Multispecific antigen-binding proteins for stimulating NK cells and uses thereof

Abstract

The present invention relates to a multispecific antigen-binding protein comprising a natural killer (NK) cell-activating cytokine and a region having affinity for a surface antigen expressed on NK cells, such as CD16A. The NK cell-activating cytokine is preferably at least one of an interleukin-21 receptor (IL21R) agonist and a 4-1BB agonist. The multispecific antigen-binding protein may further comprise an antigen-binding region that specifically binds to an NK cell-activating receptor, such as NKp46, NKp44, NKp30, NKG2D, DNAM1, and CD16A. The multispecific antigen-binding protein of the present invention can be used ex vivo or in vivo to expand and activate NK cells. The present invention further relates to the use of such multispecific antigen-binding proteins and / or ex vivo-expanded NK cells in the treatment of cancer.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the medical field, in particular to the fields of oncology, immunology and tumor immunotherapy. In particular, the present invention relates to multispecific antigen-binding proteins that stimulate and activate NK cells so that they can lyse target cells, such as tumor cells. The present invention further relates to the use of such multispecific antigen-binding proteins in the treatment of cancer. [Background technology]

[0002] Background of the Invention Cancer immunotherapy has revolutionized cancer treatment. Cancer immunotherapy is desirable because it is highly specific and can promote tumor destruction by inducing the recognition and elimination of tumor cells by the patient's own immune system. Recent advances have focused on generating or elucidating tumor antigen-specific T cell responses. These have been based on the use of immune checkpoint inhibitors that target inhibitory pathways or bispecific T cell engagers and chimeric antigen receptor (CAR) T cells that target tumor antigens. Despite these significant advances, clinical benefits have been limited to subsets of patients and specific tumor types, highlighting the need for alternative strategies.

[0003] One such alternative approach is to harness the antitumor activity of natural killer (NK) cells. NK cells are components of the innate immune system and comprise approximately 15% of circulating lymphocytes. NK cells infiltrate virtually all tissues and were originally characterized by their ability to effectively kill tumor cells without the need for prior sensitization. NK cells provide an efficient immune surveillance mechanism capable of eliminating unwanted cells, such as tumor cells or virus-infected cells. The 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 recognize and kill "self" cells that fail to express MHC / HLA antigens by activating specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express stress ligands for NK-activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit immune responses. Activated NK cells kill target cells by means similar to cytotoxic T cells, namely, via cytolytic granules containing perforin and granzymes and death receptor pathways. Activated NK cells also secrete proinflammatory cytokines, such as IFN-γ and chemokines, which promote the recruitment of other leukocytes to target tissues. NK cells respond to signals via various activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy autologous cells, their activity is inhibited through activation of killer cell immunoglobulin-like receptors (KIRs). Alternatively, when NK cells encounter foreign or cancer cells, they are activated via their activating receptors (e.g., NKG2D, NCR, DNAM1). NK cells are also activated by the constant regions of several immunoglobulins via the CD16 receptor on their surface. The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals.

[0004] Strategies based on the recruitment of cytotoxic NK cells are currently being developed. Given the lack of graft-versus-host reactions in patients receiving allogeneic NK cell infusions, NK cell-based therapies are expected to be safer than T cell therapies. Furthermore, unlike chimeric antigen receptor (CAR) T cells, administration of allogeneic CAR-engineered NK cells is not associated with the development of neurotoxicity, cytokine release syndrome (CRS), or graft-versus-host disease, and CAR-NK cell infusion does not increase circulating inflammatory cytokine concentrations above baseline levels. NK cell-based immunotherapies may be less likely to cause these adverse events because the spectrum of cytokines produced by these cells differs from that secreted by T cells. Furthermore, NK cells have a frequency approximately 1 / 10 that of T cells, and this low frequency ensures a more moderate release of cytokines, limiting the risk of CRS in patients treated with NK cell-targeting multispecific antibodies.

[0005] More recently, multifunctional antibodies called natural killer cell engagers (NKCEs) have been developed that simultaneously target tumor-associated antigens (TAAs) and activate receptors on endogenous NK cells. NKCEs are designed to enhance the interaction between NK cells and target tumor cells and increase NK cell effector function against tumor cells. Several NKCEs currently under development for clinical application are reviewed by Demaria et al. (Eur. J. Immunol. 2021. 51: 1934-1942). Summary of the Invention [Problem to be solved by the invention]

[0006] There remains a need in the art for improved multispecific antigen binding proteins for inducing NK cells with new and additional functionalities, particularly functionalities that provide therapeutic advantages over existing NKCEs. [Means for solving the problem]

[0007] Summary of the Invention In a first aspect, the present invention relates to a multispecific antigen-binding protein comprising: a) an NK cell-activating cytokine that is at least one of: i) an interleukin-21 receptor (IL21R) agonist; and ii) a 4-1BB agonist; and b) a region that has affinity for a surface antigen expressed on a natural killer (NK) cell.

[0008] In one embodiment, the multispecific antigen binding protein is a multispecific antigen binding protein wherein the IL21R agonist comprises or consists of an IL21 polypeptide or agonistic antigen-binding region that specifically binds to IL21R. In one embodiment, the multispecific antigen binding protein described herein comprises an IL21R agonist which is an IL21 polypeptide comprising an amino acid sequence having at least 50, 55, 60, 65, 38, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 70, and preferably has IL21R agonist activity as defined herein and / or preferably has affinity for IL21R as defined herein. In one embodiment, the IL21 polypeptide is an IL21 mutein that has been modified to have reduced or enhanced affinity for IL21R compared to the corresponding wild-type IL21 polypeptide. For example, an IL21 mutein that has reduced affinity for IL21R compared to the corresponding wild-type IL21 polypeptide can be an IL21 mutein having a mutation in one or more amino acids selected from the group consisting of 116, 166, 18, K72, K73, K75, K77, L13, P78, Q12, Q19, R5, R65, R76, R9, S70, S80, V69 and Y23. In one embodiment, the multispecific antigen binding protein is a multispecific antigen binding protein with an IL21R agonist valency of greater than one.

[0009] In one embodiment, the multispecific antigen-binding protein is a multispecific antigen-binding protein wherein the 4-1BB agonist comprises or consists of at least one 4-1BB ligand (4-1BBL) extracellular domain (ECD) or at least one agonistic antigen-binding region that specifically binds to 4-1BB. In one embodiment, the multispecific antigen-binding protein described herein comprises a 4-1BB agonist that comprises at least one 4-1BBL ECD comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 37, and preferably has 4-1BB agonist activity as defined herein and / or preferably has affinity for 4-1BB as defined herein. In one embodiment, the 4-1BBL ECD is a mutein that has been engineered to decrease or increase affinity for 4-1BB compared to the corresponding wild-type 4-1BBL ECD. In one embodiment, the 4-1BB agonist comprises or consists of a fusion protein comprising three 4-1BBL ECD monomers fused together in a single polypeptide chain, optionally the three 4-1BBL ECD monomers are linked by a polypeptide linker. In one embodiment, the multispecific antigen binding protein is a multispecific antigen binding protein having a 4-1BB agonist valency of greater than one.

[0010] In one embodiment, the multispecific antigen binding protein is a multispecific antigen binding protein comprising at least one IL21R agonist and at least one 4-1BB agonist.

[0011] In one embodiment, the multispecific antigen binding protein is a multispecific antigen binding protein further comprising an NK cell activating cytokine selected from the group consisting of an IL15 receptor agonist, an IL2 receptor agonist, a type I interferon (IFN-1) agonist, an IL12 receptor agonist, and an IL18 receptor agonist.

[0012] In one embodiment, the multispecific antigen-binding protein is a multispecific antigen-binding protein in which the region having affinity for a surface antigen expressed on NK cells comprises or consists of an immunoglobulin Fc region. Preferably, the Fc region is a dimeric Fc region. In one embodiment, the Fc region is an Fc region that binds to CD16A. In one embodiment, the Fc region is an Fc region that has been modified to reduce or enhance affinity for CD16A compared to a corresponding wild-type Fc region. In one embodiment, the Fc region is an Fc region that has been modified to reduce or enhance NK cell activation via CD16A binding compared to a corresponding wild-type Fc region.

[0013] In one embodiment, the multispecific antigen-binding protein further comprises c) at least one antigen-binding region that specifically binds to an NK cell-activating receptor. In one embodiment, the antigen-binding region that specifically binds to an NK cell-activating receptor is an agonistic antigen-binding region that activates the NK cell receptor. Preferably, the antigen-binding region comprises at least one immunoglobulin variable region, and more preferably, the immunoglobulin variable region comprises or consists of a Fab or an immunoglobulin single-chain variable domain (ISVD). In one embodiment, the antigen-binding region is human or humanized.

[0014] In one embodiment, the multispecific antigen-binding protein comprises two antigen-binding regions that specifically bind to an NK cell-activating receptor. The two antigen-binding regions may bind to the same NK cell-activating receptor or to at least two different NK cell-activating receptors. In one embodiment, the two antigen-binding regions are identical.

[0015] In one embodiment, the multispecific antigen binding protein is one in which the NK cell activating receptor (bound by the antigen binding region) is selected from the group consisting of NKp46, NKp30, NKG2D, CD16A, SLAMF7, NKp44, CD94-NKG2C / E, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR2DS2, KIR2DL4, KIR3DS1, CD160, NKp80, DNAM1, 2B4, CRACC, 4-BB, OX40, CRTAM, CD27, PSGL1, CD96, CD100, CEACAM1, CD59, PD-L1, Tim3 and NTB-A.

[0016] In one embodiment, the multispecific antigen binding protein is a protein in which the antigen binding region comprises a combination of complementarity determining regions (CDRs) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of: a) the CDR-H1 (SEQ ID NO:24), CDR-H2 (SEQ ID NO:25) and CDR-H3 (SEQ ID NO:26) sequences contained in SEQ ID NO:1 and the CDR-L1 (SEQ ID NO:27), CDR-L2 (SEQ ID NO:28) and CDR-L3 (SEQ ID NO:29) sequences contained in SEQ ID NO:2, and b) the CDR-H1, CDR-H2 and CDR-H3 sequences contained in SEQ ID NO:3 and the CDR-L1, CDR-L2 and CDR-L3 sequences contained in SEQ ID NO:20. Preferably, the multispecific antigen binding protein is a protein in which the antigen binding region comprises a combination of complementarity determining regions (CDRs) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of: a) the CDR-H1 (SEQ ID NO:24), CDR-H2 (SEQ ID NO:25) and CDR-H3 (SEQ ID NO:26) sequences contained in SEQ ID NO:1 and the CDR-L1 (SEQ ID NO:27), CDR-L2 (SEQ ID NO:28) and CDR-L3 (SEQ ID NO:29) sequences contained in SEQ ID NO:2. H V contained in the sequence and SEQ ID NO:2 L and b) V contained in SEQ ID NO: 3 H V contained in the sequence and SEQ ID NO: 20 L A variable light (V) selected from the group consisting of L ) and variable weight (V H ) domain combinations.

[0017] In one embodiment, the multispecific antigen-binding protein is a multispecific antigen-binding protein in which at least one NK cell-activating cytokine is conjugated to a region having affinity for a surface antigen expressed on an NK cell. In one embodiment, the at least one NK cell-activating cytokine forms a single polypeptide chain with at least one polypeptide chain of the region having affinity for a surface antigen expressed on an NK cell, and optionally the NK cell-activating cytokine is linked to the polypeptide chain of the region having affinity for a surface antigen expressed on an NK cell via a flexible linker. Preferably, the single polypeptide chain comprises, in order from N-terminus to C-terminus: i) a first NK cell-activating cytokine, ii) optionally a first flexible linker, iii) a polypeptide chain of the region having affinity for a surface antigen expressed on an NK cell, iv) optionally a second flexible linker, and v) a second NK cell-activating cytokine, and preferably the first and second NK cell-activating cytokines are different NK cell-activating cytokines. Preferably, at least one antigen-binding region that specifically binds to an NK cell activating receptor is conjugated to a region having affinity for a surface antigen expressed on an NK cell. Preferably, at least one polypeptide chain of the at least one antigen-binding region that specifically binds to an NK cell activating receptor forms a single polypeptide chain with at least one polypeptide chain of the region having affinity for a surface antigen expressed on an NK cell. Preferably, the single polypeptide chain comprises, in order from N-terminus to C-terminus, i) at least one polypeptide chain of the at least one antigen-binding region that specifically binds to an NK cell activating receptor, ii) optionally a flexible linker, and iii) a region having affinity for a surface antigen expressed on an NK cell. In one embodiment, the region having affinity for a surface antigen expressed on an NK cell is a dimeric immunoglobulin Fc region, each of the two polypeptide chains of the dimeric Fc region is linked to a CH1 domain, and each of the CH1 domains is linked to an immunoglobulin variable region that specifically binds to an NK cell activating receptor.Preferably, the two immunoglobulin variable regions bind to the same NK cell activating receptor, or the two immunoglobulin variable regions each bind to a different NK cell activating receptor. In one embodiment, the protein comprises a dimeric immunoglobulin Fc region, wherein each of the two Fc polypeptide chains is operably linked to a Fab that specifically binds to an NK cell activating receptor.

[0018] In one embodiment, the multispecific antigen-binding protein is a multispecific antigen-binding protein in which at least one NK cell-activating cytokine is conjugated to at least one antigen-binding region that specifically binds to an NK cell-activating receptor or a region that has affinity for a surface antigen expressed on an NK cell. Preferably, at least one of the NK cell-activating cytokines forms a single polypeptide chain with i) at least one polypeptide chain in the at least one antigen-binding region that specifically binds to an NK cell-activating receptor and ii) at least one polypeptide chain in the region that has affinity for a surface antigen expressed on an NK cell, optionally with a flexible linker between the agonist and at least one polypeptide chain in the region defined in i) or ii). In one embodiment, at least one of the NK cell-activating cytokines forms a single polypeptide chain with i) at least one light chain of at least one of two Fabs that specifically bind to an NK cell-activating receptor and ii) at least one of two Fc chains in a dimeric immunoglobulin Fc region, optionally with a flexible linker between the agonist and the light chain defined in i) or the Fc chain defined in ii). Preferably, at least one of the NK cell-activating cytokines is fused to: i) the N-terminus of the light chain of at least one of the two Fabs that specifically bind to the NK cell-activating receptor, optionally through a flexible linker; ii) the C-terminus of the light chain of at least one of the two Fabs that specifically bind to the NK cell-activating receptor, optionally through a flexible linker; iii) the N-terminus of the heavy chain of at least one of the two Fabs that specifically bind to the NK cell-activating receptor; and iv) the C-terminus of the heavy chain of at least one of the two Fc chains in the dimeric immunoglobulin Fc region, optionally through a flexible linker. In one embodiment, at least one of the NK cell-activating cytokines is present on at least one or both sides of the immunoglobulin structure.

[0019] In one embodiment, the multispecific antigen binding protein is heterodimeric with respect to at least one of: i) an antigen binding region that specifically binds to an NK cell activating receptor; and ii) at least one fusion NK cell activating cytokine, wherein the dimeric Fc region comprises distinct first and second polypeptide chains, the distinct first and second polypeptide chains comprising knob-into-hole modifications that facilitate association of the first and second polypeptide chains of the Fc region.

