor, hCG, IL-6 receptor, CSF1R (tumor-associated monocytes and macrophages), α-fetoprotein, E-cadherin, α-catenin, β-catenin, and γ-catenin, p120ctn, PRAME, NY-ESO-1, cdc27, adenoma-like polyposis coli protein (APC), fodrin, connexin, Examples of antigens that may be used include IL-37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, and CT-7, and c-erbB-2. In one aspect, the antigen of interest is an antigen (e.g., any one of the antigens listed above) that can be internalized into a cell when bound by a conventional human IgG1 antibody, e.g., in the presence or absence of Fcγ receptors. In one embodiment, the antigen of interest is a CD19 polypeptide; in one embodiment, the multispecific protein comprises an scFv that binds to CD19 comprising an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, or 95% identical to the sequence of an anti-CD19 scFv of the Examples herein;or comprising heavy and light chain CDR-1, 2, and 3 of the anti-CD19 heavy and light chain variable regions set forth herein.

[0103] In one embodiment, the ABD that binds to the antigen of interest is derived from (e.g., comprises the hypervariable region or CDRs of) a parent antibody that binds to the antigen of interest (e.g., a mouse antibody, a human antibody) and upon binding to its antigenic target (antigen of interest on a cell), promotes or induces downregulation or internalization of the antigen of interest into the cell. In one embodiment, the antigen of interest is a cancer antigen, such as one of the above cancer antigens known to be internalized (e.g., immunoglobulin immunoglobulin superfamily (IgSF) members, such as cytokine receptor α or β chains, Killer-Ig-like receptors, CD28 family proteins, B7-H3, B7-H4, B7-H6, KIR3DL2, PTK7, ROR1, L1-CAM, Siglec family members, EGF receptor and EGF-like receptor family members, EGFR, HER-2, integrins, anti-Mullerian hormone type II receptor, and CSF-1R). In one embodiment, the antigenic target is a polypeptide present on an immune cell capable of mediating a tumor-promoting effect, such as a monocyte or macrophage, optionally a suppressor T cell, a regulatory T cell, or a myeloid-derived suppressor cell.

[0104] In one embodiment, the non-NKp46 ABD binds to a cancer antigen, a viral antigen, a microbial antigen, or an antigen present on an infected cell (eg, virally infected) or a pro-inflammatory immune cell.

[0105] Once suitable antigen binding domains with the desired specificity and / or activity have been identified, the DNA encoding each of the ABDs can be separately placed in a suitable expression vector in an appropriate configuration along with any elements, such as DNA encoding an enzyme recognition tag, CH1, Cκ, CH2 and / or CH3 domains, and other optional elements (e.g., DNA encoding a hinge region), for transfection into a suitable host. The ABDs can be placed in one expression vector or in separate vectors, depending on which type of polypeptide is to be produced, to produce an Fc polypeptide with the desired domains operably linked to each other. The host can then be used for recombinant production of the multispecific polypeptide.

[0106] For example, a polypeptide fusion product can be made from a vector in which the first ABD of the two ABDs is operably linked to the N-terminus of the CH2 domain (e.g., directly via the heavy or light chain CH1, CK, or Cλ constant region and / or hinge region), and the CH2 domain is operably linked to the CH3 domain at its C-terminus to N-terminus. The second ABD of the two ABDs can be linked to the polypeptide at either end, or can be on a second polypeptide chain that forms a dimer, e.g., a heterodimer, with the polypeptide that contains the first ABD. The polypeptide can include a full-length Fc domain.

[0107] The multispecific polypeptide can then be produced, for example, in a suitable host cell or by any suitable synthetic process. The host cell selected for expression of the multispecific polypeptide is an important contributor to the final composition, including but not limited to, the variation in the composition of the oligosaccharide moieties decorating the protein of the immunoglobulin CH2 domain. The host cell may be of mammalian origin or may be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2 / 0, 293, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative, immortalized or transformed cell thereof. Alternatively, the host cell can be selected from a species or organism that is not capable of glycosylation of polypeptides, for example a prokaryotic cell or organism, for example a natural or engineered E. coli spp., Klebsiella spp., or Pseudomonas spp.

[0108] A variety of different protein forms can be prepared using the NKp46-binding VH-VL pairs of the present disclosure, including monomeric, heterodimeric, heterotrimeric, and tetrameric multispecific proteins, including, but not limited to, any of the various forms described in, for example, PCT Application No. PCT / EP2016 / 064537 (Innate Pharma), filed June 23, 2016, the disclosure of which is incorporated herein by reference.

[0109] Monomeric Proteins In one example, a multispecific protein comprises in a single polypeptide chain a first antigen-binding domain that binds to NKp46 (e.g., an ABD comprising a VH and VL as disclosed herein, separated by a (poly)peptide linker) and a second antigen-binding domain that binds to an antigen other than NKp46. In one embodiment, the protein or polypeptide is or comprises an scFv comprising an anti-NKp46 VH and VL of the present disclosure, or a tandem scFv comprising an scFv that binds to NKp46, and a second scFv that binds to NKp46 or an antigen of interest other than NKp46, linked by a (poly)peptide linker, optionally fused to another polypeptide or amino acid sequence.

[0110] Examples of such single-chain antigen-binding proteins include BiTE and DART protein formats. The scFv-based bispecific antibody construct known as BITE® (Bispecific T-cell Engager) utilizes a single polypeptide that contains two antigen-binding domains, each provided by a VH and VL pair, linked in tandem via a flexible linker (see, e.g., Nagorsen et al., 2009, Leukemia & Lymphoma 50:886-91; Amann et al., 2009, J Immunother 32:453-64; Baeuerle and Reinhardt, 2009, Cancer Res 69:4941-44). Another bispecific antibody, called DART® (Dual-Affinity Re-Targeting), utilizes a disulfide-stabilized diabody design (see, e.g., Moore et al., 2011, Blood 117:4542-51; Veri et al., 2010, Arthritis Rheum 62:1933-43).

[0111] In one embodiment, the single polypeptide chain further comprises an Fc domain (e.g., a full-length Fc domain or a portion thereof), optionally disposed between the first antigen-binding domain and the second antigen-binding domain.

[0112] In one aspect of any embodiment, the first antigen-binding domain and / or the second antigen-binding domain comprises an scFv, optionally, the scFv comprises a human framework amino acid sequence. In one embodiment, a monomeric bispecific Fc-derived polypeptide is provided that comprises (a) a first scFv that binds to NKp46, (b) a second scFv that binds to an antigen other than NKp46, and optionally (c) at least a portion of a human Fc domain. Optionally, the Fc domain (i) does not dimerize with another Fc-derived polypeptide, and (ii) can bind to human FcRn. Optionally, the Fc domain is disposed between the first scFv and the second scFv.

[0113] When the polypeptide fusion product comprising two ABDs and at least a portion of an Fc domain is monomeric, the CH3 domain can be positioned and / or comprise amino acid modifications to prevent CH3-CH3 dimerization. In one embodiment, the CH3 domain comprises a mutation at the dimer interface to prevent interchain CH3-CH3 dimerization. In another embodiment, the CH3 domain is a tandem CH3 domain (or the Fc domain comprises a tandem CH3 domain) to prevent interchain CH3-CH3 dimerization. Such a monomer maintains partial FcRn binding (e.g., compared to a wild-type full-length human IgG1 antibody). Optionally, the monomeric polypeptide can bind to human FcRn with moderate affinity, e.g., maintains binding to FcRn but has reduced binding to human FcRn receptors compared to a full-length wild-type human IgG1 antibody. The Fc portion can further comprise one or more amino acid modifications, e.g., in the CH2 domain, that further reduce or substantially eliminate binding to one or more Fcγ receptors.

[0114] In one configuration, a monomeric Fc derived polypeptide having at least a portion of a human Fc domain may advantageously comprise a CH2 domain and a CH3 domain, said CH3 domain comprising an altered CH3 dimer interface (e.g., a mutation in the CH3 dimer interface) to prevent dimerization with another Fc derived polypeptide. See, e.g., forms 1 and 2 of FIG. 2A). In one embodiment of any polypeptide or method herein, the CH3 domain comprises amino acid substitutions at one, two, three, four, five, six, or seven of positions L351, T366, L368, P395, F405, T407 (or Y407), and / or K409 (EU numbering as in Kabat).

[0115] Another configuration of CH3 domains that can be used for monomeric multispecific proteins is a tandem CH3 domain (see, for example, forms 3 and 4 in FIG. 2A). A tandem CH3 domain comprises a first and a second CH3 domain, where the two CH3 domains are bound to each other via non-covalent interactions. In one embodiment, the two CH3 domains are bound to each other via the CH3 dimerization interface of each CH3 domain. In one embodiment, the polypeptide chain does not dimerize with another polypeptide chain that comprises an Fc domain. An Fc domain that comprises a tandem CH3 domain interacts with the neonatal Fc receptor (FcRn) but has low or no binding to human Fcγ receptors, particularly CD16.

[0116] Multimeric Proteins Multimeric bispecific proteins, such as heterodimers, heterotrimers, and tetramers (the latter including, for example, antibodies having two heavy chains and two light chains), comprising an ABD that binds NKp46 (e.g., an ABD comprising a VH and a VL as disclosed herein) can be made according to a variety of configurations.

[0117] In one embodiment, the multimeric protein or polypeptide is a tetrameric antibody composed of two heavy chains comprising variable regions (or one, two or three CDRs thereof) from two different parent antibodies, and two light chains comprising variable regions (or one, two or three CDRs thereof) from two different parent antibodies. Such a tetramer may comprise (a) two heavy chains each comprising a variable region, a CH1 domain, a hinge, and an Fc domain, and (b) two antibody light chains each comprising a light chain variable region and a CK domain, where one heavy chain variable region binds to NKp46 together with the light chain variable region, and the other heavy chain variable region binds to an antigen of interest together with the light chain variable region.

[0118] One advantageous way of making a multimeric protein is by assembly of different polypeptide chains comprising at least one heavy or light chain variable domain fused to a human CH1 or Cκ constant domain, respectively (V-(CH1 / Cκ) unit), where the protein chains are CH1-Cκ dimerized and further linked to each other by non-covalent bonds and optionally by disulfide bonds formed between the respective CH1 and Cκ domains. In one embodiment, an isolated or purified heterodimeric or heterotrimeric protein is provided which binds to a first and a second antigen, comprising at least two or three polypeptide chains each comprising a V-(CH1 / Cκ) unit, whereby the chains are further linked to each other by non-covalent bonds and optionally by disulfide bonds between the CH1 and Cκ domains, and optionally whereby the chains are further linked to each other by non-covalent bonds between the respective variable regions and the CH3 domains of the Fc part.

[0119] The variable and constant regions can be selected and constructed such that each chain preferentially associates with its desired complementary partner chain. Thus, the resulting multimeric protein is easy to produce using conventional production methods using recombinant host cells. The selection of which VH, VL to associate with CH1 and Cκ in a unit is based on the affinity between the units to be paired to promote the formation of the desired multimer. The resulting multimer is further linked by non-covalent bonds between complementary VH and VL domains, by non-covalent bonds between complementary CH1 and Cκ domains, and optionally by disulfide bonds between complementary CH1 and Cκ domains, and / or by disulfide bonds between optional complementary hinge domains. The VH-VL association is stronger than VH-VH or VL-VL, and as a result, VH or VL can be placed adjacent to either CH1 or Cκ, as shown herein, and the resulting VC unit will preferably partner with its VC counterpart. For example, VH-Cκ preferentially pairs with VL-CH1 over VH-CH1.In addition, the inclusion of Fc domain further improves favorable chain pairing, since two Fc-containing chains are linked by non-covalent bond between CH3 domains of Fc domains.Different VC combinations can be optionally further combined into Fc pairing, thereby providing a tool for making heteromultimeric proteins.

[0120] In one embodiment, the heteromultimeric polypeptide or protein comprises a monomeric Fc domain (e.g., the second polypeptide does not comprise an Fc domain), and optionally, the Fc domain comprises a CH3 domain or a tandem CH3 domain having amino acid mutations that prevent CH3-CH3 dimerization.

[0121] In another embodiment, the heteromultimeric polypeptide or protein comprises a dimeric Fc domain capable of binding to human CD16, e.g., a human Fc domain comprising N-linked glycosylation at amino acid residue N297 (Kabat EU numbering).

[0122] Heterodimers can have, for example, the following configurations (see also examples of such proteins shown as forms 2, 11, and 12 in Figures 2A and 2C): [ka] where V of the first polypeptide chain a-1 and V of the second polypeptide chain b-1 one is a light chain variable domain and the other is a heavy chain variable domain, a-2 and V b-2 One is a light chain variable domain and the other is a heavy chain variable domain.

[0123] The heterodimer may, in another example, have the following configuration (see also an example of such a protein shown in FIG. 2B as Form 10): [ka] where V of the first polypeptide chain a-1 and V of the second polypeptide chain b-1 one is a light chain variable domain and the other is a heavy chain variable domain, a-2 and V b-2 One is a light chain variable domain and the other is a heavy chain variable domain.

[0124] The resulting heterodimer may, in another example, have the following configuration (see also the example protein shown as forms 13 and 14 in Figures 2D and 2E): [ka] where V of the first polypeptide chain a-1 and V of the second polypeptide chain b-1 one is a light chain variable domain and the other is a heavy chain variable domain, a-2 and V b-2 One is a light chain variable domain and the other is a heavy chain variable domain.

[0125] Heterotrimeric proteins can be formed, for example, by using a central (first) polypeptide chain comprising a first variable domain (V) fused to a first CH1 or CK constant region, a second variable domain (V) fused to a second CH1 or CK constant region, and an Fc domain or portion thereof disposed between the first and second variable domains (i.e., the Fc domain is disposed between the first and second V-(CH1 / CK) units). For example, the central polypeptide chain used in a heterotrimeric protein can have the following domain arrangement (from N-terminus to C-terminus): V a-1 -(CH1 or CK) a -Fc domain-V a-2 -(CH1 or CK) b .

[0126] The second polypeptide chain then has the domain arrangement (from N-terminus to C-terminus): V b-1 -(CH1 or CK) c or V b-1 -(CH1 or CK) c -Fc domain Thus, (CH1 or CK) c is the central chain (CH1 or CK) a and dimerizes to V a-1 and V b-1 and form the antigen-binding domain.

[0127] The third polypeptide chain then has the domain arrangement (from N-terminus to C-terminus): V b-2 -(CH1 or CK) d Thus, (CH1 or CK) d is the central chain (CH1 or CK) b dimerizes with the unit V a-2 and V b-2 and form the antigen-binding domain.

