A multispecific antibody that binds to CD20, NKP46, and CD16 and is conjugated to IL-2

Abstract

The present invention relates to a multispecific protein that binds to human NKp46, human CD20, human CD122 and, optionally, human CD16A. The protein according to the invention has utility in the treatment of diseases, such as cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 208,514, filed June 9, 2021, the disclosure of which is incorporated by reference in its entirety, including any drawings and sequence listing.

[0002] Sequence Listing Reference This application has been filed with a Sequence Listing in electronic format. The Sequence Listing is provided in a file entitled "NKp46-14 PCT_ST25.txt", created on June 3, 2022, and having a size of 185 KB. The information in the Electronic Format Sequence Listing is incorporated herein by reference in its entirety.

[0003] FIELD OF THEINVENTION The present disclosure relates to a multispecific binding protein comprising a first and a second antigen binding domain (ABD), a cytokine portion, and the whole or part of an immunoglobulin Fc region or a variant thereof, wherein the first ABD specifically binds to human CD20, and the second ABD specifically binds to human NKp46, and optionally the whole or part of an immunoglobulin Fc region or a variant thereof binds to human CD16. The multispecific binding protein of the present disclosure can advantageously redirect effector cells to lyse targeted CD20-expressing cells via multiple receptors. The present disclosure also relates to methods of making said binding protein, its composition, and their uses, including the treatment of diseases, including diseases involving CD20-expressing cells. [Background technology]

[0004] Natural killer (NK) cells are a subpopulation of lymphocytes involved in non-conventional immunity. NK cells provide an efficient immune surveillance mechanism by which unwanted cells, such as tumor or virus-infected cells, can be eliminated. 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 and kill cells in an MHC-unrestricted manner, particularly cells that do not express "self" MHC / HLA antigens by activating specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate the immune response.

[0005] Interest has also focused on natural killer (NK) cells due to their potential antitumor properties. WO2017114694 reports variable regions for NKp46 binding proteins for the production of multispecific proteins capable of specifically redirecting NK cells to lyse targeted target cells. However, NK cells have been shown to cause toxicity in mice through their overactivation and secretion of multiple inflammatory cytokines when IL-2 is administered with IFN-a [Rothschilds et al, Oncoimmunology. 2019;8(5)]. Additionally, NK cells have also been shown to cause toxicity of the cytokine IL-15, which also signals through IL-2Rβy [see WO2020247843, which references Guo et al, J Immunol. 2015;195(5):2353-64].

[0006] One potential solution to the immunotoxicity mediated by cytokines, such as IL-2, has been to fuse or associate it with tumor-specific antibodies. However, it was found that while IL-2 does indeed cooperate with anti-tumor antibodies in anti-tumor effects in vivo, including IL-2 and anti-tumor antigen antibodies in the same molecule showed no efficacy or toxicity advantage. The IL-2 moiety completely governs biodistribution, explaining the observation that immunocytokines that recognize unrelated antigens function comparably to tumor-specific immunocytokines when combined with antibodies [Tzeng et al. Proc Natl Acad Sci USA. 2015 Mar 17; 112(11): 3320-332]. Summary of the Invention [Problem to be solved by the invention]

[0007] Studies focusing on the effect of cytokines on NK cells have generally focused on single cytokines or simple combinations. More recently, it has been reported that IL-15, IL-18, IL-21, and IFN-α, alone and in combination, have the potential to cooperate with IL-2, and that very low concentrations of both innate and adaptive common gamma chain cytokines cooperate with equally low concentrations of IL-18 to drive rapid and strong NK cell CD25 and IFN-γ expression (Nielsen et al. Front Immunol. 2016; 7: 101). However, administration of cytokines to humans is associated with toxicity, which makes combination treatment with cytokines difficult. Furthermore, there is still little known about the potential synergy or interaction between cytokine receptor signaling pathways and other activating receptors in NK cells. Thus, there is a need for new ways to mobilize NK cells in the treatment of diseases, especially cancer.

[0008] There remains an urgent need for active agents to treat or prevent proliferative disorders such as CD20-positive B non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL) or myelodysplastic syndrome (MDS).

[0009] There is also a need for novel NK engagers with therapeutic efficacy.

[0010] There is also a need for new compounds that are easier to produce and / or administer, with no or reduced side effects, and in particular, there is a need for new compounds that have no or reduced risk of cytokine release syndrome in patients.

[0011] NK cells have the potential to mediate anti-tumor immunity. [Means for solving the problem]

[0012] The present invention arose from the discovery of functional multispecific binding proteins that bind to NKp46 and cytokine receptors (e.g., CD122) on NK cells, and optionally further bind to CD16A on NK cells, and also bind to the tumor antigen CD20 on target cells, with the ability to increase the cytotoxicity of NK cells towards target cells expressing an antigen of interest (e.g., disease-contributing cells, cancer cells).

[0013] In one embodiment, the disclosure relates to a polypeptide comprising the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 90%, 95% or 98% sequence identity thereto. The polypeptide can associate (e.g., dimerize or combine) with one or two additional polypeptides to form a binding protein (e.g., a multimeric binding protein) that specifically binds to human CD20, human NKp46, human CD122, and optionally human CD16. Also provided are multimeric (e.g., dimeric, trimeric) binding proteins comprising one, two or three such polypeptides or polypeptide chains, as well as methods of producing multimeric (e.g., dimeric, trimeric) binding proteins.

[0014] In one embodiment, the disclosure relates to a binding protein (e.g., a multimeric protein) that specifically binds to human CD20, human NKp46, human CD122, and optionally human CD16, the binding protein (e.g., a multimeric protein) comprising a first (I) polypeptide having the amino acid sequence of SEQ ID NO:1, and a second (II) polypeptide having the amino acid sequence of SEQ ID NO:70.

[0015] In one embodiment, the disclosure relates to a multimeric binding protein that specifically binds to human CD20, human NKp46, human CD122, and optionally human CD16, the multimeric binding protein comprising a first (I) polypeptide chain having the amino acid sequence of SEQ ID NO:1, a second (II) polypeptide chain having the amino acid sequence of SEQ ID NO:9, and a third (III) polypeptide chain having the amino acid sequence of SEQ ID NO:17.

[0016] In one embodiment, the disclosure relates to a multimeric binding protein that specifically binds to human CD20, human NKp46, human CD122, and optionally human CD16, the multimeric binding protein comprising a first (I) polypeptide chain having the amino acid sequence of SEQ ID NO:1, a second (II) polypeptide chain having the amino acid sequence of SEQ ID NO:73, and a third (III) polypeptide chain having the amino acid sequence of SEQ ID NO:74.

[0017] In one embodiment, the disclosure relates to a multimeric binding protein that specifically binds to human CD20, human NKp46, human CD122, and optionally human CD16, the multimeric binding protein comprising a first (I) polypeptide chain having the amino acid sequence of SEQ ID NO:66, a second (II) polypeptide chain having the amino acid sequence of SEQ ID NO:67, and a third (III) polypeptide chain having the amino acid sequence of SEQ ID NO:17.

[0018] In one embodiment, provided is a binding protein (e.g., a multimeric protein of the disclosure) comprising a first (I) polypeptide having an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1 or 66, a second (II) polypeptide having an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6, 67, 70, or 73, and, optionally, a third (III) polypeptide having an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:17 or 74.

[0019] In one embodiment, provided is a multimeric binding protein (e.g., a protein of the disclosure) comprising first and second antigen binding domains (ABDs) comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), each VH and VL comprising three complementarity determining regions (CDR1, CDR2, and CDR3); and (i) the first antigen-binding domain (ABD) specifically binds to human CD20; and - VH1 comprising CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 29 (HCDR1), SEQ ID NO: 32 (HCDR2), SEQ ID NO: 35 (HCDR3), and - VL1 comprising CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 38 (LCDR1), SEQ ID NO: 41 (LCDR2), SEQ ID NO: 44 (LCDR3) Includes; (ii) the second antigen-binding domain (ABD) specifically binds to human NKp46; and - a VH2 comprising CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 47 (HCDR1), SEQ ID NO: 50 (HCDR2), SEQ ID NO: 53 (HCDR3), and - VL2 comprising CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 56 (LCDR1), SEQ ID NO: 59 (LCDR2), SEQ ID NO: 62 (LCDR3) Including, A multimeric binding protein (eg, a protein of the present disclosure).

[0020] In certain embodiments, a multimeric binding protein according to the present disclosure comprises a mutant IL-2 polypeptide, wherein said mutant IL-2 comprises the amino acid sequence of SEQ ID NO:65.

[0021] In certain embodiments, the multimeric binding proteins of the present disclosure comprise all or a portion of an immunoglobulin Fc region or a variant thereof that binds to a human Fc-gamma receptor, wherein said all or a portion of an immunoglobulin Fc region comprises a CH2-CH3 domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6 or 14.

[0022] Also provided is a binding protein comprising a first and a second antigen binding domain (ABD), a cytokine portion and all or a portion of an immunoglobulin Fc region or a variant thereof, wherein the first ABD has a Fab structure and comprises an immunoglobulin heavy chain (VH) and an immunoglobulin light chain variable domain (VL), each of the VH and VL comprising three complementarity determining regions (CDR1, CDR2, CDR3); and (i) the first ABD specifically binds to human CD20; and - VH1 comprising CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 29 (HCDR1), SEQ ID NO: 32 (HCDR2), SEQ ID NO: 35 (HCDR3), and - VL1 comprising CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 38 (LCDR1), SEQ ID NO: 41 (LCDR2), SEQ ID NO: 44 (LCDR3) Includes; (ii) the second ABD specifically binds to human NKp46; and - a VH2 comprising CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 47 (HCDR1), SEQ ID NO: 50 (HCDR2), SEQ ID NO: 53 (HCDR3), and - VL2 comprising CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 56 (LCDR1), SEQ ID NO: 59 (LCDR2), SEQ ID NO: 62 (LCDR3) Includes; and all or part of an immunoglobulin Fc region or a variant thereof binds to a human Fc-gamma receptor. It is a binding protein.

[0023] In one embodiment, the cytokine moiety is a mutant IL-2.

[0024] In one embodiment, the first and second ABDs of the binding protein have a Fab structure. In one embodiment, the first ABD of the binding protein has a Fab structure and the second ABD of the binding protein has an scFv structure.

[0025] In one embodiment, a binding protein according to the present disclosure comprises three polypeptide chains (I), (II) and (III) forming two ABDs as defined above: V 1A -C 1A - Hinge 1 - (Fc domain) A (I) V 1B -C 1B - Hinge 2 - (Fc domain) B -L1-V 2A -C 2A (II) V 2B -C 2B - Hinge 3-L2-IL-2 (III) Including, V 1A and V 1B is the bond pair V1 of the first ABD (V H1 / V L1 ) to form; V 2A and V 2B is the bond pair V2 of the second ABD (V H2 / V L2 ) to form; C 1A and C 1B is pair C1 (CH1 / C L ) and C 2A and C 2B The pair C2 (CH1 / C L ), CH1 is immunoglobulin heavy chain constant domain 1, and C L is an immunoglobulin light chain constant domain; Hinge 1, Hinge 2 and Hinge 3 are the same or different and correspond to all or part of an immunoglobulin hinge region; (Fc domain) A and (Fc domain) B are identical or different and comprise a CH2-CH3 domain; L1 and L2 are amino acid linkers, where L1 and L2 can be different or the same; IL-2 is a mutated human interleukin-2 polypeptide or portion thereof that binds to CD122 present on NK cells.

[0026] In another embodiment, the binding protein according to the present disclosure comprises two polypeptide chains (I) and (II) forming two ABDs as defined above: V 1A -C 1A - Hinge 1 - (Fc domain) A (I) V 1B -C 1B - Hinge 2 - (Fc domain) B -L1-V 2A -L2-V 2B -L3-IL-2 (II) Including, V 1A and V 1B is the bond pair V1 of the first ABD (V H1 / V L1 ) to form; V 2A and V 2B is the bond pair V2 of the second ABD (V H2 / V L2 ) to form; C 1A and C 1B is pair C1 (CH1 / C L ), CH1 is immunoglobulin heavy chain constant domain 1, and C L is an immunoglobulin light chain constant domain; Hinge 1 and Hinge 2 are the same or different and correspond to all or part of an immunoglobulin hinge region; (Fc domain) A and (Fc domain) B are identical or different and comprise a CH2-CH3 domain; L1, L2 and L3 are amino acid linkers, where L1, L2 and L3 can be different or the same; IL-2 is a mutated human interleukin-2 polypeptide or portion thereof that binds to CD122 present on NK cells.

[0027] In one embodiment, the CH1 domain of a binding protein of the disclosure is an immunoglobulin heavy chain constant domain 1 comprising the amino acid sequence of SEQ ID NO:12.

[0028] In one embodiment, the C of the binding protein of the present disclosure K The domain is an immunoglobulin kappa light chain constant domain (C K ).

[0029] In one embodiment, the (Fc domain) of the binding protein of the present disclosure A comprises a CH2-CH3 domain corresponding to the amino acid sequence of SEQ ID NO:6.

[0030] In one embodiment, the (Fc domain) of the binding protein of the present disclosure B comprises a CH2-CH3 domain corresponding to the amino acid sequence of SEQ ID NO:14.

[0031] In one embodiment, the hinge 1 domain of a binding protein of the disclosure has the amino acid sequence of SEQ ID NO:5.

[0032] In one embodiment, the hinge 2 domain of a binding protein of the disclosure has the amino acid sequence of SEQ ID NO:13.

[0033] In one embodiment, the hinge 3 domain of a binding protein of the disclosure has the amino acid sequence of SEQ ID NO:19.

[0034] In one embodiment, the linker L1 of the binding protein of the disclosure has the amino acid sequence of SEQ ID NO:15.

[0035] In one embodiment, the linker L2 of the binding protein of the present disclosure has the amino acid sequence of any one of SEQ ID NOs: 20-23.

[0036] In certain embodiments, a binding protein of the disclosure has an Fc domain or variant thereof at residue N297 according to the Kabat numbering including N-linked glycosylation. Preferably, the Fc domain or variant thereof of the binding protein of the disclosure binds to a human CD16A (FcγRIII) polypeptide.

[0037] In one embodiment, the binding protein of the present disclosure comprises at least two polypeptide chains linked by at least one disulfide bridge. Preferably, the polypeptide chains (I) and (II) of the binding protein of the present disclosure are C 1A and hinge 2, two disulfide bridges between hinge 1 and hinge 2, and polypeptide chains (II) and (III) are connected by a disulfide bridge between hinge 3 and C2B are linked by one disulfide bridge between

[0038] In one embodiment, the V of the binding protein of the present disclosure 1A Domain is V L1 and V 1B Domain is V H1 It is.

[0039] In one embodiment, the V of the binding protein of the present disclosure 2A Domain is V H2 and V 2B Domain is V L2 It is.

[0040] In one embodiment, the C of the binding protein of the present disclosure 1A The domain is C K And C 1B The domain is CH1.

[0041] In one embodiment, the C of the binding protein of the present disclosure 2A The domain is C K And C 2B The domain is CH1.

[0042] In an alternative embodiment, the C of the binding protein of the present disclosure 2A The domain is CH1 and C 2B The domain is C K It is.

[0043] In one embodiment, the binding protein of the disclosure comprises: (a) corresponds to the amino acid sequences of SEQ ID NOs: 11 and 3, respectively H1 and V L1 , and / or (b) V corresponding to the amino acid sequences of SEQ ID NOs: 93 and 95, respectively H2 and V L2 Includes.

[0044] In one embodiment, the mutant IL-2 binding proteins of the disclosure exhibit reduced binding to CD25 compared to wild-type human IL-2 polypeptide.

[0045] In one embodiment, the binding variant IL-2 of the binding protein of the disclosure comprises an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOs: 24-28 and 65, or a sequence of at least 40, 50, 60, 70, 80 or 100 contiguous amino acid residues thereof.

[0046] In one embodiment, the binding protein of the disclosure comprises: - a polypeptide (I) consisting of the amino acid sequence of SEQ ID NO:1; - a polypeptide (II) consisting of the amino acid sequence of SEQ ID NO: 9; and - a polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 (III) Includes.

[0047] In an alternative embodiment, the binding protein of the present disclosure comprises: - a polypeptide (I) consisting of the amino acid sequence of SEQ ID NO:1; - a polypeptide (II) consisting of the amino acid sequence of SEQ ID NO: 73; and - a polypeptide consisting of the amino acid sequence of SEQ ID NO: 74 (III) Includes.

[0048] In one embodiment, the Fc domain of the binding protein of the present disclosure comprises a mutated N297 residue (according to Kabat numbering) to prevent it from being glycosylated. Preferably, the mutation is an N297S substitution. In a preferred embodiment, such a mutation substantially eliminates the CD16A binding of the binding protein of the present disclosure.

[0049] In one embodiment, the binding protein of the disclosure comprises: - a polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 66; - a polypeptide (II) consisting of the amino acid sequence of SEQ ID NO: 67; and - a polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 (III) Includes.

[0050] In an alternative embodiment, the binding protein of the present disclosure comprises: - a polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 66; - a polypeptide (II) consisting of the amino acid sequence of SEQ ID NO: 75; and - a polypeptide consisting of the amino acid sequence of SEQ ID NO: 74 (III) Includes.

[0051] In another embodiment, the Fc domain of a binding protein of the disclosure, L234A, L235E, G237A, A330S and / or P331S substitutions according to Kabat numbering.

[0052] Thus, one binding protein of the disclosure comprises: - a polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 68; - a polypeptide (II) consisting of the amino acid sequence of SEQ ID NO: 69; and - a polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 (III) Includes.

[0053] In an alternative embodiment, the binding protein of the present disclosure comprises: - a polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 68; - a polypeptide (II) consisting of the amino acid sequence of SEQ ID NO: 76; and - a polypeptide consisting of the amino acid sequence of SEQ ID NO: 74 (III) Includes.

[0054] In an alternative embodiment, the first ABD of a binding protein of the disclosure that binds CD20 is a Fab and the second ABD that binds NKp46 is an scFv.

[0055] In an alternative embodiment, the first ABD of the binding protein of the disclosure is a VH / VL pair.

[0056] In one embodiment, the binding protein of the disclosure comprises: - a polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 77; - a polypeptide (II) consisting of the amino acid sequence of SEQ ID NO: 78; and - a polypeptide consisting of the amino acid sequence of SEQ ID NO: 74 (III) Includes.

[0057] In an alternative embodiment, the binding protein of the present disclosure comprises: - a polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 77; - a polypeptide (II) consisting of the amino acid sequence of SEQ ID NO: 79; and - a polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 (III) Includes.

[0058] In one embodiment, the second ABD and cytokine portion of the binding protein of the disclosure have the configuration; -L1-V 2A -L2-V 2B -L3-IL-2 having V 2A and V 2B is the bond pair V2 of the second ABD (V H2 / V L2 ) to form; L1, L2 and L3 are amino acid linkers, where L1, L2 and L3 can be different or the same; IL-2 is a mutated human interleukin-2 polypeptide or portion thereof that binds to CD122 present on NK cells.

[0059] In one embodiment, the V of the binding protein of the present disclosure 2A Domain is V H2 and V 2B Domain is VL2 It is.

[0060] In one embodiment, the binding protein of the disclosure comprises: - a polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 1; and - a polypeptide consisting of the amino acid sequence of SEQ ID NO: 70 (II) Includes.

[0061] In one embodiment, the Fc domain of the binding protein of the present disclosure comprises a mutated N297 residue (according to Kabat numbering) to prevent it from being glycosylated. Preferably, the mutation is an N297S substitution. In a preferred embodiment, such a mutation substantially eliminates the CD16A binding of the binding protein of the present disclosure.

[0062] In one embodiment, the binding protein of the disclosure comprises: - a polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 66; - a polypeptide consisting of the amino acid sequence of SEQ ID NO: 71 (II) Includes.

[0063] In another embodiment, the Fc domain of a binding protein of the disclosure comprises L234A, L235E, G237A, A330S and / or P331S substitutions according to Kabat numbering.

[0064] Thus, one binding protein of the disclosure comprises: - a polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 68; - a polypeptide consisting of the amino acid sequence of SEQ ID NO: 72 (II) Includes.

[0065] Provided is a pharmaceutical composition comprising a binding protein of the disclosure and a pharma- ceutically acceptable carrier.

[0066] Also provided is an isolated nucleic acid sequence comprising a nucleotide sequence encoding a binding protein of the present disclosure, or a polypeptide chain thereof.

[0067] Provided is an expression vector comprising a nucleic acid of the disclosure, wherein the nucleic acid sequence comprises a nucleotide sequence that encodes a binding protein or a polypeptide chain thereof of the disclosure.

[0068] Provided is an isolated cell comprising a nucleic acid of the disclosure, wherein the nucleic acid sequence comprises a nucleotide sequence encoding a binding protein or a polypeptide chain thereof of the disclosure.

[0069] Provided is an isolated cell comprising an expression vector of the disclosure, wherein the expression vector comprises a nucleic acid of the disclosure, wherein the nucleic acid sequence comprises a nucleotide sequence encoding a binding protein or a polypeptide chain thereof of the disclosure.

[0070] Provided are binding proteins of the present disclosure for use as a medicament.

[0071] Also provided are binding proteins of the disclosure for use in treating a disease involving or characterized by CD20-expressing cells; and a method of treating a disease in a subject involving or characterized by CD20-expressing cells, comprising administering to the subject a binding protein of the disclosure.

[0072] In one embodiment, the disease treated by the binding proteins for use or methods of treatment of the disclosure is a hematological cancer, e.g., a hematological cancer characterized by malignant cells that express CD20.

[0073] In another embodiment, the disease treated by the binding proteins for use of the disclosure or the treatment methods of the disclosure is selected from the group consisting of B cell lymphoma, Hodgkin's or non-Hodgkin's B cell lymphoma, precursor B cell lymphoblastic leukemia / lymphoma and mature B cell neoplasms such as B cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), cutaneous follicle center lymphoma, marginal zone B cell lymphoma (MALT type, nodular type and splenic type), hairy cell leukemia, diffuse large B cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0074] In further embodiments, the disease (e.g., NHL, CLL, SLL) treated by the binding proteins for use or treatment methods of the present disclosure is characterized by cells (e.g., cancer cells) that express low levels of CD20 on their surface, or a low number of CD20-expressing cells.

[0075] In one embodiment, the multispecific protein is administered 1 to 4 times per month, optionally once every 2 weeks, optionally once every 3 weeks, optionally once every 4 weeks, optionally further, the treatment is for a period of at least 3 months, 6 months or 12 months.

[0076] Provided is a method of making a binding protein of the disclosure, comprising: (a) culturing a host cell under conditions suitable for expressing a plurality of recombinant polypeptides, said plurality of recombinant polypeptides comprising (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 66, 68, 77, 91 or 92, and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 9, 67, 69, 70, 71, 72, 73, 75, 76, 78 or 79, and, optionally (iii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 17 or 74; (b) optionally recovering the expressed recombinant polypeptide. The method includes:

[0077] In one embodiment, a method of making a binding protein of the disclosure comprises: (a) culturing a host cell under conditions suitable for expressing a plurality of recombinant polypeptides, said plurality of recombinant polypeptides comprising (i) a polypeptide comprising the amino acid sequence of SEQ ID NO:1, and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:70; (b) optionally recovering the expressed recombinant polypeptide. Includes.