[0020] In one embodiment the multispecific antigen binding protein is a multispecific antigen binding protein with at least one biological activity selected from the following: a) the multispecific antigen binding protein causes an increase in at least one NK cell activity selected from CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cytotoxicity, whereby preferably the increase is at least 0.1 fold higher compared to the increase achieved at the same effector:target cell ratio using the same NK cells and target cells that have not been contacted with the multispecific antigen binding protein; and b) the multispecific antigen binding protein causes an increase in at least one NK cell activity selected from CD107a degranulation, CD107 or CD69 expression, IFNy production, NK proliferation and NK cytotoxicity, whereby preferably the increase is at least 0.1 fold higher compared to the increase achieved at the same effector:target cell ratio using the same NK cells and target cells that have been contacted with a conventional human IgG1 monoclonal antibody having the same NK cell activating receptor-specific antigen binding region as the multispecific antigen.

[0021] In one embodiment, the multispecific antigen binding protein is characterized in that ex vivo expansion of donor NK cells by co-culture with a multispecific antigen binding protein described herein results in a) a fold increase in the number of expanded NK cells that is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or greater than the fold increase in the number of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated K562 feeder cells modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells). 0 or 5.0-fold; b) the telomere length of the expanded NK cells is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55% compared to the telomere length of fresh NK cells, and preferably the increase in telomere length of the expanded NK cells compared to the telomere length of fresh NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0-fold greater than the increase in telomere length of NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells. c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the expression level on expanded NK cells obtained by ex vivo expansion in co-culture with irradiated FC21 feeder cells; d) the secretion of at least one cytokine of TNF-α, IFN-γ and IL-6 by the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the expression level on expanded NK cells obtained by ex vivo expansion in co-culture with irradiated FC21 feeder cells; e) the cytotoxicity of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times that of the expanded NK cells obtained by ex vivo expansion in co-culture with irradiated FC21 feeder cells; and f) the cytotoxicity of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5 times that of the expanded NK cells obtained by ex vivo expansion in co-culture with irradiated FC21 feeder cells.A multispecific antigen-binding protein that generates a population of expanded NK cells having one or more characteristics selected from the following:

[0022] In one embodiment, the ex vivo expansion of donor NK cells by co-culture with a multispecific antigen binding protein described herein and co-stimulation with irradiated tumor cells or tumor cells results in a) a fold increase in the number of expanded NK cells of at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the fold increase in the number of expanded NK cells; b) a decrease in the telomere length of the expanded NK cells; is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55% compared to the telomere length of fresh NK cells, preferably by at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 fold increase in telomere length of expanded NK cells compared to the telomere length of fresh NK cells; c) on expanded NK cells d) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46, and CD16 is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times higher than that of the control group; d) the secretion of at least one cytokine of TNF-α, IFN-γ, and IL-6 by the proliferated NK cells is at least and e) the cytotoxicity of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times greater.

[0023] In a second aspect, the present invention relates to a pharmaceutical composition comprising a multispecific antigen-binding protein as described herein and a pharmaceutically acceptable carrier.

[0024] In a third aspect, the present invention provides an ex vivo method for expanding NK cells, comprising the step of contacting NK cells with a multispecific antigen binding protein or a pharmaceutical composition comprising the protein as described herein, preferably wherein the expanded NK cells are expanded such that: a) the fold increase in the number of expanded NK cells is at least 0.001, 0.002, 0.005, 0.006, 0.008, 0.009, 0.100, 0.110, 0.120, 0.130, 0.140, 0.150, 0.160, 0.170, 0.180, 0.190, 0.200, 0.210, 0.220, 0.230, 0.240, 0.250, 0.310, 0.320, 0.330, 0.340, 0.350, 0.410, 0.420, 0.430, 0.440, 0.450, 0.510, 0.520, 0.530, 0.540, 0.550, 0.610, 0.620, 0.710, 0.720, 0.810, 0.820, 0.830, 0.840, 0.850, 0.900, 0.910, 0.920, 0.930, 0.940, 0.950, 0.960, 0.970, 0.980, 0.990, 0.990, 1000, 1000, 1000, 1000, 1000, 1000, b) the telomere length of the expanded NK cells is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55% increased compared to the telomere length of fresh NK cells, preferably at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.02, 0.05, 0.1 times the increase in telomere length of NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells; , 0.2, 0.5, 1.0, 2.0 or 5.0 times the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on the expanded NK cells, which is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the expression level on expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells; d) at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the expression level on expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells; and e) the secretion of at least one cytokine is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times that of the cytokine secreted by expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells, and f) the cytotoxicity of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times that of the cytotoxicity of the expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells.The method has one or more characteristics selected from the group consisting of 1, 0.2, 0.5, 1.0, 2.0, or 5.0 times.

[0025] Furthermore, the present invention provides an ex vivo method for expanding NK cells, comprising the step of contacting NK cells with a multispecific antigen binding protein as described herein or a pharmaceutical composition comprising the protein and irradiated tumor cells or tumor cells, preferably the expanded NK cells exhibit a fold increase in the number of expanded NK cells of at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 fold, b) the telomere length of the expanded NK cells is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55% compared to the telomere length of fresh NK cells, preferably by at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 3.0 fold compared to the telomere length of fresh NK cells. or 5.0-fold; c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46, and CD16 on the proliferated NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0-fold; d) the expression level of at least one of TNF-α, IFN-γ, and IL-6 by the proliferated NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0-fold. a) cytokine secretion that is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0-fold greater; and b) cytotoxicity of the expanded NK cells that is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0-fold greater.In one embodiment the multispecific antigen binding protein causes an increase in Glut1, Glut3, CD71 and / or CD98 expression, mitochondrial mass, glycolysis rate, ratio of glycolysis rate to oxidative phosphorylation rate, metabolic fuel flexibility between glucose, glutamine and / or fatty acids, whereby preferably the increase is at least 0.05 fold higher compared to the increase achieved at the same effector:target cell ratio using the same NK cells and target cells that have not been contacted with the multispecific antigen binding protein; b) the multispecific antigen binding protein causes an increase in at least one of Glut1, Glut3, CD71 or CD98 expression, mitochondrial mass, glycolysis rate, ratio of glycolysis rate to oxidative phosphorylation rate, metabolic fuel flexibility between glucose, glutamine and fatty acids, whereby preferably the increase is at least 0.05 fold higher compared to the increase achieved at the same effector:target cell ratio using the same NK cells and target cells that have been contacted with a conventional human IgG1 monoclonal antibody having the same TAA-specific antigen binding region as the multispecific antigen.

[0026] In one embodiment, ex vivo expansion of donor NK cells by co-culture with a multispecific antigen binding protein described herein generates a population of expanded NK cells having one or more characteristics selected from the following: a) a decrease in mitochondrial mass or nutrient transporter expression on the expanded NK cells obtained by ex vivo expansion by co-culture with irradiated K562 feeder cells modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells) of at least 0.001, 0.002, 0.00 a) a mitochondrial mass or expression of at least one nutrient transporter, Glut1, Glut3, CD71, or CD98, that is at least 5, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, or 20 times greater than the glycolytic rate of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated K562 feeder cells (FC21 feeder cells) modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand. c) the ratio of glycolysis rate to oxidative phosphorylation (OxPhos) rate is at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, or 20.0 times that of the ratio of glycolysis rate to oxidative phosphorylation (OxPhos) rate of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated K562 feeder cells modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells). 0, 10.0, or 20.0 times the metabolic fuel flexibility between glucose, glutamine, and fatty acids of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated K562 feeder cells modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells); and d) the metabolic fuel flexibility between glucose, glutamine, and fatty acids is at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, or 10.0 times the metabolic fuel flexibility between glucose, glutamine, and fatty acids of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated K562 feeder cells modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells).

[0027] In a fourth aspect, the present invention relates to a multispecific antigen-binding protein as described herein, a pharmaceutical composition comprising the protein or ex vivo expanded NK cells obtained by the method described above, optionally in combination with a multispecific antigen-binding protein, for use as a medicament.

[0028] In a fifth aspect, the present invention relates to a multispecific antigen-binding protein as described herein, a pharmaceutical composition comprising the protein or ex vivo expanded NK cells obtained by the method described above, optionally in combination with the multispecific antigen-binding protein, for use in the treatment of cancer. In one embodiment, the present invention relates to a multispecific antigen-binding protein as described herein or a pharmaceutical composition comprising the protein for use in the treatment of cancer, wherein the multispecific antigen-binding protein or composition is used in combination with the adoptive transfer of immune cells, preferably the immune cells are selected from T cells and NK cells.

[0029] In a sixth aspect, the present invention relates to a method of enhancing the anti-tumor activity of NK cells in a subject, the method comprising the step of administering to the subject a multispecific antigen-binding protein as described herein, a pharmaceutical composition comprising the protein, ex vivo-expanded NK cells obtained by the method, optionally in combination with the multispecific antigen-binding protein, or a combination of the multispecific antigen-binding protein and immune cells selected from T cells and NK cells. In one embodiment of the method, the subject has cancer. In one embodiment, the present invention relates to a multispecific antigen-binding protein, a composition comprising the protein, or ex vivo-expanded NK cells, optionally in combination with the multispecific antigen-binding protein, for the above uses, wherein at least one of a) the multispecific antigen-binding protein and / or the ex vivo-expanded NK cells are administered as neoadjuvant therapy prior to primary therapy comprising at least one of surgery and radiation therapy for the cancer, and b) the multispecific antigen-binding protein and / or the ex vivo-expanded NK cells are administered as adjuvant therapy after primary therapy comprising at least one of surgery and radiation therapy for the cancer.

[0030] In a seventh aspect, the present invention relates to a nucleic acid molecule comprising one or more nucleotide sequences encoding the polypeptide chains of a multispecific antigen-binding protein as described herein. Preferably, the nucleic acid molecule is one in which the one or more nucleotide sequences are operably linked to regulatory sequences for expression of the one or more polypeptide chains in a host cell.

[0031] In an eighth aspect, the present invention relates to a host cell comprising a nucleic acid molecule as defined above.

[0032] In a ninth aspect, the present invention relates to a method of producing a multispecific antigen-binding protein as described herein comprising culturing a host cell as defined above such that one or more nucleotide sequences are expressed and the multispecific antigen-binding protein is produced. Preferably, the method further comprises the steps of recovering the multispecific antigen-binding protein and optionally formulating the multispecific antigen-binding protein with a pharmaceutically acceptable carrier.

[0033] Description of the Invention definition Various terms relating to the methods, compositions, uses, and other aspects of the present invention are used throughout the specification and claims. Such terms should be given their ordinary meaning in the art to which the invention pertains, unless otherwise specified. Other specifically defined terms should be construed in a manner consistent with the definitions provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, the preferred materials and methods are described herein.

[0034] "A," "an," and "the": These singular terms include plural referents unless the content clearly dictates otherwise. Thus, the indefinite article "a" or "an" typically means "at least one." Thus, for example, reference to a "cell" includes a combination of two or more cells, and the like.

[0035] "About" and "approximately": When referring to measurable values, such as amounts, durations, etc., these terms are meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, where such variations are appropriate for carrying out the disclosed methods. Additionally, amounts, ratios, and other numerical values ​​may be presented herein in a range format. Such range formats are used for convenience and brevity and include numerical values ​​explicitly specified as range limits, but it should be understood that they should be interpreted flexibly to include all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, as well as subranges such as about 10 to about 50, about 20 to about 100, etc.

[0036] "And / or": The term "and / or" refers to a situation in which one or more of the stated cases may occur alone or in combination with at least one of the stated cases, up to all of the stated cases.

[0037] "Comprises": This term is intended to be inclusive and open-ended, not exclusive. Specifically, this term and variations thereof mean that the specified features, steps, or components are included. These terms should not be interpreted to exclude the presence of other features, steps, or components.

[0038] Exemplary: This term means serving as an example, instance, or illustration, and should not be construed as excluding other configurations disclosed herein.

[0039] As used herein, "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer is also called a malignant neoplasm.

[0040] As used herein, "in combination with" is intended to refer to all dosage forms that provide a first drug with an additional (second, third) drug. The drugs may be administered simultaneously, separately, or sequentially, in any order. Drugs administered in combination have biological activity in the subject to which they are delivered.

[0041] As used herein, "simultaneous" administration refers to the administration of two or more drugs at the same time, but not necessarily via the same route of administration or in the form of a single combined formulation. For example, one drug may be provided orally, while the other may be provided intravenously during a patient's hospital visit. Separate includes administration of drugs in separate forms and / or at separate times, again not necessarily via the same route of administration. Sequential indicates that administration of a first drug is followed immediately or within a time period by administration of a second drug.

[0042] As used herein, "compositions," "products," or "combinations" useful in the methods of the present disclosure include those suitable for various routes of administration, including, but not limited to, intravenous, subcutaneous, intradermal, subcutaneous, intranodal, intratumoral, intramuscular, intraperitoneal, oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol, and / or parenteral or mucosal application. Compositions, formulations, and products according to the present disclosure typically comprise a drug (alone or in combination) and one or more suitable pharmaceutically acceptable excipients.

[0043] As used herein, "effective amount" refers to the amount of agent required to improve disease symptoms compared to untreated patients. The effective amount of an active agent used to practice the present invention for the therapeutic treatment of cancer varies depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the appropriate amount and administration regimen is determined by the attending physician or veterinarian. Such an amount is referred to as an "effective" amount. Thus, in the context of this disclosure, with respect to the administration of a drug that is "effective" against a disease or condition, it means that administration in a clinically relevant manner will result in a beneficial effect in at least a statistically significant proportion of patients, such as symptomatic improvement, cure, reduction of at least one disease sign or symptom, prolongation of life, improvement in quality of life, or other effect generally recognized as positive by physicians familiar with the treatment of a particular type of disease or condition.

[0044] As used herein, the term "NK cells" refers to a subpopulation of lymphocytes involved in innate immunity. NK cells can be identified by certain characteristics and biological properties, such as the expression of specific surface antigens, including CD56 and / or NKp46 on human NK cells, the absence of alpha / beta or gamma / delta TCR complexes on the cell surface, the ability to recognize and kill cells that fail to 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 various techniques known in the art, such as isolation from blood samples, cytopheresis, tissue or cell collection, etc. Protocols useful for assays involving NK cells can be found in Natural Killer Cells Protocols (2000, edited by Campbell KS and Colonna M. Humana Press, pp. 219-238).

[0045] "Sequence identity" is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as can be determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutes with the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods. The terms "sequence identity" or "sequence similarity" mean that two (poly)peptide or two nucleotide sequences, when optimally aligned, preferably over their entire length (at least for the shortest sequences in the comparison), share at least a certain percentage of sequence identity, as defined elsewhere herein, when maximized and minimized by programs such as ClustalW (1.83), GAP, or BESTFIT using default parameters to maximize matches and minimize gaps. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty of 50 (nucleotides) / 8 (proteins) and a gap extension penalty of 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). A preferred multiple alignment program for aligning the protein sequences of the present invention is ClustalW (1.83) using the Blosum matrix and default settings (gap opening penalty: 10, gap extension penalty: 0.05).Sequence alignment and scoring for percent sequence identity can be determined using computer programs such as the GCG Wisconsin Package version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or open source software such as the programs "needle" (which uses the global Needleman-Wunsch algorithm) or "water" (which uses the local Smith-Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as GAP described above or default settings (for both "needle" and "water" and for both protein and DNA alignments, the default gap opening penalty is 10.0, the default gap extension penalty is 0.5, and the default scoring matrix is ​​Blosum62 for proteins and DNAFull for DNA). When sequences have substantially different overall lengths, local alignments such as those using the Smith-Waterman algorithm are preferred. Alternatively, percent similarity or identity can be determined by searching against public databases using algorithms such as FASTA, BLAST, etc.

[0046] Optionally, when determining the degree of amino acid similarity, those skilled in the art can also take into account so-called "conservative" amino acid substitutions, as will be apparent to those skilled in the art. Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. Examples of classes of amino acid residues for conservative substitutions are shown in the table below.