[0128] An example of a resulting heterotrimeric configuration having a dimeric Fc domain (also shown in Figures 2D and 2E as configurations 5, 6, 7, and 16) has the domain arrangement: [ka] has.

[0129] One example of a resulting heterotrimeric configuration having monomeric Fc domains (also shown in Figures 2B and 2C as configurations 8, 9, and 17) has the domain arrangement: [ka] has.

[0130] Thus, in a trimeric polypeptide configuration, a first polypeptide can have two variable domains that each form an antigen binding domain with a variable domain of a separate polypeptide chain (i.e. a variable domain of a second chain and a variable domain of a third chain), a second polypeptide chain has one variable domain and a third polypeptide has one variable domain.

[0131] The trimeric polypeptide is (a) a first polypeptide chain comprising a first variable domain (V) fused to a first CH1 or CK constant region, a second variable domain (V) fused to a second CH1 or CK constant region, and an Fc domain or a portion thereof disposed between the first and second variable domains; (b) a second polypeptide chain comprising a variable domain fused at its C-terminus to a CH1 or CK constant region selected to be complementary to a first CH1 or CK constant region of the first polypeptide chain, and optionally an Fc domain, such that the first and second polypeptides form a CH1-CK heterodimer; and (c) a third polypeptide chain comprising a variable domain fused (e.g., at its C-terminus) to a CH1 or CK constant region, wherein the variable domain and constant region are selected to be complementary to a second variable domain and a second CH1 or CK constant region of the first polypeptide chain, whereby the first and third polypeptides form a CH1-CK heterodimer linked non-covalently and, optionally, further by a disulfide bond formed between the CH1 or CK constant region of the third polypeptide and the second CH1 or CK constant region of the first polypeptide, but not between the CH1 or CK constant region of the third polypeptide and the first CH1 or CK constant region of the first polypeptide. wherein the first polypeptide, the second polypeptide, and the third polypeptide form a CH1-CK heterotrimer, the first variable domain of the first polypeptide chain and the variable domain of the second polypeptide chain form an antigen-binding domain specific for a first antigen of interest, and the second variable domain of the first polypeptide chain and the variable domain of the third polypeptide chain form an antigen-binding domain specific for a second antigen of interest, one of the two antigens of interest is NKp46, and the ABD that binds to NKp46 comprises a VH-VL variable domain pair of the present disclosure.

[0132] Examples of domain arrangements of the trimeric bispecific polypeptides formed include, but are not limited to, the following:

[0133] [Table 4]

[0134] In any domain arrangement, the Fc domain may comprise a CH2-CH3 unit (full-length CH2 and CH3 domains or fragments thereof). In a heterodimer or heterotrimer comprising two chains with an Fc domain (dimeric Fc domain), the CH3 domain is capable of CH3-CH3 dimerization (e.g., wild-type CH3 domain). In a heterodimer or heterotrimer comprising only one chain with an Fc domain (monomeric Fc domain), the Fc domain is incapable of CH3-CH3 dimerization, e.g., the CH3 domain has an amino acid modification at the CH3 dimer interface or the Fc domain comprises a tandem CH3 domain that is incapable of CH3-CH3 dimerization.

[0135] In some exemplary configurations, the multispecific protein can be a tetramer, e.g., a heterodimeric tetramer having two light chains and two different heavy chains, where the heavy chains are engineered for heterodimerization. Such proteins can be prepared, for example, as described in PCT Application No. PCT / EP2016 / 064537 (Innate Pharma), filed June 23, 2016, the disclosure of which is incorporated herein by reference.

[0136] In any protein of the present disclosure, the hinge region is typically present in the polypeptide chain between the CH1 and CH2 domains and / or may be present between the CK and CH2 domains. The hinge region can optionally be replaced, for example, by a suitable linker peptide.

[0137] Protein domains described in this disclosure can optionally be designated as being N-terminal to C-terminal. For purposes of illustration, the arrangement of disclosed proteins is shown from N-terminus (left) to C-terminus. Domains can be said to be fused to one another (e.g., a domain can be said to be fused to the C-terminus of the domain to its left and / or a domain can be said to be fused to the N-terminus of the domain to its right).

[0138] The protein domains described in this disclosure can be fused to each other directly or through an intervening amino acid sequence. For example, the CH1 or CK domain is fused to the Fc domain (or its CH2 or CH3 domain) through a linker peptide, optionally a hinge region or a fragment thereof. In another example, the VH or VK domain is fused to the CH3 domain through a linker peptide. The VH and VL domains linked to each other in tandem are fused through a linker peptide (e.g., as in an scFv). The VH and VL domains linked to the Fc domain are fused through a linker peptide. The two polypeptide chains are further linked to each other by non-covalent bonds and, optionally, by interchain disulfide bonds formed between cysteine ​​residues in the complementary CH1 and CK domains (indicated by "|").

[0139] Linkers and Fc domains are described in more detail, for example, in PCT Application No. PCT / EP2016 / 064537 (Innate Pharma), filed June 23, 2016, the disclosure of which is incorporated herein by reference.

[0140] Once a multispecific protein has been generated, it can be evaluated for biological activity.

[0141] In one aspect of any of the embodiments herein, the multispecific protein is capable of inducing activation of an NKp46-expressing cell (e.g., an NK cell, a reporter cell) when incubated in the presence of an NKp46-expressing cell (e.g., a purified NK cell) and a target cell expressing an antigen of interest.

[0142] In one aspect of any of the embodiments herein, the multispecific protein is capable of inducing NKp46 signaling in NKp46-expressing cells (e.g., NK cells, reporter cells) when incubated in the presence of NKp46-expressing cells (e.g., purified NK cells) and target cells expressing an antigen of interest.

[0143] Optionally, NK cell activation or signaling is characterized by increased expression of cell surface markers of activation, such as CD107, CD69, etc.

[0144] Activity can be measured, for example, by contacting target cells and NKp46-expressing cells with each other in the presence of the multispecific polypeptide. In one example, aggregation of target cells with NK cells is measured. In another example, the multispecific protein can be evaluated for any property or activity known in the art associated with NK cell activity, for example, the ability to cause a measurable increase in a marker of cytotoxicity (CD107) or cytokine production (e.g., IFN-γ or TNF-α), an increase in intracellular free calcium levels, the ability to lyse target cells in a redirected killing assay, etc. Activity assays are further described in more detail, for example, in PCT Application No. PCT / EP2016 / 064537 (Innate Pharma), filed June 23, 2016, the disclosure of which is incorporated herein by reference.

[0145] Use of the compound Compounds according to the present disclosure that contain an antigen binding domain that binds to NKp46 can be used in a variety of applications, including, for example, binding to, detecting, removing, purifying, or modulating the activity of NKp46 polypeptide and / or cells expressing NKp46 polypeptide (e.g., NK cells).

[0146] In one aspect, there is provided the use of any of the compounds defined herein in the manufacture of a pharmaceutical preparation for the treatment or diagnosis of a mammal in need thereof. Also provided is the use of any of the compounds defined above as a medicament or as an active ingredient or substance in a medicament. In a further aspect, there is provided a method of preparing a pharmaceutical composition comprising a compound defined above to provide a solid or liquid formulation for oral administration, topical administration or injection. Such a method or process comprises at least the step of mixing the compound with a pharma- ceutically acceptable carrier.

[0147] In one aspect, a method is provided for treating, preventing or more generally influencing a given condition by providing a specific effect or for detecting a given condition using the multispecific protein described herein or a (pharmaceutical) composition comprising this multispecific protein.

[0148] For example, in one aspect, the invention provides a method of restoring or enhancing NKp46+ NK cell activity in a patient in need thereof (e.g., a patient having cancer or a viral or bacterial infection), comprising administering to said patient a multispecific protein as described herein. In one embodiment, the method relates to increasing NKp46+ lymphocyte activity in a patient having a disease in which increased lymphocyte (e.g., NK cell) activity would be beneficial or which is caused by or characterized by insufficient NK cell activity, e.g., cancer or a viral or microbial / bacterial infection.

[0149] The polypeptides described herein can be used to prevent or treat disorders that can be treated with antibodies, such as cancer, solid and non-solid tumors, hematological malignancies, infections, such as viral infections, and immune or autoimmune diseases.

[0150] In one embodiment, the antigen of interest (non-NKp46 antigen) is an antigen expressed on the surface of malignant cells of a type of cancer selected from the group consisting of: carcinomas including bladder, head and neck, breast, colon, kidney, liver, lung, ovarian, prostate, pancreatic, gastric, cervical, thyroid, and skin cancer, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, and Burckett's lymphoma; hematopoietic tumors of myeloid lineage including acute and chronic myeloid leukemia and promyelocytic leukemia; interleukin-1, including fibrosarcoma and rhabdomyosarcoma. tumors of myelogenous origin; other tumors including neuroblastoma and glioma; tumors of the central and peripheral nervous system including astrocytoma, neuroblastoma, glioma, and schwannoma; tumors of rhabdomyosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratocarcinoma, hematopoietic tumors of lymphoid lineage, such as, but not limited to, T-cell disorders including small cell and large cell types, such as T-cell and B-cell tumors including T-prolymphocytic leukemia (T-PLL); large granular lymphocytic leukemia (LGL), preferably of the T-cell type; Sézary syndrome (SS); adult T-cell leukemia-lymphoma (ATLL); a / d T-NHL hepatosplenic lymphoma; peripheral / retrothymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angioimmunoblastic T-cell lymphoma; angiocentric (nasal) T-cell lymphoma; undifferentiated (Ki 1+) large cell lymphoma; intestinal T-cell lymphoma; T-lymphoblast; and lymphoma / leukemia (T-Lbly / T-ALL).

[0151] In one embodiment, the polypeptides described herein can be used to prevent or treat a cancer selected from the group consisting of: carcinomas including bladder cancer, head and neck cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, and skin cancer, including squamous cell carcinoma; leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B cell lymphoma, T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, and Burke's lymphoma. hematopoietic tumors of lymphoid lineage, including lymphomas; hematopoietic tumors of myeloid lineage, including acute and chronic myeloid leukemia and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannoma; tumors of rhabdomyosarcoma, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratocarcinoma. Other exemplary disorders that can be treated by the present invention include hematopoietic tumors of lymphoid lineage, such as, but not limited to, T cell disorders, including small cell and large cell types, such as, T cell and B cell tumors, including, for example, T-prolymphocytic leukemia (T-PLL); large granular lymphocytic leukemia (LGL), preferably of the T cell type; Sézary syndrome (SS); adult T cell leukemia lymphoma (ATLL); a / d T-NHL hepatosplenic lymphoma; peripheral / retrothymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angioimmunoblastic T cell lymphoma; hemangiocentric (nasal) T cell lymphoma; anaplastic (Ki 1+) large cell lymphoma; intestinal T cell lymphoma; T lymphoblastic; and lymphoma / leukemia (T-Lbly / T-ALL).

[0152] In one example, the tumor antigen is an antigen expressed on the surface of lymphoma or leukemia cells, and the multispecific protein is administered to and / or used to treat an individual with lymphoma or leukemia. Optionally, the tumor antigen is selected from CD19, CD20, CD22, CD30, or CD33.

[0153] In one aspect, a method of treatment comprises administering to an individual a therapeutically effective amount of a multispecific protein as described herein, e.g., for the treatment of a disease disclosed herein, e.g., a cancer selected from the group above. A therapeutically effective amount can be any amount that has a therapeutic effect in patients with the disease or disorder (or that promotes, enhances, and / or induces such an effect in at least a substantial proportion of patients with the disease or disorder and patients with substantially similar characteristics to the patient).

[0154] In one embodiment, the multispecific proteins described herein can be used as monotherapy (without other therapeutic agents) or in combination therapy with one or more other therapeutic agents, including agents typically utilized for the particular therapeutic purpose for which the antibody is administered. The additional therapeutic agent is typically administered in an amount and treatment regimen typically used for that therapeutic agent in a monotherapy for the particular disease or condition being treated. Such therapeutic agents, when used in the treatment of cancer, include, but are not limited to, anticancer agents and chemotherapeutic agents; in the treatment of infectious diseases, include, but are not limited to, antiviral agents and antibiotics.

[0155] The proteins and / or polypeptides disclosed herein can be included in a kit. The kit can optionally further include any number of polypeptides and / or other compounds, for example, one, two, three, four, or other number of proteins and / or polypeptides and / or other compounds. It should be understood that this description of the contents of the kit is in no way limiting. For example, the kit can include other types of therapeutic compounds. Optionally, the kit also includes instructions for use of the proteins and / or polypeptides, for example, details of the methods described herein.

[0156] Also provided is a pharmaceutical composition comprising the compound as defined above. The compound can be administered in purified form together with a pharmaceutical carrier as a pharmaceutical composition. The form depends on the intended mode of administration and therapeutic or diagnostic use. The pharmaceutical carrier can be any compatible non-toxic substance suitable for delivering the compound to a patient. Pharmaceutically acceptable carriers are known in the art and include, for example, aqueous solutions, such as (sterile) water or physiological buffered saline, or other solvents or vehicles, such as glycols, glycerol, oils, such as olive oil, or injectable organic esters, alcohols, fats, waxes, and inert solids. Pharmaceutically acceptable carriers may further include physiologically acceptable compounds that act, for example, to stabilize or enhance absorption of the compound. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose, or dextran, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers or excipients. One of ordinary skill in the art will appreciate that the choice of a pharma- ceutically acceptable carrier, including a physiologically acceptable compound, will depend, for example, on the route of administration of the composition. Pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like, may also be included in the pharmaceutical composition.

[0157] The compounds can be administered parenterally. Preparations of the compounds for parenteral administration must be sterile. Sterilization is easily accomplished by filtration through sterile filtration membranes, optionally before or after lyophilization and reconstitution. Parenteral routes of administration of the compounds follow known methods, such as injection or infusion by intravenous, intraperitoneal, intramuscular, intraarterial, or intralesional routes. The compounds can be administered continuously by infusion or bolus injection. A typical composition for intravenous injection can be made up to contain 100-500 ml of sterile 0.9% NaCl or 5% glucose, optionally supplemented with 20% albumin solution, and 1 mg-10 g of the compound, depending on the particular type of compound and its required dosing schedule. Methods for preparing parenterally administrable compositions are known in the art. EXAMPLES

[0158] Example 1: Preparation of anti-huNKp46 antibody Part A: Generation of anti-huNKp46 antibodies Balb / c mice were immunized with recombinant human NKp46 extracellular domain recombinant Fc protein. Mice were first immunized intraperitoneally with 50 μg of NKp46 protein in complete Freund's adjuvant, second immunized intraperitoneally with 50 μg of NKp46 protein in incomplete Freund's adjuvant, and finally boosted intravenously with 10 μg of NKp46 protein. Immune splenocytes were fused to X63.Ag8.653 immortalized B cells 3 days after the boost and cultured in the presence of irradiated splenocytes.