[0078] These and additional advantageous aspects and features of the present invention may be further described elsewhere herein. [Brief description of the drawings]

[0079] [Figure 1] FIG. 1 shows an exemplary multispecific protein in a T5 format that binds NKp46, CD16A and CD122 on NK cells, and CD20 on tumor cells. [Figure 2A] 2A-2K show different configurations of multispecific proteins that differ in the number of polypeptide chains and in the organization of domains around the Fc domain dimer. [Figure 2B] 2A-2K show different configurations of multispecific proteins that differ in the number of polypeptide chains and in the organization of domains around the Fc domain dimer. [Figure 2C] 2A-2K show different configurations of multispecific proteins that differ in the number of polypeptide chains and in the organization of domains around the Fc domain dimer. [Figure 2D] 2A-2K show different configurations of multispecific proteins that differ in the number of polypeptide chains and in the organization of domains around the Fc domain dimer. [Figure 2E] 2A-2K show different configurations of multispecific proteins that differ in the number of polypeptide chains and in the organization of domains around the Fc domain dimer. [Figure 2F] 2A-2K show different configurations of multispecific proteins that differ in the number of polypeptide chains and in the organization of domains around the Fc domain dimer. [Figure 2G] 2A-2K show different configurations of multispecific proteins that differ in the number of polypeptide chains and in the organization of domains around the Fc domain dimer. [Figure 2H] 2A-2K show different configurations of multispecific proteins that differ in the number of polypeptide chains and in the organization of domains around the Fc domain dimer. [Figure 2I] 2A-2K show different configurations of multispecific proteins that differ in the number of polypeptide chains and in the organization of domains around the Fc domain dimer. [Figure 2J] 2A-2K show different configurations of multispecific proteins that differ in the number of polypeptide chains and in the organization of domains around the Fc domain dimer. [Figure 2K] 2A-2K show different configurations of multispecific proteins that differ in the number of polypeptide chains and in the organization of domains around the Fc domain dimer. [Diagram 3] FIG. 3 shows the % of pSTAT5 cells among CD4 T cells, Treg, CD8 T cells, and NK cells at the concentrations of recombinant interleukin-2, CD20-1-T5-NKCE4-v3, CD20-2-T5-NKCE4-v3, CD20-3-T5-NKCE4-v3, and CD20-4-T5-NKCE4-v3. All tested multispecific proteins resulted in an increase in potency in the ability to induce pSTAT5+ cells in NK cells compared to recombinant IL-2. At the same time, all tested multispecific proteins resulted in a decrease in potency in the ability to induce pSTAT5+ cells in CD4 T cells and Treg cells compared to recombinant IL-2. The multispecific proteins therefore allowed preferential activation of NK cells over Treg cells, CD4 T cells, and CD8 T cells. [Figure 4]FIG. 4 shows the binding potency of several CD20-1-T5-NKCE4, CD20-2-T5-NKCE4, CD20-3-T5-NKCE4, CD20-4-T5-NKCE4 to the RAJI cell line. The measured medium fluorescence intensity is shown on the y-axis and the concentration of the tested protein is shown on the x-axis. The CD20-2-T5-NKCE4 protein showed higher efficacy in binding to CD20+ Raji cells compared to other molecules. [Diagram 5] Figure 5 is a biacore sensorgram demonstrating the ability of CD20-2-T5A-NKCE4-v2A to selectively bind to the CD122 receptor. A CM5 chip containing immobilized anti-His antibody (210322CCe, 1002RU) was used. HuCD25-His (cycle 2), HuCD122-His (cycle 1) or HuCD132-His (cycle 4) were injected at the beginning of each cycle and captured on the chip. CD20-2-T5A-NKCE4-v2A (1 μM) was then injected at 10 μL / min for 120 s. The interaction between CD20-2-T5A-NKCE4-v2A and HuCD25-His, HuCD122-His or HuCD132-His was studied with a dissociation time of 600 s. [Figure 6] FIG. 6 shows the % of cytotoxicity induced by NK cells on the y-axis in the presence of each of several NKCE proteins and the concentration of the test protein on the x-axis. All CD20-T5-NKCE4-v3 proteins, whatever their CD20 ABD, were highly potent in their ability to mediate NK cell cytotoxicity towards tumor target cells. The IC-T5-NKCE4-v3 control molecule, which does not bind to CD20 on RAJI tumor cells, did not induce cytotoxicity. CD20-2-T5-NKCE4-v3 induced significantly better induction of NK cell cytotoxicity in RAJI tumor cells than that other molecules. [Figure 7]FIG. 7 shows tumor volume in mice following administration of 0.4 μg, 2 μg, or 10 μg of CD20-2-T13-NKCE4-v2a or CD20-1-T5-NKCE4. Tumors were implanted on day 0 and a single dose of 0.4 μg, 2 μg, or 10 μg of CD20-2-T13-NKCE4-v2a or CD20-1-T5-NKCE4 was administered on day 9. Each dot on the figure represents the tumor volume in an individual animal. A dose of 10 μg of CD20-2-T13-NKCE4-v2A or CD20-2-T5-NKCE4 showed strong efficacy as a single injection compared to vehicle alone. [Figure 8A] Figures 8A and 8B show the % of cytotoxicity induced by NK cells on the y-axis and the concentration of the tested proteins on the x-axis. All NKCE4 proteins, whatever their format, were highly potent in their ability to mediate NK cell cytotoxicity towards tumor target cells. [Figure 8B] Figures 8A and 8B show the % of cytotoxicity induced by NK cells on the y-axis and the concentration of the tested proteins on the x-axis. All NKCE4 proteins, whatever their format, were highly potent in their ability to mediate NK cell cytotoxicity towards tumor target cells. [Figure 9] Figure 9 shows the proliferation (RLU) of NK cell lines incubated with CD20-2-T13-NKCE4-v2A and CD20-1-T5-NKCE4. The data showed that CD20-2-T13-NKCE4-v2A was more potent than CD20-1-T5-NKCE4 molecules to induce NK cell proliferation. [Figure 10A] Figure 10A shows the serum concentration of CD20-NKCE4 over time after injection (day 0) in non-human primates (n=4 per condition tested). Figure 10B shows several pharmacokinetic parameters of CD20-NKCE4 protein (maximum concentration (Cmax) of CD20-NKCE4 protein in non-human primate serum and serum concentration 22 days after injection, as well as EC50 for STAT5 phosphorylation, cytotoxicity and proliferation of NK cells). [Figure 10B]Figure 10A shows the serum concentration of CD20-NKCE4 over time after injection (day 0) in non-human primates (n=4 per condition tested). Figure 10B shows several pharmacokinetic parameters of CD20-NKCE4 protein (maximum concentration (Cmax) of CD20-NKCE4 protein in non-human primate serum and serum concentration 22 days after injection, as well as EC50 for STAT5 phosphorylation, cytotoxicity and proliferation of NK cells). [Figure 11A] Figures 11A and 11B show the percentage of B cells over time and the number of B cells counted after incubation of several CD20-NKCE4 proteins in human PBMCs (CD20-2-T13-NKCE4-v2A or CD20-2-F13-NKCE3) or controls (IC-T13-NKCE4-v2A, huIL2v2A-His-BirA or no antibody) for 24 h. In contrast to the control molecules (IC-T13-NKCE4-v2A, huIL2v2A-His-BirA), CD20-2-T13-NKCE4-v2A and CD20-2-F13-NKCE3 were each able to deplete CD20+ B cells. [Figure 11B] Figures 11A and 11B show the percentage of B cells over time and the number of B cells counted after incubation of several CD20-NKCE4 proteins in human PBMCs (CD20-2-T13-NKCE4-v2A or CD20-2-F13-NKCE3) or controls (IC-T13-NKCE4-v2A, huIL2v2A-His-BirA or no antibody) for 24 h. In contrast to the control molecules (IC-T13-NKCE4-v2A, huIL2v2A-His-BirA), CD20-2-T13-NKCE4-v2A and CD20-2-F13-NKCE3 were each able to deplete CD20+ B cells. [Figure 12A]Figures 12A and 12B show the percentage of T cells over time and the number of T cells counted after incubation of several CD20-NKCE4 proteins in human PBMCs (CD20-2-T13-NKCE4-v2A or CD20-2-F13-NKCE3) or controls (IC-T13-NKCE4-v2A, huIL2v2A-His-BirA or no antibody) for 24 h. The data show that CD20-2-T13-NKCE4-v2A and CD20-2-F13-NKCE3 do not deplete non-CD20+ T cells. [Figure 12B] Figures 12A and 12B show the percentage of T cells over time and the number of T cells counted after incubation of several CD20-NKCE4 proteins in human PBMCs (CD20-2-T13-NKCE4-v2A or CD20-2-F13-NKCE3) or controls (IC-T13-NKCE4-v2A, huIL2v2A-His-BirA or no antibody) for 24 h. The data show that CD20-2-T13-NKCE4-v2A and CD20-2-F13-NKCE3 do not deplete non-CD20+ T cells. [Figure 13A] Figures 13A and 13B show the percentage of NK cells over time and the number of NK cells counted after incubation of several CD20-NKCE4 proteins in non-human primates (CD20-2-T13-NKCE4-v2A or CD20-2-F13-NKCE3) or controls (IC-T13-NKCE4-v2A, huIL2v2A-His-BirA or no antibody) for 24 h. The data show no reduction in NK cells induced by CD20-2-T13-NKCE4-v2A, suggesting no sibling killing among NK cells. [Figure 13B]Figures 13A and 13B show the percentage of NK cells over time and the number of NK cells counted after incubation of several CD20-NKCE4 proteins in non-human primates (CD20-2-T13-NKCE4-v2A or CD20-2-F13-NKCE3) or controls (IC-T13-NKCE4-v2A, huIL2v2A-His-BirA or no antibody) for 24 h. The data show no reduction in NK cells induced by CD20-2-T13-NKCE4-v2A, suggesting no sibling killing among NK cells. [Figure 14] FIG. 14 shows the concentration of several cytokines (IFN-γ, IL-6, TNF-α, IL-10, IL-8, MIP-1β, MCP-1, IL-1β) over time following injection of several CD20-NKCE4 in non-human primates. [Figure 15] FIG. 15 shows the evolution of the number of circulating B cells over time after injection of different CD20-NKCE4 proteins in non-human primates. [Figure 16A] Figures 16A, 16B and 16C show the time course evolution of B, NK and T cell populations in non-human primates upon administration of CD20-2-T13-NKCE4-v2A on days 0, 7 and 14. [Figure 16B] Figures 16A, 16B and 16C show the time course evolution of B, NK and T cell populations in non-human primates upon administration of CD20-2-T13-NKCE4-v2A on days 0, 7 and 14. [Figure 16C] Figures 16A, 16B and 16C show the time course evolution of B, NK and T cell populations in non-human primates upon administration of CD20-2-T13-NKCE4-v2A on days 0, 7 and 14. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] definition As used herein, "a" or "an" may mean one or more than one. When used in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0081] Where "comprising" is used, this may be replaced by "consisting essentially of" or "consisting of", where appropriate.

[0082] As used herein, the term "antigen-binding domain" or "ABD" refers to a domain that comprises a three-dimensional structure capable of immunospecifically binding to an epitope. Thus, in one embodiment, the domain comprises a hypervariable region, suitably the V of an antibody chain. H and / or V L Domain, if appropriate at least V H 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.

[0083] The term "antibody" herein is used in the broadest sense 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. A variety of techniques related to the production of antibodies are provided, 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 portion of a full-length antibody, such as 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 V of a single arm of an antibody), and Fv (typically the V of a single arm of an antibody). L and V Hdomain), single chain Fv (scFv), dsFv, Fd fragment (typically V H and CH1 domains), and dAbs (typically V H Domain) fragment;V H , V L , 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, as well as one or more isolated CDRs or functional paratopes in which isolated CDRs or antigen-binding residues or polypeptides can associate or link together to form a functional antibody fragment. Various types of antibody fragments are described or reviewed, for example, in Holliger and Hudson, Nat Biotechnol 2005;23,1126-1136; WO 2005040219, and U.S. Patent Publication Nos. 20050238646 and 20020161201.

[0084] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. Hypervariable regions generally comprise 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 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 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 by 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 insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid inserted after residue 52 of CDR H2 (residue 52a according to Kabat) as well as residues inserted after heavy chain FR residue 82 (such as residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the region of homology of the sequence of the antibody with the "standard" Kabat numbered sequence.

[0085] By "framework" or "FR" residues as used herein is meant the regions of an antibody variable domain excluding the regions defined as CDRs. Each antibody variable domain framework can be further divided into contiguous regions (FR1, FR2, FR3 and FR4) separated by the CDRs.

[0086] By "constant region" as defined herein is meant the constant region derived from an antibody encoded by one of the light or heavy chain immunoglobulin constant region genes.

[0087] By "constant light chain" or "light chain constant region" or "CL" as used herein is meant 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, with 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).

[0088] By "constant heavy chain" or "heavy chain constant region" as used herein is meant the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon genes, which define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For a full-length IgG antibody, the constant heavy chain as defined herein refers to the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, thus including positions 118-447, numbering according to the EU index.

[0089] As used herein, the term "CH1 domain," or "CH2 domain" refers to a H"Constant domain 1" or "constant domain 1" can be used interchangeably and refer to the corresponding heavy chain immunoglobulin constant domain 1.

[0090] As used herein, the term "CH2 domain" or "CH2 domain" refers to a H "Constant domain 2" or "constant domain 2" can be used interchangeably and refer to the corresponding heavy chain immunoglobulin constant domain 2.

[0091] As used herein, the term "CH3 domain" or "CH3 domain" refers to a H "Constant domain 3" or "constant domain 3" can be used interchangeably and refer to the corresponding heavy chain immunoglobulin constant domain 3.

[0092] As used herein, (CH2-CH3) A and (CH2-CH3) B The term "CH2-CH3," as in, therefore, refers to a polypeptide sequence that includes immunoglobulin heavy chain constant domain 2 (CH2) and immunoglobulin heavy chain constant domain 3 (CH3).

[0093] As used herein, the term "paired C(CH1 / CL)" or "paired C(C H 1 / C L ) refers to one constant heavy chain domain and one constant light chain domain [e.g., kappa (κ) or K ) or lambda (λ) class immunoglobulin light chains. Unless otherwise specified, when paired constant chain domains are not present on the same polypeptide chain, the term may therefore encompass all possible combinations. H 1 and C L The domains are therefore selected to be complementary to each other, so that they form the stable pair C(CH1 / CL).

[0094] Advantageously, the binding protein comprises multiple paired C domains, for example one "paired C1 (C H 1 / C L )" and one "pair C2 (C H 1 / C L In the case where the complementary CH1 and CL domains are included in the nucleotide sequence, each paired CH1 and CL domain is selected such that a pair is formed between a complementary CH1 domain and a complementary CL domain. H 1 and C L Examples of domains have been previously described in International Patent Applications WO 2006 / 064136 or WO 2012 / 089814 or WO 2015197593 A1.

[0095] Unless otherwise indicated, the term "pair C1 (C H 1 / C L )" or "Pair C2 (C H 1 / C L ) are identical or distinct constant heavy chain 1 domains (C H 1) and the same or a separate constant light chain domain (C L ) may refer to the separate constant pair domains (C1 and C2) formed by the pair C1 (C H 1 / C L )" or "Pair C2 (C H 1 / CL) are identical constant heavy chain 1 domains (C H 1) and the same constant light chain domain (C L ) may refer to the separate constant pair domains (C1 and C2) formed by the

[0096] As used herein, "Fab" or "Fab region" refers to a V H , CH1, V L The term Fab refers to a unit that contains the V, VL, and CL immunoglobulin domains. L -CL moiety associated with V H In addition to the unit containing the -CH1 portion, the Fab includes a crossover Fab structure in which there is a crossover or interchange between the light and heavy chain domains. For example, a Fab may have a VL -CH1 unit and associated V H Fab may refer to this region in isolation or in the context of a protein, multispecific protein or ABD, or any other embodiment outlined herein.

[0097] As used herein, "single chain Fv" or "scFv" refers to the V H and V L By Fv is meant an antibody fragment comprising V domains, which domains are present in a single polypeptide chain. Generally, an Fv polypeptide comprises a V domain that enables the scFv to form the desired structure for antigen binding. H Domain and V L The scFv domains further comprise a polypeptide linker between them. Methods for producing scFvs are well known in the art. For a review of methods for producing scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0098] As used herein, "Fv" or "Fv fragment" or "Fv region" refers to the V domain of a single antibody. L and V H By this is meant a polypeptide that comprises a domain.

[0099] By "Fc" or "Fc region" as used herein is meant 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 at the N-terminus of these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc includes immunoglobulin domains Cγ2 (CH2) and Cγ3 (CH3), and the hinge between Cγ1 and Cγ2, as appropriate. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues C226, P230, or A231 relative to its carboxyl terminus, numbering according to the EU index. Fc may refer to this region in isolation, or to this region in the context of an Fc polypeptide, as described below. By "Fc polypeptide" or "Fc-derived polypeptide" as used herein is meant a polypeptide comprising all or a portion of an Fc region. Fc polypeptides herein include, but are not limited to, antibodies, Fc fusions, and Fc fragments. Fc regions according to the invention also include variants that contain at least one modification that alters (enhances or decreases) an Fc-associated effector function. Fc regions according to the invention also include chimeric Fc regions that comprise different portions or domains of different Fc regions, e.g., derived from antibodies of different isotypes or species.

[0100] As used herein, "variable region" refers to the V domains that constitute the light chain (including kappa and lambda) and heavy chain immunoglobulin gene loci, respectively. L [including Vκ (Vκ) and Vλ] and / or V H By "variable region" is meant the region of an antibody that contains one or more Ig domains substantially encoded by either the light or heavy chain variable region (V L or V H) consists of a "framework" or "FR" region flanked by three hypervariable regions referred to as "complementarity determining regions" or "CDRs". The extent of the framework region and CDRs has been precisely defined, for example, in Kabat [see "Sequences of Proteins of Immunological Interest," E. Kabat et al., US Department of Health and Human Services, (1983)] and Chothia. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs that are primarily responsible for binding to an antigen.

[0101] As used herein, the term "domain" may be any region of a protein that is generally defined based on sequence homology or identity with respect to a distinct structural or functional entity. Thus, the term "region" as used in the context of this disclosure is broader in that it may include additional regions beyond the corresponding domain.

[0102] As used herein, the term "linker region", "linker peptide" or "linker polypeptide" or "amino acid linker" or "linker" refers to any amino acid sequence suitable for covalently linking two polypeptide domains, such as two antigen-binding domains together, and / or an Fc region to one or more variable regions, such as one or more antigen-binding domains. Although the term is not limited to a particular size or length of a polypeptide, such amino acid linkers are generally less than 50 amino acids long, preferably less than 30 amino acids long, such as 20 or less than 20 amino acids long, such as 15 or less than 15 amino acids long. Such amino acid linkers may optionally include the entire or a portion of an immunoglobulin polypeptide chain, such as the entire or a portion of an immunoglobulin hinge region. Alternatively, the amino acid linker may include a polypeptide sequence that is not derived from an immunoglobulin hinge region, or even from neither an immunoglobulin heavy chain polypeptide chain nor an immunoglobulin light chain polypeptide chain.

[0103] As used herein, an immunoglobulin hinge region, or a fragment thereof, can therefore be considered as a particular type of linker derived from an immunoglobulin polypeptide chain.

[0104] As used herein, the term "hinge region" or "hinge" generally refers to the flexible region carried by the corresponding heavy chain polypeptide and separating the Fc and Fab portions of a particular isotype of immunoglobulin, more particularly IgG, IgA or IgD isotypes. Such hinge regions are known in the art to depend on the immunoglobulin isotype under consideration. For native IgG, IgA and IgD isotypes, the hinge region is therefore C H 1 domain and C HThe two domains are separated and generally cleaved by papain digestion. On the other hand, the region corresponding to the hinge in IgM and IgE heavy chains is generally formed by an additional constant domain with lower flexibility. Additionally, the hinge region may contain one or more cysteines involved in interchain disulfide bonds. The hinge region may also contain, where applicable, a C H In addition to the FcγR binding site carried by the two domains, the hinge region may contain one or more binding sites for Fcγ receptors. Additionally, the hinge region may contain one or more post-translational modifications, such as one or more glycosylated residues depending on the isotype considered. Therefore, it is easily understood that the reference to the term "hinge" throughout this specification is not limited to a specific set of hinge sequences or to a specific structural position. Unless otherwise indicated, the hinge region more particularly considered includes the whole or part of the hinge from an immunoglobulin belonging to one isotype selected from IgG isotype, IgA isotype and IgD isotype; in particular, the IgG isotype.

[0105] The term "specifically binds to" means that an antibody or polypeptide can bind to a binding partner, such as NKp46, preferably in a competitive binding assay, when 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 for determining specific binding are described further below and are well known in the art.

[0106] When an antibody or polypeptide is said to "compete" with a particular multispecific protein or a particular monoclonal antibody (e.g., NKp46-1, -2, -4, -6 or -9 in the context of an anti-NKp46 monospecific antibody or multispecific protein), it means that the antibody or polypeptide competes with the particular multispecific protein or monoclonal antibody in a binding assay using 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.

[0107] The term "affinity" as used herein means the strength of binding of an antibody or protein to an epitope. The affinity of an antibody is measured by the dissociation constant K, defined as [Ab] x [Ag] / [Ab-Ag], 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. D The affinity constant K A is 1 / K DThe affinity of a protein is defined by the following: Preferred methods for determining the affinity of a protein can be found 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), which are incorporated herein by reference in their entirety. One preferred and standard method well known in the art for determining the affinity of a protein is the use of surface plasmon resonance (SPR) screening, e.g., by analysis using a BIAcore™ SPR analysis device.

[0108] Within the context of this invention, a "determinant" refers to a site of interaction or binding on a polypeptide.

[0109] The term "epitope" refers to an antigenic determinant, a section or region on an antigen to which an antibody or protein binds. A protein epitope may include amino acid residues that are effectively blocked by a specific antigen-binding antibody or peptide, i.e., within the "footprint" of an antibody, in addition to the amino acid residues directly involved in binding. It is the simplest form or smallest structural section on a complex antigen molecule that can combine with, for example, an antibody or receptor. Epitopes can be linear or conformational / structural. The term "linear epitope" is defined as an epitope that is composed of amino acid residues that are contiguous on a linear sequence of amino acids (primary structure). The term "conformational or structural epitope" is defined as an epitope that is composed of amino acid residues that represent separated portions of a linear sequence of amino acids that are not all contiguous and are therefore brought into close proximity to each other by the folding of the molecule (secondary, tertiary and / or quaternary structure). Conformational epitopes are dependent on the three-dimensional structure. The term "conformational" is therefore often used interchangeably with "structural." Epitopes can be identified by different methods known in the art, including but not limited to alanine scanning, phage display, X-ray crystallography, array-based oligo-peptide scanning or pepscan analysis, site-directed mutagenesis, high-throughput mutagenesis mapping, H / D-Ex mass spectrometry, homology modeling, docking, hydrogen-deuterium exchange, among others.[For example, Tong et al., Methods and Protocols for prediction of immunogenic epitopes", Briefings in Bioinformatics 8(2):96-108; Gershoni, Jonathan M; Roitburd-Berman, Anna; Siman-Tov, Dror D; Tarnovitski Freund, Natalia; Weiss, Yael (2007). "Epitope Mapping". BioDrugs 21 (3): 145-56; and Flanagan, Nina (May 15, 2011); "Mapping Epitopes with H / D-Ex Mass Spec: ExSAR Expands Repertoire of Technology Platform Beyond Protein Characterization", Genetic Engineering & Biotechnology News 31 (10)].

[0110] "Valent" or "valency" refers to the presence of a determined number of antigen-binding moieties in an antigen-binding protein. Native IgG has two antigen-binding moieties and is bivalent. A molecule that has one binding moiety for a particular antigen is monovalent with respect to that antigen.

[0111] By "amino acid modification" herein is meant an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. An example of an amino acid modification herein is a substitution. By "amino acid modification" herein is meant an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant 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 polypeptide in which a tyrosine at position 50 is replaced with a tryptophan. Amino acid substitutions are indicated by listing the residue / residue position present in the wild-type protein / residue present in the mutant protein. A "variant" of a polypeptide refers to a polypeptide having substantially the same amino acid sequence as 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 certain positions within the native amino acid sequence.