[0047] [Table 1]

[0048] Alternative conservative amino acid residue substitution classes.

[0049] [Table 2]

[0050] Alternative physical and functional classifications of amino acid residues.

[0051] [Table 3]

[0052] The term "agent" generally refers to any entity that is not normally present or present at the levels administered to a cell, tissue, or subject. An agent may be a compound or a composition. An agent may, for example, be selected from the group consisting of polynucleotides, polypeptides, small molecules, (multispecific) antigen-binding proteins, such as antibodies, and functional fragments thereof.

[0053] The term "antigen-binding domain" or "antigen-binding region" refers to a portion of an antigen-binding protein that is capable of specifically binding to an antigen or epitope. In one embodiment, an antigen-binding region is, for example, an antibody light chain variable region (V L ) and antibody heavy chain variable region (V H and Fab'. Examples of such antigen-binding regions include single-chain Fv (scFv), single-chain antibodies, Fv, single-chain Fv2 (scFv2), Fab, and Fab'. In one embodiment, the antigen-binding region is an immunoglobulin-derived antigen-binding region derived from a single domain antibody, which consists only of a heavy chain and is devoid of light chains, as known, for example, from camelids, in which the antigen-binding site is present on and formed by the single variable domain (also called "immunoglobulin single-chain variable domain" or "ISVD"). An example of such an ISVD is a camelid heavy chain antibody (V), also called a nanobody. HH), domain antibodies (dAbs), and single domains derived from shark antibodies (IgNAR domains). In other embodiments, the antigen-binding region comprises a non-immunoglobulin-derived domain capable of specifically binding to an antigen or epitope, such as darpin; affilin; anticalin, etc.

[0054] The term "antibody" as used 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 and / or immunological activity. Various techniques related to the production of antibodies are described, for example, in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). Antibodies may be human and / or humanized. "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.

[0055] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a native antibody. "Native antibody" refers to naturally occurring immunoglobulin molecules with various structures. For example, native IgG class antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two disulfide-bonded light chains and two heavy chains. Each heavy chain has, from N- to C-terminus, a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, CH3), also called a heavy chain constant region. Similarly, each light chain has, from N- to C-terminus, a variable region (VL), also called a variable light domain or light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region. The heavy chain of an antibody can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or m (IgM), some of which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0056] An "antibody fragment" comprises a portion of a full-length antibody, such as the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)s, and Fv (typically the V region of a single arm of an antibody). H Domestic and V L domain), single chain Fv (scFv), dsFv, Fd fragments (typically V H domain and CH1 domain) and dAbs (typically V H domain) fragment;V H , V L and V HThese include H- and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappabodies (e.g., Ill et al. Protein Eng 1997; 10: 949-57); camelid IgG; IgNAR; and multispecific antibody fragments formed from antibody fragments and one or more isolated CDRs or functional paratopes, where the isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together to form a functional antibody fragment. For a review of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, e.g., Plueckthun, in *The Pharmacology of Monoclonal Antibodies*, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NY, pp. 269-315 (1994); see also WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have increased in vivo half-lives, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific (see, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Various types of antibody fragments are described or reviewed in, for example, Heiliger and Hudson, Nat Biotechnol 2005; 23, 1126-1136; WO 2005 / 040219, US Patent Application Publication No. 20050238646 and US Patent Application Publication No. 20020161201.Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., CHO, E. coli, or phage), as described herein.

[0057] The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies are known in the art and are described, for example, in Harlow and Lane, "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, NY (1988); Hammerling et al., in: "Monoclonal Antibodies and T-Cell Hybridomas," Elsevier, NY (1981), pp. 563-681, both of which are incorporated herein by reference in their entireties.

[0058] As used herein, the term "monospecific" antibody indicates that the antibody portion of the multispecific antigen-binding protein described herein has one or more antigen-binding sites, each of which binds to the same epitope of the same antigen. The term "bispecific" means that the antibody portion of the multispecific antigen-binding protein described herein has at least two antigen-binding sites capable of specifically binding to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, a bispecific antigen-binding molecule can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0059] The term "valency" or "valency" as used herein refers to the presence of a specific number of binding sites in an antigen-binding molecule. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen-binding molecule.

[0060] Antibodies immunologically reactive with a specific antigen can be produced by recombinant methods, such as selection of libraries of recombinant antibodies in phage or similar vectors (see, e.g., Huse et al., Science 246: 1275-1281 (1989); Ward et al., Nature 341: 544-546 (1989); and Vaughan et al., Nature Biotech. 14: 309-314 (1996)), or by immunizing animals with the antigen or DNA encoding the antigen. Methods for producing and screening specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with the antigen of interest or cells expressing such antigen. Once an immune response is detected, for example, antibodies specific to the antigen are detected in the mouse serum, the mouse spleen is harvested, and splenocytes are isolated. The splenocytes are then fused to any suitable myeloma cells by well-known techniques. Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding the antigen. Ascites fluid, which generally contains high levels of antibodies, can be generated by inoculating mice intraperitoneally with positive hybridoma clones.

[0061] Typically, immunoglobulins have heavy and light chains. Each heavy and light chain contains a constant region and a variable region (the regions are also known as "domains"). The light and heavy chain variable regions contain four "framework" regions interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody, which are the combined framework regions of the constituent light and heavy chains, function to position and align the CDRs in three-dimensional space.

[0062] When used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al. 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, USA) and / or residues from the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol. 1987; 196: 901-917). Typically, the numbering of amino acid residues in this region is performed according to the method described by Kabat et al., supra. Expressions such as "Kabat position," "variable domain residue numbering similar to Kabat," and "according to Kabat" herein refer to this numbering system for the heavy chain variable domain or 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 insertion after residue 52 of CDR H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat numbered sequence in the region of homology.

[0063] As used herein, the term "framework" or "FR" residues refers to the regions of an antibody variable domain excluding the regions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions separated by the CDRs (FR1, FR2, FR3, and FR4).

[0064] As defined herein, the term "constant region" refers to the constant region from an antibody encoded by one of the light or heavy chain immunoglobulin constant region genes. As used herein, "constant light chain" or "light chain constant region" refers to the region of an antibody encoded by the kappa (Ck) or lambda (Cλ) light chain. The constant light chain typically comprises a single domain and, as defined herein, refers to positions 108-214 of Cκ or Cλ, numbering according to the EU index (Kabat et al., 1991, supra).

[0065] As used herein, the terms "constant heavy chain" or "heavy chain constant region" refer to the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon gene, which define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For full-length IgG antibodies, the constant heavy chain as defined herein extends from the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, and thus includes positions 118 to 447, numbered according to the EU index.

[0066] Papain digestion of an intact antibody generates two identical antigen-binding fragments called "Fab" fragments, each containing the heavy and light chain variable domains and the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Therefore, as used herein, the term "Fab fragment" or "or" refers to an antibody fragment containing a light chain fragment containing the VL domain and constant domain (CL) of the light chain and the VH domain and the first constant domain (CH1) of the heavy chain. Fab may refer to this region alone or in the context of a polypeptide, a multispecific antigen-binding protein, or an antigen-binding region, or any other embodiment outlined herein. Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab')2 fragment containing two antigen-binding sites (two Fab fragments) and part of the Fc region.

[0067] As used herein, the term "single-chain Fv" or "scFv" refers to the V H and V L Fv refers to an antibody fragment containing V domains, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide contains a V domain that enables the scFv to form the desired structure for antigen binding. H Domains and V L The scFv domains further comprise a polypeptide linker between them. Methods for generating 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, NY, pp. 269-315 (1994).

[0068] "Scaffold antigen-binding proteins" are known in the art; for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen-binding domains. See, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13: 245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008). In one aspect of the invention, the scaffold antigen binding protein is a protein A-derived molecule such as CTLA-4 (Evibody), lipocalin (Anticalin), Z domain of protein A (Affibody), A domain (Avimer / Maxibody), serum transferrin (trans-body); designed ankyrin repeat proteins (DARPins), variable domains of antibody light or heavy chains (single domain antibodies, sdAb), variable domains of antibody heavy chains (nanobodies, aVH), V NAR Fragment, fibronectin (AdNectin), C-type lectin domain (Tetranectin); neoantigen receptor beta-lactamase (V N fragments), the variable domains of human gamma-crystallin or ubiquitin (affilin molecules); Kunitz domains of human protease inhibitors, microbodies such as the knottin family of proteins, peptide aptamers and fibronectin (adnectins).

[0069] CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is primarily expressed by CD4 +It is a CD28 family receptor expressed on T cells. Its extracellular domain has a variable domain-like Ig fold. The loops corresponding to the CDRs of an antibody can be replaced with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as evibodies (e.g., U.S. Patent No. 7,166,697 B1). Evibodies are approximately the same size as the isolated variable regions of antibodies (e.g., domain antibodies). For further details, see Journal of Immunological Methods 248 (1-2), 31-45 (2001).

[0070] Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. They have a rigid beta-sheet secondary structure with several loops at the open end of the conical structure that can be engineered to bind different target antigens. Anticalins are 160-180 amino acids in size and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482: 337-350 (2000), U.S. Patent No. 7,250,297 B1, and U.S. Patent Application Publication No. 20070224633.

[0071] Affibodies are scaffolds derived from Staphylococcus aureus protein A that can be engineered to bind antigens. The domain consists of a triple helix bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details, see Protein Eng. Des. Sel. 17, 455-462 (2004) and European Patent Application Publication No. 1641818A1.

[0072] Avimers are multidomain proteins derived from the A-domain scaffold family. Natural domains of approximately 35 amino acids adopt defined disulfide bond structures. Diversity is generated by shuffling of natural mutations exhibited by the A-domain family. For further details, see Nature Biotechnology 23 (12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16 (6), 909-917 (June 2007).

[0073] Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind to different target antigens by inserting peptide sequences into permissive surface loops. Examples of engineered transferrin scaffolds include transbodies. For further details, see J. Biol. Chem 274, 24066-24073 (1999).

[0074] Designed ankyrin repeat proteins (DARPins) are derived from ankyrins, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha-helices and a beta-turn. They can be engineered to bind to different target antigens by randomizing residues in the first alpha-helix and beta-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). Further details can be found in J. Mol. Biol. 332, 489-503 (2003), PNAS 100 (4), 1700-1705 (2003), and J. Mol. Biol. 369, 1015-1028 (2007), as well as U.S. Patent Application Publication No. 20040132028A1.

[0075] Single domain antibodies are antibody fragments that consist of a single monomeric variable antibody domain. The first single variable domain is the variable domain of a camelid antibody heavy chain (nanobody or V H Furthermore, the term single variable domain antibody can be used to describe an autonomous human heavy chain variable domain (aVH) or a VH derived from a shark. NAR Contains fragments.

[0076] Fibronectin is a scaffold that can be engineered to bind to antigens. Adnectins consist of a backbone of the native amino acid sequence of the tenth domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the beta-sandwich can be engineered to enable Adnectins to specifically recognize therapeutic targets of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), U.S. Patent Application Publication No. 20080139791, WO2005056764, and U.S. Patent No. 6,818,418 B1.

[0077] Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA), containing a constrained variable peptide loop inserted into the active site. For details, see Expert Opin. Biol. Ther. 5, 783-797 (2005).

[0078] Microbodies are derived from naturally occurring microproteins 25-50 amino acids long containing three to four cysteine ​​bridges - examples of microproteins include KalataBI, conotoxins, and knottins. Microproteins have loops that can be engineered to contain up to 25 amino acids without affecting the overall fold of the microprotein. For further details on engineered knottin domains, see WO2008098796.

[0079] As used herein, the term "Fv" or "Fv fragment" or "Fv region" refers to the V domain of a single antibody. H and V L It refers to a polypeptide containing a domain.

[0080] As used herein, the term "Fc" or "Fc region" refers to a polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain. Fc may refer to this region in the context of an isolated Fc region or Fc polypeptide, as described below. As used herein, "Fc polypeptide" or "Fc-derived polypeptide" refers to 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 present invention also include variants containing at least one modification that alters (enhances or attenuates) an Fc-associated effector function. Fc regions according to the present invention also include chimeric Fc regions comprising different portions or domains of different Fc regions, e.g., derived from antibodies of different isotypes or species. 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, along with the flexible hinge N-terminal to these domains. In the case of IgA and IgM, the Fc may include the J chain. In the case of IgG, the Fc comprises immunoglobulin domains Cγ2 (CH2) and Cγ3 (CH3) and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is usually defined to include residues C226, P230, or A231 at its carboxyl terminus, numbered according to the EU index. The "CH2 domain" of a human IgG Fc region usually extends from about amino acid residue 231 to about amino acid residue 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises the stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to about amino acid residue 447 of IgG).The CH3 region herein can be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "bulge" ("knob") in one chain and a corresponding introduced "cavity" ("hole") in the other chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains can be used to promote heterodimerization of two non-identical antibody heavy chains as described herein. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region can be present or absent. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0081] The "knob-into-hole" technique has been described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protuberance ("knob") into the interface of a first polypeptide and a corresponding cavity ("hole") into the interface of a second polypeptide, such that the protuberance can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity of identical or similar size to the protuberance is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller side chain (e.g., alanine or threonine). The protuberance and cavity can be created by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc region, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc region. In a further specific embodiment, the subunit of the Fc region comprising the knob modification further comprises the amino acid substitution S354C, and the subunit of the Fc region comprising the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of disulfide bridges between the two subunits of the Fc region, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). Numbering follows the EU index in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0082] A "region equivalent to the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin as well as variants having modifications that result in substitutions, additions, or deletions, but that do not substantially reduce the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art to minimize impact on activity (see, e.g., Bowie, JU et al., Science 247:1306-10 (1990)).

[0083] The term "effector function" refers to biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0084] An "activating Fc receptor" is an Fc receptor that, upon engagement by the Fc region of an antibody, triggers signaling events that stimulate receptor-bearing cells to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89). A particular activating Fc receptor is human FcγRIIIa, also known as CD16 or CD16A (see UniProt accession number P08637, version 141). In humans, CD16 consists of two isoforms, CD16A and CD16B, encoded by two highly homologous genes. CD16A is a transmembrane protein expressed by lymphocytes and some monocytes, whereas CD16B is linked to the plasma membrane via a GPI anchor and is expressed primarily by neutrophils. Thus, when CD16 is referred to herein in relation to its expression on NK cells, CD16A is usually meant unless otherwise indicated.

[0085] As used herein, "variable region" refers to the V domains that make up the light chain immunoglobulin locus (including κ and λ) and the heavy chain immunoglobulin locus, respectively. L genes (including Vκ and Vλ) and / or V H The term "variable region" refers to 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 ) contain four conserved framework regions (FR) and three hypervariable regions (HVR). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0086] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally distinct loops ("hypervariable loops"). Naturally occurring four-chain antibodies generally contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). HVRs generally contain amino acid residues from the hypervariable loops and / or from the "complementarity-determining regions" (CDRs), the latter of which are most highly sequence variable and / or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Hypervariable regions (HVRs) are also called complementarity-determining regions (CDRs); these terms are used interchangeably herein with respect to the portions of the variable regions that form the antigen-binding region. This particular region is described in Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. Mol. Biol. 196: 901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein.The appropriate amino acid residues which encompass the CDRs as defined by each of the above-cited references are set forth below in Table A for comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. One of skill in the art can routinely determine which residues comprise a particular CDR, given the variable region amino acid sequence of an antibody.

[0087] [Table 4]

[0088] Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. One skilled in the art can unambiguously assign this "Kabat numbering" system to any variable region sequence without relying on experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody variable region follow the Kabat numbering system.