[0159] Primary Screening: Supernatants of growing clones (S / N) were tested in a primary screen by flow cytometry using a cell line expressing the human NKp46 construct on the cell surface. Briefly, in the FACS screen, the presence of reactive antibodies in the supernatants was revealed by a PE-labeled goat anti-mouse polyclonal antibody (pAb).

[0160] A selection of antibodies that bind to NKp46 were chosen and produced as full-length human IgG antibodies and bispecific antibodies. Among the variable regions evaluated for their activity in the bispecific molecules of Examples 2-13 were antibodies NKp46-1, -2, -3, -4, -6, and -9, with their respective variable regions shown in Table B herein.

[0161] Part B: Generation of humanized anti-human / anti-cynomolgus NKp46 antibodies Antibodies NKp46-1, -2, -3, and 4, having their respective variable regions as shown in Table B herein, were generated as humanized antibodies by complementarity determining region (CDR) grafting of the heavy and light chains with the amino acid sequences shown below. The antibodies were produced using CHO cells and tested for binding to human NKp46.

[0162] Each of the CDR-grafted antibodies bound to human NKp46 with good affinity. However, none of the CDR-grafted antibodies bound to cynomolgus NKp46. Having determined the epitopes on human NKp46 (see Example 13); considering the possibility that modifications affecting the conformation of the antibody variable region or the arrangement of the CDRs may enable epitopes shared with cynomolgus NKp46 to be recognized, multiple mutants were prepared for each of the CDR-grafted antibodies NKp46-1, -2, -3, -4, and -9, produced using CHO cells, and tested for binding to cynomolgus NKp46. For each of the antibodies NKp46-1, -2, -3, -4, and -9, one or more mutants were identified that allowed recognition of epitopes shared by human and cynomolgus NKp46 binding with good affinity. Based on 3D modeling studies, various heavy and light chain variable regions containing the NKp46-1 CDRs and human frameworks were designed, generated as human IgG1 antibodies, and tested for binding to cynomolgus monkey NKp46.

[0163] Antibody NKp46-1 Based on 3D modeling studies, various heavy and light chain variable regions containing NKp46-1 CDRs and human frameworks were designed, generated as human IgG1 antibodies, and tested for binding to cynomolgus NKp46. Two combinations of heavy and light chains are capable of binding to both human and cynomolgus NKp46. Heavy chain variable region "H1" and heavy chain "H3" were combined in each case with light chain "L1". These cross-linking variable regions include, for the heavy chain variable region, NKp46-1 heavy chain CDRs (underlined below), human IGHV1-69 CDRs, ... * 06 gene framework 1, 2, and 3 regions, and human IGHJ6 * The 01 gene framework 4 regions were included. Light chain variable region: NKp46-1 light chain CDRs (underlined below), human IGKV1-33 * 01 gene framework 1, 2, and 3 regions, and human IGKJ4 *01 gene framework 4 region. CDRs were selected according to Kabat numbering. The H1, H3, and L1 chains had specific amino acid substitutions (shown below in bold and underlined). L1 had a phenylalanine at Kabat light chain residue 87. H1 had a tyrosine at Kabat heavy chain residue 27, and a lysine and alanine at Kabat residues 66 and 67, respectively. In addition, H3 had a glycine at Kabat residue 37, an isoleucine at Kabat residue 48, and a phenylalanine at Kabat residue 91. NKp46-1: “H1” heavy chain variable region [ka] NKp46-1: “H3” heavy chain variable region [ka] NKp46-1: “L1” light chain variable region [ka]

[0164] Antibody NKp46-2 Based on 3D modeling studies, various heavy and light chain variable regions containing NKp46-2 CDRs and human frameworks were designed, generated as human IgG1 antibodies, and tested for binding to cynomolgus monkey NKp46. Three combinations of heavy and light chains are capable of binding to both human and cynomolgus monkey NKp46. Heavy chain variable regions "H1", "H2", and heavy chain "H3" were combined in each case with light chain "L1". Interestingly, H1L1 also had improved binding affinity compared to the parent antibody NKp46-2 with VH and VL of SEQ ID NOs: 5 and 6. These cross-linking variable regions include, for the heavy chain variable region, NKp46-2 heavy chain CDRs (underlined below), human IGHV4-30-4 * 01 gene framework 1, 2, and 3 regions, and human IGHJ1 *The 01 gene framework 4 regions were included. Light chain variable region: NKp46-2 light chain CDRs (underlined below), human IGKV1-39 * 01 gene framework 1, 2, and 3 regions, and human IGKJ4 * 01 gene framework 4 region. CDRs were selected according to Kabat numbering. The L1, H1, H2, and H3 chains had specific amino acid substitutions (shown below in bold and underlined). L1 had a valine at Kabat light chain residue 48. H1 had a tyrosine at Kabat heavy chain residue 27 and an arginine at Kabat residue 71. In addition, H2 had a methionine at Kabat residue 48 and an isoleucine at Kabat residue 67. In addition, H3 had a threonine at Kabat residue 31. NKp46-2: “H1” heavy chain variable region [ka] NKp46-2: “H2” heavy chain variable region [ka] NKp46-2: “H3” heavy chain variable region [ka] NKp46-2: “L1” light chain variable region [ka]

[0165] Antibody NKp46-3 Based on 3D modeling studies, various heavy and light chain variable regions containing NKp46-3 CDRs and human frameworks were designed, generated as human IgG1 antibodies, and tested for binding to cynomolgus monkey NKp46. Three combinations of heavy and light chains are capable of binding to both human and cynomolgus monkey NKp46. Heavy chain variable regions "H1", "H3", and heavy chain "H4" were combined in each case with light chain "L1". Interestingly, H3L1 also had improved binding affinity compared to the parent antibody NKp46-3 with VH and VL of SEQ ID NOs: 7 and 8. These cross-linking variable regions were designed to include, for the heavy chain variable region, NKp46-3 heavy chain CDRs (underlined below), human IGHV1-69 * 02 gene framework 1, 2, and 3 regions, and human IGHJ6 * The light chain variable region contained NKp46-3 light chain CDRs (underlined below), framework 1, 2, and 3 regions formed by a mosaic approach using FR1 and FR2 from IGKV3-15 and FR3 from IGKV3-11, and human IGKJ2 * 01 gene framework 4 region. CDRs were selected according to Kabat numbering. The L1, H1, H3, and H4 chains had specific amino acid substitutions (shown below in bold and underlined). L1 had a lysine at Kabat light chain residue 49. H1 had a tyrosine at Kabat heavy chain residue 27. In addition, H3 had an isoleucine at Kabat residue 48 and an alanine at Kabat residue 67. In addition, H4 had a leucine at Kabat residue 69. NKp46-3: “H1” heavy chain variable region [ka] NKp46-3: “H3” heavy chain variable region [ka] NKp46-3: “H4” heavy chain variable region [ka] NKp46-3: “L1” light chain variable region [ka]

[0166] Antibody NKp46-4 Based on 3D modeling studies, various heavy and light chain variable regions containing NKp46-4 CDRs and human frameworks were designed, generated as human IgG1 antibodies, and tested for binding to cynomolgus NKp46. The heavy chain variable region "H1" combined with the light chain "L2" was able to bind both human and cynomolgus NKp46, as well as the parent antibody. Two other antigen binding regions (one composed of "L2" and "H2" and the other composed of "L2" and "H3") were able to bind to cynomolgus NKp46 with moderate affinity, but with 1 / 10th the affinity of human NK-46. These cross-linking variable regions were designed using a mosaic approach using the NKp46-4 heavy chain CDRs (underlined below), FR1 from IGHV1646, FR3 from IGHV1-69, and a common FR2 (IGHV1-46 and IGHV1-69 share the same FR2) and human framework 1, 2, and 3 regions for the heavy chain variable region. * The light chain variable region contained the NKp46-4 light chain CDRs (underlined below), framework 1, 2, and 3 regions from IGKV1-NL1, and human IGKJ4 *01 gene framework 4 region. CDRs were selected according to Kabat numbering. The L2, H1, H2, and H3 chains had specific amino acid substitutions (shown below in bold and underlined). L2 had a phenylalanine at Kabat light chain residue 36 and a valine at Kabat residue 48. H1 had a threonine at Kabat heavy chain residue 30, an isoleucine at Kabat residue 48, and a valine at Kabat residue 93. H2 had a threonine at Kabat residue 67. In addition, H3 had a leucine at Kabat residue 69. NKp46-4: “H1” heavy chain variable region [ka] NKp46-4: “H2” heavy chain variable region [ka] NKp46-4: “H3” heavy chain variable region [ka] NKp46-4: “L2” light chain variable region [ka]

[0167] Antibody NKp46-9 Based on the 3D modeling studies, various heavy and light chain variable regions containing the NKp46-9 CDRs and human frameworks were designed, generated as human IgG1 antibodies, and tested for binding to cynomolgus NKp46. The heavy chain variable region "H2" when combined with either the light chain variable region "L1" or "L2", and the heavy chain variable region "H3" when combined with either the light chain variable region "L1" or "L2" were able to bind both human and cynomolgus NKp46 with higher affinity than the parent antibody (having VH and VL of SEQ ID NOs: 13 and 14). The "H1" chain was unable to bind cynomolgus NKp46 with any of the light chains tested, but was able to bind human NKp46 well. These cross-linking variable regions were: NKp46-9 heavy chain CDRs (underlined below), IGHV4-30-4 for the heavy chain variable region, * Human framework 1, 2, and 3 regions from 01, and human IGHJ6 * The 01 gene framework contained 4 regions. Light chain variable region: NKp46-9 light chain CDRs (underlined below), IGKV1-39 * Framework 1, 2, and 3 regions from 01 and human IGKJ2 * 01 gene framework 4 region. CDRs were selected according to Kabat numbering. The L1, L2, H1, H2, and H3 chains had specific amino acid substitutions (shown below in bold and underlined). L1 had a cysteine ​​at Kabat residue 36. L2 had a valine at Kabat light chain residue 48. H1 had an arginine at Kabat heavy chain residue 71. In addition, H2 had a tyrosine at Kabat residue 27. H3 had a methionine at Kabat residue 48 and an isoleucine at Kabat residue 67. NKp46-9: “H1” heavy chain variable region [ka] NKp46-9: “H2” heavy chain variable region [ka] NKp46-9: “H3” heavy chain variable region [ka] NKp46-9: “L1” light chain variable region [ka] NKp46-9: “L2” light chain variable region [ka]

[0168] Example 2: Identification of a bispecific antibody format that targets effector cell receptors and binds FcRn but not FcγR The goal of this experiment was to develop a novel bispecific protein format in which an Fc domain is placed in a polypeptide together with an anti-NKp46 binding domain and an anti-target antigen binding domain. The bispecific protein binds monovalently to NKp46 via its anti-NKp46 binding domain. The monomeric Fc domain maintains at least partial binding to the human neonatal Fc receptor (FcRn) but does not substantially bind to human CD16 and / or other human Fcγ receptors. As a result, the bispecific protein does not induce Fcγ-mediated (e.g., CD16-mediated) target cell lysis.

[0169] Example 2-1 Preparation of anti-CD19-IgG1-Fcmono anti-CD3 and binding analysis Because no anti-NKp46 bispecific antibodies have been generated that can show whether such a protein might be functional, CD3 was used as a model antigen instead of NKp46 to examine the function of the novel monovalent bispecific protein form prior to targeting of NK cells via NKp46.

[0170] A bispecific Fc based on an scFv specific for the tumor antigen CD19 (anti-CD19 scFv) and an scFv specific for the T cell activating receptor CD3 (anti-CD3 scFv) was used to evaluate the FcRn- and CD19-binding functions of the novel monomeric bispecific polypeptide format. The domain configuration of the final polypeptide is also referred to as the "F1" format (the star in the CH2 domain indicates the optional N297S mutation, which is not included in the polypeptides tested here).

[0171] A bispecific monomeric Fc-containing polypeptide was constructed based on an scFv specific for the tumor antigen CD19 (anti-CD19 scFv) and an scFv specific for the T-cell activating receptor CD3 (anti-CD3 scFv). The CH3 domain contained the mutations (EU numbering) L351K, T366S, P395V, F405R, T407A, and K409Y. This polypeptide has domains arranged as follows: anti-CD19-CH2-CH3-anti-CD3. A DNA sequence encoding a CH3 / VH linker peptide with the amino acid sequence STGS was designed to insert a specific Sall restriction site at the CH3-VH junction.

[0172] The CH3 domain contained the mutations (EU numbering) L351K, T366S, P395V, F405R, T407A, and K409Y. The CH2 domain was wild type CH2. The DNA and amino acid sequences of the monomeric CH2-CH3Fc portion and anti-CD19 are shown below.

[0173] The light and heavy chain DNA and amino acid sequences corresponding to the anti-CD19 scFv were as follows:

[0174] [Table 5]

[0175] The DNA sequence of the monomeric CH2-CH3 Fc portion and the final bispecific IgG1-Fcmono polypeptide (in which the final K was removed) is shown in SEQ ID NO: 117. The amino acid sequence is shown in SEQ ID NO: 2. The anti-CD19-F1-anti-CD3 complete sequence (mature protein) is shown in SEQ ID NO: 118.

[0176] Cloning and production of recombinant proteins Coding sequences were constructed by direct synthesis and / or PCR. PCR was performed with PrimeSTAR MAX DNA polymerase (Takara, #R045A) and PCR products were purified from 1% agarose gels using NucleoSpin gel and PCR clean-up kit (Macherey-Nagel, #740609.250). After purification, PCR products were quantified before In-Fusion ligation reactions as described in the manufacturer's protocol (ClonTech, #ST0345). Plasmids were obtained after miniprep preparations performed on an EVO200 (Tecan) using Nucleospin 96 Plasmid Kit (Macherey-Nagel, #740625.4). Plasmids were then sequenced for sequence verification before transfection of CHO cell lines.

[0177] CHO cells were grown in CD-CHO medium (Invitrogen) supplemented with phenol red and 6 mM GlutaMax. The day before transfection, cells were counted and seeded at 175,000 cells / ml. For transfection, cells (200,000 cells / transfection) were prepared as described in the AMAXA SF cell line kit (AMAXA, #V4XC-2032) and nucleofected using the DS137 protocol in a Nucleofector 4D instrument. All transfections were performed with 300 ng of validated plasmids. After transfection, cells are seeded in pre-warmed culture medium in 24-well plates. After 24 hours, hygromycin B was added to the culture medium (200 μg / ml). Protein expression is monitored in the medium after one week. Cells expressing the protein are then subcloned to obtain the best producing line. Subcloning was performed using 96 flat-bottom well plates, seeding cells at 1 cell / well in 200 μl of culture medium supplemented with 200 μg / ml hygromycin B. Cells were left for 3 weeks before testing the productivity of the clones.