[0112] "Conservative" amino acid substitution is a substitution in which an amino acid residue is replaced with an amino acid residue that has 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), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0113] The term "identity" or "identical" when used in the context of a relationship between 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" measures the percentage of identical matches between the smaller of two or more sequences, with gap alignments (if any) accommodated by a particular mathematical model or computer program (i.e., "algorithm"). The identity of 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).

[0114] Preferred methods for determining identity are designed to give the maximum match between the sequences tested. Methods for determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including 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)]. The BLASTX program is available from 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.

[0115] An "isolated" molecule is one that is the predominant species in a composition in which it is found with respect to the class of molecules to which it belongs (i.e., the 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%, at least about 95%, or more of the species of molecules, e.g., peptides, in the composition). Generally, a composition of polypeptides will exhibit 98%, 98%, or 99% homogeneity for the polypeptide in the context of all peptide species present in the composition, or at least with respect to substantially active peptide species in the context of the proposed use.

[0116] In the present context, "treatment" or "treating" refers to preventing, alleviating, managing, curing, or reducing one or more symptoms or clinically relevant findings of a disease or disorder, unless the context indicates otherwise. For example, "treatment" of a patient in whom symptoms or clinically relevant findings of a disease or disorder have not been identified is a preventive or prophylactic therapy, whereas "treatment" of a patient in whom symptoms or clinically relevant findings of a disease or disorder have been identified generally does not constitute a preventive therapy.

[0117] As used herein, the phrase "NK cells" refers to a subpopulation of lymphocytes involved in non-conventional immunity. NK cells can be identified because of certain characteristics and biological properties, such as the expression of specific surface antigens, including CD56 and / or NKp46 for human NK cells, the absence of alpha / beta or gamma / delta TCR complexes on the cell surface, the ability to bind to and kill cells that do not express "self" MHC / HLA antigens by activating specific cytolytic mechanisms, the ability to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit immune responses. Any of these characteristics and activities can be used to identify NK cells using methods well known in the art. Any subpopulation of NK cells is also 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 enhance the immune function of other cells. NK cells can be obtained by a variety of techniques known in the art, such as isolation from blood samples, cytapheresis, tissue or cell collection, etc. Useful protocols for assays involving NK cells can be found in Natural Killer Cells Protocols (edited by Campbell KS and Colonna M). Humana Press. pp. 219-238 (2000).

[0118] As used herein, an agent having "agonist" activity in NKp46 is an agent that can cause or increase "NKp46 signaling". "NKp46 signaling" refers to the ability of NKp46 polypeptide to activate or transmit intracellular signaling pathways. Changes in NKp46 signaling activity can be measured, for example, by assays designed to measure changes in NKp46 signaling pathways, for example by monitoring phosphorylation of signaling components, measuring the association of certain signaling components with other proteins or intracellular structures, or assays in the biochemical activity of components, for example kinases, or assays designed to measure expression of reporter genes under the control of NKp46-sensitive promoters and enhancers, or indirectly by downstream effects mediated by NKp46 polypeptides (for example, activation of specific cytolytic mechanisms in NK cells). The reporter gene can be a naturally occurring gene (for example, monitoring cytokine production) or a gene artificially introduced into cells. Other genes can be placed under the control of such regulatory elements, which thus serve to report the level of NKp46 signaling.

[0119] "NKp46" refers to a protein or polypeptide encoded by the Ncr1 gene or by a cDNA prepared from such a gene. Any naturally occurring isoforms, alleles, orthologs, 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 the 304 amino acid residues of human NKp46 (isoform a) is shown below:

[0120] [Table 1]

[0121] SEQ ID NO: 88 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.

[0122] As used herein, the terms "subject" or "individual" or "patient" are used interchangeably and can include human or non-human mammals, rodents or non-rodents.The terms include, but are not limited to, mammals, such as humans, including males, females and children, other primates (monkeys), pigs, rodents, such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats.

[0123] Polypeptide production The proteins described herein can be advantageously constructed and produced using well-known immunoglobulin-derived domains, particularly heavy and light chain variable domains, hinge region, CH1, CL, CH2 and CH3 constant domains, and wild-type or mutant cytokine polypeptides. The domains placed on a common polypeptide chain can be fused to each other, either directly or through a linker, depending on the specific domain involved. The immunoglobulin-derived domains are preferably humanized or of human origin, thereby providing a reduced risk of immunogenicity when administered to humans. As shown herein, advantageous protein formats are described that use minimal non-immunoglobulin-linked amino acid sequences (e.g., no more than 4 or 5 domain linkers, in some cases as few as 1 or 2 domain linkers, and use of short domain linkers), thereby further reducing the risk of immunogenicity.

[0124] Immunoglobulin variable domains are commonly derived from antibodies (immunoglobulin chains), e.g., associated V domains found on two polypeptide chains. L and V Hdomain, or single chain antigen-binding domain, e.g., scFv, V H Domain, V L Domain, dAb, V-NAR domain or V H In certain advantageous protein formats disclosed herein that allow the use of a wide range of variable regions directly from Fab or scFv without substantial further requirements for pairing and / or folding, the antigen-binding domains (e.g., ABD1 and ABD2) can also be easily derived from antibodies as Fab or scFv.

[0125] The term "antigen-binding protein" may be used to refer to an immunoglobulin derivative having antigen-binding properties. A binding protein comprises an immunologically functional immunoglobulin portion capable of binding to a target antigen. An immunologically functional immunoglobulin portion may comprise an immunoglobulin, or a portion thereof, a fusion peptide derived from an immunoglobulin portion, or a conjugate combining immunoglobulin portions to form an antigen-binding site. Each antigen-binding portion comprises at least necessarily one, two, or three CDRs of the immunoglobulin heavy and / or light chain from which the antigen-binding portion is derived. In some embodiments, an antigen-binding protein may consist of a single polypeptide chain (monomer). In other embodiments, an antigen-binding protein comprises at least two polypeptide chains, e.g., a multimeric protein, conveniently identified as being a dimeric protein trimeric protein. As further exemplified herein, an antigen-binding domain may advantageously comprise a VH and a VL (VH / VL pair). In some embodiments, the VH / VL pair can be incorporated into a Fab structure that further comprises a CH1 and a CL domain (CH1 / CL pair). A VH / VL pair refers to one VH and one VL domain that are associated with each other to form an antigen-binding domain. A CH1 / CL pair refers to one CH1 and one CL domain that are covalently or non-covalently, preferably non-covalently, bound to each other to form a heterodimer (e.g., a protein that can include one or more additional polypeptide chains, e.g., in a heterotrimer).

[0126] In one embodiment, the binding protein is (i) a first antigen-binding domain (ABD) comprising a variable region that specifically binds to a human CD20 polypeptide; (ii) a second antigen-binding domain (ABD) comprising a variable region that specifically binds to a human NKp46 polypeptide; (iii) a whole or part of an immunoglobulin Fc region or a variant thereof that binds to a human Fc-gamma receptor (CD16); and a cytokine portion. Includes.

[0127] Protein Format Multimeric multispecific proteins, such as heterodimers and heterotrimers, can be produced according to a variety of formats. Different domains on different polypeptide chains associate to form a multimeric protein. Thus, a wide range of protein formats can be constructed around Fc domain dimers capable of binding to human FcRn polypeptides (neonatal Fc receptors) with or without additional binding to CD16 or CD16A, depending on whether the presence of such CD16-binding ABD is desired. As shown herein, maximum enhancement of NK cell cytotoxicity can be obtained through the use of Fc moieties with substantial binding to activating human CD16 receptor (CD16A) binding; such CD16 binding can be obtained through the use of suitable CH2 and / or CH3 domains, as further described herein. In one embodiment, the Fc moiety is derived from a human IgG1 isotype constant region. The use of modified CH3 domains also contributes to the possibility of using a wide range of heteromultimeric protein structures. Thus, the protein comprises a first and a second polypeptide chain, each comprising a variable domain fused to a human Fc domain monomer (i.e., CH2-CH3 unit), optionally an Fc domain monomer comprising a CH3 domain capable of preferential CH3-CH3 heterodimerization, and the first and second chains associate via CH3-CH3 dimerization, resulting in a protein comprising an Fc domain dimer. The variable domains of each chain can be part of the same or different antigen-binding domains.

[0128] Multispecific proteins may therefore be constructed advantageously using VH and VL pairs arranged as scFv or Fab structures, with CH1 domains, CL domains, Fc domains and cytokines, and domain linkers. Preferably, the protein uses minimal non-natural sequences, e.g. minimal use of non-Ig linkers, optionally no more than 5, 4, 3, 2 or 1 domain linker that is not an antibody-derived sequence, and optionally the domain linker is no more than 15, 10 or 5 amino acid residues in length. In one embodiment, the protein comprises a CD16 ABD embodied as an Fc domain dimer.

[0129] In some embodiments, the multispecific protein (e.g., dimer, trimer) may comprise any of the following domain arrangements: the domains can be located on either two or three polypeptide chains, the NKp46 ABD is interposed between the Fc domain and the cytokine moiety [e.g., the protein has a terminal or distal cytokine receptor ABD at the C-terminus and a terminal or distal CD20 ABD at the topological N-terminus], the NKp46 ABD is connected to one of the polypeptide chains of the Fc domain dimer via a hinge polypeptide or flexible linker, and the ABD that binds the cytokine receptor is connected to the NKp46 ABD (e.g., one of the polypeptide chains when the NKp46 ABD is contained on two chains) via a flexible linker (e.g., a linker comprising G and S residues): (anti-CD20 ABD)-(Fc domain dimer)-(NKp46 ABD)-(cytokine moiety).

[0130] The cytokine moiety may be an IL2 polypeptide or a variant thereof. The Fc domain dimer may be specified as an Fc domain dimer that binds, for example, human FcRn and / or Fcγ receptor. In one embodiment, one or both of the CD20 ABD and the NKp46 ABD are formed from two variable regions present, and the variable regions that associate to form a particular ABD may be on the same polypeptide chain or on different polypeptide chains. In another embodiment, one or both of the CD20 ABD and the NKp46 ABD comprise a tandem variable region (scFv), and the other comprises a Fab structure. In another embodiment, both the antigen of interest and the NKp46 ABD comprise a Fab structure. In another embodiment, the CD20 ABD comprises a Fab structure, and the NKp46 ABD comprises a scFv structure.

[0131] In one embodiment, the binding protein of the present disclosure is a heterotrimer and comprises three polypeptide chains (I), (II) and (III) forming two ABDs, as defined above: V 1A -C 1A - Hinge 1 - (Fc domain) A (I) V 1B -C 1B - Hinge 2 - (Fc domain) B -L1-V 2A -C 2A (II) V 2B -C 2B - Hinge 3-L2-IL-2(III) Including, V 1A and V 1B is the bond pair V1(V H1 / V L1 ) to form; V 2A and V 2B is the bond pair V2(V H2 / V L2 ) to form; C 1A and C 1B is pair C1 (CH1 / C L) and C 2A and C 2B The pair C2 (CH1 / C L ), CH1 is immunoglobulin heavy chain constant domain 1, and C L is an immunoglobulin light chain constant domain; Hinge 1, Hinge 2 and Hinge 3 are the same or different and correspond to all or part of an immunoglobulin hinge region; (Fc domain) A and (Fc domain) B are identical or different and comprise a CH2-CH3 domain; L1 and L2 are amino acid linkers, where L1 and L2 can be different or the same; IL-2 is a mutated human interleukin-2 polypeptide or a portion thereof that binds to CD122 present on NK cells. In one embodiment, binding pair V1 binds to CD20 and binding pair V2 binds to NKp46.

[0132] V 1A , V 1B , V 2A , V 2B each of which is an immunoglobulin VH or VL domain, 1A and V 1B One of them is VH and the other is VL, and V 2A and V 2B One of them is VH and the other is VL.

[0133] In some embodiments, the binding proteins of the disclosure have an Fc domain or a variant thereof at residue N297 according to the Kabat numbering including N-linked glycosylation. In some embodiments, the binding proteins of the disclosure comprise an Fc domain that binds to a human CD16A polypeptide.

[0134] According to some embodiments, V1A is VL1 and V1B is VH1.

[0135] According to some embodiments, V2A is VH2 and V2B is VL2.

[0136] According to some embodiments, C1A is CK and C1B is CH1.

[0137] According to some embodiments, C2A is CK and C2B is CH1.

[0138] In some embodiments, VH1 comprises CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO:29 (HCDR1), SEQ ID NO:32 (HCDR2) and SEQ ID NO:35 (HCDR3); VL1 comprises CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO:38 (LCDR1), SEQ ID NO:41 (LCDR2) and SEQ ID NO:44 (LCDR3); VH2 comprises CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO:47 (HCDR1), SEQ ID NO:50 (HCDR2) and SEQ ID NO:53 (HCDR3), and VL2 comprises CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO:56 (LCDR1), SEQ ID NO:59 (LCDR2) and SEQ ID NO:62 (LCDR3).

[0139] In some embodiments, the binding protein comprises (a) a V corresponding to the amino acid sequence of SEQ ID NOs: 11 and 3, respectively, as shown below. H1 and V L1 and / or (b) V corresponding to the amino acid sequences of SEQ ID NOs: 93 and 95, respectively. H2 and V L2 Includes. V H1 (SEQ ID NO:11) EVQLVESGGG LVQPDRSLRL SCAASGFTFH DYAMHWVRQA PGKGLEWVST ISWNSGTIGY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TALYYCAKDI QYGNYYYGMD VWGQGTTVTV SS V L1 (SEQ ID NO:3) EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPITFGQ GTRLEIK V H2 (SEQ ID NO:93) QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYVINWVRQA PGQGLEWMGE IYPGSGTNYY NEKFKAKATI TADKSTSTAY MELSSLRSED TAVYYCARRG RYGLYAMDYW GQGTTVTVSS V L2 (SEQ ID NO:95) DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP GKAPKLLIYY TSRLHSGVPS RFSGSGSGTD FTFTISSLQP EDIATYFCQQ GNTRPWTFGG GTKVEIK

[0140] In some embodiments, the binding protein comprises (a) a V corresponding to the amino acid sequence of SEQ ID NOs: 11 and 3, respectively, or a variant thereof having at least 95% sequence identity. H1 and V L1 and / or (b) V corresponding to the amino acid sequences of SEQ ID NOs: 93 and 95, respectively, or variants thereof having at least 95% sequence identity. H2 and V L2 Includes.

[0141] In some embodiments, the binding protein comprises (a) a V corresponding to the amino acid sequence of SEQ ID NOs: 11 and 3, respectively, or a variant thereof having at least 90% sequence identity. H1 and V L1 and / or (b) V corresponding to the amino acid sequences of SEQ ID NOs: 93 and 95, respectively, or variants thereof having at least 90% sequence identity. H2 and V L2 Includes.

[0142] In some embodiments, in the heterotrimeric binding proteins of the present disclosure, CH1 is an immunoglobulin heavy chain constant domain 1 comprising the amino acid sequence of SEQ ID NO: 12; CK is an immunoglobulin kappa light chain constant domain (CK) comprising the amino acid sequence of SEQ ID NO:4; (Fc domain) A comprises a CH2-CH3 domain corresponding to the amino acid sequence of SEQ ID NO:6; (Fc domain) B comprises a CH2-CH3 domain corresponding to the amino acid sequence of SEQ ID NO: 14; Hinge 1 corresponds to the amino acid sequence of SEQ ID NO:5; Hinge 2 corresponds to the amino acid sequence of SEQ ID NO:13; Hinge 3 corresponds to the amino acid sequence of SEQ ID NO:19; L1 corresponds to the amino acid sequence of SEQ ID NO: 15; and / or L2 corresponds to any one of the amino acid sequences of SEQ ID NOs:20 to 23.

[0143] In some embodiments, the ABD that binds CD20 and the ABD that binds NKp46 each have a Fab structure.

[0144] In some embodiments, as disclosed herein below, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:1, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:9, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:17. First Polypeptide Chain (I) (SEQ ID NO:1) EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPITFGQ GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGECDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD ​​IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K Second Polypeptide Chain (II) (SEQ ID NO:9) EVQLVESGGG LVQPDRSLRL SCAASGFTFH DYAMHWVRQA PGKGLEWVST ISWNSGTIGY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TALYYCAKDI QYGNYYYGMD VWGQGTTVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKRV EPKSCDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GSTGSQVQLV QSGAEVKKPG SSVKVSCKAS GYTFSDYVIN WVRQAPGQGL EWMGEIYPGS GTNYYNEKFK AKATITADKS TSTAYMELSS LRSEDTAVYY CARRGRYGLY AMDYWGQGTT VTVSSRTVAA PSVFIFPPSD EQLKSGTASV VCLLNNFYPR EAKVQWKVDN ALQSGNSQES VTEQDSKDST YSLSSTLTLS KADYEKHKVY Third Polypeptide Chain (III) (SEQ ID NO: 17) DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP GKAPKLLIYY TSRLHSGVPS RFSGSGTD FTFTISSLQP EDIATYFCQQ GNTRPWTFGG GTKVEIKAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHSGSSSS GSSSSGSSSS TKKTQLQLEH LLLDLQMILN GINNYKNPKL TAMLTKKFYM PKKATELKHL QCLEEELKPL EEVLNLAQSK NFHLRPRDLI SNINVIVLEL KGSETTFMCE YADETATIVE FLNRWITFAQ SIISTLT

[0145] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:1, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:9, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:17, or variants thereof having at least 95% sequence identity.

[0146] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:1, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:9, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:17, or variants thereof having at least 90% sequence identity.

[0147] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:1, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:9, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:17, or variants thereof having at least 80% sequence identity.

[0148] The binding protein having the first, second and third polypeptide chains described above is the protein format shown in Figure 1 and Figure 2A (CD20-2-T5-NKCE4).

[0149] In another embodiment, C 2A is CH1 and C 2B is C K It is.

[0150] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:1, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:73, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:74.

[0151] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:1, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:73, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:74, or variants thereof having at least 95% sequence identity.

[0152] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:1, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:73, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:74, or variants thereof having at least 90% sequence identity.

[0153] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:1, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:73, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:74, or variants thereof having at least 80% sequence identity.

[0154] A binding protein having the above-mentioned first, second and third polypeptide chains (C 2A is CH1 and C2B is C K ) are arranged in the format shown in FIG. 2G (CD20-2-T25-NKCE4).

[0155] In another embodiment, the binding protein of the present disclosure has residue N297 (according to Kabat numbering) of the Fc domain mutated to prevent it from being glycosylated. In a preferred embodiment, said mutation is an N297S substitution. Advantageously, said mutation substantially eliminates CD16A binding.

[0156] According to some embodiments, C2A is CK and C2B is CH1.

[0157] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:66, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:67, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:17.

[0158] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:66, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:67, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:17, or variants thereof having at least 95% sequence identity.

[0159] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:66, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:67, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:17, or variants thereof having at least 90% sequence identity.

[0160] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:66, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:67, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:17, or variants thereof having at least 80% sequence identity.

[0161] An example of such a protein format is presented in Figure 2B (CD20-2-T6-NKCE4).

[0162] In an alternative embodiment, C 2A is CH1 and C 2B is C K It is.

[0163] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:66, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:75, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:74.

[0164] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:66, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:75, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:74, or variants thereof having at least 95% sequence identity.

[0165] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:66, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:75, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:74, or variants thereof having at least 90% sequence identity.

[0166] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:66, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:75, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:74, or variants thereof having at least 80% sequence identity.

[0167] An example of such a protein format is presented in Figure 2H (CD20-2-T26-NKCE4).

[0168] In another embodiment, the binding protein has an Fc domain that includes a L234A, L235E, G237A, A330S and / or P331S substitution according to the Kabat numbering.

[0169] According to some embodiments, C2A is CK and C2B is CH1.

[0170] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:68, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:69, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:17.

[0171] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:68, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:69, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:17, or variants thereof having at least 95% sequence identity.

[0172] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:68, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:69, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:17, or variants thereof having at least 90% sequence identity.

[0173] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:68, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:69, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:17, or variants thereof having at least 80% sequence identity.

[0174] An example of such a protein format is presented in FIG. 2C (CD20-2-T6B3-NKCE4).

[0175] In an alternative embodiment, C2A is CH1 and C2B is CK.

[0176] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:68, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:76, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:74.

[0177] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 68 or a variant thereof having at least 95% sequence identity, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 76 or a variant thereof having at least 95% sequence identity, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 74 or a variant thereof having at least 95% sequence identity.

[0178] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 68 or a variant thereof having at least 90% sequence identity, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 76 or a variant thereof having at least 90% sequence identity, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 74 or a variant thereof having at least 90% sequence identity, or a variant thereof having at least 90% sequence identity.

[0179] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 68 or a variant thereof having at least 80% sequence identity, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 76 or a variant thereof having at least 80% sequence identity, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 74 or a variant thereof having at least 80% sequence identity.

[0180] An example of such a protein format is presented in Figure 2I (CD20-2-T26B3-NKCE4).

[0181] In other embodiments, a binding protein of the disclosure comprises an ABD that binds CD20 which is a VH / VL pair and an ABD that binds NKp46 which is a Fab.

[0182] According to some embodiments, C2A is CK and C2B is CH1.

[0183] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:77, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:79, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:17.

[0184] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 77 or a variant thereof having at least 90% sequence identity, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 79 or a variant thereof having at least 95% sequence identity, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17 or a variant thereof having at least 95% sequence identity.

[0185] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 77 or a variant thereof having at least 90% sequence identity, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 79 or a variant thereof having at least 90% sequence identity, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17 or a variant thereof having at least 90% sequence identity.

[0186] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 77 or a variant thereof having at least 80% sequence identity, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 79 or a variant thereof having at least 80% sequence identity, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17 or a variant thereof having at least 80% sequence identity, or a variant thereof having at least 80% sequence identity.

[0187] An example of such a protein format is presented in Figure 2K (CD20-2-T195-NKCE4).

[0188] In an alternative embodiment, C 2A is CH1 and C 2B is C K It is.

[0189] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:77, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:78, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:74.

[0190] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 77, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 78, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 74, or variants thereof having at least 95% sequence identity.

[0191] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 77 or a variant thereof having at least 90% sequence identity, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 78 or a variant thereof having at least 90% sequence identity, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 74 or a variant thereof having at least 90% sequence identity.

[0192] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 77 or a variant thereof having at least 80% sequence identity, a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 78 or a variant thereof having at least 80% sequence identity, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 74 or a variant thereof having at least 80% sequence identity.

[0193] An example of such a protein format is presented in Figure 2J (CD20-2-T175-NKCE4).

[0194] In another embodiment, the binding protein of the present disclosure is a heterodimer and comprises two polypeptide chains (I) and (II) forming two ABDs, as defined above: V 1A -C 1A - Hinge 1 - (Fc domain) A (I) V 1B -C 1B - Hinge 2 - (Fc domain) B -L1-V 2A -L2-V 2B -L3-IL-2(II) Including, V 1A and V 1B is the bond pair V1(V H1 / V L1 ) to form; V 2A and V2B forms an scFv; C 1A and C 1B is pair C1 (CH1 / C L ) and C 2A and C 2B The pair C2 (CH1 / C L ), CH1 is immunoglobulin heavy chain constant domain 1, and C L is an immunoglobulin light chain constant domain; Hinge 1, Hinge 2 and Hinge 3 are the same or different and correspond to all or part of an immunoglobulin hinge region; (Fc domain) A and (Fc domain) B are identical or different and comprise a CH2-CH3 domain; L1 and L2 are amino acid linkers, where L1 and L2 can be different or the same; IL-2 is a mutated human interleukin-2 polypeptide or a portion thereof that binds to CD122 present on NK cells. In one embodiment, binding pair V1 binds to CD20 and binding pair V2 binds to NKp46.