[0089] With the exception of CDR1 in VH, CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also comprise "specificity-determining residues" or "SDRs," which are residues that contact the antigen. SDRs are contained within regions of CDRs referred to as abbreviated CDRs or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDRL2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, amino acid residues 50-55 of L2, amino acid residues 89-96 of L3, amino acid residues 31-35B of H1, amino acid residues 50-58 of H2, and amino acid residues 95-102 of H3 (see Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0090] As used herein, in the context of an antigen-binding molecule (e.g., an antibody), the term "affinity matured" refers to an antigen-binding molecule derived from a reference antigen-binding molecule, e.g., by mutation, that binds to the same antigen as the reference antibody, preferably to the same epitope, and has a higher affinity for the antigen than the reference antigen-binding molecule. Affinity maturation generally involves modifying one or more amino acid residues in one or more CDRs of the antigen-binding molecule. Typically, the affinity-matured antigen-binding molecule binds to the same epitope as the initial reference antigen-binding molecule.

[0091] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and m, respectively.

[0092] A "blocking" or "antagonist" antibody is one that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. As used herein, an "agonist antibody" is an antibody that mimics at least one functional activity of a polypeptide of interest.

[0093] The term "specifically binds" refers to the number of different antigens or antigenic determinants that a particular antigen-binding region or antigen-binding protein can bind. The specificity of an antigen-binding protein can be determined based on affinity and / or avidity. The equilibrium constant for the dissociation of an antigen from an antigen-binding protein (K D Affinity, expressed as 1 / K, is a measure of the binding strength between an antigenic determinant and an antigen-binding site on an antigen-binding protein. Alternatively, affinity can be expressed as 1 / K D The affinity constant (K A ) Affinity can be determined in a manner known per se, depending on the specific combination of antigen-binding protein of interest and antigen. As used herein, avidity is understood to refer to the strength of binding of a target molecule to multiple binding sites by a larger complex of binding agents, i.e., the strength of binding in multivalent binding. Avidity relates to both the affinity between an antigenic determinant and its antigen-binding site on the antigen-binding protein and the valency, i.e., the number of binding sites present on the antigen-binding protein. Affinity, on the other hand, refers to a simple monovalent receptor-ligand system.

[0094] Typically, the antigen-binding region or region of the multispecific antigen-binding protein of the invention that has affinity for a surface antigen expressed on NK cells has a molecular weight of about 10 -6 ~10 -12 M or less, preferably 10 -8 ~10 -12 The dissociation constant (K D ) and / or at least 10 -6 M or 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9M, e.g., at least 10 -10 , 10 -11 , 10 -12 It binds to its target molecule (antigen) with a binding affinity of M or greater. 10 -4 Any K above M (i.e., less than 100 μM) D A value of 10 is generally considered to represent non-specific binding. Thus, an antigen-binding region that "specifically binds" to an antigen, as can be determined as described below, has a value of 10 -4 K below M D An antigen-binding domain is an antigen-binding domain that binds to an antigen with an affinity of less than 800, 400, 200, 100, 50, 10, or 5 nM, more preferably less than 1 nM, e.g., less than 500, 200, 100, 50, 10, or 5 pM, of a multispecific antigen-binding protein of the invention. A variety of methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present invention (see, e.g., 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)). Specific exemplary embodiments are described below.

[0095] "K D " or "K DThe "value" can be measured using an ELISA as described in the Examples herein, or by using a surface plasmon resonance assay using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ) with an immobilized antigen CM5 chip of approximately 10-50 response units (RU) at 25°C. Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) in 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of coupled protein. Following antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of antibody or Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25 μl / min. The association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (K) is calculated using a simple one-to-one Langmuir binding model (BIAcore evaluation software version 3.2) and by simultaneously fitting the association and dissociation sensorgrams. D ) is the ratio k off / k on See, for example, Chen, Y., et al., (1999) J. Mol Biol 293: 865-881. If the on-rate is 10 by the surface plasmon resonance assay described above, 6 M -1 S -1Above 1000 kJ / s, the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25°C in the presence of increasing antigen concentrations, as measured with a spectrometer such as a stopped-flow integrated spectrophotometer (Aviv Instruments) or an 8000 Series SLM-Aminco spectrophotometer (ThermoSpectronic) equipped with a stirred red cuvette.

[0096] The term "humanized antibody" or "humanized immunoglobulin" refers to an immunoglobulin comprising a human framework, at least one, and preferably all, complementarity-determining regions (CDRs) from a non-human antibody, and any constant regions present that are substantially identical to human immunoglobulin constant regions, i.e., at least about 85%, at least 90%, and at least 95% identical. Thus, all portions of a humanized immunoglobulin, except optionally for the CDRs, are substantially identical to corresponding portions of one or more native human immunoglobulin sequences. Often, framework residues in the human framework regions are substituted with corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at specific positions. See, e.g., Queen et al., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 (each incorporated by reference in its entirety). Antibodies can be modified by techniques such as CDR grafting (EP 239,400; PCT Publication WO 91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering, or resurfacing (EP 592,106; EP 519,596; Padlan, Mol. Immunol., 28: 489-498 (1991); Studnicka et al., Prot. Eng. 7: 805-814 (1994); Roguska et al., Proc. Natl. Acad. Sci. 91: 969-973 (1993)). (1994) and chain shuffling (U.S. Patent No. 5,565,332), which are incorporated herein by reference in their entireties.

[0097] One class of antigen-binding regions for use in the present invention corresponds to the amino acid sequence of a naturally occurring single variable domain but is "humanized", i.e., one or more amino acid residues in the amino acid sequence of said naturally occurring single variable domain sequence have been replaced by a V residue from a conventional four-chain antibody of human origin. H It comprises an immunoglobulin single variable domain (ISVD) having an amino acid sequence by substituting one or more of the amino acid residues present at corresponding positions in the domain. This can be done in a manner known per se, which will be clear to the skilled artisan, for example, as described by Jones et al. (Nature 321: 522-525, 1986); Riechmann et al. (Nature 332: 323-329, 1988); Presta (Curr. Op. Struct. Biol. 2: 593-596, 1992), Vaswani and Hamilton (Ann. Allergy, Asthma and Immunol., 1: 105-115, 1998); Harris (Biochem. Soc. Transactions, 23: 1035-1038, 1995); Hurle and Gross (Curr. Op. Biotech., 5: 428-433, 1994) and V. H This can be done based on certain prior art on humanization, including, for example, Vincke et al. (2009, J. Biol. Chem. 284: 3273-3284). Again, it should be noted that such humanized single variable domains of the present invention can be obtained by any suitable method known per se, and are therefore not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring single variable domains as starting material.

[0098] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0099] For purposes herein, an "acceptor human framework" is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework or human consensus framework "derived from" a human immunoglobulin framework may comprise the same amino acid sequence thereof or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0100] As an alternative to humanization, human antibodies can be generated. By "human antibody" is meant an antibody containing fully human light and heavy chains and constant regions, produced by any known standard method. For example, transgenic animals (e.g., mice) are available that, upon immunization, can produce a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining region PH gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies after immunization. See, e.g., Jakobovits et al., Proc. Nat. Acad. Sci. USA, 90: 255-1 (1993); Jakobovits et al., Nature, 362: 255-258 (1993). Alternatively, phage display technology (McCafferty et al., Nature 348: 552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro from donor-derived immunoglobulin variable (V) domain gene repertoires. According to this technology, antibody V domain genes are cloned in frame into either the major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of B cells. Phage display can be performed in a variety of formats. For a review, see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3: 564-571 (1993). Human antibodies can also be generated by in vitro activated B cells or SCID mice whose immune systems have been reconstituted with human cells.Once a human antibody is obtained, its encoding DNA sequence can be isolated, cloned, and introduced into an appropriate expression system, preferably a cell line of mammalian origin, which is then expressed and released into culture medium from which the antibody can be isolated.

[0101] As used herein, the term "tumor-associated antigen" (TAA) refers to any antigen, including, but not limited to, proteins, glycoproteins, gangliosides, carbohydrates, and lipids associated with cancer. Such antigens may be expressed on malignant cells or in the tumor microenvironment, such as tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrates. The term TAA specifically includes homologs of wild-type TAAs that differ from the wild-type TAAs as a result of tumor-specific mutations (which may be patient-specific or shared), resulting in altered amino acid sequences, i.e., so-called neoantigens.

[0102] The terms "nucleic acid construct" or "nucleic acid vector" are understood herein to mean an artificial nucleic acid molecule resulting from the use of recombinant DNA technology. Thus, the term "nucleic acid construct" does not include naturally occurring nucleic acid molecules, although a nucleic acid construct may include (parts of) naturally occurring nucleic acid molecules. The terms "expression vector" or "expression construct" refer to a nucleic acid molecule capable of effecting expression of a nucleotide sequence or gene in a host cell or host organism compatible with such expression vector or construct. These expression vectors typically contain regulatory sequence elements operably linked to the nucleotide sequence to be expressed to effect its expression. Such regulatory elements usually include at least appropriate transcriptional regulatory sequences and optionally a 3' transcription termination signal. Additional elements necessary or useful for effecting expression, such as expression enhancer elements, may also be present. The expression vector can be introduced into a suitable host cell and effect expression of the coding sequence in an in vitro cell culture of the host cell. While the expression vector is suitable for replication in the host cell or organism of the present invention, the expression construct is usually integrated into the genome of the host cell for maintenance. Techniques for introducing nucleic acids into cells are well established in the art, and any appropriate technique can be used depending on the particular situation. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as adenovirus, AAV, lentivirus, or vaccinia. For microorganisms, such as bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophages. The introduced nucleic acid may be on an extrachromosomal vector within the cell, or the nucleic acid may be integrated into the genome of the host cell. Integration may be facilitated by including sequences within the nucleic acid or vector that facilitate recombination with the genome, according to standard techniques. After introduction, the nucleic acid may be expressed to produce the encoded fusion protein.In some embodiments, host cells (which may include cells that have actually been transformed, although the cells are more likely to be the progeny of transformed cells) may be cultured in vitro under conditions for expression of the nucleic acid such that the encoded fusion protein polypeptide is produced, and where an inducible promoter is used, expression may require activation of the inducible promoter.

[0103] As used herein, the term "promoter" or "transcriptional regulatory sequence" refers to a nucleic acid fragment that functions to control transcription of one or more coding sequences, is located upstream in the direction of transcription of a transcription start site of the coding sequence, and is structurally identified by the presence of binding sites for DNA-dependent RNA polymerase, a transcription initiation site and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those of skill in the art that act to directly or indirectly regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example, by the application of a chemical inducer.

[0104] The term "selectable marker" is a term familiar to those skilled in the art and is used herein to describe any genetic entity that, when expressed, can be used to select one or more cells containing the selectable marker. The term "reporter" can be used interchangeably with marker, but is primarily used to refer to a visible marker, such as green fluorescent protein (GFP). Selectable markers can be dominant, recessive, or bidirectional.

[0105] As used herein, the term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous, and, where necessary to link two protein-coding regions, contiguous and in reading frame.

[0106] The terms "protein" and "polypeptide" are used interchangeably and refer to molecules made up of chains of amino acids, without reference to a particular mode of action, size, three-dimensional structure, or origin.

[0107] The term "signal peptide" (sometimes called signal sequence) refers to a short peptide (usually 16-30 amino acids long) present at the N-terminus of most newly synthesized proteins destined for the secretory pathway. At the end of the signal peptide, there is usually a stretch of amino acids that is recognized and cleaved by a signal peptidase during or after translocation (from the cytosol to the secretory pathway, i.e., ER) to generate a free signal peptide and the mature protein. Signal peptides are highly heterogeneous, and many prokaryotic and eukaryotic signal peptides are functionally interchangeable, even between different species, although the efficiency of protein secretion can depend on the signal peptide. Suitable signal peptides are generally known in the art and can be found, for example, in Kaell et al. (2004 J. Mol. Biol. 338: 1027-1036) and von Heijne (1985, J. Mol. Biol. 184 (1): 99-105).

[0108] The term "gene" refers to a DNA fragment comprising a region (transcribed region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell and operably linked to appropriate regulatory regions (e.g., a promoter). A gene usually comprises several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end) that includes a polyadenylation site. "Gene expression" refers to the process by which a DNA region operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into RNA that is biologically active, i.e., can be translated into a biologically active protein or peptide.

[0109] The term "homologous," when used to indicate the relationship between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that the nucleic acid or polypeptide molecule is naturally produced by a host cell or organism of the same species, preferably the same type or strain. When homologous to a host cell, the nucleic acid sequence encoding the polypeptide is typically (but not necessarily) operably linked to a different (heterologous) promoter sequence and, if applicable, a different (heterologous) secretion signal sequence and / or terminator sequence from those in its natural environment. It is understood that regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. When used to indicate the relationship between two nucleic acid sequences, the term "homologous" means that one single-stranded nucleic acid sequence can hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization can depend on many factors, including the amount of identity between the sequences and hybridization conditions, such as temperature and salt concentration, as discussed below.

[0110] The term "heterologous," when used with reference to a nucleic acid (DNA or RNA) or protein, refers to a nucleic acid or protein that does not naturally occur as part of the organism, cell, genome, or DNA or RNA sequence in which it is found, or that is found in a location or position within a cell, genome, or DNA or RNA sequence that is different from that in which it is found in nature. A heterologous nucleic acid or protein is not endogenous to the cell into which it is introduced, but is obtained from another cell, or is synthetically or recombinantly produced. Generally, although not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly, exogenous RNA encodes proteins that are not normally expressed in the cell in which the exogenous RNA is present. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or foreign proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is encompassed herein by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e., combinations in which at least two of the combined sequences are heterologous to each other.

[0111] Detailed Description of the Invention The present invention arises, in part, from the observation that multispecific antigen-binding proteins comprising an NK cell-activating cytokine that triggers at least one of the interleukin-21 receptor and 4-1BB on NK cells and comprising a region with affinity for a surface antigen expressed on NK cells, e.g., an Fc region, can induce NK cell hyperfunction, including NK cell proliferation, resistance to the tumor microenvironment, and an enhanced ability to mediate lysis of target cells, and a prolonged duration of these abilities (Figure 1). Such abilities can be further enhanced when the multispecific antigen-binding protein comprises at least one antigen-binding region that specifically binds to an NK cell-activating receptor.

[0112] Multispecific antigen-binding proteins In a first aspect, the present invention relates to a multispecific antigen-binding protein. In one embodiment, the multispecific antigen-binding protein comprises a) an NK cell-activating cytokine, and ii) a 4-1BB agonist, and b) a region having affinity for a surface antigen expressed on natural killer (NK) cells. The NK cell-activating cytokine is preferably at least one of i) an interleukin-21 receptor (IL21R) agonist, and ii) a 4-1BB agonist. The region having affinity for the NK cell surface antigen enables the multispecific antigen-binding protein to bind to NK cells, after which the NK cell-activating cytokine can induce a hyperactive phenotype in the NK cells. In one embodiment, the multispecific antigen-binding protein further comprises c) at least one antigen-binding region that specifically binds to an NK cell-activating receptor.

[0113] Thus, in one embodiment, the multispecific antigen binding protein comprises: a) an NK cell activating cytokine that is at least one of: i) an interleukin-21 receptor (IL21R) agonist, and ii) a 4-1BB agonist; b) a region having affinity for a surface antigen expressed on an NK cell; and c) at least one antigen binding region that specifically binds to an NK cell activating receptor, the NK cell activating receptor.