[0178] Recombinant proteins containing IgG1-Fc fragments are purified using Protein A beads (-rProteinA Sepharose fast flow, GE Healthcare, ref.:17-1279-03). Briefly, cell culture supernatants were concentrated, clarified by centrifugation and injected onto a Protein A column to capture recombinant Fc-containing proteins. Proteins were eluted at acidic pH (citric acid 0.1 M pH 3), immediately neutralized with TRIS-HCL pH 8.5 and dialyzed against 1x PBS. Recombinant scFvs containing the "six his" tag were purified by affinity chromatography using cobalt resin. Other recombinant scFvs were purified by size-exclusion chromatography (SEC).

[0179] Example 2-2: Binding analysis of anti-CD19-IgG1-Fcmono-anti-CD3 to B221, JURKAT, HUT78, and CHO cell lines Cells were harvested and stained with cell supernatant of anti-CD19-F1-anti-CD3 producing cells for 1 hour at 4° C. After washing twice with staining buffer (PBS 1X / BSA 0.2% / EDTA 2mM), cells were stained with goat anti-human (Fc)-PE antibody (IM0550 Beckman Coulter-1 / 200) for 30 minutes at 4° C. After two washes, staining was acquired on a BD FACS Canto II and analyzed using FlowJo software.

[0180] The expression of CD3 and CD19 was also controlled by flow cytometry. Cells were harvested and stained with 5 μl of anti-CD3-APC and 5 μl of anti-CD19-FITC antibodies in PBS 1X / BSA 0.2% / EDTA 2mM buffer for 30 min at 4° C. After two washes, staining was acquired on a BD FACS Canto II and analyzed using FlowJo software.

[0181] The anti-CD19-F1-anti-CD3 protein binds to CD3 cell lines (HUT78 and JURKAT cell lines) and to CD19 cell lines (B221 cell line) but not to the CHO cell line used as a negative control.

[0182] Example 2-3: Aggregation of T and B cells with purified anti-CD19-F1-anti-CD3 Purified anti-CD19-F1-anti-CD3 was tested in a T / B cell aggregation assay to assess whether the antibody functions in agglutinating CD19- and CD3-expressing cells.

[0183] The results are shown in Figure 1. The top panel shows that anti-CD19-F1-anti-CD3 does not cause aggregation in the presence of B221 (CD19) or JURKAT (CD3) cell lines, but does aggregate cells when both B221 and JURKAT cells are incubated simultaneously, illustrating that the bispecific antibody is functional. The bottom panel shows a no antibody control.

[0184] Example 2-4: Binding of bispecific monomeric Fc polypeptides to FcRn Affinity testing by surface plasmon resonance (SPR) Biacore T100 General Procedures and Reagents SPR measurements were performed at 25°C on a Biacore T100 instrument (Biacore GE Healthcare). In all Biacore experiments, acetate buffer (50 mM acetate pH 5.6, 150 mM NaCl, 0.1% surfactant p20) and HBS-EP+ (Biacore GE Healthcare) served as running and regeneration buffers, respectively. Sensorgrams were analyzed with Biacore T100 Evaluation software. Recombinant mouse FcRn was purchased from R&D Systems.

[0185] Immobilization of FcRn Recombinant FcRn protein was covalently immobilized to the carboxyl groups of the dextran layer on the Sensor Chip CM5. The chip surface was activated with EDC / NHS (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (Biacore GE Healthcare)). FcRn protein was diluted to 10 μg / ml in binding buffer (10 mM acetic acid, pH 5.6) and injected until the appropriate immobilization level (i.e., 2500 RU) was reached. The remaining activated groups were deactivated using 100 mM ethanolamine pH 8 (Biacore GE Healthcare).

[0186] Affinity testing Monovalent affinity studies were performed according to the Single Cycle Kinetic (SCK) protocol. Five serial dilutions of soluble analytes (antibodies and bispecific molecules) from 41.5 to 660 nM were added to FcRn (without regeneration) and allowed to dissociate for 10 min before regeneration. For each analyte, all sensorgrams were fitted using a 1:1 SCK binding model.

[0187] result Anti-CD19-F1-anti-CD3, whose CH2-CH3 domains are placed between two antigen binding domains (here two scFvs), was evaluated to determine whether such a bispecific monomeric Fc protein could maintain binding to FcRn and thus have an improved in vivo half-life compared to conventional bispecific antibodies. Results showed that FcRN binding was maintained, and the model suggests a 1:1 ratio (one FcRn for each monomeric Fc) rather than a 2:1 ratio (two FcRn for each antibody) for normal IgG.

[0188] Affinity was assessed using SPR for a chimeric full-length antibody with a human IgG1 constant region. The monomeric Fc maintained significant monomeric binding to FcRn (monomeric Fc: KD=194 nM affinity; full-length antibody with bivalent binding: KD=15.4 nM affinity).

[0189] Example 3: Generation of anti-CD19x anti-NKp46 bispecific monomeric Fc domain polypeptides It was unclear whether activating receptors on NK cells could contribute to the lysis of target cells, and furthermore, because anti-NKp46 antibodies can block NKp46, it was unclear whether cytotoxicity could be mediated by NKp46. Therefore, we investigated whether the bispecific protein form could cause triggering of NKp46 and whether it could induce NKp46 agonism in the absence of target cells, thereby resulting in inappropriate NK activation away from the target and / or reduced overall activity against the target cells.

[0190] A novel bispecific protein format was developed as a single chain protein that binds to FcRn but not to FcγR. In addition, multimeric proteins were developed that contain two or three polypeptide chains and in which the Fc domain remains monomeric, which are compatible for use with antibody variable regions that do not maintain binding to their targets when converted to scFv format. The scFv format can be conveniently used for screening antibodies, and by including at least one binding region as a F(ab) structure, any anti-target (e.g., anti-tumor) antibody variable region can be directly expressed and tested in the bispecific construct as a F(ab) format in the bispecific protein, regardless of whether the antibody maintains binding as a scFv, thereby allowing a simple screening and increasing the number of available antibodies. These formats in which the Fc domain remains monomeric have the advantage of maintaining maximum conformational flexibility and may allow optimal binding to NKp46 or target antigens as shown below.

[0191] Different constructs were made for use in preparing bispecific antibodies using variable domains from scFvs specific for the tumor antigen CD19 described in Example 2-1 and different variable regions from an antibody specific for the NKp46 receptor shown in Example 1. Also made were variable regions from the commercially available antibody Bab281 (an mIgG1 available from Beckman Coulter, Inc. (Brea, CA, USA) (see also Pessino et al, J. Exp. Med, 1998, 188(5):953-960 and Sivori et al, Eur J Immunol, 1999.29:1656-1666)) specific for the NKp46 receptor as anti-NKp46.

[0192] In order for the Fc domain to remain monomeric in a single chain polypeptide or in a multimer in which only one chain has an Fc domain, CH3-CH3 dimerization was prevented by two different strategies: (1) the use of CH3 domains containing specific mutations (EU numbering), namely, L351K, T366S, P395V, F405R, T407A, and K409Y; or (2) the use of tandem CH3 domains in which the tandem CH3 domains are separated by a flexible linker linked to each other, which prevents interchain CH3-CH3 dimerization. The DNA and amino acid sequences of the monomeric CH2-CH3 Fc moieties containing the point mutations identified above were the same as in Example 2-1. The DNA and amino acid sequences of the monomeric CH2-CH3-linker-CH3 Fc moieties with tandem CH3 domains are shown in Figures 2A-2D.

[0193] The light and heavy chain DNA and amino acid sequences of the anti-CD19 scFv were also the same as in Example 2-1. The proteins were cloned, produced, and purified as in Example 2-1. The light and heavy chain DNA and amino acid sequences of various anti-NKp46 scFvs are shown below.

[0194] [Table 6]

[0195] [Table 7]

[0196] Form 1 (F1) (anti-CD19-IgG1-Fcmono-anti-NKp46 (scFv)) The domain structure of Form 1 (F1) is shown in Figure 2A. A bispecific Fc-containing polypeptide was constructed based on an scFv specific for the tumor antigen CD19 (anti-CD19 scFv) and an scFv specific for the NKp46 receptor. This polypeptide is a single chain polypeptide with the domains arranged (N-terminus to C-terminus) as follows: (Vκ-VH) 抗CD19 -CH2-CH3-(VH-Vκ)抗NKp46

[0197] A DNA sequence encoding a CH3 / VH linker peptide with the amino acid sequence STGS was designed to insert a specific Sall restriction site at the CH3-VH junction. In the domain arrangement of the final polypeptide shown in FIG. 2 (the star in the CH2 domain indicates the optional N297S mutation), the anti-CD3 scFv is replaced by the anti-NKp46 scFv. The (Vκ-VH) unit contains a linker between the VH and Vκ domains. The protein was cloned, produced, and purified as in Example 2-1. The amino acid sequences of the bispecific polypeptides (full sequences (mature proteins)) are shown in the corresponding SEQ ID NOs listed in Table 2B below.

[0198] [Table 8]

[0199] Form 2 (F2): CD19-F2-NKp46-3 The domain structure of the F2 polypeptide is shown in Figure 2A. The DNA and amino acid sequences of the monomeric CH2-CH3 Fc portion are similar to those in Example 2-1, which also contains the CH3 domain mutations (mutations (EU numbering) L351K, T366S, P395V, F405R, T407A, and K409Y). The heterodimer is composed of: (1) A first (central) polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: (Vκ-VH) 抗CD19 -CH2-CH3-VH 抗NKp46 -CH1, and (2) a second polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: VK 抗NKp46 -CK.

[0200] The (Vκ-VH) unit was composed of a VH domain, a linker, and a Vκ unit (i.e., scFv). As with other forms of the bispecific polypeptide of the invention, a DNA sequence encoding a CH3 / VH linker peptide with the amino acid sequence STGS was designed to insert a specific Sall restriction site at the CH3-VH junction. The protein was cloned, produced, and purified as in Example 2-1. The amino acid sequences of the first and second chains of the F2 protein are shown in SEQ ID NOs: 140 and 141.

[0201] Form 3 (F3): CD19-F3-NKp46-3 The domain structure of the F3 polypeptide is shown in Figure 2 A. The DNA and amino acid sequences of the CH2-CH3 Fc portion contained tandem CH3 domains that linked two CH3 domains to each other on the same polypeptide chain, thereby preventing dimerization between different bispecific proteins.

[0202] The single polypeptide chain has domains arranged (N-terminus to C-terminus) as follows: (Vκ-VH) 抗CD19 -CH2-CH3-CH3-(VH-Vκ) 抗NKp46

[0203] The (VκK-VH) unit was composed of a VH domain, a linker, and a Vκ unit (scFv). The protein was cloned, produced, and purified as in Example 2-1. The bispecific protein was purified from cell culture supernatant by affinity chromatography using prot-A beads, analyzed, and purified by SEC. The protein showed a high production yield of 3.4 mg / L and a simple SEC profile for the purified protein. The amino acid sequence of the F3 protein is shown in SEQ ID NO: 142.

[0204] Form 4 (F4): CD19-F4-NKp46-3 The domain structure of the F4 polypeptide is shown in Figure 2A. The DNA and amino acid sequences of the CH2-CH3 Fc portion contain a tandem CH3 domain like form F3, and further contain the N297S mutation that prevents N-linked glycosylation and abolishes FcγR binding. The protein was cloned, produced, and purified as in Example 2-1. The bispecific protein was purified from cell culture supernatant by affinity chromatography using prot-A beads, analyzed, and purified by SEC. The protein showed a good production yield of 1 mg / L, and the purified protein showed a simple SEC profile. The amino acid sequence of the F4 protein with the NKp46-3 variable domain is shown in SEQ ID NO: 143.

[0205] Form 8 (F8) The domain structure of the F8 polypeptide is shown in FIG. 2B. The DNA and amino acid sequences of the monomeric CH2-CH3 Fc portion are similar to form F2 and also contain CH3 domain mutations (mutations (EU numbering) L351K, T366S, P395V, F405R, T407A, and K409Y, as well as the N297S mutation, which prevents N-linked glycosylation and further abolishes FcγR binding). Three mutants of the F8 protein were generated: (a) one in which the cysteine ​​residues in the hinge region were left intact (wild type, called F8A), (b) a second in which the cysteine ​​residues in the hinge region were replaced by serine residues (F8B), and (c) a third containing the linker sequence GGGSS replacing the hinge residues DKTHTCPPCP (F8C). Mutants F8B and F8C offered an advantage in generation because they prevented the formation of central chain homodimers. This heterotrimer is composed of: (1) A first (central) polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: V H 抗CD19 -CH1-CH2-CH3-VH 抗NKp46 -Cκ, and (2) a second polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: VK 抗NKp46-CH1, and (3) a third polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: Vκ 抗CD19 -Cκ.

[0206] The protein was cloned, produced and purified as in Example 2-1. The bispecific protein was purified from cell culture supernatant by affinity chromatography using prot-A beads, analyzed and purified by SEC. The protein showed a high production yield of 3.7mg / L (F8C), and the purified protein also showed a simple SEC profile. The amino acid sequences of the three chains of F8 protein (C mutant) with NKp46-3 variable region are shown in SEQ ID NOs: 144, 145 and 146.

[0207] Form 9 (F9): CD19-F9-NKp46-3 The F9 polypeptide is a trimeric polypeptide with a central polypeptide chain and two polypeptide chains each linked to the central chain by CH1-Cκ dimerization. The domain structure of the trimeric F9 protein is shown in FIG. 2B, where the bond between the CH1 and Cκ domains is an interchain disulfide bond. The two antigen-binding domains have an F(ab) structure that allows the use of this antibody whether it maintains its function in scFv form or not. The DNA and amino acid sequences of the CH2-CH3 Fc portion contain a tandem CH3 domain similar to that of form F4, and a CH2 domain containing a N297S substitution. Three mutants of the F9 protein were generated: (a) the first one in which the cysteine ​​residues in the hinge region were left intact (wild type, called F9A), (b) the second one in which the cysteine ​​residues in the hinge region were replaced by serine residues (F9B), and (c) the third one containing the linker sequence GGGSS replacing the hinge residues DKTHTCPPCP (F9C). Mutants F9B and F9C offered an advantage in engineering by preventing the formation of homodimers of the central chain, which are composed of: (1) A first (central) polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: V H 抗CD19 -CH1-CH2-CH3-CH3-V H 抗NKp46 -Cκ, and (2) a second polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: Vκ 抗NKp46 -CH1, and (3) a third polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: Vκ 抗CD19 -Cκ.