[0195] In some embodiments, the binding protein comprises a first polypeptide chain (I) (disclosed herein below) comprising the amino acid sequence of SEQ ID NO:1, and a second polypeptide chain (II) (disclosed herein below) comprising the amino acid sequence of SEQ ID NO:70. First Polypeptide Chain (I) (SEQ ID NO:1) EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPITFGQ GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGECDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD ​​IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K Second Polypeptide Chain (II) (SEQ ID NO:70) EVQLVESGGG LVQPDRSLRL SCAASGFTFH DYAMHWVRQA PGKGLEWVST ISWNSGTIGY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TALYYCAKDI QYGNYYYGMD VWGQGTTVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKRV EPKSCDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GSTGSQVQLV QSGAEVKKPG SSVKVSCKAS GYTFSDYVIN WVRQAPGQGL EWMGEIYPGS GTNYYNEKFK AKATITADKS TSTAYMELSS LRSEDTAVYY CARRGRYGLY AMDYWGQGTT VTVSSVEGGS GGSGGSGGSG GVDDIQMTQS PSSLSASVGD RVTITCRASQ DISNYLNWYQ QKPGKAPKLL IYYTSRLHSG VPSRFSGSGS GTDFTFTISS LQPEDIATYF CQQGNTRPWT FGGGTKVEIK GSSSSGSSSS GSSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTAMLT KKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFAQSIIST LT

[0196] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:1, or a variant thereof having at least 95% sequence identity, and a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:70, or a variant thereof having at least 95% sequence identity.

[0197] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:1, or a variant thereof having at least 90% sequence identity, and a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:70, or a variant thereof having at least 90% sequence identity.

[0198] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:1, or a variant thereof having at least 80% sequence identity, and a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:70, or a variant thereof having at least 80% sequence identity.

[0199] An example of such a protein format is presented in Figure 2D (CD20-2-T13-NKCE4).

[0200] In another embodiment, the binding protein of the present disclosure has residue N297 (according to Kabat numbering) of the Fc domain mutated to prevent it from being glycosylated. In a preferred embodiment, said mutation is an N297S substitution. Advantageously, said mutation substantially eliminates CD16A binding.

[0201] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:66, and a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:71.

[0202] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 66, or a variant thereof having at least 95% sequence identity, and a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof having at least 95% sequence identity.

[0203] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof having at least 90% sequence identity, and a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof having at least 90% sequence identity.

[0204] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 66, or a variant thereof having at least 80% sequence identity, and a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof having at least 80% sequence identity.

[0205] An example of such a protein format is presented in FIG. 2E (CD20-2-T14-NKCE4).

[0206] In another embodiment, the binding protein has an Fc domain that includes a L234A, L235E, G237A, A330S and / or P331S substitution according to the Kabat numbering.

[0207] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO:68, and a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO:72.

[0208] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 68, or a variant thereof having at least 95% sequence identity, and a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 72, or a variant thereof having at least 95% sequence identity.

[0209] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 68, or a variant thereof having at least 90% sequence identity, and a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 72, or a variant thereof having at least 90% sequence identity.

[0210] In some embodiments, the binding protein comprises a first polypeptide chain (I) comprising the amino acid sequence of SEQ ID NO: 68, or a variant thereof having at least 80% sequence identity, and a second polypeptide chain (II) comprising the amino acid sequence of SEQ ID NO: 72, or a variant thereof having at least 80% sequence identity.

[0211] An example of such a protein format is presented in Figure 2F (CD20-2-T14B3-NKCE4).

[0212] CD20 ABD CD20 is a cell surface protein present on most B-cell neoplasms and not present on otherwise similar-looking T-cell neoplasms.CD20-positive cells may also be found in cases of Hodgkin's disease, myeloma, and thymoma.CD20 is the target of monoclonal antibody (mAb) rituximab, ofatumumab, ocrelizumab, genmab, obinutuzumab, ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, and tositumomab, all of which are active agents in the treatment of all B-cell lymphomas and leukemias.

[0213] In one embodiment, the ABD that binds to a CD20 polypeptide of a binding protein of the disclosure comprises a VH and VL pair provided in Table 2 below:

[0214] [Table 2]

[0215] In one embodiment, the ABD that binds to a CD20 polypeptide of a binding protein of the disclosure comprises a VH comprising three CDRs (HCDR1, HCDR2 and HCDR3) and a VL comprising three CDRs (LCDR1, LCDR2 and LCDR3).

[0216] In another aspect of any embodiment herein, any of the heavy and light chain CDR1, CDR2 and CDR3 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 a particular CDR or set of CDRs listed in the following corresponding SEQ ID NOs or tables, which summarize the sequences of the CDRs according to the IMGT, Kabat and Chothia definition system:

[0217] [Table 3]

[0218] In some embodiments, the first ABD of the binding protein specifically binds to a human CD20 polypeptide, and - VH1 comprising CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 29 (HCDR1), SEQ ID NO: 32 (HCDR2), SEQ ID NO: 35 (HCDR3), and - VL1 comprising CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 38 (LCDR1), SEQ ID NO: 41 (LCDR2), SEQ ID NO: 44 (LCDR3) Includes.

[0219] NKp46 As discussed herein, the binding proteins of the disclosure comprise an ABD that binds to a human NKp46 polypeptide (and ABD).

[0220] In one embodiment, the second ABD of the binding protein comprises a VH comprising CDRs 1, 2 and 3 of the amino acid sequences of SEQ ID NO: 47 (HCDR1), SEQ ID NO: 50 (HCDR2), SEQ ID NO: 53 (HCDR3), optionally with one, two, three or more amino acids in the CDRs replaced by different amino acids; and a VL comprising CDRs 1, 2 and 3 of the amino acid sequences of SEQ ID NO: 56 (LCDR1), SEQ ID NO: 59 (LCDR2), SEQ ID NO: 62 (LCDR3), optionally with one, two, three or more amino acids in the CDRs replaced by different amino acids.

[0221] Thus, the second ABD of the binding protein of the present disclosure can bind to a region spanning the D1 and D2 domains (at the boundary of the D1 and D2 domains, the D1 / D2 junction) of the NKp46 polypeptide of SEQ ID NO: 1. In some embodiments, the VH / VL pair of the second ABD of the binding protein is 10 -8 Less than M, 10 -9 Less than M or 10 -10 K under M D In some embodiments, the multispecific protein has an affinity (KD) for human NKp46, as determined by SPR, of 1-100 nM, optionally 1-50 nM, optionally 1-20 nM, optionally about 10 or 15 nM.

[0222] In one embodiment, the multispecific protein (or its NKp46-binding ABD or VH / VL pair, e.g., when configured in a multispecific protein or as a conventional full-length antibody) binds to NKp46 at substantially the same region, site or epitope on NKp46 as antibody NKp46-1. In one embodiment, all key residues of the epitope are in the segment corresponding to domains D1 or D2. In one embodiment, the antibody or multispecific protein binds to residues present in the D2 domain in addition to those present in the D1 domain. In one embodiment, the antibody binds to an epitope comprising 1, 2, 3, 4, 5, 6, 7 or more residues in the segment corresponding to the D1 / D2 junction of the NKp46 polypeptide of SEQ ID NO:88. In one embodiment, the antibody or multispecific protein binds to NKp46 at the D1 / D2 domain junction and binds to an epitope comprising or consisting of one, two, three, four or five of residues K41, E42, E119, Y121 and / or Y194.

[0223] The amino acid sequences of the heavy and light chain variable regions of NKp46-1 are presented in Table 4 below.

[0224] [Table 4]

[0225] The NKp46-binding multispecific protein binds to essentially the same epitope or determinant as the monoclonal antibody NKp46-1, and optionally the antibody comprises the hypervariable region of the antibody NKp46-1. In any embodiment herein, the antibody NKp46-1 may be characterized by its amino acid sequence and / or the nucleic acid sequence encoding it. In one embodiment, the antibody comprises the Fab or F(ab')2 portion of NKp46-1.

[0226] In one embodiment, the NKp46-binding ABD comprises a humanized VH / VL of antibody NKp46-1. Based on 3D modeling studies, different heavy and light chain variable regions containing NKp46-1 CDRs and human frameworks, produced as human IgG1 antibodies, were designed and tested for binding to cynomolgus NKp46. Two combinations of heavy and light chains were able to bind to both human and cynomolgus NKp46: heavy chain variable region "H1" and heavy chain "H3", in each case combined with light chain "L1". These cross-linking variable regions included the NKp46-1 heavy chain CDRs (underlined below), the human IGHV1-69*06 gene framework 1, 2 and 3 regions and the human IGHJ6*01 gene framework 4 region for the heavy chain variable region; the NKp46-1 light chain CDRs (underlined below), the human IGKV1-33*01 gene framework 1, 2 and 3 regions and the human IGKJ4*01 gene framework 4 region for the light chain variable region. The CDRs were selected according to Kabat numbering. The H1, H3 and L1 chains had unique amino acid substitutions (bold and underlined below). 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. H3 additionally had a glycine at Kabat residue 37, an isoleucine at Kabat residue 48, and a phenylalanine at Kabat residue 91.

[0227] [Table 5]

[0228] According to one embodiment, the antibody comprises the three CDRs of the heavy chain variable region of NKp46-1, or a humanized version thereof (NKp46-1H1 or NKp46-1H3). Also provided is a polypeptide further comprising one, two or three of the CDRs of the light chain variable region of NKp46-1, or a humanized version thereof (NKp46-1L1 or NKp46-1L1). Optionally, any one or more of the light or heavy chain CDRs may contain one, two, three, four or five or more amino acid modifications (e.g., substitutions, insertions or deletions).

[0229] The multispecific protein or NKp46-binding ABD can be, for example: (a) the heavy chain variable region of NKp46-1 (SEQ ID NO: 16), in which one, two, three or more amino acids may be optionally substituted with different amino acids; (b) the light chain variable region NKp46-1 (SEQ ID NO: 18), in which one, two, three or more amino acids may be optionally substituted with different amino acids; or (a) the heavy chain variable region of NKp46-1H1 (SEQ ID NO:93), in which one, two, three or more amino acids may be optionally substituted with different amino acids; (b) the light chain variable region of NKp46-1L1 (SEQ ID NO:95), in which one, two, three or more amino acids may be optionally substituted with different amino acids; or (a) the heavy chain variable region of NKp46-1H3 (SEQ ID NO:94), optionally in which one, two, three or more amino acids are substituted with different amino acids; (b) the light chain variable region of NKp46-1L1 (SEQ ID NO: 95), in which one, two, three or more amino acids may be optionally substituted with different amino acids. may include.

[0230] In some embodiments, the multispecific protein or NKp46 binding ABD comprises: (a) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequence of NKp46-1 as shown in the table below, where appropriate one, two, three or more amino acids in the CDRs may be replaced by different amino acids; (b) The light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences of NKp46-1 as shown in the table below, where appropriate one, two, three or more amino acids in the CDRs may be replaced by different amino acids. may include.

[0231] In one embodiment, the above-mentioned CDRs are according to Kabat numbering, e.g., as shown in the table below. In one embodiment, the above-mentioned CDRs are according to Chothia numbering, e.g., as shown in the table below. In one embodiment, the above-mentioned CDRs are according to IMGT numbering, e.g., as shown in the table below.

[0232] In another aspect of any embodiment herein, any of the heavy and light chain CDR1, CDR2 and CDR3 may be characterized by its sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids and / or as having an amino acid sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with a particular CDR or set of CDRs listed in the corresponding SEQ ID NO or table below.

[0233] The sequences of the CDRs according to the IMGT, Kabat and Chothia definition systems are summarized in Table 6 below.

[0234] [Table 6]

[0235] IL2 part In some embodiments, the cytokine portion of the binding protein of the disclosure is a mutant interleukin-2 polypeptide.

[0236] The cytokine portion can be a fragment comprising at least 20, 30, 40, 50, 60, 70, 80, or 100 consecutive amino acids of a human interleukin-2 polypeptide. In certain embodiments, the IL-2 polypeptide is a variant of a human cytokine that comprises one or more amino acid modifications (e.g., amino acid substitutions) compared to wild-type IL-2, e.g., to decrease binding affinity to receptors present on non-NK cells, e.g., Treg cells, CD4 T cells, CD8 T cells.

[0237] Optionally, signaling is assessed by contacting IL-2 (e.g., as a recombinant protein domain or within a multispecific protein of the disclosure) with NK cells and measuring signaling, e.g., measuring STAT phosphorylation in NK cells.

[0238] In one embodiment, the IL-2 or CD122 specific ABD binds to its receptor with a binding affinity (KD) of about 1 nm to about 200 nm, optionally about 1 nm to about 100 nm, optionally about 10 nM to about 200 nM, optionally about 10 nM to about 100 nM, optionally about 15 nM to about 100 nM, as determined by SPR.

[0239] The CD122-binding ABD is advantageously a mutant or modified IL-2 polypeptide having reduced binding (e.g., reduced or eliminated binding affinity, e.g., as determined by SPR) to CD25 (IL-2Rα) compared to wild-type human interleukin-2. Such mutant or modified IL-2 polypeptides are also referred to herein as "IL2v" or "non-alpha IL-2". The CD122-binding ABD may be optionally identified as having a binding affinity to human CD122 that is substantially equivalent to that of wild-type human IL-2. The CD122-binding ABD may be optionally identified as having a binding affinity to CD122 and / or an ability to induce CD122 signaling that is substantially equivalent to that of wild-type human IL-2. In one embodiment, the CD122-binding ABD has a reduction in binding affinity to CD25 that is greater than the reduction in binding affinity to CD122, for example at least a 1-log, 2-log or 3-log reduction in binding affinity to CD25 and less than a 1-log reduction in binding affinity to CD122.

[0240] It is believed that IL-2 binds to IL-2Rβ (CD122) in the form of a monomeric IL-2 receptor (IL-2R) and subsequently recruits IL-2Rγ (CD132; also referred to as common γ chain) subunits. In cells that do not express CD25 on their surface, the binding to CD122 (e.g., reduced binding) can therefore be appropriately specified as being in or to the CD122:CD132 complex. CD122 (or CD122:CD132 complex) can be appropriately specified as being present on the surface of NK cells. In cells that express CD25 on their surface, it is believed that IL-2 binds to CD25 (IL-2Rα) in the form of a monomeric IL-2 receptor and subsequently associates with the subunits IL-2Rβ and IL-2Rγ. Binding to CD25 (eg, reduced binding, partially reduced binding) may thus be specified as being binding at or to the CD25:CD122 complex or the CD25:CD122:CD132 complex, as appropriate.

[0241] In the multispecific proteins herein, the multispecific protein may optionally be specified as being configured and / or in a conformation (or capable of assuming a conformation) such that, when the multispecific protein is bound to NKp46 (and optionally further to CD16) at the surface of a cell (e.g., an NK cell, a CD122+CD25- cell), the CD122 ABD (e.g., IL2v) is capable of binding to CD122 at the surface of said cell. Optionally, the multispecific protein:CD122 complex is further capable of binding to CD132 at the surface of said cell.

[0242] The CD122 ABD or IL2v can be a modified IL-2 polypeptide, for example a monomeric IL-2 polypeptide modified by introducing one or more amino acid substitutions, insertions or deletions that reduce binding to CD25.

[0243] In some embodiments, when selectively reduced binding to CD25 is desired, the IL-2 polypeptide may be modified by conjugating or associating it with one or more other additional molecules, such as polymers or (poly)peptides, which result in further reduced or eliminated binding to CD25. For example, wild-type or mutant IL-2 polypeptides may be modified or further modified by conjugating another moiety to the IL-2 polypeptide that shields, masks, binds to, or interacts with the CD25-binding site of human IL-2, thereby reducing binding to CD25. In some examples, a molecule, such as a polymer (e.g., a PEG polymer), is conjugated to the IL-2 polypeptide to shield or mask the epitope on IL-2 that is bound by CD25, for example by introduction (e.g., substitution) to place an amino acid containing a specialized chemical hook at a unique site on the IL-2 polypeptide. In another example, a wild-type or mutant IL-2 polypeptide is conjugated to an anti-IL-2 monoclonal antibody or antibody fragment that binds to or interacts with the CD25 binding site of human IL-2, thereby reducing binding to CD25.

[0244] In any embodiment, the IL2 polypeptide may be a full-length IL-2 polypeptide or may be an IL-2 polypeptide fragment, so long as the fragment or IL2v comprising it retains the specified activity (e.g., retains at least partial CD122 binding compared to a wild-type IL-2 polypeptide).

[0245] As provided herein, an IL2v polypeptide may advantageously comprise an IL-2 polypeptide containing one or more amino acid mutations designed to reduce its ability to bind to human CD25 (IL-2Rα) while retaining at least at least a partial, or optionally substantially complete, ability to bind to human CD122.

[0246] Various IL2v or non-alpha IL-2 moieties have been described that reduce the activation bias of IL-2 on CD25+ cells. Such IL2v have reduced binding to IL-2Rα and maintain at least partial binding to IL-2Rβ. Several IL2v polypeptides have been described, many with mutations in the amino acid residue regions 35-72 and / or 79-92 of the IL-2 polypeptide. For example, reduced affinity for IL-2Rα may be obtained by substituting one or more of the following residues in the sequence of a wild-type IL-2 polypeptide: R38, F42, K43, Y45, E62, P65, E68, V69, and L72 (amino acid residue numbering refers to the mature IL-2 polypeptide shown in SEQ ID NO:27). Wild-type mature human IL-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 27) "IL-2p" wild type mature IL-2 with appropriate deletion of the three N-terminal residues APA: SSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:28)

[0247] An exemplary IL2v (also referred to in the Examples herein as IL2v) can have the amino acid sequence of wild type IL-2 with five amino acid substitutions T3A, F42A, Y45A, L72G and C125A, as shown below, optionally with a deletion of the three further N-terminal residues APA: APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 24)

[0248] As few as one or two mutations can reduce binding to IL-2Rα and IL-2Rβ. For example, as exemplified in the multispecific protein herein, an IL2v polypeptide having two amino acid substitutions R38A and F42K in the wild-type human IL-2 amino acid sequence exhibited suitably reduced binding to IL-2Rα while retaining binding to IL-2Rβ, resulting in a highly active multispecific protein referred to herein as IL2v2. IL2v2 (R38A / F42K replacement): [ka]

[0249] In one embodiment, the IL2v2 polypeptide can further include the substitution C125A (with respect to the wild-type mature human IL-2 of SEQ ID NO:27), which is referred to herein as IL2v2A. IL2V2A (R38A / F42K / C125A replacement): [ka]

[0250] In one embodiment, the IL2v polypeptide has a wild-type IL-2p amino acid sequence with three amino acid substitutions R38A, F42K and T41A (with respect to the wild-type mature human IL-2 of SEQ ID NO:27), as shown below, which is referred to herein as IL2v3: IL2v3 (R38A / T41A / F42K replacement): [ka]

[0251] Thus, in one embodiment, the IL2 variant comprises at least one or at least two amino acid modifications (e.g., substitutions, insertions, deletions) compared to the human wild-type IL-2 polypeptide. In one embodiment, the IL2v comprises an R38 substitution (e.g., R38A) and an F42 substitution (e.g., F42K) compared to the human wild-type IL-2 polypeptide. In one embodiment, the IL2v comprises an R38 substitution (e.g., R38A), an F42 substitution (e.g., F42K) and a T41 substitution (e.g., T41A) compared to the human wild-type IL-2 polypeptide. In one embodiment, the IL2v comprises a T3 substitution (e.g., T3A), an F42 substitution (e.g., F42A), a Y45 substitution (e.g., Y45A), an L72 substitution (e.g., L72G) and a C125 substitution (e.g., C125A) compared to the human wild-type IL-2 polypeptide. Optionally, IL2v comprises an amino acid sequence identical to, or at least 70%, 80%, 90%, 95%, 98% or 99% identical to, the polypeptide of SEQ ID NO: 24-26 or 65. Optionally, IL2v comprises a fragment of a human IL-2 polypeptide, the fragment having an amino sequence identical to, or at least 70%, 80%, 90%, 95%, 98% or 99% identical to, a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 24-26 or 65.

[0252] Any combination of positions may be modified. In some embodiments, the IL-2 variant contains two or more modifications. In some embodiments, the IL-2 variant contains three or more modifications. In some embodiments, the IL-2 variant contains four, five, or six or more modifications.

[0253] IL2 variant polypeptides can contain, for example, 2, 3, 4, 5, 6 or 7 amino acid modifications (e.g., substitutions). For example, U.S. Patent No. 5,229,109 (the disclosure of which is incorporated herein by reference) provides a human IL2 polypeptide with R38A and F42K substitutions. U.S. Patent No. 9,447,159 (the disclosure of which is incorporated herein by reference) describes a human IL2 polypeptide with T3A, F42A, Y45A, and L72G substitutions. U.S. Pat. No. 9,266,938 (the disclosure of which is incorporated herein by reference) describes human IL2 polypeptides having substitutions at residue L72 (e.g., L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K), residue F42 (e.g., F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, and F42K); and residue Y45 (e.g., Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K), including, for example, the triple mutation F42A / Y45A / L72G to reduce or eliminate affinity for the IL-2Rα receptor. Still further, WO 2020 / 057646 (the disclosure of which is incorporated herein by reference) relates to amino acid sequences of IL-2v polypeptides that include amino acid substitutions in various combinations among amino acid residues K35, T37, R38, F42, Y45, E61 and E68.Still further, WO2020252418 (the disclosure of which is incorporated herein by reference) relates to amino acid sequences of IL-2v polypeptides in which at least one amino acid residue position R38, T41, F42, F44, E62, P65, E68, Y107, or C125 is substituted with another amino acid, e.g., the amino acid substitutions include substitutions of L19D, L19H, L19N, L19P, L19Q, L19R, L19S, L19Y at position 19, substitutions of R38A, R38F, R38G at position 38, substitutions of T41A, T41G, and at position 42; F44G and F44V at position 44; E62A, E62F, E62H and E62L at position 62; P65A, P65E, P65G, P65H, P65K, P65N, P65Q, P65R at position 65; E68E, E68F, E68H, E68L and E68P at position 68; Y107G, Y107H, Y107L and Y107V at position 107; and C125I at position 125 and Q126E at position 126. The numbering of the positions is relative to wild type mature human IL-2.

[0254] The modified IL-2 may have a lower binding affinity to its receptor, and optionally the modified IL-2 may be identified as exhibiting a KD for binding to CD25 or to the CD25:CD122:CD132 complex that is within 1-log, optionally 2-log, optionally 3-log of the KD of a wild-type human IL-2 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 27). The modified IL-2 may be identified as exhibiting less than 20%, 30%, 40% or 50% of the binding affinity to CD25 or the CD25:CD122:CD132 complex as compared to the wild-type human IL-2 polypeptide. The IL2 may be identified as exhibiting at least 50%, 70%, 80% or 90% of the binding affinity to CD122 or the CD122:CD132 complex as compared to the wild-type human IL-2 polypeptide. In some embodiments, IL2 exhibits at least 50%, 60%, 70% or 80% but less than 100% binding affinity to CD122 or the CD122:CD132 complex compared to a wild-type human IL-2 polypeptide. In some embodiments, IL2v exhibits less than 50% binding affinity to CD25 and at least 50%, 60%, 70% or 80% binding affinity to CD122 compared to a wild-type IL-2 polypeptide.

[0255] The difference in binding affinity of wild-type and disclosed mutant polypeptides to CD25 and CD122 and complexes thereof can be measured, for example, in standard surface plasmon resonance (SPR) assays that measure the affinity of protein-protein interactions, with which one of skill in the art is familiar.

[0256] Exemplary IL2 variant polypeptides have one or more, two or more, or three or more CD25 affinity-reducing amino acid substitutions relative to a wild-type mature IL-2 polypeptide having the amino acid sequence of SEQ ID NO: 27. In one embodiment, an exemplary IL2v polypeptide comprises one or more, two or more, or three or more substituted residues selected from the following group: Q11, H16, L18, L19, D20, D84, S87, Q22, R38, T41, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, I92, T123, Q126, S127, I129, and S130.

[0257] In one embodiment, an exemplary IL2 mutant polypeptide has one, two, three, four, five or more of the following amino acid residue positions substituted with another amino acid: R38, T41, F42, F44, E62, P65, E68, Y107, or C125.