[0114] NK cell-activating cytokines Thus, the multispecific antigen-binding proteins described herein comprise at least one NK cell-activating cytokine. In one embodiment, the NK cell-activating cytokine is at least one of i) an interleukin-21 receptor (IL21R) agonist, and ii) a 4-1BB agonist. In one embodiment, the multispecific antigen-binding protein comprises at least an IL21R agonist. In one embodiment, the multispecific antigen-binding protein comprises at least a 4-1BB agonist. Also, in one embodiment, the multispecific antigen-binding protein comprises at least both an IL21R agonist and a 4-1BB agonist. In a further embodiment, the multispecific antigen-binding proteins described herein may comprise an additional NK cell-activating cytokine in addition to at least one of an IL21R agonist and a 4-1BB agonist. Such additional NK cell-activating cytokine may be selected from the group consisting of an IL15 receptor agonist, an IL2 receptor agonist, an IL12 receptor agonist, and an IL18 receptor agonist, as described in further detail below.

[0115] Thus, in one embodiment, the multispecific antigen binding proteins described herein comprise at least an interleukin-21 receptor (IL21R) agonist.

[0116] Interleukin-21 (IL21) is a protein encoded by the IL21 gene (Entrez Gene ID: 59067) in humans. IL21 is a cytokine that has potent regulatory effects on cells of the immune system, including natural killer (NK) cells, and induces cell division / proliferation in its target cells. The amino acid sequence of the human IL21 precursor (including its signal sequence) is described in NCBI Accession Nos. NP_001193935 and NP_068575, the disclosures of which are incorporated herein by reference. IL21 (mature / processed) comprises amino acids 30-153 of NP_001193935 or amino acids 30-162 of NP_068575 (i.e., SEQ ID NO: 38). IL21 exerts its effects on target cells via the IL-21 receptor (IL21R), which is expressed on the surface of T, B, and NK cells. IL21R is structurally similar to receptors for other type I cytokines, such as IL-2R or IL-15, and requires dimerization with the common gamma chain (γc) to bind to IL-21. IL21R is encoded in humans by the IL21R gene (Entrez Gene ID: 50615). The amino acid sequence of human IL21R is set forth in NCBI accession numbers NP_068570, NP_851564, and NP_851565, the disclosures of which are incorporated herein by reference.

[0117] As used herein, an "IL21R agonist" is an agent that has "agonist" activity on the IL21 receptor, which means an agent that can cause or increase "IL21R signaling." "IL21R signaling" refers to the ability of IL21R, which is expressed on the surface of, for example, T, B, and NK cells, and, when triggered by its natural ligand, IL21, to activate or transduce intracellular signaling pathways. "Natural ligand IL21" is understood herein as human wild-type IL21 comprising or consisting of the amino acid sequence set forth above. Upon binding to IL-21, the IL-21 receptor acts via the Jak / STAT pathway, utilizing Jak1 and Jak3 as well as STAT3 homodimers to activate its target genes. IL21R agonist activity, i.e., changes in IL21R signaling activity, can be measured indirectly, for example, by assays designed to measure changes in the IL21R signaling pathway, such as assays that measure the association of specific signaling components with other proteins or intracellular structures by monitoring the phosphorylation of the signaling components, or assays designed to measure the biochemical activity of components such as kinases, or assays designed to measure the expression of a reporter gene under the control of an IL21R-sensitive promoter and enhancer, or downstream effects mediated by IL21R (e.g., activation of specific cytolytic machinery in NK cells). A suitable cell-based assay for the biological activity of IL21R agonists is described, for example, in Maurer et al. (mAbs. 2012; 4 (1): 69-83), in which a mouse pre-B cell line is transfected with both human IL21R and a STAT-responsive luciferase reporter gene. IL21R agonist activity can be determined using this cell line by measuring the level of STAT3 phosphorylation using anti-pSTAT3 antibody-conjugated beads and / or by detecting luciferase luminescence when the cell line is contacted with an IL21R agonist.The natural ligand IL21 can serve as a positive control in assays for IL21R agonist activity and can also be used as a reference for the amount of IL21R agonist activity of a given non-natural IL21R agonist, for example a multispecific antigen binding protein described herein that comprises an IL21R agonist.

[0118] In one embodiment, the multispecific antigen binding proteins described herein comprise an IL21R agonist that has reduced IL21R agonist activity compared to human wild-type IL21. In one embodiment, the IL21R agonist has IL21R agonist activity that is 2, 5, 10, 20, 50, 100, 200, 500, or 1000 times less than the activity of human wild-type IL21.

[0119] In one embodiment, the multispecific antigen binding proteins described herein comprise an IL21R agonist that has enhanced IL21R agonist activity compared to human wild-type IL21. In one embodiment, the IL21R agonist has IL21R agonist activity that is 2, 5, 10, 20, 50, 100, 200, 500, or 1000 times greater than the activity of human wild-type IL21.

[0120] In one embodiment, the multispecific antigen binding protein described herein comprises an IL21R agonist which is an IL21 polypeptide comprising an amino acid sequence with at least 50, 55, 60, 65, 38, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 70 and which preferably has IL21R agonist activity as defined above and / or preferably has affinity for IL21R as defined below.

[0121] In one embodiment, the multispecific antigen binding proteins described herein comprise an IL21R agonist that has reduced or enhanced affinity for IL21R compared to human wild-type IL21. The affinity of an IL21R agonist for IL21R can be assayed using methods commonly known in the art, such as surface plasmon resonance.

[0122] In one embodiment, a multispecific antigen binding protein described herein comprises an IL21R agonist that has reduced affinity for IL21R compared to human wild-type IL21. In one embodiment, the affinity of the IL21R agonist for IL21R is 2, 5, 10, 20, 50, 100, 200, 500, 1000, 10000 or 100000 times less than the affinity of human wild-type IL21.

[0123] In one embodiment, a multispecific antigen binding protein described herein comprises an IL21R agonist that has enhanced affinity for IL21R compared to human wild-type IL21. In one embodiment, the affinity of the IL21R agonist for IL21R is 2, 5, 10, 20, 50, 100, 200, 500, or 1000 times greater than the affinity of human wild-type IL21.

[0124] In one embodiment, the multispecific antigen-binding protein described herein comprises an IL21R agonist that is IL21 or a fragment thereof that has IL21R agonist activity. Preferably, the IL21R agonist is human IL21 or a fragment thereof that has IL21R agonist activity. In one embodiment, the IL21R agonist is an IL21 mutein that has reduced affinity for IL21R compared to human wild-type IL21. IL21 muteins that have reduced affinity for IL21R compared to human wild-type IL21 are described in Shen et al. (Front Immunol. 2020; 11: 832). Thus, in one embodiment, the IL21R agonist is an IL21 mutein having one or more amino acid mutations (i.e., amino acid substitutions, deletions or insertions) selected from the group consisting of I16, I66, I8, K72, K73, K75, K77, L13, P78, Q12, Q19, R5, R65, R76, R9, S70, S80, V69 and Y23 (amino acid positions referring to positions in SEQ ID NO: 38 or the corresponding positions in an IL-21 allelic variant). Preferably, the IL21R agonist is selected from the group consisting of I8A, K72D, K73A, K75D, K77D, L13D, P78D, Q12A, Q19D, R5A, R65D, R76A, R9A, S70E, S80G, V69D, Y23D, I16E, I66G, I8D, K72G, K73D, K75G , K77G, P79D, Q12D, R5D, R65G, R76D, R9D, S70G, S80P, V69G, I66P, I8E, K72P, K 73E, K75P, K77P, Q12E, R5E, R65P, R76E, R9E, S70P, V69P, I8G, K73G, Q12N, R5G, IL21 muteins comprising one or more amino acid substitutions selected from the group consisting of R76G, R9G, S70Y, I8N, K73H, Q12S, R5H, R76H, R9H, I8S, K73I, Q12T, R5I, R76I, R9I, K73N, Q12V, R5K, R76K, R9K, K73P, R5L, R76L, R9L, K73Q, R5M, R76M, R9M, K73S, R5N, R76N, R9N, K73V, R5Q, R76P, R9Q, R5S, R76Q, R9S, R5T, R76S, R9T, R5V, R76T, R9V, R5Y, R76V, R9Y and R76Y.

[0125] In one embodiment, the IL21R agonist is an IL21 mutein having an affinity of 0.028-0.099 nM for human IL21R-Fc in Table 2 of Shen et al. (2020; supra). In one embodiment, the IL21R agonist is an IL21 mutein having an affinity of 0.10-0.29 nM for human IL21R-Fc in Table 2 of Shen et al. (2020; supra). In one embodiment, the IL21R agonist is an IL21 mutein having an affinity of 0.30-0.99 nM for human IL21R-Fc in Table 2 of Shen et al. (2020; supra). In one embodiment, the IL21R agonist is an IL21 mutein having an affinity of 1.0-2.9 nM for human IL21R-Fc in Table 2 of Shen et al. (2020; supra). In one embodiment, the IL21R agonist is an IL21 mutein with an affinity of greater than 2.9 nM for human IL21R-Fc in Table 2 of Shen et al. (2020; supra).

[0126] In one embodiment, the multispecific antigen-binding protein described herein comprises an IL21R agonist that is an antigen-binding region that specifically binds to IL21R and has IL21R agonist activity. The antigen-binding region may be an antigen-binding region as described herein above.

[0127] In one embodiment, the multispecific antigen binding protein described herein comprises more than one IL21R agonist as described above. Thus, in one embodiment, the multispecific antigen binding protein has an IL21R agonist valency of greater than 1. The IL21R agonist valency of the multispecific antigen binding protein can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0128] In one embodiment, the multispecific antigen binding proteins described herein comprise at least a 4-1BB agonist.

[0129] 4-1BB is a member of the tumor necrosis factor receptor family. Its alternative names are tumor necrosis factor receptor superfamily member 9 (TNFRSF9), CD137, and induced by lymphocyte activation (ILA). 4-1BB is encoded by the TNFRSF9 gene (Entrez Gene ID: 3604). The amino acid sequence of human 4-1BB is described in NCBI accession number NP_001552, the disclosure of which is incorporated herein by reference. 4-1BB is known as a costimulatory immune checkpoint molecule. 4-1BB is expressed by activated T cells of both CD4+ and CD8+ lineages and on activated NK cells. NK cells with increased 4-1BB expression are known to be highly active against target cells (e.g., tumor cells) that express the 4-1BB ligand. 4-1BB ligand (4-1BBL), also known as TNFSF9 or CD137L, is a protein encoded in humans by the TNFSF9 gene (Entrez Gene ID: 8744). The amino acid sequence of human 4-1BBL is set forth in NCBI accession number NP_003802, the disclosure of which is incorporated herein by reference. The 4-1BB / 4-1BBL complex consists of three 4-1BB monomers bound to trimeric 4-1BBL. Each 4-1BB monomer binds two 4-1BBLs via their cysteine-rich domains (CRDs). Interaction between 4-1BB and a second 4-1BBL is required to stabilize their interaction.

[0130] As used herein, a "4-1BB agonist" refers to an agent having agonistic activity at 4-1BB, which means an agent capable of inducing or increasing "4-1BB signaling." "4-1BB signaling" refers to the ability of 4-1BB, which is expressed on the surface of T, B, and NK cells, to activate or transduce intracellular signaling pathways when triggered by its natural ligand, 4-1BBL. A "natural 4-1BB ligand" is herein understood as the extracellular domain (ECD) of human wild-type 4-1BBL comprising or consisting of the amino acid sequence at positions 71 to 254 of the amino acid sequence of human 4-1BBL (i.e., SEQ ID NO: 37). Accordingly, a 4-1BBL extracellular domain (ECD) is herein understood as a polypeptide comprising or consisting of the amino acid sequence at positions 71 to 254 of human 4-1BBL or a fragment thereof having 4-1BB agonistic activity.

[0131] 4-1BB agonist activity, i.e., changes in 4-1BB signaling activity, can be measured indirectly by assays designed to measure changes in the 4-1BB signaling pathway, for example, by monitoring the phosphorylation of signaling components, assays measuring the association of specific signaling components with other proteins or intracellular structures, or the biochemical activity of components such as kinases, or downstream effects mediated by 4-1BB (e.g., production of specific cytokines). A suitable cell-based assay for the in vitro biological activity of 4-1BB agonists is described, for example, in Zhang et al. (Clin Cancer Res, 2007; 13 (9): 2758-2767), and uses the measurement of IL-2 production from aseptically removed splenocytes from BALB / c mice in microtiter plates pre-coated with anti-CD3 monoclonal antibody (145-11C clone). Other suitable cell-based assays for the in vitro biological activity of 4-1BB agonists are described in WO 2016 / 075278, Example 6 (see, e.g., Example 6.1). The natural 4-1BB ligand, 4-1BBL ECD trimer, is described in Fellermeier et al. (Oncoimmunol. 2016, 5 (11): e1238540), and for example, a 4-1BBL ECD trimer comprising the amino acid sequence of SEQ ID NO: 36 or an anti-CD137 agonist antibody (e.g., antibody 2A, EEpstein et al., Tumor necrosis imaging and treatment of solid tumors. In: V.P. Torchilin, editor. Handbook of targeted delivery of imaging agents, Vol. 16. Boca Raton: CRC Press; 1995. p. 259.) can serve as a positive control in assays for 4-1BB agonist activity and can also be used as a measure of the amount of 4-1BB agonist activity of a given non-natural 4-1BB agonist, such as the multispecific antigen-binding proteins described herein that comprise a 4-1BB agonist.

[0132] In one embodiment, the multispecific antigen binding proteins described herein comprise a 4-1BB agonist that has reduced 4-1BB agonist activity compared to human wild-type 4-1BBL or anti-4-1BB agonist antibody 2 A. In one embodiment, the 4-1BB agonist has 4-1BB agonist activity that is 2, 5, 10, 20, 50, 100, 200, 500, or 1000 times less than the ECD of human wild-type 4-1BBL or anti-4-1BB agonist antibody 2A.

[0133] In one embodiment, the multispecific antigen binding proteins described herein comprise a 4-1BB agonist that has enhanced 4-1BB agonist activity compared to human wild-type 4-1BBL, hi one embodiment, the 4-1BB agonist has 4-1BB agonist activity that is 2, 5, 10, 20, 50, 100, 200, 500, or 1000 times greater than the ECD of human wild-type 4-1BBL or the anti-4-1BB agonist antibody 2A.

[0134] In one embodiment, a multispecific antigen binding protein described herein comprises at least one 4-1BBL ECD comprising an amino acid sequence having at least 50, 55, 60, 65, 37, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 70, and preferably has 4-1BB agonist activity as defined above and / or comprises a 4-1BB agonist preferably having affinity for 4-1BB as defined below.

[0135] In one embodiment, the multispecific antigen-binding proteins described herein comprise a 4-1BB agonist that has reduced or enhanced affinity for 4-1BB compared to the ECD of human wild-type 4-1BBL. The affinity of the 4-1BB agonist for 4-1BB can be assayed using methods commonly known in the art, such as surface plasmon resonance.

[0136] In one embodiment, the multispecific antigen binding protein described herein comprises a 4-1BB agonist that has reduced affinity for 4-1BB compared to human wild-type 4-1BBL. In one embodiment, the affinity of the 4-1BB agonist for 4-1BB is 2, 5, 10, 20, 50, 100, 200, 500, 1000, or 10,000 times less than the ECD of human wild-type 4-1BBL or the anti-4-1BB agonist antibody 2A.

[0137] In one embodiment, the multispecific antigen binding proteins described herein comprise a 4-1BB agonist that has enhanced affinity for 4-1BB compared to human wild-type 4-1BBL. In one embodiment, the affinity of the 4-1BB agonist for 4-1BB is 2, 5, 10, 20, 50, 100, 200, 500, or 1000 times greater than the ECD of human wild-type 4-1BBL or the anti-4-1BB agonist antibody 2A.