[0208] The proteins were cloned, produced, and purified as in Example 2-1. The bispecific proteins were purified from cell culture supernatants by affinity chromatography using prot-A beads, analyzed, and purified by SEC. The proteins showed high production yields of 8.7 mg / L (F9A) and 3.0 mg / L (F9B), and the purified proteins also showed simple SEC profiles.

[0209] The amino acid sequences of the three chains of F9 protein variant F9A are shown in SEQ ID NOs: 147, 148, and 149. The amino acid sequences of the three chains of F9 protein variant F9B are shown in SEQ ID NOs: 150, 151, and 152. The amino acid sequences of the three chains of F9 protein variant F9C are shown in SEQ ID NOs: 153, 154, and 155.

[0210] Form 10 (F10): CD19-F9-NKp46-3 The F10 polypeptide is a dimeric protein with a central polypeptide chain and a second polypeptide chain linked to the central chain by CH1-Cκ dimerization. The domain structure of the dimeric F10 protein is shown in FIG. 2B, where the bond between the CH1 and Cκ domains is an interchain disulfide bond. One of the two antigen-binding domains has a Fab structure and the other is an scFv. The DNA and amino acid sequences of the CH2-CH3 Fc portion contain a tandem CH3 domain as shown in form F4, and a CH2 domain containing a N297S substitution. Three mutants of the F10 protein were also generated: (a) the first one in which the cysteine ​​residues in the hinge region were left intact (wild type, called F10A), (b) the second one in which the cysteine ​​residues in the hinge region were replaced by serine residues (F10B), and (c) the third one containing the linker sequence GGGSS replacing the hinge residues DKTHTCPPCP (F10C). The mutants F10B and F10C offered the advantage of being engineered to prevent the formation of homodimers of the central chain. (Vκ-V H ) unit is V H The heterodimer was composed of the following: (1) A first (central) polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: V H 抗CD19 -CH1-CH2-CH3-CH3-(V H -Vκ) 抗NKp46 , and (2) a second polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: Vκ 抗CD19 -Cκ.

[0211] These proteins were cloned, produced, and purified as in Example 2-1. The bispecific proteins were purified from cell culture supernatants by affinity chromatography using prot-A beads, analyzed, and purified by SEC. The proteins showed good production yields of 2 mg / L (F10A), and the purified proteins also showed simple SEC profiles. The amino acid sequences of the two chains of the F10A protein variant are shown in SEQ ID NO: 156 (second chain) and 157 (first chain). The amino acid sequences of the two chains of the F10B protein variant are shown in SEQ ID NO: 158 (second chain) and 159 (first chain). The amino acid sequences of the two chains of the F10C protein variant are shown in SEQ ID NO: 160 (second chain) and 161 (first chain).

[0212] Form 11 (F11): CD19-F11-NKp46-3 The domain structure of the F11 polypeptide is shown in Figure 2C. This heterodimeric protein is similar to F10, except that the structures of the antigen-binding domains are reversed. One of the two antigen-binding domains has a Fab-like structure and the other is an scFv. This heterodimer is composed of: (1) A first (central) polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: (Vκ-V H ) 抗CD19 -CH2-CH3-CH3-VH 抗NKp46 -Cκ, and (2) a second polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: Vκ 抗NKp46 -CH1.

[0213] The protein was cloned, produced and purified as in Example 2-1. The bispecific protein was purified from cell culture supernatant by affinity chromatography using prot-A beads, analyzed and purified by SEC. The protein showed a good production yield of 2mg / L, and the purified protein also showed a simple SEC profile. The amino acid sequences of the two chains of F11 protein are shown in SEQ ID NO: 162 (first chain) and SEQ ID NO: 163 (second chain).

[0214] Form 12 (F12): CD19-F12-NKp46-3 The domain structure of the dimeric F12 polypeptide is shown in Figure 2C, where the bond between the CH1 and Cκ domains is a disulfide bond. This heterodimeric protein is similar to F11, but the CH1 and Cκ domains in the F(ab) structure are reversed. This heterodimer is composed of: (1) A first (central) polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: (Vκ-V H ) 抗CD19 -CH2-CH3-CH3-V H 抗NKp46 -CH1, and (2) a second polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: Vκ 抗NKp46 -Cκ.

[0215] The protein was cloned, produced and purified as in Example 2-1. The bispecific protein was purified from cell culture supernatant by affinity chromatography using prot-A beads, analyzed and purified by SEC. The protein showed a good production yield of 2.8 mg / L, and the purified protein also showed a simple SEC profile. The amino acid sequences of the two chains of the F12 protein are shown in SEQ ID NO: 164 (first chain) and SEQ ID NO: 165 (second chain).

[0216] Form 17 (F17): CD19-F17-NKp46-3 The domain structure of the trimeric F17 polypeptide is shown in FIG. 2C, with the linkage between the CH and Cκ domains being a disulfide bond. The heterodimeric protein is similar to F9, but contains the V H The Vκ and Vκ domains, and the CH1 and Cκ domains in the C-terminal F(ab) structure, are inverted relative to their partners, respectively. This heterotrimer is composed of: (1) A first (central) polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: V H 抗CD19 -CH1-CH2-CH3-CH3-Vκ 抗NKp46 -CH1, and (2) a second polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: V H 抗NKp46 -Cκ, and (3) a third polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: Vκ 抗CD19 -Cκ.

[0217] In addition, three mutants of F17 protein were made: (a) the first one (wild type, called F17A) in which the cysteine ​​residues in the hinge region remain intact; (b) the second one (F10B) in which the cysteine ​​residues in the hinge region are replaced by serine residues; and (c) the third one (F17C) containing the linker sequence GGGSS replacing the hinge residue DKTHTCPPCP. The proteins were cloned, produced, and purified as in Example 2-1. The amino acid sequences of the three chains of F17B protein are shown in SEQ ID NOs: 166, 167, and 168.

[0218] Example 4: Bispecific NKp46 antibody format with dimeric Fc domain A novel protein construct with a dimeric Fc domain was developed that shares many of the advantages of the monomeric Fc domain protein of Example 3, but binds to FcRn with high affinity. Various protein forms were generated with low or abolished binding to FcγR (including CD16), for example, binding affinity to human CD16 within 1-log of the binding affinity of wild-type human IgG1 antibody as assessed by SPR (see, for example, methods of Example 16). The various polypeptide forms were analyzed using a 3D-PCR method (V H Heterodimeric proteins comprising a central chain with -(CH1 / Cκ)-CH2-CH3-) or (Vκ-(CH1 or Cκ)-CH2-CH3-) units have been tested and compared to investigate their functionality. One or both of the CH3 domains are fused, optionally via an intervening amino acid sequence or domain, to a variable domain (a single variable domain linked to a variable domain on a separate polypeptide chain), a tandem variable domain (e.g., scFv), or a single variable domain capable of binding antigen as a single variable domain. The two chains associate by CH1-Cκ dimerization to form a disulfide-linked dimer, or when associated with a third chain to form a trimer.

[0219] Various constructs were made using variable domain DNA and amino acid sequences derived from the scFv specific for the tumor antigen CD19 described in Example 2-1 and different variable regions from the antibody specific for NKp46 revealed in Example 1 for use in preparing bispecific antibodies. Proteins were cloned, produced and purified as in Example 2-1. The domain structures are shown in Figures 2A-6D.

[0220] Form 5 (F5): CD19-F5-NKp46-3 The domain structure of the trimeric F5 polypeptide is shown in Figure 2D, where the interchain bonds between the hinge domains (shown diagrammatically between the CH1 / Cκ and CH2 domains on the chains) and between the CH1 and Cκ domains are interchain disulfide bonds. The heterotrimer is composed of: (1) A first (central) polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: V H 抗CD19 -CH1-CH2-CH3-V H 抗NKp46 -Cκ, and (2) a second polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: Vκ 抗CD19 -CK-CH2-CH3, and (3) a third polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: Vκ 抗NKp46 -CH1.

[0221] The protein was cloned, produced, and purified as in Example 2-1. The bispecific protein was purified from cell culture supernatant by affinity chromatography using prot-A beads, analyzed, and purified by SEC. The protein showed a high production yield of 37 mg / L, and the purified protein also showed a simple SEC profile. The amino acid sequences of the three polypeptide chains are shown in SEQ ID NOs: 169 (second chain), 170 (first chain), and 171 (third chain).

[0222] Form 6 (F6): CD19-F6-NKp46-3 The domain structure of the heterotrimeric F6 polypeptide is shown in FIG. 2D. The F6 protein is the same as F5, but contains an N297S substitution to prevent N-linked glycosylation. The protein was cloned, produced, and purified as in Example 2-1. The dual-specific protein was purified from cell culture supernatant by affinity chromatography using prot-A beads, analyzed, and purified by SEC. The protein showed a high production yield of 12 mg / L, and the purified protein showed a simple SEC profile. The amino acid sequences of the three polypeptide chains are shown in SEQ ID NOs: 172 (second chain), 173 (first chain), and 174 (third chain).

[0223] Form 7 (F7): CD19-F7-NKp46-3 The domain structure of the heterotrimeric F7 polypeptide is shown in Figure 2D. The F7 protein consists of a central chain and a Vκ 抗NKp46 - The same as F6, except that it has a cysteine ​​to serine substitution in the CH1 and Cκ domains linked to the Fc domain at their C-terminus to prevent the formation of a minor population of dimeric species with the CH1 chain. The protein was cloned, produced, and purified as in Example 2-1. The bispecific protein was purified from cell culture supernatant by affinity chromatography using prot-A beads, analyzed, and purified by SEC. The protein showed a high production yield of 11 mg / L, and the purified protein showed a simple SEC profile. The amino acid sequences of the three polypeptide chains are shown in SEQ ID NOs: 175 (second chain), 176 (first chain), and 177 (third chain).

[0224] Form 13 (F13): CD19-F13-NKp46-3 The domain structure of the dimeric F13 polypeptide is shown in Figure 2D, where the interchain bonds between the hinge domains (shown between the CH1 / Cκ and CH2 domains on the chains) and between the CH1 and Cκ domains are interchain disulfide bonds. This heterodimer is composed of: (1) A first (central) polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: V H 抗CD19 -CH1-CH2-CH3-(V H -Vκ) 抗NKp46 , and (2) a second polypeptide chain having domains arranged (N-terminus to C-terminus) as follows: Vκ 抗CD19 -Cκ-CH2-CH3.

[0225] (V H -Vκ) units are V H It consisted of a domain, a linker, and a Vκ unit (scFv).

[0226] The protein was cloned, produced, and purified as in Example 2-1. The bispecific protein was purified from cell culture supernatant by affinity chromatography using prot-A beads, analyzed, and purified by SEC. The protein showed a high production yield of 6.4 mg / L, and the purified protein showed a simple SEC profile. The amino acid sequences of the two polypeptide chains are shown in SEQ ID NO: 178 (second chain) and 179 (first chain).

[0227] Form 14 (F14): CD19-F14-NKp46-3 The domain structure of the dimeric F14 polypeptide is shown in FIG. 2E. The F14 polypeptide is a dimeric polypeptide that shares the structure of the F13 form, but instead of the wild-type Fc domain (CH2-CH3), the F14 bispecific form has the CH2 domain mutation N297S to eliminate N-linked glycosylation. The protein was cloned, produced, and purified as in Example 2-1. The bispecific protein was purified from cell culture supernatant by affinity chromatography using prot-A beads, analyzed, and purified by SEC. The protein showed a high production yield of 2.4 mg / L, and the purified protein showed a simple SEC profile. The amino acid sequences of the two polypeptide chains are shown in SEQ ID NO: 180 (second chain) and 181 (first chain).

[0228] Form 15 (F15): CD19-F15-NKp46-3 The domain structure of the trimeric F15 polypeptide is shown in FIG. 2E. The F15 polypeptide is a trimeric polypeptide that shares the structure of the F6 form but differs due to the reversal of the N-terminal VH-CH1 and Vκ-Cκ units between the central and second chains. The protein was cloned, produced, and purified as in Example 2-1. The bispecific protein was purified from cell culture supernatant by affinity chromatography using prot-A beads, analyzed, and purified by SEC. The protein showed a good production yield of 0.9 mg / L and a simple SEC profile for the purified protein. The amino acid sequences of the three polypeptide chains are shown in SEQ ID NO: 182 (second chain), 183 (first chain), and 184 (third chain).

[0229] Form 16 (F16): CD19-F16-NKp46-3 The domain structure of the trimeric F16 polypeptide is shown in Figure 2E. The F16 polypeptide is a trimeric polypeptide that shares the structure of the F6 form but differs due to the reversal of the C-terminal VH-CK and Vκ-CH1 units between the central and second chains. The protein was cloned, produced, and purified as in Example 2-1. The amino acid sequences of the three polypeptide chains are shown in SEQ ID NO: 185 (second chain), 186 (first chain), and 187 (third chain).

[0230] Form T5 (T5) The domain structure of the trimeric T5 polypeptide is shown in FIG. 2F. The T5 polypeptide is a trimeric polypeptide that shares the structure of the F5 form, but differs by the fusion of an scFv unit at the C-terminus of the third chain (the chain lacking the Fc domain). Thus, the protein has two antigen-binding domains for the antigen of interest and one for NKp46, and binds to CD16 via its Fc domain. The protein was cloned, produced, and purified as in Example 2-1. The T5 protein had two antigen-binding domains that bind human CD20 (and bind to different epitopes on CD20) derived from different antibodies. The first anti-CD20 ABD was the V-terminal fragment of the parent antibody GA101 (GAZYVA®, Gazyvaro®, obinutuzumab, Roche Pharmaceuticals). H and V L The second anti-CD20 ABD included the V-formula of the parent antibody rituximab (Rituxan®, Mabthera®, Roche Pharmaceuticals). H and V L The third antigen-binding domain binds to human NKp46. The amino acid sequences of the three chains of the T5 protein are shown below (rituximab sequences are bold and underlined, anti-GA101 sequences are underlined, and anti-NKp46 sequences are italicized):

[0231] GA101-T5-Ritux-NKp46 Polypeptide 1 (SEQ ID NO: 188) [ka] Polypeptide 2 (SEQ ID NO: 189) [ka] Polypeptide 3: (SEQ ID NO: 190) [ka]

[0232] Form T6 (T6) The domain structure of the trimeric T6 polypeptide is shown in FIG. 2F. The T6 polypeptide is a trimeric polypeptide that shares the structure of the F6 form, but differs by the fusion of an scFv unit at the C-terminus of the third chain (the chain lacking the Fc domain). This trimeric protein contains two antigen-binding domains for the antigen of interest and one for NKp46, and does not bind to CD16 via its Fc domain due to the N297 substitution. The protein was cloned, produced, and purified as in Example 2-1. The T6 protein contains two antigen-binding domains that bind to human CD20. The first anti-CD20 ABD is the V-terminal domain of the parent antibody GA101. H and V L and the second anti-CD20 ABD comprises the V of rituximab. H and V L The amino acid sequences of the three chains of the T6 protein are shown in SEQ ID NOs:191, 192, and 193.