[0258] In one embodiment, reduced affinity for CD25 or a protein complex containing it (e.g., the CD25:CD122:CD132 complex) may be obtained by substituting one or more of the following residues in the sequence of the wild-type mature IL-2 polypeptide: R38, F42, K43, Y45, E62, P65, E68, V69, and L72.

[0259] In yet another example, an IL-2 polypeptide is modified by connecting, fusing, binding, or associating it with one or more other additional compounds, chemical compounds, polymers (e.g., PEG), or polypeptides or polypeptide chains that result in reduced binding to CD25. For example, a wild-type IL-2 polypeptide or fragment thereof can be modified by connecting it with a CD25-binding peptide or polypeptide, including, but not limited to, an anti-IL-2 monoclonal antibody or antibody fragment thereof, that binds to or interacts with the CD25 binding site of human IL-2, thereby reducing binding to CD25.

[0260] In other examples, an IL-2 polypeptide or fragment thereof can be modified by attaching thereto a moiety of interest (e.g., a compound, a chemical compound, a polymer, a linear or branched PEG polymer) that is covalently attached to a natural or unnatural amino acid introduced at a selected position. Such modified interleukin-2 (IL-2) polypeptides can include at least one unnatural amino acid at a position on the polypeptide that reduces binding between the modified IL-2 polypeptide and CD25 but retains significant binding to the CD122:CD132 signaling complex, the reduced binding to CD25 compared to the binding between a wild-type IL-2 polypeptide and CD25. The unnatural amino acid may be located at any one or more of residues K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107 of IL-2. As disclosed in PCT Publication Nos. WO 2019 / 028419 and WO 2019 / 014267, the disclosures of which are incorporated herein by reference, the unnatural amino acid may be incorporated into the modified IL-2 polypeptide by an orthogonal tRNA synthetase / tRNA pair. The unnatural amino acid may include, for example, a lysine analog, an aromatic side chain, an azide group, an alkyne group, or an aldehyde or ketone group. The modified IL-2 polypeptides can then be covalently attached to a water soluble polymer, lipid, protein, or peptide through the unnatural amino acid.Examples of suitable polymers include polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamides), poly(hydroxyalkylmethacrylates), poly(saccharides), poly(a-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholines), or combinations thereof, or polysaccharides such as dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES).

[0261] Constant domain The constant region domains may be derived from any suitable human antibody, particularly a human antibody of the gamma isotype, including the constant heavy (CH1) and light chain (CL, Cκ or Cλ) domains, hinge domain, CH2 and CH3 domains.

[0262] With respect to the heavy chain constant domain, "CH1" generally refers to positions 118-220 according to the EU index in Kabat. Depending on the context, the CH1 domain (e.g., as shown in the domain arrangement) can optionally include residues extending into the hinge region such that the CH1 includes at least a portion of the hinge region. For example, when positioned C-terminal on a polypeptide chain and / or at the C-terminus of an Fc domain and / or within a Fab structure that is or is C-terminal to an Fc domain, the CH1 domain can optionally include at least a portion of the hinge region, e.g., the CH1 domain can include at least the upper hinge region, e.g., the upper hinge region of a human IgG1 hinge, and optionally further, the terminal threonine of the upper hinge can be replaced by a serine. Such a CH2 domain can thus include the amino acid sequence: EPKSCDKTHS at its C-terminus. Exemplary human CH1 domain amino acid sequences include: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV (SEQ ID NO: 12) Exemplary human Cκ domain amino acid sequences include: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4)

[0263] In some exemplary configurations, the multispecific protein may be a heterodimer or heterotrimer comprising one or two Fabs (e.g., one Fab binds NKp46 and the other binds CD20) in which the variable regions, CH1 and / or CL domains have been engineered by introducing amino acid substitutions in a knobs-into-hole or electrostatic steering approach to promote the desired chain pairing of the CH1 domain with the CK domain. In some exemplary configurations, the multispecific protein may be a heterodimer or heterotrimer comprising one or two Fabs (e.g., one Fab binds NKp46 and the other binds CD20) in which the Fabs have a VH / VL crossover (VH and VL replace each other) or a CH1 / CL crossover (CH1 and CL replace each other) and the CH1 and / or CL domains contain amino acid substitutions to promote correct chain association by knobs-into-hole or electrostatic steering.

[0264] "CH2" generally refers to positions 237-340 according to the EU index in Kabat, and "CH3" generally refers to positions 341-447 according to the EU index in Kabat. The CH2 and CH3 domains can be derived from any suitable antibody. Such CH2 and CH3 domains can be used as wild-type domains or can serve as the basis for modified CH2 or CH3 domains. Optionally, the CH2 and / or CH3 domains can be of human origin or can include those of another species (e.g., rodent, rabbit, non-human primate), or can include modified or chimeric CH2 and / or CH3 domains, e.g., those that include portions or residues from different CH2 or CH3 domains from antibodies of different antibody isotypes or species. Exemplary human IgG1 CH2 domain amino acid sequences include the following: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 7) Exemplary human IgG1 CH3 domain amino acid sequences include the following: GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8)

[0265] In any of the domain arrangements, the Fc domain monomer 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 monomer (i.e., the heterodimer or heterotrimer comprises an Fc domain dimer), the CH3 domain has the ability of CH3-CH3 dimerization (e.g., it comprises a wild-type CH3 domain or a CH3 domain with a modification to promote the desired CH3-CH3 dimerization). The Fc domain may further comprise a C-terminal lysine (K) as appropriate (see SEQ ID NO: 6). An exemplary human IgG1 CH2-CH3 (Fc) domain amino acid sequence includes the following: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6) or APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 14)

[0266] In some exemplary configurations, the multispecific protein may be a heterodimer, heterotrimer, or heterotetramer, in which the polypeptide chains are engineered for heterodimerization between each other to generate the desired protein. In embodiments in which the desired chain pairing is not driven by CH1-Cκ dimerization or enhanced pairing is desired, the chains may contain constant or Fc domains with amino acid modifications (e.g., substitutions) that favor preferential heterodimerization of two different chains over homodimerization of two identical chains.

[0267] In some embodiments, a "knobs-into-holes" approach is used, in which domain interfaces (e.g., the CH3 domain interface of an antibody Fc region) are mutated so that the antibody preferentially heterodimerizes. These mutations create changes in charge polarity between interfaces (e.g., Fc dimer interfaces), such that co-expression of electrostatically matched chains (e.g., Fc-containing chains) supports favorable attractive interactions, thereby promoting the formation of desired heterodimers (e.g., Fc heterodimers), while unfavorable repulsive charge interactions suppress undesirable heterodimers (e.g., Fc homodimers). See, for example, the mutations and approaches reviewed in Brinkmann and Kontermann, 2017 MAbs, 9(2): 182-212, the disclosure of which is incorporated herein by reference. For example, one heavy chain contains a T366W substitution, and a second heavy chain contains a T366S, L368A, and Y407V substitution. See, e.g., Ridgway et al. (1996) Protein Eng., 9, pp. 617-621; Atwell (1997) J. Mol. Biol., 270, pp. 26-35; and WO 2009 / 089004, the disclosures of which are incorporated herein by reference. In another approach, one heavy chain comprises a F405L substitution and the second heavy chain comprises a K409R substitution. See, e.g., Labrijn et al. (2013) Proc. Natl. Acad. Sci. USA, 110, pp. 5145-5150. In another approach, one heavy chain comprises T350V, L351Y, F405A and Y407V substitutions, and a second heavy chain comprises T350V, T366S, K392L and T394W substitutions.See, for example, Von Kreudenstein et al., (2013) mAbs 5:646-654.In another approach, one heavy chain comprises both K409D and K392D substitutions, and a second heavy chain comprises both D399K and E356K substitutions.See, e.g., Gunasekaran et al., (2010) J. Biol. Chem. 285:19637-19646. In another approach, one heavy chain comprises D221E, P228E and L368E substitutions, and the second heavy chain comprises D221R, P228R and K409R substitutions. See, e.g., Strop et al., (2012) J. Mol. Biol. 420: 204-219. In another approach, one heavy chain comprises S364H and F405A substitutions, and the second heavy chain comprises Y349T and T394F substitutions. See, e.g., Moore et al., (2011) mAbs 3: 546-557. In another approach, one heavy chain comprises an H435R substitution and the second heavy chain may or may not comprise a substitution, as appropriate. See, for example, U.S. Pat. No. 8,586,713. When such heteromultimeric antibodies have Fc regions derived from human IgG2 or IgG4, the Fc regions of these antibodies can be engineered to contain amino acid modifications that allow CD16 binding. In some embodiments, the antibody may comprise a mammalian antibody-type N-linked glycosylation at residue N297 (Kabat EU numbering).

[0268] In some embodiments, one or more pairs of disulfide bonds, such as A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C, are introduced into the Fc region to increase stability, for example, to a loss of stability caused by other Fc modifications. Additional examples include introducing K338I, A339K, and K340S mutations to enhance Fc stability and aggregation resistance (Gao et al, 2019 Mol Pharm. 2019;16:3647).

[0269] In some embodiments, where a multispecific protein is intended to have reduced binding to human Fc gamma receptors, in some embodiments, where a multispecific protein is intended to have reduced binding to human CD16A polypeptides (and optionally further reduced binding to CD32A, CD32B and / or CD64), the Fc domain is a human IgG4 Fc domain, and optionally further, the Fc domain comprises a S228P mutation to stabilize the hinge disulfide.

[0270] In embodiments, where the multispecific protein is intended to have reduced binding to human CD16A polypeptide (and optionally further reduced binding to CD32A, CD32B and / or CD64), the CH2 and / or CH3 domains (or the Fc domains comprising same) may comprise modifications to reduce or eliminate binding to FcγRIIIA (CD16). For example, a CH2 mutation at residue N297 (Kabat numbering) in an Fc domain dimeric protein can substantially eliminate CD16A binding. However, the skilled artisan will appreciate that other configurations may be implemented. For example, substitutions into human IgG1 or IgG2 residues at positions 234-237 and / or into residues at positions 327, 330 and 331 have been shown to greatly reduce binding to Fcγ receptors and therefore ADCC and CDC. Furthermore, Idusogie et al. (2000) J. Immunol. 164(8):4178-84 demonstrated that alanine substitutions at different positions, including K322, significantly reduced complement activation.

[0271] In one embodiment, the asparagine (N) at Kabat heavy chain residue 297 can be substituted with a residue other than asparagine, such as serine.

[0272] In one embodiment, the Fc domain modified to reduce binding to CD16A comprises substitutions in the Fc domain at Kabat residues 234, 235, 237, 330 and 331. In one embodiment, the Fc domain is of the human IgG1 subtype. Amino acid residues are designated according to the EU numbering according to Kabat.

[0273] In one embodiment, the Fc domain modified to reduce binding to CD16A comprises amino acid modifications (e.g., substitutions) at one or more of Kabat residues 233-237, as well as amino acid modifications (e.g., substitutions) at Kabat residues 330 and / or 331. One example of such an Fc domain comprises substitutions at Kabat residues L234, L235, G237, A330 and P331 (e.g., L234A / L235E / G237A / A330S / P331S).

[0274] In one embodiment, the Fc domain with low or reduced binding to CD16A comprises a human IgG1 Fc domain, wherein the CH2-CH3 domain has the following amino acid sequence (human IgG1 with an N297S substitution), or an amino acid sequence at least 90%, 95% or 99% identical thereto. APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 89)

[0275] In one embodiment, the Fc domain that has been modified to reduce binding to CD16A comprises a CH2-CH3 domain having the following amino acid sequence, or an amino acid sequence at least 90%, 95% or 99% identical thereto, but retaining the amino acid residues at Kabat positions 234, 235, 237, 330 and 331 (underlined): [ka]

[0276] Any of the above Fc domain sequences may further include a C-terminal lysine (K), as in the naturally occurring sequence, as appropriate.

[0277] In certain embodiments herein where binding to CD16 (CD16A) is desired, the CH2 and / or CH3 domains (or the Fc domains comprising same) may be wild-type domains or may contain one or more amino acid modifications (e.g., amino acid substitutions) that increase binding to human CD16 and optionally another receptor, such as FcRn. Optionally, the modifications do not substantially reduce or eliminate the ability of the Fc-derived polypeptide to bind to neonatal Fc receptors (FcRn), such as human FcRn. Exemplary modifications include modified human IgG1-derived constant regions that contain at least one amino acid modification (e.g., substitution, deletion, insertion) and / or altered type of glycosylation, e.g., hypofucosylation. Such modifications can affect interactions with the Fc receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRI (CD64), FcγRIIA (CD32A) and FcγRIII (CD 16) are activating (i.e., immune system enhancing) receptors, and FcγRIIB (CD32B) is an inhibitory (i.e., immune system attenuating) receptor. The modification may, for example, increase the binding of the Fc domain to FcγRIIIa on effector (e.g., NK) cells and / or decrease binding to FcγRIIB. Examples of modifications are provided in PCT Publication WO 2014 / 044686, the disclosure of which is incorporated herein by reference. Specific mutations (in the IgG1 Fc domain) that affect (enhance) FcγRIIIa or FcRn binding are also described below.

[0278] [Table 7]

[0279] In some embodiments, the multispecific protein comprises a variant Fc region that comprises at least one amino acid modification (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH2 and / or CH3 domains of the Fc region, which modification enhances binding to a human CD16 polypeptide. In other embodiments, the multispecific protein comprises at least one amino acid modification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH2 domain of the Fc region at amino acids 237-341 or in the lower hinge-CH2 region comprising residues 231-341. In some embodiments, the multispecific protein comprises at least two amino acid modifications (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications), at least one such modification being in the CH3 region and at least one such modification being in the CH2 region. Also included are amino acid modifications in the hinge region. In one embodiment, included are amino acid modifications in the CH1 domain, appropriately in the upper hinge region including residues 216-230 (Kabat EU numbering).Any suitable functional combination of Fc modifications may be used, e.g., those described in U.S. Patent Nos. 7,632,497; 7,521,542; 7,425,619; 7,416,727; 7,371,826; 7,355,008; 7,335,742; 7,332,581; 7,183,387; 7,122,637; 6,821,505 and 6,737,056; and / or PCT Publication Nos. WO 2011 / 109400; WO 2008 / 105886; WO 2008 / 0029 33;WO 2007 / 021841;WO 2007 / 106707;WO 06 / 088494;WO 05 / 115452;WO 05 / 110474;WO 04 / 1032269;WO 00 / 42072;WO 06 / 088494;WO 07 / 024249;WO 05 / 047327;WO 04 / 099249 and WO 04 / 063351; and / or Lazar Any combination of the different Fc modifications disclosed in any of: Presta, LG et al. (2006) Proc. Nat. Acad. Sci. USA 103(11): 405-410; Presta, LG et al. (2002) Biochem. Soc. Trans. 30(4):487-490; Shields, RL et al. (2002) J. Biol. Chem. 26; 277(30):26733-26740 and Shields, RL et al. (2001) J. Biol. Chem. 276(9):6591-6604 may be made.

[0280] In some embodiments, the multispecific protein comprises an Fc domain that comprises at least one amino acid modification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to a wild-type Fc region, such that the molecule has enhanced binding affinity for human CD16 relative to the same molecule comprising a wild-type Fc region, and optionally the variant Fc region comprises any of the following: 221, 239, 243, 247, 255, 256, 258, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, and / or 439 (Kabat EU numbering).

[0281] In one embodiment, the multispecific protein comprises an Fc domain comprising at least one amino acid modification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to a wild-type Fc region, such that the molecule has enhanced binding affinity for human CD16 relative to a molecule comprising a wild-type Fc region, and optionally the variant Fc region comprises a substitution at any one or more of positions 239, 298, 330, 332, 333 and / or 334 (e.g., an S239D, S298A, A330L, I332E, E333A and / or K334A substitution), and optionally the variant Fc region comprises a substitution at residues S239 and I332, such as an S239D and I332E substitution (Kabat EU numbering).

[0282] In some embodiments, the multispecific protein comprises an Fc domain comprising an N-linked glycosylation at Kabat residue N297. In some embodiments, the multispecific protein comprises an Fc domain comprising an altered glycosylation pattern that increases binding affinity to human CD16. Such carbohydrate modification can be achieved, for example, by expressing a nucleic acid encoding the multispecific protein in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery are known in the art and can be used as host cells to express recombinant antibodies, thereby producing antibodies with altered glycosylation. See, for example, Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as EP 1176195; PCT Publication WO 06 / 133148; WO 03 / 035835; WO 99 / 54342, each of which is incorporated herein by reference in its entirety. In one embodiment, the multispecific protein contains one or more hypofucosylated constant regions. Such multispecific proteins may or may not contain amino acid alterations and / or may be expressed or synthesized or processed under conditions that result in hypofucosylation. In one aspect, the multispecific protein composition comprises a multispecific protein as described herein, in which at least 20, 30, 40, 50, 60, 75, 85, 90, 95% or substantially all of the antibody species in the composition have a constant region that comprises a core carbohydrate structure lacking fucose (e.g., complex, hybrid and high mannose structures). In one embodiment, provided is a multispecific protein composition that does not contain an N-linked glycan that comprises a core carbohydrate structure with fucose. The core carbohydrate is preferably a glycan at Asn297.

[0283] Optionally, a multispecific protein comprising an Fc domain dimer may be characterized by having a binding affinity for a human CD16A polypeptide within 1-log of that of a conventional human IgG1 antibody, as assessed, for example, by surface plasmon resonance.

[0284] In one embodiment, a multispecific protein comprising an Fc domain dimer in which the Fc domain has been engineered to enhance Fc receptor binding may be characterized by having a binding affinity for a human CD16A polypeptide that is at least 1-log higher than that of a conventional or wild-type human IgG1 antibody, as assessed, for example, by surface plasmon resonance.

[0285] In one embodiment, a multispecific protein comprising an Fc domain dimer may be characterized by having a binding affinity for a human FcRn (neonatal Fc receptor) polypeptide that is within 1-log of that of a conventional human IgG1 antibody, as assessed, for example, by surface plasmon resonance.

[0286] Optionally, the multispecific protein comprising an Fc domain dimer exhibits a 10 fold increase in binding affinity when assessed by surface plasmon resonance (e.g., as in the Examples herein, SPR measurements performed on a Biacore T100 instrument (Biacore GE Healthcare) using a bispecific antibody immobilized on a Sensor Chip CM5 and serial dilutions of a soluble CD16 polypeptide injected over the immobilized bispecific antibody). -5 M (10 μmolar concentration) or less, 10 -6 The antibody may be characterized by a Kd for binding (monovalent) to a human Fc receptor polypeptide (e.g., CD16A) of less than M (1 μmolar).

[0287] Connections and Linkers In general, there are many suitable linkers that can be used in multispecific proteins, including traditional peptide bonds, produced by recombinant technology. In some embodiments, the linker is a "domain linker", which is used to link together any two domains outlined herein. Adjacent protein domains can be identified as being connected or fused to each other by a domain linker. An exemplary domain linker is a (poly)peptide linker, optionally a flexible (poly)peptide linker. A peptide linker or polypeptide linker, used interchangeably herein, may have a subsequence derived from a particular domain, such as the hinge, CH1 or CL domain, or may primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should be of sufficient length to link two molecules in such a way that they adopt the correct conformation relative to each other so as to retain the desired activity. In one embodiment, the linker is about 1-50 amino acids in length, preferably about 2-30 amino acids in length. In one embodiment, linkers of 4 to 20 amino acids in length may be used, with about 5 to about 15 amino acids having use in some embodiments. While any suitable linker may be used, in many embodiments the linker (e.g., a flexible linker) will be, for example, (GS) n , (GSGGS) n , (GGGGS) n , (GSSS) n , (GSSSS) n and (GGGS) nGlycine-serine polypeptides or polymers, glycine-alanine polypeptides, alanine-serine polypeptides, and other flexible linkers, including n = 1, n = 2, n = 3, n = 4, n = 5, n = 6, n = 7, n = 8, n = 9, n = 10, n = 11, n = 12, n = 13, n = 14, n = 15, n = 16, n = 17, n = 18, n = 19, n = 20, n = 21, n = 22, n = 23, n = 24, n = 25, n = 26, n = 27, n = 28, n = 29, n = 30, n = 31, n = 32, n = 33, n = 34, n = 35, n = 36, n = 37, n = 38, n = 39, n = 39, n = 32, n = 35, n = 36, n = 37, n = 38, n = 39 ... n , for example, (G2S), (G2S)2, (G2S)3, (G2S)4, (G2S)5, (G2S)6, (G2S)7 or (G2S)8, or, for example, (G3S), where n is an integer from 1 to 15. n In one embodiment, a peptide linker is used that comprises the domain linker (G4S) n In some embodiments, the (GS2) peptide is an integer from 1 to 10, optionally from 1 to 6, optionally from 1 to 4. n , (GS3) n or (GS4) n For example, n=1 to 20, e.g., (GS2), (GS2)2, (GS2)3, (GS3)1, (GS3)2, (GS3)3, (GS4)1, (GS4)2, (GS4)3, e.g., n is an integer from 1 to 15. In one embodiment, the domain linker is (GS4) n peptide, for example, n is an integer from 1 to 10, optionally 1 to 6, optionally 1 to 4. In one embodiment, the domain linker comprises a C-terminal GS dipeptide, for example the linker comprises (GS4) and has the amino acid sequence GSSSS (SEQ ID NO: 20), GSSSSGSSSS (SEQ ID NO: 21), GSSSSGSSSSGS (SEQ ID NO: 22) or GSSSSGSSSSGSSSS (SEQ ID NO: 23).

[0288] Any of the peptide or domain linkers may be specified to contain at least 4 residues, at least 5 residues, at least 10 residues, at least 15 residues, at least 20 residues, or more residues in length, in other embodiments, the linker contains 2-4 residues, 2-4 residues, 2-6 residues, 2-8 residues, 2-10 residues, 2-12 residues, 2-14 residues, 2-16 residues, 2-18 residues, 2-20 residues, 2-22 residues, 2-24 residues, 2-26 residues, 2-28 residues, 2-30 residues, 2-50 residues, or 10-50 residues in length.

[0289] Exemplary polypeptide linkers may include sequence fragments from the CH1 or CL domain; for example, the first 4-12 or 5-12 amino acid residues of the CL / CH1 domain are particularly useful for use in linking scFv moieties. Linkers can be derived from immunoglobulin light chains, such as CK or Cλ. Linkers can be derived from immunoglobulin heavy chains of any isotype, including, for example, Cy1, Cy2, Cy3, Cy4 and Cμ. Linker sequences may also be derived from other proteins, such as Ig-like proteins (e.g., TCR, FcR, KIR), sequences derived from hinge regions, and other naturally occurring sequences from other proteins. In certain domain configurations, V H and V L The domains are linked in tandem to another domain (e.g., scFv), separated by a linker peptide, which is then fused to the N- or C-terminus of the Fc domain (or its CH2 domain). Such tandem variable regions or scFvs can be connected to the Fc domain via a hinge region or part thereof, an N-terminal fragment of the CH1 or CL domain, or a glycine- and serine-containing flexible polypeptide linker.

[0290] The Fc domain may be connected to other domains via immunoglobulin-derived sequences or via non-immunoglobulin sequences, including any suitable linking amino acid sequence. Advantageously, immunoglobulin-derived sequences can be easily used between the CH1 or CL domain and the Fc domain, particularly when the CH1 or CL domain is fused at its C-terminus to the N-terminus of the Fc domain (or CH2 domain). An immunoglobulin hinge region or a portion of the hinge region can be, and typically is, present between the CH1 and CH2 domains on the polypeptide chain. A hinge or a portion of the hinge region can also be placed on the polypeptide chain between the CL (e.g., Cκ) domain and the CH2 domain of the Fc domain, when the CL is adjacent to the Fc domain on the polypeptide chain. However, it is understood that the hinge region can be replaced as appropriate, for example by a suitable linker peptide, such as a flexible polypeptide linker.