[0138] In one embodiment, the multispecific antigen-binding proteins described herein comprise a 4-1BB agonist comprising or consisting of the ECD of 4-1BBL or a fragment thereof having 4-1BB agonist activity. Preferably, the 4-1BB agonist is human 4-1BBL or a fragment thereof having 4-1BB agonist activity. In one embodiment, the 4-1BB agonist is a mutein of the ECD of 4-1BBL that has reduced affinity for 4-1BB compared to the ECD of human wild-type 4-1BBL or the anti-4-1BB agonist antibody 2A.

[0139] In one embodiment, the multispecific antigen-binding proteins described herein include a 4-1BB agonist that comprises or consists of a fusion protein comprising three 4-1BBL ECD monomers fused to a single polypeptide chain, for example, as described in Fellermeier et al. (2016, supra). In one embodiment, the three 4-1BBL ECD monomers are linked by a polypeptide linker. In one embodiment, the three 4-1BBL ECD monomers are linked by a polypeptide linker selected from the group consisting of (GGGGS)4, GGGSGGG, GGSGGGGSGG, and G, of which (GGGGS)4 is preferred. Other suitable flexible polypeptide linkers are described below. In one embodiment, the multispecific antigen-binding proteins described herein include a 4-1BB agonist that comprises or consists of a fusion protein comprising three 4-1BBL ECD monomers fused to a single polypeptide chain, for example, comprising the amino acid sequence of SEQ ID NO: 36.

[0140] In one embodiment, the multispecific antigen-binding protein described herein comprises a 4-1BB agonist comprising three 4-1BBL ECD monomers present in two or more polypeptide chains of the multispecific antigen-binding protein. For example, two 4-1BBL ECD monomers may be fused together into a single polypeptide chain, optionally connected together via a polypeptide linker as described above, which is part of the first polypeptide chain of the multispecific antigen-binding protein, and the third 4-1BBL ECD monomer is part of the second polypeptide chain of the multispecific antigen-binding protein, for example, as described in WO 2016 / 075278. The first and second polypeptide chains of the multispecific antigen-binding protein may each comprise a heavy chain and a light chain, or vice versa, whereby the 4-1BBL ECD is preferably fused to the N-terminus of the variable domain. Alternatively, the first and second polypeptide chains of the multispecific antigen-binding protein may be two chains comprising two heavy chains, whereby the 4-1BBL ECD is preferably fused to the C-terminus of the constant domain.

[0141] In one embodiment, the multispecific antigen-binding proteins described herein comprise a 4-1BB agonist comprising an antigen-binding region that specifically binds to 4-1BB and has 4-1BB agonist activity. Antibodies against 4-1BB are described, for example, in WO2005035584, WO2006088464, and U.S. Patent Application Publication No. 2006188439.

[0142] In one embodiment, the multispecific antigen binding proteins described herein comprise more than one 4-1BB agonist as described above. Thus, in one embodiment, the multispecific antigen binding protein has a 4-1BB agonist valence of greater than 1. The 4-1BB agonist valence of the multispecific antigen binding protein may be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0143] In a preferred embodiment, the multispecific antigen binding proteins described herein comprise both at least one IL21R agonist as described herein above and at least one 4-1BB agonist as described herein above.

[0144] In further embodiments, the multispecific antigen-binding proteins described herein may comprise, in addition to at least one of an IL21R agonist and a 4-1BB agonist, an additional NK cell-activating cytokine selected from the group consisting of an IL15 receptor agonist, a type I interferon (IFN-1) agonist, an IL2 receptor agonist, an IL12 receptor agonist, and an IL18 receptor agonist. In one embodiment, the IL15 receptor agonist is an IL15 polypeptide or agonistic antigen-binding region that specifically binds to the IL15 receptor. In one embodiment, the IL2 receptor agonist is an IL2 polypeptide or agonistic antigen-binding region that specifically binds to the IL2 receptor. In one embodiment, the IL12 receptor agonist is an IL12 polypeptide or agonistic antigen-binding region that specifically binds to the IL12 receptor. In one embodiment, the IL18 receptor agonist is an IL18 polypeptide or agonistic antigen-binding region that specifically binds to the IL18 receptor.

[0145] Regions with affinity for surface antigens expressed on NK cells In one embodiment, the multispecific antigen-binding proteins described herein may further comprise a region with affinity for a surface antigen expressed on NK cells. Thus, the presence of such a region with affinity for a surface antigen expressed on NK cells in the multispecific antigen-binding protein is optional.

[0146] In one embodiment of the multispecific antigen-binding protein, the region with affinity for a surface antigen expressed on NK cells comprises or consists of an immunoglobulin Fc region, or at least a portion thereof, that binds to the type III Fcγ receptor (FcγRIIIa), also referred to herein as CD16A, expressed on (human) NK cells. In one embodiment, the immunoglobulin Fc region comprises at least one of a CH2 domain and a CH3 domain. In one embodiment, the immunoglobulin Fc region comprises at least one of the CH2 and CH3 domains and a hinge region. In one embodiment, the immunoglobulin Fc region comprises or consists of a hinge region and the CH2 and CH3 domains. In one embodiment, the immunoglobulin Fc region is a dimeric Fc region, or at least a portion thereof, that binds to CD16A.

[0147] In one embodiment, the Fc region or portion thereof that binds to CD16A is a wild-type region or portion thereof.

[0148] In one embodiment, an Fc region or a portion thereof that binds to CD16A can be modified to enhance or reduce its binding affinity to CD16A. Within the Fc region, CD16A binding is mediated by the hinge region and CH2 domain. For example, in human IgG1, interaction with CD16 is primarily focused on amino acid residues D265-E269, N297-T299, A327-I332, L234-S239 in the CH2 domain and the carbohydrate residue N-acetyl-D-glucosamine (see Sondermann et al., 2000 Nature, 406 (6793): 267-273). Based on known domains, mutations can be selected to enhance or reduce binding affinity to CD16A, such as by using a phage display library or a yeast surface display cDNA library, or can be designed based on the known three-dimensional structure of the interaction.

[0149] Thus, in one embodiment, if a multispecific antigen-binding protein is intended to have increased affinity for CD16A, the CD16A-binding Fc region or portion thereof may contain a modification that increases affinity for CD16A. Thus, the CD16A-binding Fc region or portion thereof may contain one or more amino acid modifications (e.g., amino acid substitutions, deletions, or insertions) that increase binding to (human) CD16A and, optionally, to another receptor, such as FcRn. Exemplary modifications include modified human IgG1-derived constant regions containing at least one amino acid modification (e.g., substitution, deletion, or insertion) and / or glycosylation, e.g., hypofucosylation. The modifications can, for example, increase binding of the Fc region to FcyRIIIa (CD16A) on NK cells. Examples of modifications are provided in U.S. Pat. No. 10,577,419, the disclosure of which is incorporated herein by reference. Particular mutations (in the IgG1 Fc region) that enhance FcyRIIIa (CD16A) binding include E333A, S239D / I332E and S239D / A330L / I332E.

[0150] In one embodiment, the multispecific antigen binding protein comprises a CD16A-binding Fc region or portion thereof 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 improved binding affinity for (human) CD16A relative to a molecule comprising the 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., S239D, S298A, A330L, I332E, E333A and / or K334A substitutions), and optionally the variant Fc region comprises substitutions at residue S239 and residue I332, such as a S239D substitution and an I332E substitution (Kabat EU numbering).

[0151] In one embodiment, the multispecific antigen-binding protein comprises a CD16A-binding Fc region or portion thereof comprising an altered glycosylation pattern that increases binding affinity to (human) CD16A. Such carbohydrate modifications 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 1,176,195; WO 06 / 133148; WO 03 / 035835; WO 99 / 54342, each of which is incorporated by reference in its entirety. In one embodiment, a multispecific antigen-binding protein comprises one or more hypofucosylated constant regions. Such multispecific antigen-binding proteins may or may not comprise amino acid alterations and / or may be expressed or synthesized or processed under conditions that result in hypofucosylation. In one aspect, in a composition comprising a multispecific antigen-binding protein as described herein, at least 20, 30, 40, 50, 60, 75, 85, 90, 95% or substantially all of the multispecific antigen-binding protein have constant regions comprising a core carbohydrate structure lacking fucose (e.g., complex, hybrid and high mannose structures). In one embodiment, a multispecific antigen-binding protein is provided that does not comprise an N-linked glycan comprising a core carbohydrate structure having fucose. The core carbohydrate is preferably the glycan at Asn297.

[0152] In one embodiment, the multispecific antigen binding protein comprising a CD16A-binding Fc region or portion thereof that has been engineered to have increased binding affinity to CD16A has a binding affinity to human CD16A that is at least 1, 2 or 3 logs greater than the binding affinity of a conventional human IgG1 antibody or a wild-type human IgG1 antibody, as assessed, for example, by surface plasmon resonance.

[0153] In another embodiment, if a multispecific antigen-binding protein is intended to have reduced affinity for CD16A, the CH2 and / or CH3 domains, the Fc region that binds CD16A, or portions thereof, it may contain modifications that reduce affinity for CD16A. For example, a CH2 mutation in a dimeric Fc region protein at residue N297 (Kabat numbering) can eliminate CD16A binding. Other modifications of the Fc region that reduce or eliminate binding to CD16A include the L234A / L235A modification, also known as the "LALA" modification, as exemplified herein. Modifications of the Fc region that reduce or eliminate binding to CD16A may be useful in multispecific antigen-binding proteins to reduce or avoid NK cell fratricide. The lack of NK cell fratricide may be an advantageous feature of the multispecific antigen-binding proteins described herein. NK cell crosslinking with NK cells or other immune cells is expected to reduce the therapeutic effect of NK cell involvement. Most importantly, cross-linking of NK cells with one or more NK cells or other immune cells via bivalent or multivalent interactions with FcRy or in combination with a second immune cell antigen (e.g., NKp46, NKG2D, NKp30, or SLAMF7) can result in immune cell activation.This may result in the induction of target cell-driven fratricide or immune cell killing (e.g., NK-NK cytolysis), ultimately leading to efficient NK cell depletion in vivo, as previously described for the CD16-directed murine IgG antibody (3G8), the CD38-directed antibody daratumumab, and other approaches (Choi et al. 2008 Immunology 124 (2) 215-22; DOI: 10.111 l / j.l365-2567. 2007. 02757.x; Yoshida 2010 Front. Microbiol 1: 128 DOI: 10.3389 / fmicb.2010.00128; Wang et al. 2018 Clin Cancer Res, 24 (16): 4006-4017; DOI: 10.1158 / 1078-0432.CCR-17-3117;His et al 2008;Nakamura 2013 PNAS; 110 (23) 9421-9426; DOI: 10.1073 / pnas.1300140110;Breman et al 2018 Front Immunol, 12 (9) 2940; DOI: 10.3389 / fimmu.2018.02940).

[0154] Those skilled in the art will appreciate that other configurations for modifying the Fc region can be implemented. For example, substitution of human IgG1 or IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 has been shown to significantly reduce binding to Fcy 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.

[0155] In one embodiment, the multispecific antigen binding protein comprising a CD16A-binding Fc region or portion thereof that has been modified to have reduced binding affinity to CD16A has a binding affinity to human CD16A that is at least 1, 2 or 3 logs lower than the binding affinity of a conventional or wild-type human IgG1 antibody, as assessed, for example, by surface plasmon resonance.

[0156] In one embodiment, the multispecific antigen binding protein comprises an Fc region having an amino acid sequence with at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% amino acid identity to an Fc region in at least one of SEQ ID NOs: 1, 3, 11-19 and 23, and preferably having one or more of the structural and / or functional characteristics described above.

[0157] Antigen-binding region that specifically binds to NK cell-activating receptors In one embodiment, the multispecific antigen binding protein described herein further comprises an antigen binding region that specifically binds to an NK cell activating receptor.

[0158] The antigen-binding region used in the multispecific antigen-binding proteins described herein can be derived from any of a variety of immunoglobulin or non-immunoglobulin scaffolds, such as affibodies based on the Z domain of Staphylococcal protein A, engineered Kunitz domains, monobodies or adnectins based on the 10th extracellular domain of human fibronectin III, lipocalin-derived anticalins, DARPins (designed ankyrin repeat domains), affilins, multimerized LDLR-A modules, avimers, or cysteine-rich notin peptides. See, for example, Gebauer and Skerra (2009) Current Opinion in Chemical Biology 13: 245-255, the disclosure of which is incorporated herein by reference. In an alternative embodiment, the antigen-binding region that specifically binds to an NK cell-activating receptor is a ligand, e.g., a peptide ligand, that specifically binds to and preferably activates an NK cell-activating receptor.

[0159] In preferred embodiments, the antigen-binding regions used in the multispecific antigen-binding proteins described herein comprise or consist of immunoglobulin variable regions, which are variable domains typically derived from antibodies (immunoglobulin chains), such as the related VVs found on two polypeptide chains such as those present in a Fab. L Domain and V H Alternatively, an immunoglobulin variable region may comprise or consist of a variable domain in the form of a scFv, V H Domain, V L A single-chain antigen-binding domain such as a dAb, V-NAR domain, or V H The immunoglobulin variable region used in the multispecific antigen-binding proteins described herein may comprise or consist of an immunoglobulin single chain variable domain (ISVD), such as an H domain. The immunoglobulin variable region used in the multispecific antigen-binding proteins described herein may be a human or humanized immunoglobulin variable region or an immunoglobulin single chain variable domain as defined above.

[0160] In one embodiment of the multispecific antigen-binding proteins described herein, the antigen-binding region that specifically binds to an NK cell-activating receptor is an agonistic antigen-binding region that activates the NK cell receptor. As used herein, an antigen-binding region with "agonistic" activity at an NK cell-activating receptor is an agent that can cause or increase "NK cell-activating receptor-mediated signaling." "NK cell-activating receptor-mediated signaling" refers to the ability of an NK cell-activating receptor to activate or transduce an intracellular signaling pathway. Changes in NK cell-activating receptor signaling activity can be measured indirectly, for example, by assays designed to measure changes in the NK cell-activating receptor signaling pathway, such as assays that measure the association of specific signaling components with other proteins or intracellular structures by monitoring the phosphorylation of the signaling component, or the biochemical activity of components such as kinases, or assays designed to measure the expression of a reporter gene under the control of an NK cell-activating receptor-sensitive promoter and enhancer, or downstream effects mediated by the NK cell-activating receptor polypeptide (e.g., activation of specific cytolytic machinery in NK cells). The reporter gene can be a naturally occurring gene (e.g., monitoring cytokine production) or a gene artificially introduced into the cell. Other genes may be placed under the control of such regulatory elements and thus serve to report the level of NK cell activating receptor signaling activity.

[0161] In one embodiment, the antigen-binding region that specifically binds to an NK cell activating receptor is an antigen-binding region derived from an immunoglobulin or non-immunoglobulin scaffold as defined above. Preferably, the antigen-binding region that specifically binds to an NK cell activating receptor comprises or consists of at least one immunoglobulin variable region. More preferably, the antigen-binding region that specifically binds to an NK cell activating receptor comprises or consists of a Fab that specifically binds to an NK cell activating receptor or an immunoglobulin single-chain variable domain (ISVD) that specifically binds to an NK cell activating receptor. In one embodiment, the antigen-binding region that specifically binds to an NK cell activating receptor is an antigen-binding region derived from an immunoglobulin or non-immunoglobulin scaffold as defined above. Preferably, the antigen-binding region that specifically binds to an NK cell activating receptor comprises or consists of a Fab that specifically binds to an NK cell activating receptor or an immunoglobulin single-chain variable domain (ISVD) that specifically binds to an NK cell activating receptor. In one embodiment, the antigen-binding region that specifically binds to an NK cell activating receptor is an antigen-binding region derived from an immunoglobulin or non-immunoglobulin scaffold as defined above. -4 K below M D It is an antigen-binding region that binds to the NK cell activating receptor at a specific value.