[0233] Form T9B (T9B) The domain structure of the trimeric T9B polypeptide is shown in FIG. 2F. The T9B polypeptide is a trimeric polypeptide that shares the structure of the F9 form (F9B mutant), but differs by the fusion of an scFv unit at the C-terminus of the free CH1 domain (on the third chain). This protein contains two antigen-binding domains for the antigen of interest and one for NKp46, but does not bind to CD16 via its Fc domain due to the monomeric Fc domain and / or N297 substitution. The trimeric protein as described above was cloned, produced, and purified as in Example 2-1. The T9B protein had two antigen-binding domains that bind to human CD20. The first anti-CD20 ABD was the V of the parent antibody GA101. H and V L and the second anti-CD20 ABD comprises the V of the parent antibody rituximab. H and V L The amino acid sequences of the three chains of the T9B protein are shown below.

[0234] GA101-T9B-Ritux-NKp46 Polypeptide 2: (SEQ ID NO: 195) [ka] Polypeptide 1: (SEQ ID NO: 194) [ka] Polypeptide 3 (SEQ ID NO:196): [ka]

[0235] Form T11(T1):CD19-T11-NKp46-3 The domain structure of the dimeric T11 polypeptide is shown in FIG. 2F. The T11 polypeptide is a trimeric polypeptide that shares the structure of the F11 form, but differs by the fusion of an scFv unit at the C-terminus of the free CH1 domain. This dimeric protein contains two antigen-binding domains for the antigen of interest and one for NKp46, but does not bind to CD16 via its Fc domain due to the monomeric Fc domain and / or N297 substitution. The protein was cloned, produced, and purified as in Example 2-1. The T11 protein contains two antigen-binding domains that bind to human CD20. The first anti-CD20 ABD is the V-terminal domain of the parent antibody GA101. H and V L and the second anti-CD20 ABD comprises the V of rituximab. H and V L The amino acid sequences of the two chains of the T11 protein are shown below.

[0236] GA101-T11-Ritux-NKp46 Polypeptide 1 (SEQ ID NO:197): [ka] Polypeptide 2 (SEQ ID NO:198): [ka]

[0237] Example 5: NKp46 binding affinity by bispecific proteins by surface plasmon resonance (SPR) Bacore T100 General Procedures and Reagents SPR measurements were performed at 25°C on a Biacore T100 instrument (Biacore GE Healthcare). In all Biacore experiments, HBS-EP+ (Biacore GE Healthcare) and NaOH 10 mM served as running buffer and regeneration buffer, respectively. Sensorgrams were analyzed with Biacore T100 Evaluation software. Protein A was purchased from (GE Healthcare). Human NKp46 recombinant protein was cloned, produced, and purified at Innate Pharma.

[0238] Protein A immobilization Protein A protein was covalently immobilized to the carboxyl groups of the dextran layer on the Sensor Chip CM5. The chip surface was activated with EDC / NHS (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (Biacore GE Healthcare)). Protein A was diluted to 10 μg / ml in binding buffer (10 mM acetic acid, pH 5.6) and injected until the appropriate immobilization level (i.e., 2500 RU) was reached. The remaining activated groups were deactivated using 100 mM ethanolamine pH 8 (Biacore GE Healthcare).

[0239] Binding Tests First, the bispecific protein was tested in the format F1 described in Example 2 with different anti-NKp46 variable regions from NKp46-1, NKp46-2, NKp46-3, or NKp46-4 antibodies. Next, the antibodies were tested as different formats F3, F4, F5, F6, F9, F10, F11, F13, F14 with the anti-NKp46 variable region from the NKp46-3 antibody and compared to the NKp46-3 antibody as a full length human IgG1.

[0240] The bispecific proteins were captured on a Protein A chip at 1 μg / mL and recombinant human NKp46 protein was injected over the captured bispecific antibodies at 5 μg / mL. For blank subtraction, the cycle was run again to replace the NKp46 protein with running buffer.

[0241] The Bab281 antibody was separately tested for binding to NKp46 by SPR and further tested by flow cytometry using a cell line expressing a human NKp46 construct on the cell surface. For FACS screening, the presence of reactive antibodies in the supernatants was revealed by a PE-labeled goat anti-mouse polyclonal antibody (pAb). SPC and FACS results showed that the Bab281-based antibody did not bind to the NKp46 cell line or to the NKp46 Fc protein. Bab281 lost binding to its target when present in a dual-specific form.

[0242] Affinity testing Monovalent affinity studies were performed following the regular Capture-Kinetic protocol recommended by the manufacturer (Biacore GE Healthcare kinetic wizard). Seven serial dilutions of human NKp46 recombinant protein ranging from 62.5 to 400 nM were injected sequentially over the captured bispecific antibodies and allowed to dissociate for 10 min before regeneration. The entire set of sensorgrams was fitted using a 1:1 kinetic binding model.

[0243] result SPR showed that bispecific polypeptides of form F1 with NKp46-1, 2, 3, and 4 scFv binding domains bound to NKp46, whereas other bispecific polypeptides with scFvs of other anti-NKp46 antibodies did not maintain NKp46 binding. Binding domains that did not maintain binding in the monomeric bispecific form initially bound to NKp46 but lost binding upon conversion to the bispecific form. All of the bispecific polypeptides of forms F1, F2, F3, F4, F5, F6, F9, F10, F11, F13, F14 maintained binding to NKp46 when using the NKp46-3 variable region. The monovalent affinities and kinetic association and dissociation rate constants are shown in Table 3 below.

[0244] [Table 9]

[0245] Example 6: Binding of NK cells to Daudi tumor targets with Fc-containing NKp46xCD19 bispecific protein Bispecific antibodies with monomeric Fc domains and domains arranged according to the single chain F1 or dimeric F2 formats described in Example 3, and NKp46 binding regions based on NKp46-1, NKp46-2, NKp46-3, or NKp46-4 were tested for their functional ability to induce NK cells to lyse CD19-positive tumor target cells (the well-characterized B lymphoblastoid cell line Daudi). The F2 protein further contained the variable region of NKp46-9, which loses binding to NKp46 in the scFv format but maintains binding in the F(ab)-like format of F2.

[0246] Briefly, the cytolytic activity of (a) resting human NK cells and (b) the human NKp46-transfected human NK cell line KHYG-1 was measured in a typical 4-hour incubation in U-bottom 96-well plates. 51 The Cr release assay was performed on Daudi cells. 51 Cr(50μCi(1.85MBq) / 1×10 6hNK46-transfected KHYG-1 cells were labeled with 100 mM acetylcholine (HAT) and then mixed with hNK46-transfected KHYG-1 in the presence of different concentrations of monomeric bispecific antibodies at effector / target ratios equal to 50 for KHYG-1, 10 (for F1 protein) or 8.8 (for F2 protein) for resting NK cells. After brief centrifugation and 4 h incubation at 37° C., samples of the supernatant were removed and transferred to a LumaPlate (Perkin Elmer Life Sciences, Boston, MA) and incubated for 4 h. 51 Cr release was measured with a TopCount NXT beta detector (PerkinElmer Life Sciences, Boston, MA). All experimental conditions were analyzed in triplicate and the percentage of specific lysis was determined as follows: 100 x (mean cpm experimental release - mean cpm spontaneous release) / (mean cpm total release - mean cpm spontaneous release). The percentage of total release was obtained by lysis of target cells with 2% Triton X100 (Sigma) and spontaneous release corresponds to target cells in medium (no effectors or Abs).

[0247] result In the KHYG-1 hNKp46 NK experimental model, each bispecific antibody NKp46-1, NKp46-2, NKp46-3, NKp46-4, or NKp46-9 induced specific lysis of Daudi cells by the human KHYG-1 hNKp46 NK cell line compared to negative controls (human IgG1 isotype control (IC) and CD19 / CD3 bispecific antibody), thereby indicating that these antibodies induce lysis of Daudi target cells by KHYG-1 hNKp46 via CD19 / NKp46 cross-linking.

[0248] When resting NK cells were used as effectors, each bispecific antibody NKp46-1, NKp46-2, NKp46-3, NKp46-4, or NKp46-9 similarly induced specific lysis of Daudi cells by human NK cells compared to a negative control (human IgG1 isotype control (IC) antibody), thereby indicating that these antibodies induce lysis of Daudi target cells by human NK cells by CD19 / NKp46 cross-linking. Rituximab (RTX, chimeric IgG1) was used as a positive control for ADCC (antibody-dependent cellular cytotoxicity) by resting human NK cells. The maximum response obtained with RTX (at 10 μg / ml in this assay) was 21.6% specific lysis, illustrating that the bispecific antibodies have high target cell lysis activity. The results of the experiments with resting NK cells are shown in Figure 3A for the single chain F1 protein and in Figure 3B for the dimeric Fc protein.

[0249] Example 7: Comparison of full-length anti-NKp46 mAb with depleting anti-tumor mAb: NKp46xCD19 bispecific protein prevents non-specific NK activation These studies aimed to determine whether bispecific antibodies could mediate NKp46-mediated NK activation against cancer target cells without triggering non-specific NK cell activation.

[0250] NKp46xCD19 bispecific proteins with a configuration according to the F2 format described in Example 3 with anti-NKp46 variable domains from NKp46-1, NKp46-2, NKp46-3, NKp46-4, or NKp46-9 were compared with: (a) a full-length monospecific anti-NKp46 antibody (human IgG1-like NKp46-3), and (b) An anti-CD19 antibody such as a full-length human IgG1 as an ADCC inducing antibody control comparator.

[0251] The experiment further included the following as controls: rituximab, an anti-CD20 ADCC-inducing antibody control of a target antigen with high expression levels; the anti-CD52 antibody alemtuzumab, a human IgG1 that binds to the CD52 target present on both target cells and NK cells; and a negative control isotype control therapeutic antibody (a human IgG1 that does not bind to a target present on target cells (HUG1-IC)).

[0252] Various proteins were tested for their functional effects on NK cell activation in the presence of CD19-positive tumor target cells (Daudi cells), in the presence of CD19-negative CD16-positive target cells (HUT78 T lymphoma cells), and in the absence of target cells.

[0253] Briefly, NK activation was tested by assessing the expression of CD69 and CD107 on NK cells by flow cytometry. Assays were performed in 96U well plates in complete RPMI, 150 μL final / well. Effector cells were freshly purified NK cells from donors. Target cells were Daudi (CD19-positive), HUT78 (CD19-negative), or K562 (NK activation control cell line). In addition to the K562 positive control, the following three conditions were tested: >NK cells only >NK cells vs Daudi (C19+) >NK cells vs. HUT78 (CD19-) The effector:target (E:T) ratio was 2.5:1 (50,000E:20,000T) and the antibody dilution range started at 10 μg / mL at 1 / 4 dilution (n=8 concentrations). Antibodies, target cells, and effector cells were mixed, spun at 300g for 1 min, incubated at 37°C for 4 h, spun at 500g for 3 min, washed twice with staining buffer (SB), 50 μL of staining Ab mix was added, incubated at 300g for 30 min, washed twice with SB resuspended pellet with CellFix, stored at 4°C overnight, and fluorescence revealed with a Canto II (HTS).

[0254] result 1. NK cells only The results are shown in Figure 4A. In the absence of target antigen-expressing cells, none of the bispecific anti-NKp46xanti-CD19 antibodies (containing each of the variable regions of NKp46-1, NKp46-2, NKp46-3, NKp46-4, and NKp46-9) activated NK cells as assessed by CD69 or CD107 expression. Full-length anti-CD19 also did not activate NK cells. However, full-length anti-NKp46 antibodies caused detectable activation of NK cells. Alemtuzumab also induced very high levels of NK cell activation. An isotype control antibody did not induce activation.

[0255] 2. NK cells vs. Daudi (CD19+) The results are shown in Figure 4B. In the presence of target antigen-expressing cells, the bispecific anti-NKp46xanti-CD19 antibodies (containing each of the binding domains of NKp46-1, NKp46-2, NKp46-3, NKp46-4, and NKp46-9) all activated NK cells. Full-length anti-CD19 showed only very low activation of NK cells at best. Neither full-length anti-NKp46 antibodies nor alemtuzumab showed a substantial increase in activation beyond that observed in the presence of NK cells alone. Figure 4 shows that the full-length anti-NKp46 antibodies showed similar levels of baseline activation observed in the presence of NK cells alone. Alemtuzumab also induced a similar level of NK cell activation to that observed in the presence of NK cells alone, and at the higher antibody concentration (ET 2.5:1) in this setting, activation was higher than with the bispecific anti-NKp46xanti-CD19 antibodies. The isotype control antibody did not induce activation.

[0256] 3. NK cells vs. HUT78 (CD19-) The results are shown in Figure 4C. In the presence of target antigen-negative HUT78 cells, bispecific anti-NKp46xanti-CD19 antibodies (containing each of the variable regions of NKp46-1, NKp46-2, NKp46-3, NKp46-4, and NKp46-9) all activated NK cells. However, full-length anti-NKp46 antibodies and alemtuzumab caused detectable activation of NK cells at a similar level observed in the presence of NK cells alone. An isotype control antibody did not induce activation.

[0257] In conclusion, bispecific anti-NKp46 proteins are able to activate NK cells in a target cell-specific manner, unlike full-length monospecific anti-NKp46 antibodies and full-length antibodies of depleting IgG isotypes that similarly activate NK cells in the absence of target cells. NK cell activation achieved with anti-NKp46 bispecific proteins was higher than that observed with full-length anti-CD19 IgG1 antibodies.

[0258] Example 8: Comparative efficacy of depleting antitumor mAb:NKp46xCD19 bispecific protein at low ET ratios These studies aimed to determine whether bispecific antibodies could mediate NKp46-mediated NK cell activation against cancer target cells at low effector:target ratios. The ET ratio used in this example was 1:1, which is believed to be closer to the setting that would be encountered in vivo than the 2.5:1 ET ratio used in Example 7 or the 10:1 ET ratio in Example 6.

[0259] NKp46xCD19 bispecific proteins with a configuration according to the F2 format described in Example 3 with anti-NKp46 variable domains from NKp46-1, NKp46-2, NKp46-3, NKp46-4, or NKp46-9 were compared with: (a) a full-length monospecific anti-NKp46 antibody (human IgG1-like NKp46-3), and (b) An anti-CD19 antibody such as a full-length human IgG1 as an ADCC-inducing antibody control.