[0291] The NKp46 ABD and the C122 ABD (e.g., cytokine) are advantageously linked to the remainder of the multispecific protein (e.g., to its constant domain or Fc domain) via a flexible linker (e.g., a polypeptide linker) (e.g., between or within the ABD and the Fc domain) that leads to less structural rigidity or stiffness compared to a conventional (e.g., wild-type full-length human IgG) antibody. For example, the multispecific protein may have a structural or flexible linker between the NKp46 ABD and the constant domain or Fc domain that allows for an increased range of domain movement compared to the two ABDs in a conventional (e.g., wild-type full-length human IgG) antibody. In particular, the structural or flexible linker may be configured to confer greater intrachain domain movement to the antigen-binding site compared to the antigen-binding site in a conventional human IgG1 antibody. Rigidity or domain / interchain domain motion can be determined, for example, by computer modeling, electron microscopy, spectroscopy, such as nuclear magnetic resonance (NMR), X-ray crystallography, or sedimentation velocity analytical ultracentrifugation (AUC) to measure or compare the radius of rotational motion of proteins including linkers or hinges. A test protein or linker may have a lower rigidity than a comparison protein if the test protein has a value obtained from one of the tests described in the preceding sentence that is at least 5%, 10%, 25%, 50%, 75%, or 100% different from the value of the comparison protein, e.g., an IgG1 antibody or hinge. A cytokine may be fused to the C-terminus of the CH3 domain by a linker, e.g., any of SEQ ID NOs: 20-23.

[0292] In one embodiment, the multispecific protein may have a structured or flexible linker between the NKp46 ABD and the Fc domain that allows the NKp46 ABD and the ABD that binds CD20 to have a spacing between said ABDs of less than about 80 angstroms, less than about 60 angstroms, or including a range of about 40-60 angstroms.

[0293] At its C-terminus, the Fc domain (or its CH3 domain) may be connected to the N-terminus of the NKp46 ABD or cytokine polypeptide via a polypeptide linker, such as a glycine-serine containing linker, suitably a linker having the amino acid sequence STGS (SEQ ID NO: 15).

[0294] In certain embodiments, the CH1 or CL domain of a Fab (e.g., NKp46 ABD) is fused at its C-terminus to the N-terminus of a cytokine via a flexible polypeptide linker, e.g., a glycine-serine containing linker. Preferably, the linker has a length of at least 4 amino acid residues, and optionally the linker has a length of 5, 6, 7, 8, 9 or 10 amino acid residues.

[0295] In certain embodiments, the NKp46 ABD is placed at the C-terminus of the Fc domain, and NKp46 is positioned in the multispecific protein between the Fc domain and the cytokine polypeptide. The NKp46 ABD is connected or fused at its N-terminus (N-terminus of the VH or VL domain) to the C-terminus of the Fc domain via a linker of sufficient length (e.g., glycine and serine containing linkers, linkers with the sequence STGS, flexible polypeptide linkers) so that the NKp46-binding ABD folds and / or orients in a manner that allows it to bind to Nkp46 on the surface of a NK cell, while at the same time having sufficient distance and range of motion relative to the adjacent Fc domain (or more generally the remainder of the multispecific protein) so that the Fc domain can be simultaneously found by CD16 expressed on the surface of the same NK cell. Additionally, when the NKp46 ABD is placed between the Fc domain and the cytokine polypeptide in the multispecific protein, the C-terminus of the VH or VL of the scFv NKp46 ABD, or the CH1 or CL domain of the Fab NKp46 ABD, is connected or fused to the N-terminus of the cytokine polypeptide via a flexible linker (e.g., a flexible polypeptide linker) of sufficient length to allow the NKp46-binding ABD to fold and / or orient in a manner that allows it to bind to Nkp46 on the surface of a NK cell, while providing sufficient distance and range of motion for an adjacent cytokine polypeptide, such that the cytokine polypeptide can also be simultaneously bound by its cytokine receptor expressed on the surface of the NK cell. Preferably, the linker has a chain length of at least 4 amino acid residues, and optionally the linker has a length of 5, 6, 7, 8, 9 or 10 amino acid residues.

[0296] In a tandem variable region (e.g., scFv), two V domains (e.g., V H Domains and V LThe ABD domains are generally linked together by a linker of sufficient length to allow the ABD to fold in a manner that allows it to bind to the antigen it is intended to bind. Exemplary linkers include linkers that include glycine and serine residues, such as the amino acid sequence GEGTSTGSGGSGGSGGAD (SEQ ID NO: 96). In another specific embodiment, the V of the scFv H Domains and V L The domains are linked together by the amino acid sequence (G4S)3.

[0297] In one embodiment, the (poly)peptide linker used to link the VH or VL domain of the scFv to the CH2 domain of the Fc domain comprises a fragment of the CH1 or CL domain and / or the hinge region. For example, the N-terminal amino acid sequence of CH1 can be fused to the variable domain to mimic the native structure of the wild-type antibody as closely as possible. In one embodiment, the linker comprises an amino acid sequence from the hinge domain or the N-terminal CH1 amino acid. In one embodiment, the linker peptide mimics a canonical VK-CK elbow junction, for example, the linker comprises or consists of the amino acid sequence RTVA.

[0298] In one embodiment, the hinge region used to connect the C-terminus of the CH1 or CK domain (e.g., the CH1 or CK domain of a Fab) to the N-terminus of the CH2 domain may be a fragment of the hinge region (e.g., a truncated hinge region without cysteine ​​residues) or may contain one or more amino acid modifications that remove (e.g., replace with another amino acid or delete) a cysteine ​​residue, optionally both cysteine ​​residues in the hinge region. Removal of cysteines may be useful to prevent undesired disulfide bond formation, e.g., the formation of disulfide bridges in monomeric polypeptides.

[0299] "Hinge" or "hinge region" or "antibody hinge region" herein refers to a flexible polypeptide or linker between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 220, and the IgG CH2 domain begins at residue EU position 237. Thus, for IgG, the hinge generally comprises positions 221 (D221 in IgG1) to 236 (G236 in IgG1), numbering according to the EU index as in Kabat. References to specific amino acid residues within constant region domains found in a polypeptide are defined according to Kabat in the context of an IgG antibody, unless otherwise indicated or otherwise contradicted by context.

[0300] For example, the hinge domain may comprise the amino acid sequence: DKTHTCPPCP (SEQ ID NO:5), or an amino acid sequence at least 60%, 70%, 80% or 90% identical thereto; EPKSCDKTHTCPPCP (SEQ ID NO:13), or an amino acid sequence at least 60%, 70%, 80% or 90% identical thereto; or EPKSCDKTHS (SEQ ID NO:19), or an amino acid sequence at least 60%, 70%, 80% or 90% identical thereto.

[0301] Polypeptide chains that dimerize and associate with each other through non-covalent bonds may or may not be additionally linked by interchain disulfide bonds formed between the respective CH1 and Cκ domains and / or between the respective hinge domains on the chains. The CH1, Cκ and / or hinge domains (or other suitable linking amino acid sequences) can be configured as appropriate to facilitate the desired pairing of the chains and to allow interchain disulfide bonds to form between the chains to avoid undesired or incorrect disulfide bond formation. For example, if two polypeptide chains to be paired each have a CH1 or Cκ adjacent to a hinge domain, the polypeptide chains can be configured to reduce (or completely eliminate) the number of cysteines available for interchain disulfide bond formation between the respective CH1 / Cκ-hinge segments. For example, the amino acid sequence of each CH1, Cκ and / or hinge domain can be modified to remove cysteine ​​residues in both the CH1 / Cκ and hinge domains of the polypeptide; thereby, the CH1 and Cκ domains of the two dimerizing chains associate via non-covalent interactions.

[0302] In another example, the CH1 or Cκ domain adjacent to the hinge domain (e.g., at the N-terminus) contains a cysteine ​​capable of forming an interchain disulfide bond, and the hinge domain located C-terminal to the CH1 or Cκ contains a deletion or substitution of one or both cysteines of the hinge (e.g., Cys 239 and Cys 242 when numbered for a human IgG1 hinge according to Kabat).

[0303] In another example, the CH1 or Cκ domain adjacent to the hinge domain (e.g., at the N-terminus) contains a deletion or substitution at a cysteine ​​residue capable of forming an interchain disulfide bond, and the hinge domain located C-terminal to the CH1 or Cκ contains one or both cysteines of the hinge (e.g., Cys 239 and Cys 242 when numbered for a human IgG1 hinge according to Kabat).

[0304] In another example, the hinge region is derived from an IgM antibody. In such an embodiment, the CH1 / CK pairing mimics the Cμ2 domain homodimerization in an IgM antibody. For example, the CH1 or Cκ domain adjacent to the hinge domain (e.g., at the N-terminus) contains deletions or substitutions in cysteines capable of forming interchain disulfide bonds, and the IgM hinge domain located at the C-terminus of CH1 or Cκ contains one or both cysteines of the hinge.

[0305] Activity Test Multispecific proteins can be evaluated for biological activity, such as antigen binding, ability to induce proliferation of NK cells, induce target cell lysis by NK, and / or induce any unique signaling activity induced by NK cells, such as cytokine production or cell surface expression of a marker of activation, to induce NK cell activation. In one embodiment, provided is a method for evaluating biological activity of a multispecific protein of the present disclosure, such as antigen binding, ability to induce target cell lysis and / or unique signaling activity induced thereby. It is understood that if the unique contribution or activity of one of the components of a multispecific protein (e.g., NKp46 binding ABD, ABD that binds to an antigen of interest, Fc domain, cytokine receptor ABD, etc.) is evaluated, the multispecific format can be produced in a suitable format that allows evaluation of the component (e.g., domain) of interest. The present disclosure also provides such methods for use in testing, evaluating, creating and / or producing multispecific proteins. For example, if the contribution or activity of a cytokine is being evaluated, a multispecific protein can be produced as a protein with the cytokine and another protein in which the cytokine has been modified to delete it or otherwise modulate its activity (e.g., the two multispecific proteins have otherwise the same or equivalent structure) and tested in the assay of interest. For example, if the contribution or activity of an anti-NKp46 ABD is being evaluated, a multispecific protein can be produced as a protein with the ABD and another protein in which the ABD is absent or is replaced by an ABD that does not bind to NKp46 (e.g., an ABD that binds to an antigen not present in the assay system), the two multispecific proteins have otherwise the same or equivalent structure, and the two multispecific proteins are tested in the assay of interest.In another example, where the contribution or activity of an anti-CD20 ABD is being evaluated, a multispecific protein can be produced as a protein with an ABD and another protein in which the ABD is absent or is replaced by an ABD that does not bind to CD20 (e.g., an ABD that binds to an antigen not present in the assay system, an ABD that binds a different tumor antigen), the two multispecific proteins otherwise having the same or equivalent structure, and the two multispecific proteins are tested in the assay of interest.

[0306] In one aspect of any of the embodiments described herein, the multispecific protein has the ability to induce activation of an NKp46-expressing cell (e.g., an NK cell, a reporter cell) when the protein is incubated in the presence of an NKp46-expressing cell (e.g., a purified NK cell) and a target cell expressing CD20 (e.g., a tumor cell).

[0307] In one aspect of any embodiment described herein, the multispecific protein has the ability to induce NKp46 signaling in NKp46-expressing cells (e.g., NK cells, reporter cells) when the protein is incubated in the presence of NKp46-expressing cells (e.g., purified NK cells) and target cells expressing an antigen of interest. In one aspect of any embodiment described herein, the multispecific protein has the ability to induce CD16A signaling in CD16A- and NKp46-expressing cells (e.g., NK cells, reporter cells) when the protein is incubated in the presence of CD16A- and NKp46-expressing cells (e.g., purified NK cells) and target cells expressing CD20.

[0308] Optionally, NK cell activation or signaling is in characterized by increased expression of cell surface markers of activation, such as CD107, CD69, Sca-1 or Ly-6A / E, KLRG1, and the like.

[0309] In one aspect of any of the embodiments described herein, the multispecific protein has the ability to induce an increase in CD137 present on the cell surface of NKp46- and / or CD16-expressing cells (e.g., NK cells, reporter cells) when the protein is incubated in the presence of NKp46- and / or CD16-expressing cells (e.g., purified NK cells), optionally in the absence of target cells.

[0310] In one aspect of any of the embodiments described herein, the multispecific protein has the ability to activate or enhance the proliferation of NK cells at least 10-fold, at least 50-fold, or at least 100-fold compared to the same multispecific protein lacking a cytokine receptor ABD (e.g., CD122 ABD). Optionally, the multispecific protein exhibits an EC50 for activating or enhancing the proliferation of NK cells that is at least 10-fold, 50-fold, or 100-fold lower than the EC50 for activating or enhancing the proliferation of CD25-expressing T cells.

[0311] In one aspect of any embodiment described herein, the multispecific protein has the ability to activate or enhance the proliferation of NK cells over CD25-expressing T cells by at least 10-fold, at least 50-fold, or at least 100-fold. Optionally, the CD25-expressing T cells are CD4 T cells, optionally Treg cells, or CD8 T cells.

[0312] Enhancement of cytokine receptor-mediated activation or proliferation in cells (e.g., NK cells, CD4 T cells, CD8 T cells, or Treg cells) by cytokine receptor ABD-containing proteins can be determined by measuring the expression of pSTAT or cell proliferation markers (e.g., Ki67) in the cells after treatment with a multispecific protein. Enhancement of IL-2R pathway-mediated activation or proliferation in cells (e.g., NK cells, CD4 T cells, CD8 T cells, or Treg cells) by CD122 ABD-containing proteins can be determined by measuring the expression of pSTAT5 or cell proliferation marker Ki67 in the cells after treatment with a multispecific protein. IL-2 and IL-15 lead to phosphorylation of STAT5 protein, which is involved in cell proliferation, survival, differentiation, and apoptosis. Phosphorylated STAT5 (pSTAT5) translocates to the nucleus and regulates the transcription of target genes, including CD25. STAT5 is also required for NK cell survival, which is tightly regulated by the JAK-STAT signaling pathway. In one aspect of any embodiment described herein, the multispecific protein has the ability to induce STAT5 signaling in NKp46-expressing cells (e.g., NK cells) when the protein is incubated in the presence of NKp46-expressing cells (e.g., purified NK cells). In one aspect of any embodiment described herein, the multispecific protein has the ability to cause an increase in expression of pSTAT5 in NK cells over CD25-expressing T cells that is at least 10-fold, at least 50-fold, or at least 100-fold lower. Optionally, the multispecific protein has an EC50 for inducing expression of pSTAT5 in NK cells that is at least 10-fold, 50-fold, or 100-fold lower than its EC50 for inducing expression of pSTAT5 in CD25-expressing T cells. 50 Shows.

[0313] The activity can be measured, for example, by contacting NKp46-expressing cells (or CD25-expressing cells, depending on the assay) with a multispecific polypeptide, optionally further in the presence of target cells (e.g., tumor cells). In some embodiments, the activity is measured, for example, by contacting target cells and NK cells (i.e., NKp46-expressing cells) with each other in the presence of a multispecific polypeptide. NKp46-expressing cells may be used as purified NK cells or NKp46-expressing cells, or as NKp46-expressing cells within a population of peripheral blood mononuclear cells (PBMCs). The target cells can be cells expressing an antigen of interest, optionally tumor cells.

[0314] In one example, multispecific proteins can be assessed for their ability to cause a measurable increase in any property or activity known in the art to be associated with NK cell activity, such as markers 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, e.g., in a redirected killing assay.

[0315] In the presence of target cells (target cells expressing an antigen of interest) and NK cells expressing NKp46, the multispecific protein has the ability to cause an increase in a property or activity associated with NK cell activity in vitro (e.g., NK cell cytotoxicity, CD107 expression, IFNγ production, activation of target cell killing). For example, a multispecific protein according to the invention can be selected based on its ability to increase NK cell activity by greater than about 20%, preferably at least about 30%, at least about 40%, at least about 50%, or greater, compared to that achieved with the same effector:target cell ratio using the same NK cells and target cells that have not been contacted with the multispecific protein, as measured by an assay that detects NK cell activity, e.g., an assay that detects expression of NK activation markers or detects NK cell cytotoxicity, e.g., an assay that detects CD107 or CD69 expression, IFNγ production, or a classical in vitro chromium release assay of cytotoxicity. Exemplary protocols for detecting NK cell activation and for cytotoxicity assays are described in, in addition to the examples herein, for example, in Pessino et al, J. Exp. Med, 1998, 188 (5): 953-960; Sivori et al, Eur J Immunol, 1999. 29:1656-1666; Brando et al, (2005) J. Leukoc. Biol. 78:359-371; El-Sherbiny et al, (2007) Cancer Research 67(18):8444-9; and Nolte-'t Hoen et al, (2007) Blood 109:670-673). In the classical in vitro chromium release test of cytotoxicity, target cells are incubated for 1 h prior to the addition of NK cells. 51 Cr, and then killing is measured by the release of cytochrome P450 from the cells into the medium as a result of killing. 51The release of Cr is estimated to be proportional to the release of Cr. Optionally, a multispecific protein according to the invention may be selected for or characterized by its ability to have NK cell activity towards a target cell, i.e., a greater ability to induce lysis of the target cell, as measured by an assay of NK cell activity (e.g., an assay that detects NK cell-mediated lysis of a target cell expressing the antigen of interest), compared to a conventional human IgG1 antibody that binds the same antigen of interest.

[0316] As shown herein, the different ABDs of a multispecific protein contribute to the overall activity of the multispecific protein, which ultimately manifests a strong anti-tumor activity in vivo. The test method exemplified herein allows for the in vitro evaluation of the activity of different individual ABDs of a multispecific protein by generating mutants of the multispecific protein lacking a particular ABD and / or using cells lacking a receptor for a particular ABD. As shown herein, the multispecific protein according to the present disclosure, when it does not contain a cytokine receptor ABD (e.g., CD122 ABD) and has an Fc domain that does not bind to CD16, does not substantially induce NKp46 signaling (and / or resulting NK activation) of NK cells when the protein is not bound to an antigen of interest on a target cell (e.g., in the absence of an antigen of interest and / or a target cell). Thus, the monovalent NKp46-binding component of a multispecific protein does not itself cause NKp46 signaling. Thus, in the case of a multispecific protein having an Fc domain that binds CD16, such a multispecific protein can be produced in a configuration in which the cytokine receptor ABD (e.g., CD122 ABD) has been inactivated (e.g., modified, masked or deleted, thereby eliminating its ability to bind IL-2R), and the protein can be assessed for its ability to induce NKp46 signaling or NKp46-mediated NK cell activation by testing the effect of this multispecific protein on NKp46 expression by CD16-negative NK cells. Optionally, the multispecific protein will elicit NKp46 signaling or NKp46-mediated NK cell activation when the multispecific protein is incubated with such NKp46-expressing CD16-negative cells (e.g., purified NK cells or purified reporter cells) in the absence of target cells. + CD16 - The antibodies may be characterized as not substantially inducing (or increasing) NKp46 signaling by NK cells, reporter cells.

[0317] In one aspect of any embodiment herein, a multispecific protein may be characterized, for example, by: (a) the multispecific protein has the ability to induce cytokine receptor (e.g., CD122) signaling in NKp46-expressing cells (e.g., NK cells) when incubated in the presence of the NKp46-expressing cells (e.g., purified NK cells) (e.g., as determined by assessing STAT signaling, e.g., assessing STAT phosphorylation); (b) having the ability to induce NK cells to lyse target cells when incubated in the presence of NK cells expressing NKp46 (and optionally further CD16) and CD20 expressing cells; and (c) where the multispecific protein has been modified to lack a cytokine receptor ABD (e.g., CD122 ABD) or comprises an inactivated cytokine receptor ABD, it lacks NK cell activation or cytotoxicity and / or lacks agonist activity at NKp46 when incubated with NK cells (CD16-negative NK cells, as appropriate, NKp46-expressing NK cells that do not express CD16), as appropriate, purified NK cells, in the absence of target cells.

[0318] Use of the compound In one embodiment, provided is the use of any of the multispecific proteins and / or cells expressing said proteins (or their polypeptide chains) to manufacture a pharmaceutical preparation for the treatment, prevention or diagnosis of a disease in a mammal in need thereof. Also provided is the use of any of the compounds defined above as a medicament or an active ingredient or substance in a medicament. In a further embodiment, the present invention provides a method of preparing a pharmaceutical composition containing a compound defined herein to provide a solid or liquid formulation for administration (e.g., by subcutaneous or intravenous injection). Such a method or process comprises at least the step of mixing the compound with a pharma- ceutically acceptable carrier.

[0319] In any embodiment herein, the multispecific protein according to the present disclosure may advantageously be administered at a dose of 1 μg to 1 mg per kg of body weight, suitably 0.05 to 0.5 mg per kg of body weight. The multispecific protein may advantageously be administered 1 to 4 times per month, preferably 1 to 2 times per month, such as once per week, once every 2 weeks, once every 3 weeks or once every 4 weeks. Suitably, administration is by intravenous infusion or subcutaneous administration.

[0320] In one embodiment, provided is a method of treating, preventing or more generally affecting a predefined condition or detecting a certain condition in an individual by using or administering a multispecific protein or antibody described herein, or a (pharmaceutical) composition comprising same.

[0321] For example, in one embodiment, the present invention provides a method for the administration of NKp46-expressing cells, particularly NKp46, to a patient in need thereof (e.g., a patient with cancer). + NK cells (e.g. NKp46 + CD16 + NK cells, NKp46 + CD16 - The present invention provides a method of selectively restoring or enhancing the activity and / or proliferation of NK cells over CD25-expressing lymphocytes, e.g., CD4 T cells, CD8 T cells, Treg cells, in a patient in whom increased lymphocyte (e.g., NK cell) activity is beneficial or who has a disease caused or characterized by insufficient NK cell activity, e.g., cancer. + Lymphocytes (e.g. NKp46 + CD16 + NK cells, NKp46 + CD16 - The aim of this study is to increase the activity of immune cells (NK cells).

[0322] In another aspect, the present invention provides an improved method for treating NKp46 in a patient in need thereof (e.g., a patient with cancer). + NK cells (e.g. NKp46 + CD16 + NK cells, NKp46 + CD16 - The present invention provides a method for restoring or enhancing the activity and / or proliferation of NKp46+ lymphocytes (e.g., NK cells) in a patient in whom increased lymphocyte (e.g., NK cell) activity is beneficial or who has a disease caused or characterized by insufficient NK cell activity, e.g., cancer, the activity and / or proliferation of NKp46+ lymphocytes (e.g., NKp46+ lymphocytes) in a patient in whom increased lymphocyte (e.g., NK cell) activity is beneficial or who has a disease caused or characterized by insufficient NK cell activity, e.g., cancer. + CD16 + The aim of this study is to increase the activity of immune cells (NK cells).

[0323] In another embodiment, the subject multispecific proteins may be used or administered in combination with immune cells, particularly NK cells, derived from the patient to be treated or from a different donor, and these NK cells may be administered to a patient in need thereof, e.g., a patient with a disease in which increased lymphocyte (e.g., NK cell) activity would be beneficial or which is caused or characterized by insufficient NK cell activity, e.g., cancer, or a viral or microorganism, e.g., bacterial or parasitic infection. Because NK cells (unlike CAR-T cells) do not express TCRs, these NK cells, even if derived from a different donor, do not induce GVHD responses [see, e.g., Glienke et al., "Advantages and applications of CAR-expressing natural killer cells", Front. Pharmacol. 6, Art. 21:1-6 (2015); Hermanson and Kaufman, Front. Immunol. 6, Art. 195:1-6 (2015)].