[0162] In one embodiment, the antigen-binding region that specifically binds to an NK cell activating receptor comprises or consists of a human or humanized immunoglobulin variable region or an immunoglobulin single chain variable domain as defined above.

[0163] In one embodiment, the multispecific antigen-binding proteins described herein comprise two antigen-binding regions that specifically bind to an NK cell-activating receptor. In multispecific antigen-binding proteins comprising two antigen-binding regions that specifically bind to an NK cell-activating receptor, the two antigen-binding regions can bind to the same NK cell-activating receptor or to at least two different NK cell-activating receptors. In one embodiment of a multispecific antigen-binding protein comprising two antigen-binding regions that specifically bind to an NK cell-activating receptor, the two antigen-binding regions are identical. Thus, with respect to the two antigen-binding regions that specifically bind to an NK cell-activating receptor, the multispecific antigen-binding proteins described herein can be homodimeric or heterodimeric antigen-binding proteins.

[0164] In one embodiment, the multispecific antigen binding proteins described herein comprise an antigen binding region that specifically binds to an NK cell activating receptor selected from the group consisting of NKp46, NKp30, NKG2D, CD16A, SLAMF7, NKp44, CD94-NKG2C / E, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR2DS2, KIR2DL4, KIR3DS1, CD160, NKp80, DNAM1, 2B4, CRACC, 4-BB, OX40, CRTAM, CD27, PSGL1, CD96, CD100, CEACAM1, CD59, PD-L1, Tim3 and NTB-A, of which NKp46, NKp30, NKG2D, CD16A and SLAMF7 are preferred.

[0165] In one embodiment, the multispecific antigen-binding proteins described herein comprise an antigen-binding region that specifically binds to an NK cell-activating receptor that is a natural cytotoxicity receptor (NCR). Natural cytotoxicity receptors are type 1 transmembrane proteins of the immunoglobulin superfamily that mediate NK killing and IFNγ release upon stimulation. They bind to viral ligands such as hemagglutinin and hemagglutinin-neuraminidase, as well as several bacterial and cellular ligands associated with tumor growth, such as PCNA. Natural cytotoxicity receptors include NKp46, NKp44, and NKp30. In one embodiment, the multispecific antigen-binding proteins described herein comprise an antigen-binding region that specifically binds to an NK cell-activating receptor that is an NCR selected from the group consisting of NKp46, NKp44, and NKp30.

[0166] "NKp46" refers to a protein or polypeptide encoded by the Ncr1 gene or a cDNA prepared from such a gene. NKp46 is also known as NCR1, CD335 (cluster of differentiation), NKP46, NK-p46, and LY94. Any naturally occurring isoform, allele, ortholog, or variant is encompassed by the term NKp46 polypeptide (e.g., an NKp46 polypeptide that is 90%, 95%, 98%, or 99% identical to SEQ ID NO: 50, or a contiguous sequence of at least 20, 30, 50, 100, or 200 amino acid residues thereof). The 304 amino acid residue sequence of human NKp46 (isoform a) is shown in SEQ ID NO: 50, which corresponds to NCBI Accession No. NP_004820, the disclosure of which is incorporated herein by reference. The human NKp46 mRNA sequence is set forth in NCBI Accession No. NM_004829, the disclosure of which is incorporated herein by reference.

[0167] "NKp44" refers to a protein or polypeptide encoded by the Ncr2 gene or a cDNA prepared from such a gene. NKp44 is also known as NCR2, CD336 (cluster of differentiation 336), NKP44, NK-p44, LY95, and dJ149M18.1. Any naturally occurring isoform, allele, ortholog, or variant is encompassed by the term NKp44 polypeptide (e.g., an NKp44 polypeptide 90%, 95%, 98%, or 99% identical to SEQ ID NO: 51, or a contiguous sequence of at least 20, 30, 50, 100, or 200 amino acid residues thereof). The 276 amino acid residue sequence of human NKp46 is shown in SEQ ID NO: 51, which corresponds to NCBI Accession No. NP_004819, the disclosure of which is incorporated herein by reference. The human NKp46 mRNA sequence is set forth in NCBI Accession No. NM_004828, the disclosure of which is incorporated herein by reference.

[0168] "NKp30" refers to a protein or polypeptide encoded by the Ncr3 gene or a cDNA prepared from such a gene. NKp30 is also known as NCR3 and CD337 (cluster of differentiation 337). Any naturally occurring isoform, allele, ortholog, or variant is encompassed by the term NKp30 polypeptide (e.g., an NKp30 polypeptide 90%, 95%, 98%, or 99% identical to SEQ ID NO: 52, or a contiguous sequence of at least 20, 30, 50, 100, or 200 amino acid residues thereof). The 201 amino acid residue sequence of human NKp30 is shown below in SEQ ID NO: 52, which corresponds to NCBI Accession No. NP_667341, the disclosure of which is incorporated herein by reference. The human NKp30 mRNA sequence is set forth in NCBI Accession No. NM_147130, the disclosure of which is incorporated herein by reference.

[0169] NKG2D is an activating receptor (transmembrane protein) belonging to the NKG2 family of C-type lectin-like receptors. In humans, NKG2D is encoded by the KLRK1 gene. NKG2D recognizes induced self-proteins from the MIC and RAET1 / ULBP families that appear on the surface of stressed, malignantly transformed, and infected cells. "NKG2D" refers to a protein or polypeptide encoded by the KLRK1 gene or a cDNA prepared from such a gene. NKG2D is also known as KLRK1, CD314 (cluster of differentiation 314), D12S2489E, KLR, NKG2-D, natural killer group 2D, killer cell lectin-like receptor K1, and killer cell lectin-like receptor K1. Any naturally occurring isoform, allele, ortholog, or variant is encompassed by the term NKG2D polypeptide (e.g., an NKG2D polypeptide that is 90%, 95%, 98%, or 99% identical to SEQ ID NO: 53, or a contiguous sequence of at least 20, 30, 50, 100, or 200 amino acid residues thereof). The 216 amino acid residue sequence of human NKG2D is set forth in SEQ ID NO: 53, which corresponds to NCBI Accession No. NP_001186734, the disclosure of which is incorporated herein by reference. The human NKG2D mRNA sequence is set forth in NCBI Accession No. NM_007360, the disclosure of which is incorporated herein by reference.

[0170] "DNAM-1" is an approximately 65 kDa glycoprotein expressed specifically on the surface of NK cells. It is a member of the immunoglobulin superfamily containing two Ig-like domains of the V set. DNAM-1 mediates cell adhesion to other cells bearing its ligands, CD112 and CD155, and cross-linking DNAM-1 with antibodies causes cell activation. "DNAM-1" refers to a protein or polypeptide encoded by the KLRK1 gene or a cDNA prepared from such a gene. DNAM-1 is also known as CD226 (cluster of differentiation 226), DNAM-1, DNAM1, PTA1, and TLiSA1. Any naturally occurring isoform, allele, ortholog, or variant is encompassed by the term DNAM-1 polypeptide (e.g., a DNAM-1 polypeptide 90%, 95%, 98%, or 99% identical to SEQ ID NO: 54 or a contiguous sequence of at least 20, 30, 50, 100, or 200 amino acid residues thereof). The 336 amino acid residue sequence of human DNAM-1 is shown in SEQ ID NO: 54, which corresponds to NCBI accession number NP_006557, the disclosure of which is incorporated herein by reference. The human DNAM-1 mRNA sequence is set forth in NCBI accession number NM_006566, the disclosure of which is incorporated herein by reference.

[0171] As described above, CD16A is an immunoglobulin gamma Fc region receptor (FcγRIIIa) expressed on NK cells and through which NK cells recognize IgG bound to the surface of pathogen-infected or TAA-expressing target cells. Any naturally occurring isoform, allele, ortholog, or variant is encompassed by the term CD16A polypeptide (e.g., a CD16A polypeptide 90%, 95%, 98%, or 99% identical to SEQ ID NO: 55, or a contiguous sequence of at least 20, 30, 50, 100, or 200 amino acid residues thereof). The 254 amino acid residue sequence of human CD16A is shown in SEQ ID NO: 55, which corresponds to UniProt Accession No. P08637, the disclosure of which is incorporated herein by reference.

[0172] "SLAMF7" refers to a protein encoded by the human SLAMF7 gene in humans. Isoform 1 of SLAMF7 mediates NK cell activation via an SH2D1A-independent extracellular signal-regulated ERK-mediated pathway. SLAMF7 is also known as CD319 (cluster of differentiation 319), 19A, CRACC, and CS1. Any naturally occurring isoform, allele, ortholog, or variant is encompassed by the term SLAMF7 polypeptide (e.g., an SLAMF7 polypeptide 90%, 95%, 98%, or 99% identical to SEQ ID NO: 56, or a contiguous sequence of at least 20, 30, 50, 100, or 200 amino acid residues thereof). The 335 amino acid residue sequence of human SLAMF7 is shown in SEQ ID NO: 56, which corresponds to UniProt Accession No. Q9NQ25-1, the disclosure of which is incorporated herein by reference.

[0173] In one embodiment, the multispecific antigen-binding protein described herein comprises at least one antigen-binding region obtained / obtainable from a monoclonal antibody against an NK cell-activating receptor known in the art. In one embodiment, at least one antigen-binding region comprises at least six CDR sequences obtained / obtainable from a monoclonal antibody against an NK cell-activating receptor, as known in the art. In one embodiment, at least one antigen-binding region comprises a variable light (V) sequence obtained / obtainable from a monoclonal antibody against an NK cell-activating receptor, as known in the art. L ) domain and variable weight (V H) domain sequence. Many examples of monoclonal antibodies against NK cell activating receptors have been described in the art. Anti-NKp46 monoclonal antibodies are described in WO 2011 / 086179, WO 2016 / 209021 and Gauthier et al. (2019, Cell 177, 1701-1713) or WO 2016 / 207278, such as NKp46-1, -2, -3, -4, -6 or -9. Anti-NKG2D monoclonal antibodies are described in WO 2009 / 077483, WO 2018 / 148447, WO 2019 / 157366, WO 2018 / 148445, WO 2018 / 152518, and WO 2019 / 195409, and comprise the heavy and light chain sequences of SEQ ID NOs: 16 and 20, respectively. Monoclonal antibodies against NKG2A are described, for example, in WO 2008 / 009545, WO 2009 / 092805, WO 2016 / 032334, WO 2020 / 094071, and WO 2020 / 102501. Monoclonal antibodies against NKp30 are described, for example, in WO 2020 / 172605. Monoclonal antibodies against DNAM-1 are described, for example, in WO 2013 / 140787. Examples of anti-SLAMF7 monoclonal antibodies include elotuzumab and others described in U.S. Patent Application Publication No. 2018208653. Monoclonal antibodies against 4-1BB (CD137) are described, for example, in WO 2005 / 035584, WO 2006 / 088464, and U.S. Patent Application Publication No. 2006188439. Monoclonal antibodies against OX40 are described, for example, in WO 2007 / 062245, U.S. Patent Application Publication No. 2010136030, U.S. Patent Application Publication No. 2019100596, WO 2013 / 008171, and WO 2013 / 028231. Monoclonal antibodies against CD96 are described, for example, in WO 2019 / 091449.Monoclonal antibodies against CD160 are described, for example, in U.S. Patent Application Publication Nos. 2012003224 and 2013122006. Monoclonal antibodies against KIR2DS1-5 are described, for example, in WO 2016 / 031936.

[0174] Thus, in one embodiment, the multispecific antigen binding protein described herein comprises a combination of complementarity determining regions (CDRs) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of: a) the CDR-H1 (SEQ ID NO: 24), CDR-H2 (SEQ ID NO: 25) and CDR-H3 (SEQ ID NO: 26) sequences contained in SEQ ID NO: 1 and the CDR-L1 (SEQ ID NO: 27), CDR-L2 (SEQ ID NO: 28) and CDR-L3 (SEQ ID NO: 29) sequences contained in SEQ ID NO: 2; and b) the CDR-H1, CDR-H2 and CDR-H3 sequences contained in SEQ ID NO: 3 and the CDR-L1, CDR-L2 and CDR-L3 sequences contained in SEQ ID NO: 20.

[0175] In one embodiment, the multispecific antigen binding protein described herein comprises a) the V contained in SEQ ID NO:1 H V contained in the sequence and SEQ ID NO:2 L and b) V contained in SEQ ID NO: 3 H V contained in the sequence and SEQ ID NO: 20 L Variable weight (V) sequences selected from the group consisting of H ) and variable light (V L ) domain combinations.

[0176] In one embodiment, the multispecific antigen binding protein described herein comprises a combination of heavy and light chains selected from the group consisting of: a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2; and b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.

[0177] Structure of a multispecific antigen-binding protein In one embodiment, in the multispecific antigen-binding proteins described herein, at least the NK cell-activating cytokine is conjugated to a region having affinity for a surface antigen expressed on NK cells. Conjugation of the cytokine to this region is understood to mean that they are covalently bound to each other. The cytokine can be chemically crosslinked to this region using a crosslinking agent for linking two proteinaceous molecules, as is well known in the art. Several commercially available crosslinking reagents exist for preparing protein or peptide bioconjugates. Many of these crosslinking agents allow for dimeric homo- or heteroconjugation of biological molecules via free amine or sulfhydryl groups in protein side chains. Other crosslinking methods include coupling with hydrazide moieties via carbohydrate groups. It is preferable to use a crosslinker with heterofunctional specificity to crosslink the NK cell-activating cytokine to the NK cell-binding region. In one embodiment, the crosslinker includes a flexible spacer to provide freedom of movement or mobility of the two moieties relative to each other.

[0178] However, in one embodiment, at least one NK cell-activating cytokine is conjugated to the region having affinity for a surface antigen expressed on an NK cell by being included in a single polypeptide chain. Because the region having affinity for a surface antigen expressed on an NK cell may comprise two polypeptide chains, such as a dimeric Fc region of an antibody, in one embodiment, at least one polypeptide chain of the region having affinity for a surface antigen expressed on an NK cell forms a single polypeptide chain with at least one NK cell-activating cytokine. In one embodiment, the NK cell-activating cytokine is linked to the polypeptide chain of the region having affinity for a surface antigen expressed on an NK cell through a flexible linker. In one embodiment, the single polypeptide chain comprises two or more NK cell-activating cytokines, optionally linked to each other or to the polypeptide chain of the region having affinity for a surface antigen expressed on an NK cell through a flexible linker. The flexible linker may be an immunoglobulin hinge region or may be a linker as described below.

[0179] Thus, in one embodiment, the multispecific antigen-binding protein described herein comprises a single polypeptide chain comprising, in order from N-terminus to C-terminus: i) a first NK cell-activating cytokine, ii) optionally a first flexible linker, iii) a polypeptide chain of a region having affinity for a surface antigen expressed on an NK cell, iv) optionally a second flexible linker, and v) a second NK cell-activating cytokine, preferably wherein the first and second NK cell-activating cytokines are different NK cell-activating cytokines. The first and second NK cell-activating cytokines may be NK cell-activating cytokines and may be selected from an IL21R agonist and a 4-1BB agonist as defined herein above.

[0180] In one embodiment, the multispecific antigen-binding protein described herein further comprises at least one antigen-binding region that specifically binds to an NK cell-activating receptor. In one embodiment, the at least one antigen-binding region that specifically binds to an NK cell-activating receptor is conjugated to a region that has affinity for a surface antigen expressed on an NK cell. Preferably, the antigen-binding region that specifically binds to an NK cell-activating receptor is conjugated to at least one polypeptide chain of a region that has affinity for a surface antigen expressed on an NK cell. As described above, conjugation of two regions is understood to mean that they are covalently bound to each other, which can be achieved by chemically cross-linking them to each other using a cross-linking agent for linking two proteinaceous molecules, as is well known in the art.