[0260] The experiments further included the following controls: Rituximab (anti-CD20 ADCC-inducing antibody control of a target antigen with high expression levels); anti-CD52 antibody Alemtuzumab (human IgG1 that binds to the CD52 target present on both target cells and NK cells), and a negative control isotype control therapeutic antibody (human IgG1 that does not bind to the target present on target cells (HUG1-IC)). Various proteins were tested for their functional impact on NK cell activation, as assessed by CD69 or CD107 expression in the presence of CD19 positive tumor target cells (Daudi cells), CD19 negative CD16 positive target cells (HUT78 T lymphoma cells), and in the absence of target cells. These experiments were performed similarly to Example 7, except that the ET ratio was 1:1.

[0261] result The results are shown in Figure 5 (Figure 5A: CD107 and Figure 5B: CD69). In the presence of target antigen-expressing cells, bispecific anti-NKp46xanti-CD19 antibodies (containing each of the variable regions of NKp46-1, NKp46-2, NKp46-3, NKp46-4, and NKp46-9) all activated NK cells in the presence of Daudi cells.

[0262] The activation induced by the bispecific anti-NKp46xanti-CD19 antibody in the presence of Daudi cells was much more potent than the full-length human IgG1 anti-CD19 antibody, an ADCC-inducing antibody with low activity in this setting. Moreover, in this low E:T ratio setting, the activation induced by the bispecific anti-NKp46xanti-CD19 antibody was as potent as the anti-CD20 antibody rituximab, with differences only observed at the highest concentration, which was 10-fold higher than the concentration at which differences were observed at an ET ratio of 2.5:1.

[0263] In the absence of target cells or in the presence of target antigen-negative HUT78 cells, full-length anti-NKp46 antibodies and alemtuzumab demonstrated similar levels of baseline activation to that observed in the presence of Daudi cells. Anti-NKp46 x anti-CD19 antibodies did not activate NK cells in the presence of HUT78 cells.

[0264] In conclusion, bispecific anti-NKp46 proteins can activate NK cells in a target cell-specific manner and at lower effector:target ratios are more effective than conventional human IgG1 antibodies in mediating NK cell activation.

[0265] Example 9: Mechanism of action of NKp46 NKp46xanti-CD19 bispecific antibodies with configurations according to the F2, F3, F5, or F6 formats described in Examples 3 or 4, having anti-NKp46 variable domains from NKp46-3, were compared to rituximab (an anti-CD20 ADCC-inducing antibody), and a human IgG1 isotype control antibody for the functional ability to induce CD16- / NKp46+ NK cell lines to lyse CD19-positive tumor target cells.

[0266] Briefly, the cytolytic activity of the CD16- / NKp46+ human NK cell line KHYG-1 was assayed in a typical 4-h culture in U-bottom 96-well plates. 51 The Cr release assay was performed. Daudi cells or B221 cells 51 Cr(50μCi(1.85MBq) / 1×10 6 cells) and then diluted 1 / 5 (n=8 concentrations) to 10 -7 The antibodies were mixed with KHYG-1 at an effector / target ratio equal to 50:1 in the presence of a range of dilutions starting from 10 mol / mL.

[0267] After brief centrifugation and incubation at 37° C. for 4 hours, 50 μL of the supernatant was removed and transferred to a LumaPlate (Perkin Elmer Life Sciences, Boston, Mass.) 51Cr release was measured with a TopCount NXT beta detector (PerkinElmer Life Sciences, Boston, MA). All experimental conditions were analyzed in triplicate and the percentage of specific lysis was determined as follows: 100 x (mean cpm experimental release - mean cpm spontaneous release) / (mean cpm total release - mean cpm spontaneous release). The percentage of total release was obtained by lysis of target cells with 2% Triton X100 (Sigma) and spontaneous release corresponds to target cells in medium (no effectors or Abs).

[0268] result The results are shown in Figure 6A (KHYG-1 vs Daudi) and Figure 6B (KHYG-1 vs B221). In the KHYG-1 hNKp46 NK experimental model, each NKp46xCD19 bispecific protein (forms F2, F3, F5, and F6) induced specific lysis of Daudi or B221 cells by the human KHYG-1 hNKp46 NK cell line, but not Rituximab and human IgG1 isotype control (IC) antibodies.

[0269] Example 10: Comparative efficacy of bispecific antibodies versus conventional IgG antibodies using fresh human NK cells The NKp46xCD19 bispecific protein binding to human CD16 with a configuration according to the F5 format with anti-NKp46 variable domains from NKp46-3 was compared to the same bispecific antibody in the F6 format (lacking CD16 binding) and a human IgG1 isotype anti-CD19 antibody, as well as a human IgG1 isotype control antibody, for the functional ability to induce purified NK cells to lyse CD19-positive Daudi tumor target cells.

[0270] Briefly, the cytolytic activity of freshly purified human NK cells from EFS Buffy Coat was assayed in a typical 4-h culture in U-bottom 96-well plates. 51 The Cr release assay was performed. Daudi cells or HUT78 cells (negative control cells that do not express CD19) were used. 51Cr and then mixed with NK cells at an effector / target ratio equal to 10:1 in the presence of a range of test antibodies starting at 10 μg / ml at a 1 / 10 dilution (n=8 concentrations).

[0271] After brief centrifugation and incubation at 37°C for 4 hours, 50 μl of the supernatant was removed and transferred to a LumaPlate (Perkin Elmer Life Sciences, Boston, MA). 51 Cr release was measured with a TopCount NXT beta detector (PerkinElmer Life Sciences, Boston, MA). All experimental conditions were analyzed in triplicate and the percentage of specific lysis was determined as follows: 100 x (mean cpm experimental release - mean cpm spontaneous release) / (mean cpm total release - mean cpm spontaneous release). The percentage of total release was obtained by lysis of target cells with 2% Triton X100 (Sigma) and spontaneous release corresponds to target cells in medium (no effectors or Abs).

[0272] The results are shown in Figure 7. CD19-F6-NKp46 (F6 form of the bispecific protein), in which the Fc domain does not bind to CD16 due to the N297 substitution, is as potent as the full-length IgG1 anti-CD19 antibody in mediating NK cell lysis of Daudi target cells, which is remarkable considering that the control IgG1 anti-CD19 antibody binds bivalently to CD19 and that anti-CD19 is bound by CD16. Surprisingly, CD19-F6-NKp46 (F5 form of the protein), in which the Fc domain additionally binds to CD16, is much more potent in mediating lysis of Daudi target cells than the full-length IgG1 anti-CD19 antibody or the F6 form of the bispecific protein. At comparable levels of target cell lysis, CD19-F6-NKp46 was at least 1000 times more potent than the full-length anti-CD19 IgG1.

[0273] Example 11: Binding of various dual specific forms to FcRn The affinity of various antibody formats to human FcRN was tested by surface plasmon resonance (SPR) by covalently immobilizing recombinant FcRn protein to the carboxyl groups of the dextran layer on a Sensor Chip CM5, as described in Examples 2-6.

[0274] A chimeric full-length anti-CD19 antibody with a human IgG1 constant region and a NKp46xCD19 bispecific protein with the anti-NKp46 variable domain from NKp46-3 (NKp46-2 in F2) configured according to the F3, F4, F5, F6, F9, F10, F11, F13, or F14 format described in Examples 3 or 4 were tested for each analyte and all sensorgrams were fitted using a steady-state or 1:1 SCK binding model.

[0275] The results are shown in Table 4 below. Bispecific proteins with dimeric Fc domains (forms F5, F6, F13, F14) bound to FcRn with affinities similar to those of full-length IgG1 antibodies. Bispecific proteins with monomeric Fc domains (F3, F4, F9, F10, F11) also showed binding to FcRn, but at a lower level than bispecific proteins with dimeric Fc domains.

[0276] [Table 10]

[0277] Example 12: Binding to FcγR To assess whether such bispecific monomeric Fc proteins can maintain binding to Fcγ receptors, various multimeric Fc proteins were evaluated.

[0278] SPR measurements were performed at 25°C on a Biacore T100 instrument (Biacore GE Healthcare). In all Biacore experiments, HBS-EP+ (Biacore GE Healthcare) and 10 mM NaOH, 500 mM NaCl served as running and regeneration buffers, respectively. Sensorgrams were analyzed with Biacore T100 Evaluation software. Recombinant human FcRs (CD64, CD32a, CD32b, CD16a, and CD16b) were cloned, produced, and purified.

[0279] F5 and F6 bispecific antibodies CD19-F5-NKp46-3 or CD19-F6-NKp46-3 were covalently immobilized to the carboxyl groups of the dextran layer on the sensor chip CM5. The chip surface was activated with EDC / NHS (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (Biacore GE Healthcare)). The bispecific antibodies were diluted to 10 μg / ml in binding buffer (10 mM acetic acid, pH 5.6) and injected until the appropriate immobilization level (i.e., 800-900 RU) was reached. Deactivation of the remaining activated groups was performed with 100 mM ethanolamine pH 8 (Biacore GE Healthcare).

[0280] Monovalent affinity studies were evaluated following a typical kinetic wizard (manufacturer's recommendations). Serial dilutions of soluble analyte (FcR) from 0.7 to 60 nM for CD64 and 60 to 5000 nM for all other FcRs were injected over the immobilized bispecific antibodies and allowed to dissociate for 10 min before regeneration. All sensorgram sets were fitted using a 1:1 kinetic binding model for CD64 and a steady-state affinity model for all other FcRs.

[0281] Full-length wild-type human IgG1 bound to all cynomolgus monkey and human Fcγ receptors, whereas CD19-F6-NKp46-3 bispecific antibody did not bind to any receptor. On the other hand, CD19-F5-NKp46-3 bound to each of the human receptors CD64 (KD=0.7nM), CD32a (KD=846nM), CD32b (KD=1850nM), CD16a (KD=1098nM), and CD16b (KD=2426nM). Conventional human anti-IgG1 antibodies have comparable binding to Fc receptors (KDs shown in the table below).

[0282] [Table 11]

[0283] Example 13: Epitope mapping of anti-NKp46 antibodies A. Competition Assay The competition assay was carried out by surface plasmon resonance (SPR) according to the method described below.

[0284] SPR measurements were performed at 25°C on a Biacore T100 instrument (Biacore GE Healthcare). In all Biacore experiments, HBS-EP+ (Biacore GE Healthcare) and NaOH 10 mM NaCl 500 mM served as running and regeneration buffers, respectively. Sensorgrams were analyzed with Biacore T100 Evaluation software. Anti-6xHis tagged antibody was purchased from QIAGEN. Human 6xHis tagged NKp46 recombinant protein (NKp46-His) was cloned, produced, and purified at Innate Pharma.

[0285] Anti-His antibodies were covalently immobilized to the carboxyl groups of the dextran layer on the Sensor Chip CM5. The chip surface was activated with EDC / NHS (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (Biacore GE Healthcare)). Protein A and anti-His antibodies were diluted to 10 μg / ml in binding buffer (10 mM acetic acid, pH 5.6) and injected until the appropriate immobilization level (i.e., 2000-2500 RU) was reached. The remaining activated groups were deactivated using 100 mM ethanolamine pH 8 (Biacore GE Healthcare).

[0286] Normal human IgG1 chimeric parent antibodies with NKp46 binding regions matching NKp46-1, NKp46-2, NKp46-3, or NKp46-4 were used in competition studies performed with an anti-6xHis tagged antibody chip.

[0287] Bispecific antibodies having an NKp46 binding region based on NKp46-1, NKp46-2, NKp46-3, or NKp46-4 at 1 μg / mL were captured on a Protein A chip, and recombinant human NKp46 protein was injected at 5 μg / mL along with a second test bispecific antibody from the NKp46-1, NKp46-2, NKp46-3, or NKp46-4 group.

[0288] None of NKp46-1, NKp46-2, NKp46-3, or NKp46-4 compete with each other for binding to NKp46, and each of these antibodies represents a different epitope.

[0289] B. Binding to NKp46 mutants To define the epitope of anti-NKp46 antibodies, we designed NKp46 mutants defined by one, two, or three amino acid substitutions exposed on the molecular surface on two domains of NKp46. This approach generated 42 mutants transfected in Hek-293T cells, as shown in the table below. Both targeted amino acid mutations in Table 5 below are shown using the numbering of SEQ ID NO: 1 (also in accordance with the numbering used in Jaron-Mendelson et al. (2012) J. Immunol. 88(12):6165-74).

[0290] [Table 12]

[0291] Generation of mutants NKp46 mutants were generated by PCR. Amplified sequences were run on an agarose gel and purified using the Macherey Nagel PCR Clean-Up Gel Extraction kit (reference number 740609). Two or three purified PCR products generated for each mutant were then ligated into an expression vector using the ClonTech InFusion system. Vectors containing the mutant sequences were prepared in Miniprep and sequenced. After sequencing, vectors containing the mutant sequences were prepared in Miniprep using the Promega PureYield™ Plasmid Midiprep System. HEK293T cells were grown in DMEM medium (Invitrogen), transfected with the vectors using Invitrogen's Lipofectamine 2000, and incubated at 37° C. in a CO2 incubator for 24 hours before testing for transgene expression.

[0292] Flow cytometric analysis of anti-NKp46 binding to HEK293T transfected cells All anti-NKp46 antibodies were tested for binding to their respective mutants by flow cytometry. Initial experiments were performed to determine antibodies that lost binding to one or several mutants at a certain concentration (10 μg / ml). To confirm loss of binding, titrations of antibodies were performed for antibodies whose binding was expected to be affected by NKp46 mutants (1-0, 1-0.01-0.001 μg / ml).

[0293] result Antibody NKp46-1 had reduced binding to mutant 2, which has mutations at residues K41, E42, and E119 (numbered as in NKp46 wild type), compared to wild type NK46. Similarly, NKp46-1 also had reduced binding to the additional mutant Supp7, which has mutations at residues Y121 and Y194.

[0294] Antibody NKp46-3 had poor binding to mutant 19, which has mutations at residues I135 and S136. Similarly, NKp46-1 also had poor binding to the additional mutant Supp8, which has mutations at residues P132 and E133.

[0295] Antibody NKp46-4 had poor binding to mutant 6, which has mutations at residues R101 and V102. Similarly, NKp46-1 also had poor binding to the additional mutant Supp6, which has mutations at residues E104 and L105.