[0324] In one embodiment, the multispecific proteins disclosed herein that mediate NK cell activation, proliferation, tumor infiltration and / or target cell lysis via multiple activating receptors on effector cells, including NKp46, CD16 and CD122, are administered to effector cells or tumor-infiltrating effector cells (e.g., NKp46 + Individuals whose NK cells are poorly functioning, exhausted or suppressed, e.g., expression and / or upregulation of one or more inhibitory receptors (e.g., TIM-3, PD1, CD96, TIGIT, etc.), or downregulation or low levels of CD16 expression (e.g., elevated proportions of NKp46 + CD16 - The method may be advantageously used for the treatment of patients who have a significant population of effector cells characterized by the presence of NK cells.

[0325] The multispecific polypeptides described herein can be used to prevent or treat disorders that can be treated with antibodies, such as cancer, hematological malignancies, and inflammatory or autoimmune disorders.

[0326] In one embodiment, the multispecific protein is used to prevent or treat a cancer characterized by CD20 expressing cells selected from the group consisting of lymphomas, preferably B-cell non-Hodgkin's lymphoma (NHL), and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, b) small non-cleaved cell lymphoma / Burkitt lymphoma (including endemic Burkitt lymphoma, sporadic Burkitt lymphoma, and non-Burkitt lymphoma), c) marginal zone lymphoma (including extranodal marginal zone B-cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B-cell lymphoma, and splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphoma (including B-cell diffuse large cell lymphoma, ... lymphoma (DLCL), diffuse mixed cell lymphoma, immunoblastic lymphoma, primary mediastinal B-cell lymphoma, angiocentric lymphoma-pulmonary B-cell lymphoma], f) hairy cell leukemia, g) lymphocytic lymphoma, Waldenstrom's macroglobulinemia, h) acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasma cell neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma, j) Hodgkin's disease.

[0327] In one embodiment, the multispecific protein is used to prevent or treat cancers characterized by CD20 expressing cells, said cancers being solid tumors, preferably solid non-hematological (non-lymphoid) tumors. Such solid tumors include non-hematological malignancies with B cell involvement, i.e., B cells are involved in a "tumor-promoting" response. Such solid tumors are typically characterized by palpable tumors, typically at least 0.5 mm in diameter, more typically at least 1.0 mm in diameter. Examples include colorectal cancer, liver cancer, breast cancer, lung cancer, head and neck cancer, gastric cancer, testicular cancer, prostate cancer, ovarian cancer, uterine cancer and others. These cancers may be at an early stage (pre-cancer), intermediate stage (stages I and II), or advanced stage, including metastatic solid tumors. These solid tumors are preferably cancers where B cells induce a tumor-promoting response, i.e., the presence of B cells is involved in tumor initiation, maintenance or metastasis.

[0328] In one example, the tumor antigen is an antigen expressed on the surface of a lymphoma or leukemia cell, and the multispecific protein is administered to and / or used to treat an individual with lymphoma or leukemia.

[0329] In one embodiment, the multispecific polypeptides of the invention described herein may be used to prevent or treat cancers characterized by tumor cells expressing CD20, to which the multispecific proteins of the disclosure specifically bind.

[0330] In one embodiment, the method of treatment comprises administering to an individual a multispecific protein as described herein in a therapeutically effective amount, e.g., for the treatment of any of the diseases disclosed herein, e.g., the cancers identified above. A therapeutically effective amount may be any amount that has a therapeutic effect in patients with a 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 said patients).

[0331] The multispecific proteins may be used with or without the preceding step of detecting expression of an antigen of interest (e.g., a tumor antigen) on target cells in a biological sample obtained from an individual (e.g., a biological sample containing cancer cells, cancer tissue, or cancer-adjacent tissue). In another embodiment, the disclosure provides a method of treating or preventing cancer in an individual in need thereof, comprising: a) detecting a cell (e.g., a tumor cell) in a sample from an individual that expresses an antigen of interest (e.g., an antigen of interest to which the multispecific protein specifically binds via the ABD of the antigen of interest); and b) once it has been determined that the sample contains cells expressing the antigen of interest, optionally at a level at least corresponding to a reference level (e.g. corresponding to an individual who derives a substantial benefit from the multispecific protein) or optionally at a level that is increased compared to the reference level (e.g. corresponding to a healthy individual or an individual who does not derive a substantial benefit from the proteins described herein), administering to the individual a multispecific protein of the disclosure that binds (e.g. via its Fc domain) to the antigen of interest, NKp46, a cytokine receptor (e.g. CD122), and optionally CD16A. The present invention provides a method comprising:

[0332] In some embodiments, the multispecific protein is used to treat tumors characterized by low levels of cell surface expression of CD20. Thus, the tumor or cancer can be characterized by cells expressing low levels of CD20. Optionally, the level of CD20 is less than 100,000 copies of CD20 per cancer cell. In some embodiments, the level of tumor antigen is less than 90,000, less than 75,000, less than 50,000, or less than 40,000 copies of CD20 per cancer cell. Optionally, the use further comprises detecting the level of CD20 in one or more cancer cells of the subject.

[0333] In one embodiment, the disclosure provides a method of treating or preventing a disease (e.g., cancer) in an individual in need thereof, comprising: a) detecting expression (e.g., cell surface expression) of a CD20 polypeptide in cancer cells in a sample from an individual (e.g., in the circulation or in the tumor environment); and b) once expression of CD20 on the cancer cells has been determined, administering to the individual a multispecific protein of the present disclosure. The present invention provides a method comprising:

[0334] In one embodiment, the disclosure provides a method of treating or preventing a disease (e.g., cancer) in an individual in need thereof, comprising: a) detecting expression of a CD20 polypeptide in cancer cells in a sample from an individual (e.g., in the circulation or in the tumor environment); and b) upon determination of low expression of CD20 on the cancer cells, optionally a reduced level of low expression compared to a reference level (e.g., a level corresponding to a reference level for low cell surface expression; a level corresponding to an individual who does not derive substantial benefit from a therapeutic agent (e.g., an available anti-CD20 antibody, e.g., rituximab, or another approved anti-CD20 agent), administering to the individual a multispecific protein of the disclosure. Optionally, low expression corresponds to less than 100,000 copies of CD20 per cancer cell. In some embodiments, low expression corresponds to less than 90,000, less than 75,000, less than 50,000, or less than 40,000 copies of CD20 per cancer cell.

[0335] The multispecific proteins may be used with or without a preceding step of detecting or characterizing NK cells from the individual to be treated. Optionally, in one embodiment, the invention provides a method of treating or preventing cancer in an individual in need thereof, comprising: a) detecting NK cells (e.g., tumor-infiltrating NK cells) in a tumor sample (or within the tumor and / or adjacent tissue) from an individual; and b) once it has been determined that the tumor or tumor sample is characterized by a low number or activity of NK cells, as appropriate, at a reduced level or number compared to a reference level (e.g., at a level corresponding to an individual that would derive no, low or insufficient benefit from conventional IgG antibody therapy, e.g., a conventional IgG1 antibody that binds to the same cancer antigen), administering to the individual a multispecific protein of the present disclosure. The present invention provides a method comprising:

[0336] In some embodiments, the individual has a tumor characterized by a CD16 (e.g., CD16A)-deficient tumor microenvironment. Optionally, the method of treatment using a multispecific protein comprises detecting the expression level of CD16 in a sample (e.g., a tumor sample) from the individual. Detecting CD16 optionally comprises detecting the level of CD16A or CD16B. In some embodiments, the CD16-deficient microenvironment is assessed in a patient who has undergone hematopoietic stem cell transplantation. Optionally, the CD16-deficient microenvironment comprises a population of infiltrating NK cells, and the infiltrating NK cells have less than 50% expression of CD16 compared to control NK cells. In some embodiments, the infiltrating NK cells have less than 30%, less than 20%, or less than 10% expression of CD16 compared to control NK cells. Optionally, the CD16-deficient microenvironment comprises a population of infiltrating NK cells, and at least 10% of the infiltrating NK cells have reduced expression of CD16 compared to control NK cells. In some embodiments, at least 20%, at least 30%, or at least 40% of the infiltrating NK cells have reduced expression of CD16 compared to control NK cells.

[0337] Optionally, in one embodiment, provided is a method of treating or preventing cancer in an individual in need thereof, comprising: a) detecting CD16 expression in cells (e.g., tumor-infiltrating NK cells) from a tumor or tumor sample (e.g., within the tumor and / or adjacent tissue) from an individual; and b) once it has been determined that the tumor or tumor sample is characterized by a CD16-deficient microenvironment, administering to the individual a multispecific protein of the present disclosure. The method includes:

[0338] Optionally, in one embodiment, provided is a method of treating or preventing cancer in an individual in need thereof, comprising: a) detecting CD16 expression on the surface of NK cells (e.g., tumor-infiltrating NK cells) in a tumor sample (or within the tumor and / or adjacent tissue) from an individual; and b) The tumor or tumor sample expresses CD16 at an increased level or number, as appropriate, compared to a reference level. - Once it has been determined that the individual is characterized by an elevated proportion of NK cells, administering to the individual a multispecific protein of the present disclosure. The method includes:

[0339] In one embodiment, the disclosure provides a method of treating or preventing a disease (e.g., cancer) in an individual in need thereof, comprising: a) detecting cell surface expression of one or more inhibitory receptors on immune effector cells (e.g., NK cells, T cells) in a sample from an individual (e.g., in the circulation or in the tumor environment); and b) optionally, once cell surface expression of one or more inhibitory receptors on immune effector cells at an increased level compared to a reference level (e.g., at an increased level compared to a healthy individual, an individual not suffering from immune exhaustion or suppression, or an individual who does not derive substantial benefit from the proteins described herein), administering to the individual a multispecific protein of the present disclosure. The present invention provides a method comprising:

[0340] In one embodiment, the disclosure provides a method of treating or preventing a disease (e.g., cancer) in an individual in need thereof, comprising: a) detecting cell surface expression of an NKG2D polypeptide on immune effector cells (e.g., NK cells, T cells) in a sample from an individual (e.g., in the circulation or in the tumor environment); and b) optionally, administering to the individual a multispecific protein of the disclosure upon determination of decreased cell surface expression of an NKG2D polypeptide on immune effector cells at a decreased level compared to a reference level (e.g., at an increased level compared to a healthy individual, an individual not suffering from immune exhaustion or suppression, or an individual who does not derive substantial benefit from the proteins described herein); The present invention provides a method comprising:

[0341] In one embodiment, the multispecific proteins may be used as a monotherapy (without other therapeutic agents) or in a combination treatment with one or more other therapeutic agents.

[0342] The multispecific proteins can also be included in kits, e.g. (i) a pharmaceutical composition comprising a multispecific protein; (ii) a pharmaceutical composition comprising a multispecific protein and a further therapeutic agent, and optionally instructions for administering said multispecific protein together with said further therapeutic agent. The composition may be included in a kit comprising:

[0343] The pharmaceutical composition may be specified as including a pharma- ceutically acceptable carrier, if appropriate. The multispecific protein may be specified as being present in a therapeutically effective amount adapted for use in any of the methods herein, if appropriate. The kit may also include instructions, including, for example, an administration schedule, to enable a practitioner (e.g., a doctor, a nurse, or a patient) to administer the composition contained therein to a patient with cancer. In any embodiment, the kit may include instructions for administering the multispecific protein, if appropriate, other therapeutic agents. The kit may also include a syringe.

[0344] Optionally, the kit includes multiple packages of single-dose pharmaceutical compositions, each containing an effective amount of the multispecific protein and optionally another therapeutic agent for single administration. The equipment or device required for administering the pharmaceutical composition may also be included in the kit. For example, the kit may provide one or more pre-filled syringes containing a certain amount of the multispecific protein.

[0345] In one embodiment, the present invention provides a kit for treating cancer or tumor in a human patient suffering from cervical cancer, comprising: (a) a dose of a multispecific protein that specifically binds human CD20, human NKp46, human CD122, and optionally CD16A, said protein comprising a first (I) polypeptide chain comprising the amino acid sequence of SEQ ID NO:1, and a second (II) polypeptide chain comprising the amino acid sequence of SEQ ID NO:70; and / or (b) the dose of another therapeutic agent, if appropriate; and / or (c) optionally, instructions for using the multispecific protein, and optionally, the anti-HER antibody and / or the chemotherapeutic agent, in any of the methods described herein. A kit is provided, comprising:

[0346] In some embodiments, the multispecific protein comprises a first (I) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 1, and a second (II) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the multimeric protein is administered every 1, 2, 3 or 4 weeks at a dose comprised between 1 μg / kg body weight and 1 mg / kg body weight.

[0347] The kit may further contain any number of polypeptides and / or other compounds, for example, 1, 2, 3, 4, or any other number of multispecific proteins and / or other compounds, as appropriate.It is understood that this description of the contents of the kit is not limiting in any way.For example, the kit may contain other types of therapeutic compounds.As appropriate, the kit also includes instructions for using the polypeptide, for example, detailing the methods described herein, for example, in the detection or treatment of a specific disease state.

[0348] Also provided are pharmaceutical compositions comprising the subject multispecific proteins and, optionally, other compounds as defined above. The multispecific proteins and, optionally, other compounds may be administered as pharmaceutical compositions in purified form together with a pharmaceutical carrier. The form depends on the intended mode of administration and the therapeutic or diagnostic application. The pharmaceutical carrier may be any compatible non-toxic substance suitable for delivering the compound to a patient. Pharmaceutically acceptable carriers are well 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. The pharmaceutically acceptable carrier may further contain a physiologically acceptable compound that acts, for example, to stabilize the compound or increase its absorption. 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. The choice of a pharma- ceutically acceptable carrier, including a physiologically acceptable compound, is known to those skilled in the art and depends, for example, on the route of administration of the composition. Pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like, may also be incorporated into the pharmaceutical composition.

[0349] The multispecific proteins according to the invention may be administered parenterally. Preparations of the compounds for parenteral administration must be sterile. Sterilization is readily achieved by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution as appropriate. Parenteral routes for administration of the compounds follow known methods, such as injection or infusion by intravenous, intraperitoneal, intramuscular, intraarterial, or intralesional routes. The compounds may be administered continuously by infusion or by bolus injection. A typical composition for intravenous infusion may be made up to contain 100-500 ml of sterile 0.9% NaCl or 5% glucose, appropriately supplemented with 20% albumin solution and 1 mg-10 g of the compound, depending on the particular type of compound and its required dosing regimen. Methods for preparing parenterally administrable compositions are well known in the art. EXAMPLES

[0350] Preparation of multispecific proteins The domain structure of an exemplary "T5" format multispecific protein used in the examples is shown in Figure 1 and Figure 2A. Figure 1 shows the domain linkers, such as hinge and glycine-serine linkers, and interchain disulfide bridges. The domain structure of an exemplary "T6" format, which has a N297S mutation to substantially eliminate CD16A binding but is otherwise equivalent to format T5, is shown in Figure 2B. To construct T5 chain L (also referred to as chain 3), the CK domain normally associated with the NKp46-1 VK domain in the NKp46-binding ABD was replaced by the CH1 domain (cross-mab version). The T25 (Figure 2G) format differs from the T5 format by the replacement of CH1 and CK in the NKp46-binding ABD such that the CK domain normally associated with the NKp46-1 VK domain and the CH1 normally associated with the VH remain associated with them. To ensure correct pairing between chain L (chain 3) and chain H (chain 1) and proper disulfide bond formation between the H and L chains, the upper hinge residues of human IgG1 were added at the C-terminus of the CH1 domain of chain L, upstream of the linker connecting chain L to the IL-2 variant. Other protein formats are shown in Figures 2A-2K.

[0351] The sequences encoding each polypeptide chain for each multispecific antigen-binding protein were inserted between the HindIII and BamHI restriction sites in the pTT-5 vector. The three vectors (prepared as endotoxin-free midipreps or maxipreps) were used to co-transfect EXPI-293F cells (Life Technologies) in the presence of PEI (37°C, 5% CO2, 150 rpm). The cells were used to transfect 1x10 6Seeding was performed in culture flasks at a density of 10000 cells / ml (EXPI293 medium, Gibco). As a reference, DNA ratios of 0.1 μg / ml (polypeptide chain I), 0.4 μg / ml (polypeptide chain II) or 0.8 μg / ml (polypeptide chain III) were used for the "T5" construct. Valproic acid (final concentration 0.5 mM), glucose (4 g / L) and tryptone N1 (0.5%) were added. Supernatants were harvested after 6 days and passed through Stericup filters with 0.22 μm pores.

[0352] Multispecific antigen-binding proteins were purified from the harvested supernatant using rProtein A Sepharose Fast Flow (GE Healthcare, ref. 17-1279-03). Size exclusion chromatography (SEC) purification was then performed and proteins eluted at the expected size were finally filtered on a 0.22 μm device. [Example 1]

[0353] CD20-T5-NKCE4 is potent to selectively promote IL2R activation in NK cells Heterotrimeric proteins CD20-1-T5-NKCE4, CD20-2-T5-NKCE4, CD20-3-T5-NKCE4, CD20-4-T5-NKCE4 containing one C-terminal portion of mutant IL-2 were prepared and evaluated for their ability to promote IL-2 R activation in NK cells, CD4 T cells, CD8 T cells and Treg cells. The heterotrimeric proteins incorporate a cytokine portion that is a mutant form of human interleukin-2 with the amino acid sequence of SEQ ID NO: 26, including the deletion of the three first residues and the substitutions R38A, T41A, F42K, which confer a reduced binding affinity to CD25 compared to wild-type human IL-2. The heterotrimeric proteins incorporate a further Fc domain suitable for binding to CD16A, an ABD that binds to a site on the D1 / D2 domain of NKp46, and a VH / VL pair of SEQ ID NO: 16 and 18 that forms one ABD that binds to CD20. The following different ABDs that bind to CD20 were evaluated: - CD20-1-T5-NKCE4-v3, comprising a VH comprising the amino acid sequence of SEQ ID NO: 82 and a VL comprising the amino acid sequence of SEQ ID NO: 83; - CD20-2-T5-NKCE4-v3, comprising a VH comprising the amino acid sequence of SEQ ID NO: 11 and a VL comprising the amino acid sequence of SEQ ID NO: 3; - CD20-3-T5-NKCE4-v3, comprising a VH comprising the amino acid sequence of SEQ ID NO: 84 and a VL comprising the amino acid sequence of SEQ ID NO: 85; - CD20-4-T5-NKCE4-v3, comprising a VH comprising the amino acid sequence of SEQ ID NO: 86 and a VL comprising the amino acid sequence of SEQ ID NO: 87.

[0354] The sequences of the VH / VL domains used in these examples are shown in Table 8 below.

[0355] [Table 8]

[0356] As shown in Figure 1 and Figure 2A, the heterotrimeric protein was constructed according to the T5 protein format.

[0357] Briefly, 1M / well of purified PBMCs were seeded in 96-well plates and treated with increasing doses of CD20-1-T5-NKCE4, CD20-2-T5-NKCE4, CD20-3-T5-NKCE4, CD20-4-T5-NKCE4 or recombinant IL-2 (1.33 × 10 -5 M~133 nM) for 20 min in a 37°C, 5.5% CO2 incubator. STAT5 phosphorylation was then analyzed by flow cytometry on NK cells (CD3-CD56+), CD8 T cells (CD3+ CD8+), CD4 T cells (CD3+ CD4+ FoxP3-) and Tregs (gated on CD3+ CD4+ CD25+ FoxP3+).

[0358] The results are shown in Figure 3, which shows the percentage of pSTAT5 cells among NK cells, CD4 T cells, CD8 T cells, and Treg cells on the y-axis and the concentration of the tested protein on the x-axis. Recombinant human IL-2 promotes the activation of IL-2 receptors in each tested cell, but CD20-T5-NKCE4 showed significantly lower activation of CD4 T cells and Treg cells. IL-2R activation in CD8 T cells by CD20-T5-NKCE4 was at a similar level to recombinant IL-2. However, CD20-T5-NKCE4 resulted in an approximately 1-log increase in the percentage of pSTAT5+ cells among NK cells compared to recombinant IL-2 in NK cells. Thus, CD20-T5-NKCE4 protein allows selective activation of NK cells over Treg cells, CD4 T cells, and CD8 T cells. [Example 2]

[0359] CD20-2-T5-NKCE4 binding to RAJI tumor cells The heterotrimeric proteins CD20-1-T5-NKCE4-v3, CD20-2-T5-NKCE4-v3, CD20-3-T5-NKCE4-v3, CD20-4-T5-NKCE4-v3, described in Example 1, were evaluated for their ability to bind to RAJI cells by flow cytometry. Different proteins were tested for binding to RAJI cells (CD20 expressing cells).

[0360] In short, 10 5 RAJI cells were incubated with normal mouse serum for saturation for 10 min at 4° C. RAJI cells were then incubated with increasing doses of CD20-T5-NKCE4 for 30 min at 4° C. After two washes, CD20-T5-NKCE4 bound to RAJI cells was revealed using a secondary goat anti-human IgG(H+L) APC antibody and flow cytometry analysis.

[0361] The results are shown in Figure 4, which shows the measured median fluorescence intensity on the y-axis and the concentration of the tested protein on the x-axis. The binding affinities of the different heterotrimeric proteins to tumor cells were comparable, with the exception of CD20-2-T5-NKCE4, which exhibited significantly stronger binding to RAJI tumor cells compared to the other proteins.

[0362] The CD20-2 ABD conferred particularly strong binding to the NKCE4 protein. [Example 3]

[0363] CD20-2-T5 NKCE4-v2A selectively binds to CD122 The heterotrimeric protein CD20-2-T5A-NKCE4-v2A, comprising a first polypeptide chain of SEQ ID NO:91, a second polypeptide chain of SEQ ID NO:9, and a third polypeptide chain of SEQ ID NO:17, was assessed for its ability to bind to the IL-2 receptors CD25, CD122 and CD132 via an SPR-Biacore instrument.

[0364] Briefly, a biacore instrument was used with a CM5 chip containing immobilized anti-His antibodies. At the beginning of each cycle, the ligands HuCD122-His (cycle 1), HuCD25-His (cycle 2) or HuCD132-His (cycle 4) were injected at a dilution of 15 mg / ml and captured on the chip. The protein CD20-2-T5A-NKCE4-v2A (1 μM) was then injected using a flow rate of 10 μL / min for 120 s. The interaction between CD20-2-T5A-NKCE4-v2A and HuCD25-His, HuCD122-His or HuCD132-His was studied using a dissociation time of 600 s. The chip was then regenerated with NaOH 10 mM for 10 s at a flow rate of 40 μL / min.

[0365] A portion of the sensorgram of this experiment is shown in FIG. 5. This sensorgram reveals the response measured during injection of CD20-2-T5A-NKCE4-v2A protein. In detail, during cycle 1 (ligand=HuCD122-His), injection of 15 μg / mL CD122-His for 120 s at 10 μL / min induced a response of +154.0 RU. Then injection of 1 μM CD20-2-T5A-NKCE4-v2A for 120 s at 10 μL / min induced a response of +34.4 RU. Finally, a residual response lower than 1 RU was observed after regeneration with NaOH 10 mM for 10 s at 40 μL / min. During cycle 2 (ligand=HuCD25-His), injection of 15 μg / mL CD25-His for 120 s at 10 μL / min generated a response of +80 RU. Next, injection of 1 μM CD20-2-T5A-NKCE4-v2A for 120 s at 10 μL / min induced a response of −7 RU. Finally, a residual response lower than 5 RU was observed after regeneration with NaOH 10 mM for 10 s at 40 μL / min. During cycle 4 (ligand=HuCD132-His), injection of 15 μg / mL CD132-His for 120 s at 10 μL / min induced a response of +41 RU. Next, injection of 1 μM CD20-2-T5A-NKCE4-v2A for 120 s at 10 μL / min induced a response of −6 RU. A residual response lower than 5 RU was observed after regeneration with NaOH 10 mM for 10 s at 40 μL / min.