[0296] In this study, we identified the epitopes of anti-NKp46 antibodies (NKp46-1, NKp46-3, and NKp46-4). The epitopes of NKp46-4, NKp46-3, and NKp46-1 are in the D1 domain, D2 domain, and D1 / D2 junction of NKp46, respectively. R101, V012, E104, and L105 are essential residues for NKp46 binding and are defined as part of the epitope of NKp46-4. The epitope of NKp46-1 includes residues K41, E42, E119, Y121, and Y194. The epitope of NKp46-3 includes residues P132, E133, I135, and S136.

[0297] Headings and subheadings are used herein for convenience only and should not be construed as limiting the invention in any way. Any combination of the above-mentioned elements in all possible variations is encompassed by the present invention unless otherwise stated herein or the context dictates otherwise. The ranges of values ​​stated herein merely serve as a shorthand method of individually stating each separate value falling within the range, unless otherwise stated herein, and each separate value is encompassed herein as if it were individually stated herein. Unless otherwise stated, all precise values ​​stated herein represent the corresponding approximate values ​​(e.g., all precise exemplary values ​​stated with respect to a particular factor or measurement can be considered to indicate the corresponding approximate measurement, modified by "about" where appropriate). All methods described herein can be performed in any suitable order, unless otherwise stated herein or the context dictates otherwise.

[0298] The use of any examples or exemplary language (e.g., "such as") described herein is merely to make the invention more clear and does not limit the scope of the invention, unless specifically stated. No language herein should be construed as indicating any element as essential to the practice of the invention, unless specifically stated.

[0299] Descriptions herein of any aspect or embodiment of the invention using words, e.g., words referring to one or more elements, are intended to support similar aspects or embodiments of the invention that "consist of," "consist essentially of," or "substantially include" that particular element or elements, unless specifically stated otherwise or the context clearly dictates otherwise (e.g., a composition described herein as including a particular element will be understood to also be a description of a composition consisting of that element, unless specifically stated otherwise or the context clearly dictates otherwise).

[0300] This invention includes all modifications and equivalents of the subject matter recited in the embodiments or claims described herein to the maximum extent permitted by applicable law.

[0301] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0302] Although the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, it will be apparent to those skilled in the art from the teachings of the invention that certain variations and modifications can be made without departing from the spirit or scope of the appended claims.

Claims

1. An antigen-binding domain that binds to a human NKp46 polypeptide, (a) a VH comprising the amino acid sequence of SEQ ID NO: 199 or 200 (NKp46-1 H1 or H3 variable domain), and a VL comprising the amino acid sequence of SEQ ID NO: 201 (NKp46-1 L1 variable domain); (b) a VH comprising the amino acid sequence of SEQ ID NO: 202, 203, or 204 (NKp46-2 H1, H2, or H3 variable domain), and a VL comprising the amino acid sequence of SEQ ID NO: 205 (NKp46-2 L1 variable domain); (c) a VH comprising the amino acid sequence of SEQ ID NO: 206, 207, or 208 (NKp46-3 H1, H3, or H4 variable domain), and a VL comprising the amino acid sequence of SEQ ID NO: 209 (NKp46-3 L1 variable domain); (d) a VH comprising the amino acid sequence of SEQ ID NO: 210, 211, or 212 (NKp46-4 H1 variable domain), and a VL comprising the amino acid sequence of SEQ ID NO: 213 (NKp46-4 L2 variable domain); (e) a VH comprising the amino acid sequence of SEQ ID NO: 215 (NKp46-9 H2 variable domain), and a VL comprising the amino acid sequence of SEQ ID NO: 217 or 218 (NKp46-9 L1 or L2 variable domain); or (f) a VH comprising the amino acid sequence of SEQ ID NO: 216 (NKp46-9 H3 variable domain), and a VL comprising the amino acid sequence of SEQ ID NO: 217 or 218 (NKp46-9 L1 or L2 variable domain). An antigen-binding domain comprising a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of:

2. A protein or polypeptide comprising the antigen-binding domain of claim 1.

3. The composition of claim 2 , wherein the protein is an antibody.

4. The composition of claim 2 , wherein the protein is a multispecific antigen-binding protein.

5. The composition of any one of claims 1 to 4, wherein the VH and the VL are located on a single polypeptide chain and are fused to each other via a peptide linker.

6. The composition of any one of claims 1 to 4, wherein the VH is located on a first polypeptide chain within the protein and the VL is located on a second polypeptide chain within the protein.

7. The composition of claim 3 , wherein the protein is a full-length IgG antibody or a fragment thereof that maintains binding to the NKp46 polypeptide.

8. 6. The composition of any one of claims 1 to 5, wherein the protein is a multispecific protein further comprising a second antigen-binding domain that binds to an antigen of interest, and wherein the multispecific protein is capable of causing an NKp46-expressing NK cell to lyse a target cell expressing the antigen of interest.

9. NKp46-4 H2L2, NKp46-4 H3L2, NKp46-9 H2L1, NKp46-9 H2L2, NKp46-9 H3L1, or NKp46-9 H4L1. A protein comprising VH and VL domains that comprise amino acid sequences that are at least 80%, 90%, 95%, or 98% identical to the respective VH and VL domains of any one of H3L2.

10. A protein or polypeptide comprising an antigen-binding domain that binds to a human NKp46 polypeptide and a non-human primate NKp46 polypeptide, said antigen-binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising framework sequences of human origin, and an immunoglobulin light chain variable region (VL) comprising framework sequences of human origin, said protein comprising: (a) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:3, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:4 (NKp46-1); (b) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:5, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:6 (NKp46-2); (c) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 7, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 8 (NKp46-3); or (d) a VH comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 9, and a VL comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 10 (NKp46-4); or (e) VH comprising an amino acid sequence at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 13, and VL comprising an amino acid sequence at least 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 14 (NKp46-9). A protein or polypeptide comprising a VH and VL combination selected from the group consisting of:

11. The protein is (a) a VH comprising CDR1, 2, and 3 of a VH having the amino acid sequence of SEQ ID NO: 3 and FR1, 2, and 3 of a human IGHV1-69 gene segment, and a VL comprising CDR1, 2, and 3 of a VL having the amino acid sequence of SEQ ID NO: 4 and FR1, 2, and 3 of a human IGKV1-33 gene segment; (b) a VH comprising CDR1, 2, and 3 of a VH having the amino acid sequence of SEQ ID NO:5 and FR1, 2, and 3 of a human IGHV4-30-4 gene segment, and a VL comprising CDR1, 2, and 3 of a VL having the amino acid sequence of SEQ ID NO:6 and FR1, 2, and 3 of a human IGKV1-39 gene segment; (c) a VH comprising CDR1, 2, and 3 of a VH having the amino acid sequence of SEQ ID NO: 7 and FR1, 2, and 3 of a human IGHV1-69 gene segment, and a VL comprising CDR1, 2, and 3 of a VL having the amino acid sequence of SEQ ID NO: 8 and FR1, 2, and 3 of a human IGKV3-11 and / or IGKV3-15 gene segment; (d) a VH comprising CDR1, 2, and 3 of a VH having the amino acid sequence of SEQ ID NO:9 and FR1, 2, and 3 of a human IGHV1-46 and / or IGHV1-69 gene segment, and a VL comprising CDR1, 2, and 3 of a VL having the amino acid sequence of SEQ ID NO:10 and FR1, 2, and 3 of a human IGKV1-NL1 gene segment; (e) a VH comprising CDR1, 2, and 3 of a VH having the amino acid sequence of SEQ ID NO: 13 and FR1, 2, and 3 of a human IGHV4-30-4 gene segment, and a VL comprising CDR1, 2, and 3 of a VL having the amino acid sequence of SEQ ID NO: 14 and FR1, 2, and 3 of a human IGKV1-39 gene segment; The composition according to any one of claims 1 to 10, comprising a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of:

12. The antigen binding domain is selected from the group consisting of the antibodies NKp46-1 H1L1, NKp46-1 H3L1, NKp46-2 H1L1, NKp46-2 H2L1, NKp46-2 H3L1, NKp46-3 H1L1, NKp46-3 H3L1, NKp46-3 H4L1, NKp46-4 H1L2, NKp46-4 H2L2, NKp46-4 H3L2, NKp46-9 H2L1, NKp46-9 H2L2, NKp46-9 H3L1, or NKp46-9 12. The composition of claim 10 or 11, comprising a VH and VL domain that comprise amino acid sequences that are at least 80%, 90%, 95%, or 98% identical to the respective VH and VL domains of any one of H3L2.

13. The antigen-binding domain or the protein or polypeptide comprising same comprises: a. a mutant NKp46 polypeptide having mutations at residues R101, V102, E104, and / or L105 compared to binding to wild-type NKp46; b. a mutant NKp46 polypeptide having a mutation in any one or more of residues K41, E42, E119, Y121, and / or Y194 compared to said binding to wild-type NKp46; and c. A mutant NKp46 polypeptide having a mutation in any one or more of residues P132, E133, I135, and / or S136, compared to said binding to wild-type NKp46. The composition according to any one of claims 1 to 12, having low binding to a mutant NKp46 polypeptide selected from the group consisting of:

14. The antigen-binding domain according to any one of claims 1 to 13, wherein the VH and VL domains are present on the same polypeptide chain.

15. The antigen-binding domain of any one of claims 1 to 14, wherein the VH and VL domains are separated by a peptide linker.

16. 16. The protein of any one of claims 1 to 15, which is a multispecific protein further comprising a second antigen-binding domain that binds to an antigen of interest, said multispecific protein being capable of causing an NKp46-expressing NK cell to lyse a target cell expressing said antigen of interest.

17. The multispecific protein of claim 16 , which consists of a single polypeptide chain.

18. 18. The multispecific protein of claim 16 or 17, wherein the antigen binding domain that binds to a NKp46 polypeptide and the antigen binding domain that binds to an antigen of interest each comprise a VH fused to a VL via a linker.

19. 19. The multispecific protein of claim 16 or 18, which is a multimeric polypeptide selected from a dimer, trimer, or tetramer.

20. The composition of any one of claims 1 to 19, wherein the protein comprises at least a portion of an Fc domain and has reduced binding to a human CD16 polypeptide compared to a full-length wild-type human IgG1 antibody.

21. The composition of any one of claims 1 to 19, wherein the protein comprises at least a portion of an Fc domain, the Fc domain being dimeric and maintaining binding to human CD16.

22. 22. The composition of any one of claims 1 to 16 or 18 to 21, wherein the protein is a heteromultimeric protein comprising at least two different polypeptide chains, each comprising at least one heavy or light chain variable domain and an Fc domain fused to a human CH1 or Cκ constant domain (V-(CH1 / Cκ) unit), wherein the protein chains are CH1-Cκ dimerized and further linked to each other by non-covalent bonds, and optionally by disulfide bonds formed between the respective CH1 and Cκ domains and by non-covalent bonds between the CH3 domains of the respective Fc portions.

23. 23. The protein of claim 22, wherein both the first and second polypeptide chains comprise a hinge domain at the junction of the CH1 and / or Cκ domain and the Fc domain.

24. The composition of any one of claims 1 to 23, wherein the antigen-binding domain, polypeptide or multispecific protein binds monovalently to the NKp46 polypeptide.

25. the protein is a heterodimer, and (a) Domain arrangement: V ａ－１ - (CH1 or CK) ａ -Fc domain-V ａ－２ -V ｂ－２ and a first polypeptide having (b) Domain Arrangement: V ｂ－１ - (CH or CK) ｂ - Fc domain A second polypeptide chain having Including, V ａ－１ and V ｂ－１ one is a light chain variable domain and the other is a heavy chain variable domain, ａ－２ and V ｂ－ one of the two is a light chain variable domain and the other is a heavy chain variable domain; (CH1 or CK) ｂ is the above (CH1 or CK) on the central chain ａ and V ｂ－１ is the V of the central chain ａ－１ together with V to form an antigen-binding domain; ａ -2 and V ｂ－２ The composition of any one of claims 1 to 16 or 18 to 21, wherein said first and second antigen-binding domains are linked together to an antigen-binding domain.

26. the protein is a heterotrimer, and (a) Domain arrangement: V ａ－１ - (CH1 or CK) ａ -Fc domain-V ａ－２ - (CH1 or CK) ｂ a first polypeptide chain having the following structure: (b) Domain Arrangement: V ｂ－１ - (CH1 or CK) ｃ - Fc domain and a second polypeptide chain having (c) Domain Arrangement: V ｂ－２ - (CH1 or CK) ｄ A third polypeptide chain having Including, V ａ－１ and V ｂ－１ one is a light chain variable domain and the other is a heavy chain variable domain, ａ－２ and V ｂ－２ one is a light chain variable domain and the other is a heavy chain variable domain; (CH1 or CK) ｃ is the above (CH1 or CK) on the central chain ａ and V ａ－１ and the V ｂ－１ forms the antigen-binding domain; and (CH1 or CK) ｄ is the (CH1 or CK) on the central chain ｂ dimerizes with the V ａ－２ and the V ｂ－２ The composition according to any one of claims 1 to 16 or 18 to 21, wherein said composition forms an antigen-binding domain.

27. V ａ－２ and V ｂ－２ The composition of claim 25 or 26, wherein together they form an antigen-binding domain that binds to NKp46.

28. Domain Placement: 【Chemistry 1】 The protein according to any one of claims 25 to 27, having the formula:

29. Domain Placement: 【Chemistry 2】 The protein according to any one of claims 25 to 27, having the formula:

30. Domain Placement: 【Chemistry 3】 The protein according to any one of claims 25 to 27, having the formula:

31. The composition according to any one of claims 1 to 30, wherein the human NKp46 polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO:

1.

32. 32. The composition of any one of claims 1 to 31, wherein the protein binds to macaca fascicularis (cynomolgus monkey) NKp46 polypeptide.

33. 33. The composition of any one of claims 1 to 32, wherein the protein competes for binding to a human NKp46 polypeptide with a monoclonal antibody having the VH and VL domains of NKp46-1, NKp46-2, NKp46-3, NKp46-4, or NKp46-9.

34. A pharmaceutical composition comprising a compound according to any one of claims 1 to 33 and a pharma- ceutically acceptable carrier.

35. 34. An isolated nucleic acid encoding a protein or a polypeptide chain thereof according to any one of claims 1 to 33.

36. A recombinant cell producing the composition of any one of claims 1 to 33.

37. 37. A method for producing a protein, comprising culturing a cell according to claim 36 under conditions suitable for expression of a protein according to any one of claims 1 to 33, and recovering said protein.

38. Use of a polypeptide or composition according to any one of claims 1 to 34 as a medicament for the treatment of a disease.

39. 35. A method of treating a disease in a subject, comprising administering to said subject a composition according to any one of claims 1 to 34.

40. 40. The method or use of claim 38 or 39, wherein the disease is cancer, an infectious disease, or an inflammatory or autoimmune disease.

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