[0366] As shown in Figure 5, no binding was observed between CD20-2-T5A-NKCE4-v2A and the receptors CD25 (interleukin 2 receptor alpha) and CD132 (interleukin 2 receptor gamma). The sensorgrams, however, show that CD20-2-T5A-NKCE4-v2A can bind to CD122 (interleukin 2 receptor beta). [Example 4]

[0367] Affinity of CD20-2-NKCE4-v2A for CD122 Heterotrimeric proteins CD20-2-T5A-NKCE4-v2, CD20-2-T6AB3-NKCE4-v2A, and dimeric protein CD20-2-T13A-NKCE4-v2A were produced and their affinity to CD122 was studied through SPR-Biacore.

[0368] CD20-2-T5A-NKCE4-v2 comprises a first polypeptide chain of SEQ ID NO: 91, a second polypeptide chain of SEQ ID NO: 9, and a third polypeptide chain of SEQ ID NO: 17. CD20-2-T6AB3-NKCE4-v2A comprises a first polypeptide chain of SEQ ID NO: 92, a second polypeptide chain of SEQ ID NO: 69, and a third polypeptide chain of SEQ ID NO: 17. CD20-2-T13A-NKCE4-v2A comprises a first polypeptide chain of SEQ ID NO: 91 and a second polypeptide chain of SEQ ID NO: 70.

[0369] Briefly, a biacore instrument was used with a CM5 chip containing immobilized anti-His antibodies. The ligand HuCD122-His was injected at a dilution of 15 mg / ml and captured on the chip. The proteins CD20-2-T5A-NKCE4-v2, CD20-2-T6AB3-NKCE4-v2A, CD20-2-T13A-NKCE4-v2A were then injected in the concentration range 31.25 nM to 1 μM for 120 s using a flow rate of 10 μL / min. The interaction between these proteins and HuCD122-His was studied using a dissociation time of 600 s. The chip was then regenerated with NaOH 10 mM for 10 s at a flow rate of 40 μL / min.

[0370] The data were analyzed under the steady-state model, which was deemed to be the most accurate for the appearance of the sensorgrams, and the different KDs so calculated are shown in Table 9 below.

[0371] [Table 9]

[0372] According to the steady-state response fit, it can be concluded that NKCE formats that additionally differ in their cytokine portion (IL-2v2 having the amino acid sequence of SEQ ID NO:25 and IL2v2A having the amino acid sequence of SEQ ID NO:65) show comparable affinity for CD122 (IL2Rβ). [Example 5]

[0373] CD20-2 NKCE4 is the best inducer of cytotoxicity in RAJI tumor cells In this experiment, NKCE proteins were evaluated for their ability to induce killing of RAJI tumor cells (CD20+) by NK cells from a human donor at an effector:target ratio of 10:1 in a standard 4-hour cytotoxicity assay using calcein release as a readout.

[0374] As in Example 1, the CD20-T5-NKCE4-v3 protein exhibited several CD20 ABDs: - CD20-1-T5-NKCE4-v3, comprising a VH comprising the amino acid sequence of SEQ ID NO: 91 and a VL comprising the amino acid sequence of SEQ ID NO: 92; - CD20-2-T5-NKCE4-v3, comprising a VH comprising the amino acid sequence of SEQ ID NO: 11 and a VL comprising the amino acid sequence of SEQ ID NO: 3; - CD20-3-T5-NKCE4-v3, comprising a VH comprising the amino acid sequence of SEQ ID NO: 93 and a VL comprising the amino acid sequence of SEQ ID NO: 94; - CD20-4-T5-NKCE4-v3, comprising a VH comprising the amino acid sequence of SEQ ID NO: 95 and a VL comprising the amino acid sequence of SEQ ID NO: 96. - IC-T5-NKCE4-v3, which has the same structure as the other proteins, except that the CD20 ABD is replaced by a VH / VL pair that does not bind to the protein present in this experiment.

[0375] As shown in Figure 1 and Figure 2A, the heterotrimeric protein was constructed according to the T5 protein format.

[0376] Briefly, freshly purified NK cells from healthy donors were co-cultured with Raji tumor cells previously loaded with calcein at a ratio of 10:1. The cells were incubated with the above test proteins (6.6 × 10 -_6 The cells were incubated with 100 μg / ml of 100 mM PBS (a dose of ∼66 nM) in a 37°C, 5.5% CO2 incubator for 4 h. Cytotoxicity was monitored by assessing calcein release.

[0377] The results are shown in Figure 6, which shows the % of cytotoxicity induced by NK cells on the y-axis and the concentration of the tested protein on the x-axis. All CD20-T5-NKCE4-v3 proteins, whatever their CD20 ABD, were highly potent in their ability to mediate NK cell cytotoxicity towards tumor target cells. However, CD20-2-T5-NKCE4-v3 induced significantly better induction of NK cell cytotoxicity in RAJI tumor cells.

[0378] The different EC50 of cytotoxicity for each molecule was calculated for NK cells isolated from the blood of four different donors and is shown in Table 10 below.

[0379] [Table 10] [Example 6]

[0380] CD20-2-T5-NKCE4-v3 exhibits potent antitumor activity in vivo In this experiment, a single injection of 0.4 μg, 2 μg or 10 μg of NK cell engager protein CD20-2-T13-NKCE4-v2A or CD20-2-T5-NKCE4 was evaluated for its in vivo antitumor activity in a mouse model of human cancer. CD20-2-T13-NKCE4-v2a is a heterodimeric protein comprising a first polypeptide chain of the amino acid sequence of SEQ ID NO: 1 and a second polypeptide chain of the amino acid sequence of SEQ ID NO: 70, and binds to NKp46, CD122, CD20 and CD16A. CD20-1-T5-NKCE4 is a heterotrimeric protein comprising a first polypeptide chain of the amino acid sequence of SEQ ID NO: 101, a second polypeptide chain of the amino acid sequence of SEQ ID NO: 102, and a third polypeptide chain of the amino acid sequence of SEQ ID NO: 103, and binds to NKp46, CD122, CD20 and CD16A.

[0381] Briefly, CB17 SCID mice were injected subcutaneously with 5 × 10 6 RAJI cells were transplanted. Nine days after transplantation, mice were treated with a single intravenous injection of 0.4, 2, or 10 μg of CD20-2-T13-NKCE4-v2A or CD20-2-T5-NKCE4 and PBS as vehicle. Tumor volumes were measured 26 days after transplantation.

[0382] The results are shown in Figure 7. Each dot represents the tumor volume in an individual animal. A dose of 10 μg CD20-2-T13-NKCE4-v2A or CD20-2-T5-NKCE4 showed strong efficacy as a single injection compared to vehicle alone. [Example 7]

[0383] Different formats of NKCE4 can induce cytotoxicity in RAJI tumor cells. In this experiment, several additional formats of NKCE4 incorporating both the CD20-2 and NKp46 binding domains of the VH / VL pairs of SEQ ID NOs: 16 and 18 were evaluated for their ability to induce cytotoxicity in RAJI tumor cells. The test proteins included in this experiment were: - CD20-2-T5-NKCE4-v2 is a heterotrimeric protein comprising a first polypeptide chain of the amino acid sequence of SEQ ID NO: 1, a second polypeptide chain of the amino acid sequence of SEQ ID NO: 9, and a third polypeptide chain of the amino acid sequence of SEQ ID NO: 98. CD20-2-T5-NKCE4-v2 contains, from N-terminus to C-terminus, an anti-CD20 VH / VL pair (Fab), an Fc domain dimer that binds to CD16, an NKp46 VH / VL pair (Fab), IL2v2. - CD20-2-T5A-NKCE4-v2 is a heterotrimeric protein comprising a first polypeptide chain of the amino acid sequence of SEQ ID NO: 91, a second polypeptide chain of the amino acid sequence of SEQ ID NO: 9, and a third polypeptide chain of the amino acid sequence of SEQ ID NO: 98. CD20-2-T5A-NKCE4-v2 contains, from N-terminus to C-terminus, an anti-CD20 VH / VL pair (Fab), an Fc domain dimer that binds to CD16, an NKp46 VH / VL pair (Fab), IL2v2. - CD20-2-T13A-NKCE4-v2 is a heterodimeric protein comprising a first polypeptide chain of the amino acid sequence of SEQ ID NO: 91 and a second polypeptide chain of the amino acid sequence of SEQ ID NO: 99. CD20-2-T13A-NKCE4-v2 contains, from N-terminus to C-terminus, an anti-CD20 VH / VL pair (Fab), an Fc domain dimer that binds to CD16, an NKp46 scFv, and IL2v2. - CD20-2-T6AB3-NKCE4-v2 is a heterotrimeric protein comprising a first polypeptide chain of the amino acid sequence of SEQ ID NO: 92, a second polypeptide chain of the amino acid sequence of SEQ ID NO: 69, and a third polypeptide chain of the amino acid sequence of SEQ ID NO: 98. CD20-2-T13AB3-NKCE4-v2 contains, from N-terminus to C-terminus, an anti-CD20 VH / VL pair (Fab), an Fc domain dimer mutated to abolish CD16 binding, NKp46 (Fab), IL2v2. - CD20-2-T14A-NKCE4-v2A is a heterodimeric protein comprising a first polypeptide chain of the amino acid sequence of SEQ ID NO: 92 and a second polypeptide chain of the amino acid sequence of SEQ ID NO: 71. CD20-2-T14A-NKCE4-v2A contains, from N-terminus to C-terminus, an anti-CD20 VH / VL pair (Fab), an Fc domain dimer mutated to abolish CD16 binding, an NKp46 scFv, IL2v2A. - CD20-2-T175-NKCE4-v2 is a heterotrimeric protein comprising a first polypeptide chain of the amino acid sequence of SEQ ID NO: 77, a second polypeptide chain of the amino acid sequence of SEQ ID NO: 78, and a third polypeptide chain of the amino acid sequence of SEQ ID NO: 100. CD20-2-T175-NKCE4-v2 contains, from N-terminus to C-terminus, an anti-CD20 VH / VL pair, an Fc domain dimer that binds to CD16, NKp46(Fab), IL2v2A. - CD20-2-T195-NKCE4-v2 is a heterotrimeric protein comprising a first polypeptide chain of the amino acid sequence of SEQ ID NO: 77, a second polypeptide chain of the amino acid sequence of SEQ ID NO: 79, and a third polypeptide chain of the amino acid sequence of SEQ ID NO: 98. CD20-2-T195-NKCE4-v2 contains, from N-terminus to C-terminus, an anti-CD20 VH / VL pair, an Fc domain dimer that binds to CD16, NKp46(Fab), IL2v2.

[0384] Briefly, freshly purified NK cells from donors were rested overnight in complete medium. The rested NK cells were then co-cultured with Raji tumor cells previously loaded with calcein release at a ratio of 10 to 1. The cells were then incubated with the above test proteins [6.1×10-6 to 1 nM doses, 10-5 to 10 2 Doses from 6.1×10-6 to 61nMdoses from 10-5 to 10 2 M)] for 4 h in a 37° C., 5.5% CO2 incubator. Cytotoxicity is monitored by assessing calcein release.

[0385] The results are shown in Figures 8A and 8B, which show the % of cytotoxicity induced by NK cells on the y-axis and the concentration of the tested proteins on the x-axis. All NKCE4 proteins, whatever their format, were highly potent in their ability to mediate NK cell cytotoxicity towards tumor target cells. [Example 8]

[0386] Comparison of induction of IL2R signaling in NK cells In this experiment, the potency of two multispecific proteins to induce proliferation of NK cell lines in a bioassay system was evaluated.

[0387] The test proteins were as follows: - a heterodimeric protein, CD20-2-T13-NKCE4-v2A, comprising a first polypeptide chain of the amino acid sequence of SEQ ID NO: 1 and a second polypeptide chain of the amino acid sequence of SEQ ID NO: 70. CD20-2-T13-NKCE4-v2A contains, from N-terminus to C-terminus, an anti-CD20 VH / VL pair (Fab), an Fc domain dimer that binds to CD16, an NKp46 scFv, IL2v2A; - a heterotrimeric protein containing from N-terminus to C-terminus the anti-CD20 VH / VL pair (Fab) according to SEQ ID NO: 82 and 83, an Fc domain dimer that binds to CD16, NKp46 scFv, IL2v, CD20-1-T5-NKCE4-IL2v.

[0388] Briefly, 10,000 KHYG-1 cells, which express NKp46 and were modified to express high levels of CD16, were incubated with serial dilutions of test molecules from 7.5ug / mL to 0.01ng / mL for 50h. Cell proliferation was assessed using Cell Titer Glo (RLU on the y-axis). The results are shown in Figure 9. The data showed that CD20-2-T13-NKCE4v2A was more potent than CD20-1-T5-NKCE4-IL2v multispecific protein to induce NK cell proliferation. [Example 9]

[0389] Administration of CD20-NKCE4 to non-human primates In this experiment, several formats of NKCE4 incorporating both the CD20-1 and NKp46 binding domains of the VH / VL pairs of SEQ ID NOs: 82 and 83 were tested.

[0390] The test proteins included in this experiment were: - CD20-1-T5-NKCE4 is a heterotrimeric protein comprising a first polypeptide chain of the amino acid sequence of SEQ ID NO: 101, a second polypeptide chain of the amino acid sequence of SEQ ID NO: 102, and a third polypeptide chain of the amino acid sequence of SEQ ID NO: 103. CD20-1-T5-NKCE4 contains, from N-terminus to C-terminus, an anti-CD20 VH / VL pair (Fab), an Fc domain dimer that binds to CD16, an NKp46 scFv, an IL2v; - CD20-1-T6-NKCE4 is a heterotrimeric protein comprising a first polypeptide chain of the amino acid sequence of SEQ ID NO: 104, a second polypeptide chain of the amino acid sequence of SEQ ID NO: 105, and a third polypeptide chain of the amino acid sequence of SEQ ID NO: 106. CD20-1-T6-NKCE4 contains from N-terminus to C-terminus an anti-CD20 VH / VL pair (Fab), an Fc domain dimer mutated to abolish CD16 binding, an NKp46 scFv, an IL2v.

[0391] The first multispecific protein CD20-1-T5-NKCE4 was Fc-competent and had a CH2-CH3 domain capable of binding to CD16A, while the second multispecific protein CD20-1-T6-NKCE4 was Fc-incompetent and had a CH2-CH3 domain with a mutation that prevented binding to CD16A. These multispecific proteins were then administered intravenously to non-human primates at doses of 0.05 mg / kg body weight and 0.5 mg / kg body weight for Fc-competent CD20-1-T5-NKCE4, and at a dose of 0.5 mg / kg for Fc-incompetent CD20-1-T6-NKCE4. Each of these three settings was administered to a cohort of four non-human primates (compared to vehicle as a control).

[0392] The pharmacokinetics of these multispecific proteins were followed by measuring their serum concentrations over time, and the results are shown in Figure 10A.

[0393] Evaluable terminal half-lives were calculated for three anti-drug antibody-free animals treated with CD20-1-T5-NKCE4 (Fc-competent), one at 0.05 mg / kg and two at 0.5 mg / kg. The terminal half-life for the 0.05 mg / kg dose was 146 hours (6 days). The terminal half-life for the 0.5 mg / kg dose was 311 hours (13 days) and 175 hours (7 days). The in vivo stability results indicate that the multispecific protein is compatible with administration to humans, which may be, for example, every 2, 3 or 4 weeks.

[0394] The concentration at which CD20-1-T5-NKCE4 protein induces pSTAT5 expression in NK cells, cytotoxicity against NK cells, and proliferation of NK cells was determined in vitro in human PBMCs and compared to the maximum serum concentration of CD20-1-T5-NKCE4 and serum concentration of CD20-1-T5-NKCE4 at day 22 post-injection administered at 0.5 mg / kg body weight. The results are shown in FIG. 10B. The results show that serum concentration of CD20-1-T5-NKCE4 administered at 0.5 mg / kg body weight at day 22 was significantly higher than the EC50 for pSTAT5 induction, NK cell-mediated cytotoxicity, and NK cell proliferation, respectively. 50 indicates that the value remained at or above that value.

[0395] The release of several cytokines (IFN-γ, IL-6, TNF-α, IL-10, IL-8, MIP-1β, MCP-1, IL-1β) was also monitored after injection of the multispecific protein. The results are shown in FIG. 14. A transient increase in the release of IFN-γ, IL-6, IL-10, IL-8, MIP-1β, MCP-1 was observed during the first 5 days after injection, which then decreased to baseline. The multispecific protein had no effect on TNF-α and IL-1β release. In conclusion, the CD20-specific NKCE4 molecule induced low systemic cytokine release in non-human primates, but provided a strong depletion of CD20-expressing B cells, as shown in FIG. 15. [Example 10]

[0396] Study of immune cell populations upon administration of CD20-NKCE4 to non-human primates The multispecific protein was administered intravenously or subcutaneously to non-human primates at a dose of 0.5 mg per kg of body weight administered weekly (i.e., days 0, 7, and 14 of the experiment). Two non-human primates (2258 and 2261) received a dose of CD20-2-T13-NKCE4-v2A intravenously, while one (2262) received a dose by subcutaneous injection.

[0397] The number of circulating B, T and NK cells was followed over time by flow cytometry. The results are shown in Figure 16A, Figure 16B and Figure 16C. Administration of the multispecific protein CD20-2-T13-NKCE4-v2A induced a strong and persistent depletion (about 100%) of B cells. Additionally, a moderate expansion / contraction of T and NK cell populations was observed, especially after the first administration of the multispecific protein (day 0), but no depletion was observed for these cell populations.

[0398] CD20-2-T13-NKCE4-v2A-induced depletion of B, T and NK cell subsets was further monitored in vitro in human PBMCs and compared to an isotype control multispecific protein that binds CD16A and NKp46 (IC-T13-NKCE4-v2A), a multispecific protein that binds CD20, CD16, NKp46 but does not contain an IL-2 moiety (CD20-2-F13-NKCE3), and a control IL-2 protein (IL-2 linked to His and BirA protein tags).

[0399] Proteins tested included: - CD20-2-T13-NKCE4-v2A is a heterodimeric protein comprising a first polypeptide chain of the amino acid sequence of SEQ ID NO: 1 and a second polypeptide chain of the amino acid sequence of SEQ ID NO: 70. CD20-2-T13-NKCE4-v2A contains, from N-terminus to C-terminus, an anti-CD20 VH / VL pair (Fab), an Fc domain dimer that binds to CD16, an NKp46 scFv, IL2v2A; - IC-T13-NKCE4-v2A, which has the same structure as CD20-2-T13-NKCE4-v2A, but in which the CD20 ABD is replaced by an isotype control (CD20 VH / VL is replaced by a VH / VL pair that does not bind to any protein present in the experiment). - CD20-2-F13-NKCE3 is a heterodimeric protein comprising a first polypeptide chain of the amino acid sequence of SEQ ID NO: 1 and a second polypeptide chain of the amino acid sequence of SEQ ID NO: 97. CD20-2-F13-NKCE3 contains, from N-terminus to C-terminus, an anti-CD20 VH / VL pair (Fab), an Fc domain dimer that binds to CD16, and an NKp46 scFv.

[0400] The results are shown in Figures 11A and 11B for B cells, 12A and 12B for T cells, and 13A and 13B for NK cells. As shown in Figures 11A and 11B, -1 The multispecific proteins CD20-2-T13-NKCE4-v2A and CD20-2-F13-NKCE3 in the nM range were able to deplete nearly 100% of B cells. Neither IL2 nor IC-T13-NKCE4-v2A was able to induce B cell depletion. As shown in Figures 12A, 12B, 13A and 13B, none of the tested proteins was able to induce significant T cell or NK cell depletion.

[0401] The results show that CD20-2-T13-NKCE4-v2A is highly potent in its ability to deplete B cells without inducing NK cell kin killing.

[0402] All headings and sub-headings 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 indicated herein or otherwise clearly contradicted by context. The description of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually described herein. Unless otherwise indicated, all precise values ​​provided herein are representative of the corresponding approximate value (e.g., all precise exemplary values ​​provided for a particular factor or measurement may also be considered to provide the corresponding approximate measurement, modified by "about", where appropriate). All methods described herein may be carried out in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by context.

[0403] The use of any and all examples or exemplary language (e.g., "for example") provided herein is merely intended to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. Nothing in this specification should be construed as indicating that any element is essential to the practice of the invention unless expressly stated.

[0404] A description herein of any aspect or embodiment of the invention using such terminology with reference to one or more elements is intended to provide support for similar aspects or embodiments of the invention that "consist of," "consist essentially of," or "substantially include" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as including a particular element should be understood to also describe a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0405] This invention includes all modifications and equivalents of the subject matter recited in the embodiments or claims presented in this application to the maximum extent permitted by applicable law.

[0406] All publications and patent applications referenced 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.

[0407] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

1. A multimeric binding protein that specifically binds to human CD20, human NKp46, human CD122, and optionally CD16A, a) a first (I) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 1 and a second (II) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70; b) a first (I) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 1, a second (II) polypeptide chain having the amino acid sequence of SEQ ID NO: 9, and a third (III) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17; c) a first (I) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 1, a second (II) polypeptide chain having the amino acid sequence of SEQ ID NO: 73, and a third (III) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 74; or, d) a first (I) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 66, a second (II) polypeptide chain having the amino acid sequence of SEQ ID NO: 67, and a third (III) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17 The multimeric binding protein comprising.

2. The binding protein according to claim 1, comprising a first (I) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 1, a second (II) polypeptide chain having the amino acid sequence of SEQ ID NO: 9, and a third (III) polypeptide chain comprising the amino acid sequence of SEQ ID NO:

17.

3. The binding protein according to claim 1, comprising a first (I) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 1, a second (II) polypeptide chain having the amino acid sequence of SEQ ID NO: 73, and a third (III) polypeptide chain comprising the amino acid sequence of SEQ ID NO:

74.

4. The binding protein according to claim 1, comprising a first (I) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 66, a second (II) polypeptide chain having the amino acid sequence of SEQ ID NO: 67, and a third (III) polypeptide chain comprising the amino acid sequence of SEQ ID NO:

17.

5. - The polypeptide (I) consists of the amino acid sequence of SEQ ID NO: 1; - The polypeptide (II) consists of the amino acid sequence of SEQ ID NO: 9; and - The polypeptide (III) consists of the amino acid sequence of SEQ ID NO: 17, the multimeric binding protein according to claim 2.

6. The multimeric binding protein according to claim 1, comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 1 and a polypeptide containing the amino acid sequence of SEQ ID NO:

70.

7. - The polypeptide (I) consists of the amino acid sequence of SEQ ID NO: 1; and - The polypeptide (II) consists of the amino acid sequence of SEQ ID NO: 70, the multimeric binding protein according to claim 1.

8. A pharmaceutical composition comprising the multimeric binding protein according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

9. An isolated nucleic acid molecule comprising the nucleotide sequence encoding the multimeric binding protein according to any one of claims 1 to 7 or its polypeptide chain.

10. An isolated cell comprising the nucleic acid molecule according to claim 9.

11. Use as a medicament in the treatment of diseases of the multimeric binding protein according to any one of claims 1 to 7.

12. The multimer-binding protein for use according to claim 11, wherein the disease is selected from the group consisting of B-cell lymphoma, Hodgkin or non-Hodgkin B-cell lymphoma, precursor B-cell lymphoblastic leukemia / lymphoma and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), cutaneous follicular center lymphoma, marginal zone B-cell lymphoma (MALT type, nodular type and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, plasmacytoma, multiple myeloma, post-transplant lymphoproliferative disorder, Waldenström macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

13. The multimer-binding protein for use according to claim 11, wherein the disease is characterized by cells expressing low levels of CD20.

14. The multimer-binding protein for use according to claim 11, wherein the multimer protein is administered once per month to 4 times per month, or once every 3 or 4 weeks, and optionally the treatment is carried out over a period of at least 3 months, 6 months or 12 months.

15. A method for producing the multimer-binding protein according to any one of claims 1 to 7, comprising: (a) culturing a host cell under conditions suitable for expressing a plurality of recombinant polypeptides, wherein the plurality of recombinant polypeptides comprises (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 66, and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 9, 67, 70 or 73, and optionally (iii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 17 or 74; (b) optionally, recovering the expressed recombinant polypeptide The method comprising the above steps.

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