[0181] However, in one embodiment, at least one antigen-binding region that specifically binds to an NK cell activating receptor is contained in a single polypeptide chain, thereby conjugating it to a region that has affinity for a surface antigen expressed on an NK cell. H Chain and V L In one embodiment, at least one polypeptide chain in the antigen-binding region that specifically binds to an NK cell-activating receptor forms a single polypeptide chain with at least one polypeptide chain in the region having affinity for a surface antigen expressed on an NK cell. Similarly, the region having affinity for a surface antigen expressed on an NK cell may also comprise two polypeptide chains, such as a dimeric Fc region of an antibody, and in one embodiment, at least one polypeptide chain in the region having affinity for a surface antigen expressed on an NK cell forms a single polypeptide chain with at least one polypeptide chain in the antigen-binding region that specifically binds to an NK cell-activating receptor.

[0182] Thus, in one embodiment, the multispecific antigen-binding proteins described herein comprise a single polypeptide chain comprising, in order from N-terminus to C-terminus: i) at least one polypeptide chain in at least one antigen-binding region that specifically binds to an NK cell activating receptor, ii) optionally a flexible linker, and iii) (at least one polypeptide chain of) a region with affinity for a surface antigen expressed on an NK cell. The flexible linker may be an immunoglobulin hinge region or may be a linker as described below.

[0183] In one embodiment, the region having affinity for a surface antigen expressed on an NK cell is a dimeric immunoglobulin Fc region, wherein each of the two polypeptide chains of the Fc region is linked to a CH1 domain, and each of the CH1 domains is linked to an immunoglobulin variable region that specifically binds to an NK cell activating receptor. The dimeric immunoglobulin Fc region is preferably a dimer of Fc regions that bind to CD16A as described hereinabove. The immunoglobulin variable region can be any of scFv, V H Domain, V L domain or dAb, V-NAR domain or V H In one embodiment, the immunoglobulin variable region linked to the CH1 domain can be an immunoglobulin single chain variable domain (ISVD), such as an H domain. In one embodiment, the immunoglobulin variable region linked to the CH1 domain can be a V domain linked to a Cκ or Cλ domain. L Domain and paired V H In this embodiment, preferably, H and V L Together the domains bind specifically to the NK cell activating receptor.

[0184] In one embodiment, the two immunoglobulin variable regions bind to the same NK cell activating receptor, or the two immunoglobulin variable regions each bind to a different NK cell activating receptor. Thus, with respect to their specificity for an NK cell activating receptor, the multispecific antigen-binding proteins described herein may be homodimers, with two identical immunoglobulin variable regions both binding to the same NK cell activating receptor. Alternatively, the multispecific antigen-binding proteins described herein may be heterodimers, with respect to their specificity for an NK cell activating receptor, with each of the two immunoglobulin variable regions each binding to a different NK cell activating receptor. In embodiments in which the multispecific antigen-binding protein is bispecific for an NK cell activating receptor, it is preferred that one of the two immunoglobulin variable regions is an immunoglobulin single-chain variable domain and the other immunoglobulin variable region is not an immunoglobulin single-chain variable domain. Next, assembly of heterodimeric antibody heavy chains can be achieved by expressing two different antibody heavy chain sequences in the same cell, which may result in the assembly of homodimers as well as heterodimers of each antibody heavy chain. Promotion of preferential assembly of heterodimers can be achieved by incorporating different mutations into the CH3 domain of each antibody heavy chain constant region, as shown in U.S. Patent Application Nos. 13 / 494,870, 16 / 028850, 11 / 533,709, 12 / 875,015, 13 / 289,934, 14 / 773,418, 12 / 811,207, 13 / 866,756, 14 / 647,480, 14 / 830,336, and WO 2019 / 195409. For example, mutations can be made in the CH3 domain based on human IgG1 to incorporate different pairs of amino acid substitutions within the first and second polypeptides that allow the two chains to selectively heterodimerize with one another.For example, in a CH3 domain containing amino acid substitutions, the CH3 domain interface of an antibody Fc region has been mutated to create an altered charge polarity across the Fc dimer interface such that co-expression of electrostatically matched Fc chains favors favorable attractive interactions, thereby promoting desired Fc heterodimer formation, while unfavorable repulsive charge interactions suppress undesired Fc homodimer formation.

[0185] In one embodiment, a "knobs-into-holes" approach is used, in which the CH3 domain interface of an antibody Fc region is mutated to cause the antibody to preferentially form heterodimers (including an additional light chain). These mutations create altered charge polarity across the Fc dimer interface, such that coexpression of electrostatically matched Fc chains favors favorable attractive interactions, thereby promoting desired Fc heterodimer formation, while unfavorable repulsive charge interactions suppress undesired Fc homodimer formation. For example, one heavy chain contains a T366W substitution and the second heavy chain contains T366S, L368A, and Y407V substitutions (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 contains a F405L substitution and the second heavy chain contains 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 contains T350V, L351Y, F405A, and Y407V substitutions and the second heavy chain contains T350V, T366S, K392L, and T394W substitutions (see, e.g., Von Kreudenstein et al., (2013) mAbs 5: 646-654). In another approach, one heavy chain contains both K409D and K392D substitutions, and the second heavy chain contains both D399K and E356K substitutions (see, e.g., Gunasekaran et al., (2010) J. Biol. Chem. 285: 19637-19646). In another approach, one heavy chain contains D221E, P228E, and L368E substitutions, and the second heavy chain contains 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 H435R substitution, and the second heavy chain optionally comprises or does not comprise substitutions (see, e.g., U.S. Patent 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 enable CD16 binding. In some embodiments, the antibodies can comprise mammalian antibody-type N-linked glycosylation at residue N297 (Kabat EU numbering).

[0186] In one preferred embodiment, the multispecific antigen-binding proteins described herein comprise a dimeric immunoglobulin Fc region that is a dimer of the CD16A-binding Fc regions described above, wherein each of the two Fc polypeptide chains is operably linked to a Fab that specifically binds to an NK cell-activating receptor. Thus, independent of the presence of an NK cell-activating cytokine, a multispecific antigen-binding protein comprising such a dimeric Fc linked to two Fabs forms an immunoglobulin structure, such as a conventional IgG immunoglobulin.

[0187] The multispecific antigen-binding proteins described herein, which comprise an antigen-binding region that specifically binds to an NK cell-activating receptor, therefore also comprise at least one NK cell-activating cytokine. In one embodiment, at least one of the NK cell-activating cytokines is conjugated to at least one antigen-binding region that specifically binds to an NK cell-activating receptor or a region with affinity for a surface antigen expressed on NK cells. As noted above, conjugation of two proteinaceous entities is understood to mean that they are covalently linked to each other; this can be done by chemical cross-linking using a cross-linking agent to link the two proteinaceous molecules, as is well known in the art, and the cross-linking agent can comprise a flexible spacer.

[0188] However, in one embodiment, the at least one NK cell-activating cytokine forms at least one single polypeptide chain of: i) at least one polypeptide chain in at least one antigen-binding region that specifically binds to an NK cell-activating receptor, and ii) (at least one polypeptide chain of) a region that has affinity for a surface antigen expressed on an NK cell. In one embodiment, a flexible linker (described below) is present between the agonist and the region defined in i) or ii).

[0189] In one embodiment, at least one NK cell-activating cytokine forms i) a light chain in at least one of the two Fabs that specifically bind to an NK cell-activating receptor, and ii) at least one single polypeptide chain in at least one of the two Fc chains in the dimeric immunoglobulin Fc region. In one embodiment, a flexible linker (described below) is present between the agonist and the light chain defined in i) or the Fc chain defined in ii).

[0190] In one embodiment, at least one NK cell-activating cytokine is fused to at least one of: i) the N-terminus of the light chain of at least one of the two Fabs that specifically binds to an NK cell-activating receptor, optionally through a flexible linker; ii) the C-terminus of the light chain of at least one of the two Fabs that specifically binds to an NK cell-activating receptor, optionally through a flexible linker; iii) the N-terminus of the heavy chain of at least one of the two Fabs that specifically binds to an NK cell-activating receptor; and iv) the C-terminus of the heavy chain of at least one of the two Fc chains in the dimeric immunoglobulin Fc region, optionally through a flexible linker, whereby the flexible linker may be as described below.

[0191] In one embodiment where the multispecific antigen-binding protein comprises a dimeric antigen-binding protein (homo- or hetero-) as described above, the dimer may comprise at least one NK cell-activating cytokine on only one of the two monomers in the dimer, or the dimer may comprise at least one NK cell-activating cytokine on each (i.e., both) of the two monomers in the dimer. Thus, in one embodiment where the multispecific antigen-binding protein comprises an immunoglobulin structure, at least one of the NK cell-activating cytokines may be present on at least one or both sides of the immunoglobulin structure. In embodiments where at least one NK cell-activating cytokine is present on the dimer or each of the two monomers in the immunoglobulin structure, the multispecific antigen-binding protein may comprise, for example, an IL21R agonist on both monomers, a 4-1BB agonist on both monomers, or an IL21R agonist on the first monomer and a 4-1BB agonist on the second monomer. Where the multispecific antigen-binding protein comprises heterodimeric heavy chains, the above-described "knob-into-hole" technique can be applied, whereby the CH3 domain of one chain is engineered to have a "protuberance" ("knob") and the second chain is engineered to have a corresponding "cavity" ("hole").

[0192] Thus, in one embodiment, the multispecific antigen binding protein described herein is heterodimeric with respect to at least one of: i) an antigen binding region that specifically binds to an NK cell activating receptor; and ii) at least one fusion NK cell activating cytokine, wherein the dimeric Fc region comprises distinct first and second polypeptide chains that comprise knobs-into-hole modifications that facilitate association of the first and second polypeptide chains of the Fc region.

[0193] Suitable linker amino acid sequences for linking various functional domains and regions in the multispecific antigen-binding proteins described herein are known in the art (e.g., Chen et al., 2013, Adv Drug Deliv Rev. 65 (10): 1357-1369). Linker amino acid sequences can be rigid but are usually flexible. Flexible linkers are usually applied when the linked domains require some degree of movement or interaction. They are generally composed of small non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Due to their small size, these amino acids provide flexibility and allow mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing unfavorable interactions between the linker and the protein moiety. A preferred flexible linker has a sequence consisting mainly of a stretch of Gly and Ser residues (a "GS" linker). An example of a preferred (and widely used) flexible linker is (GGGGS) n (SEQ ID NO: 30). By adjusting the copy number "n," the length of this GS linker can be optimized to achieve appropriate separation of functional domains or maintain necessary inter-domain interactions. Specific examples of GS linkers include (GGGGS)4 (SEQ ID NO: 31), GGGSGGG (SEQ ID NO: 32), GGSGGGGSGG (SEQ ID NO: 33), and G. In addition to the GS linker, many other flexible linkers have been designed for recombinant fusion proteins. These flexible linkers are also rich in small or polar amino acids such as Gly and Ser, but may also contain additional amino acids such as Thr and Ala to maintain flexibility and polar amino acids such as Lys and Glu to improve solubility, such as the flexible linkers KESGSVSSEQLAQFRSLD (SEQ ID NO: 34) and EGKSSGSGSESKST (SEQ ID NO: 35), which have been applied to the construction of biologically active scFvs.

[0194] In one embodiment, the multispecific antigen binding protein described herein is a multispecific antigen binding protein exemplified herein, e.g., AVC-009, AVC-010, AVC-011, AVC-012, AVC-013, AVC-014 or AVC-015 (see Tables 1.1.1 and 1.1.2) or a derivative thereof, wherein the anti-NKp46 variable heavy (V H ) domain and variable light (V L ) domain is a variable heavy (V) domain derived from another monoclonal antibody against the NK cell activating receptor. H ) domain and variable light (V L ) domain.

[0195] In one embodiment, the multispecific antigen-binding protein described herein comprises: a) a first heavy chain of a monoclonal antibody against an NK cell-activating receptor as described above, preferably wherein a first NK cell-activating cytokine is fused to the C-terminus of the first heavy chain, optionally via a flexible linker; b) a second heavy chain of a monoclonal antibody against an NK cell-activating receptor, preferably wherein a second NK cell-activating cytokine is fused to the C-terminus of the second heavy chain, optionally via a flexible linker; and c) a first light chain and a second light chain of a monoclonal antibody against an NK cell-activating receptor, optionally wherein a first or second NK cell-activating cytokine is fused to the C-terminus of the light chain, optionally via a flexible linker, and preferably wherein the amino acid sequences of the Fc regions of the first and second heavy chains comprise knob-into-hole modifications that facilitate association of the first and second heavy chains. In one embodiment, the first NK cell-activating cytokine is a 4-1BB agonist as described above, preferably a fusion protein comprising three 4-1BBL ECD monomers fused together in a single polypeptide chain as described above, or a single 4-1BBL ECD monomer as described above. In one embodiment, the second NK cell-activating cytokine is an IL21R agonist as described above, preferably an IL21R polypeptide as described above. In one embodiment, the monoclonal antibody against an NK cell-activating receptor is a monoclonal antibody against NKp46 as described herein. In one embodiment, the Fc region of the heavy chain comprises a modification that reduces affinity for CD16A, e.g., a "LALA" modification, as described above.

[0196] In one embodiment, the multispecific antigen binding protein (AVC-009) comprises a) a first heavy chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 11; b) a second heavy chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 12; and c) a first light chain and a second light chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO:2.

[0197] In one embodiment, the multispecific antigen binding protein (AVC-010) comprises a) a first heavy chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 13, b) a second heavy chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 14, and c) a first light chain and a second light chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 2.

[0198] In one embodiment, the multispecific antigen binding protein (AVC-011) comprises a) a first heavy chain and a second heavy chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 15, and b) a first light chain and a second light chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 21.

[0199] In one embodiment, the multispecific antigen binding protein (AVC-012) comprises a) a first heavy chain and a second heavy chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 39, and b) a first light chain and a second light chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 21.

[0200] In one embodiment, the multispecific antigen binding protein (AVC-013) comprises a) a first heavy chain and a second heavy chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 18, and b) a first light chain and a second light chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO:2.

[0201] In one embodiment, the multispecific antigen-binding protein described herein is a multispecific antigen-binding protein comprising first and second polypeptide chains of an Fc region as described above, wherein preferably a first NK cell-activating cytokine is fused to the N-terminus of at least one polypeptide chain of the Fc region, optionally through a flexible linker, and preferably a second NK cell-activating cytokine is fused to the C-terminus of at least one polypeptide chain of the Fc region, optionally through a flexible linker. In one embodiment, the first NK cell-activating cytokine is an IL21R agonist as described above, preferably an IL21R polypeptide as described above. In one embodiment, the second NK cell-activating cytokine is a 4-1BB agonist as described above, preferably a fusion protein comprising three 4-1BBL ECD monomers fused together in a single polypeptide chain as described above or a single 4-1BBL ECD monomer as described above. In one embodiment, the Fc region of the heavy chain comprises a modification, e.g., a "LALA" modification, that reduces affinity for CD16A as described above. In one embodiment, the multispecific antigen binding protein (AVC-014) comprises a) first and second polypeptide chains comprising an amino acid sequence having at least 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 19. In one embodiment, the multispecific antigen binding protein (AVC-015) comprises a) first and second polypeptide chains comprising an amino acid sequence having at least 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 40.

[0202] Biological activities of multispecific antigen-binding proteins The multispecific antigen binding proteins described herein may have one or more biological activities including, for example, antigen binding, binding to NK cells, ability to recruit NK cells to target cells, activation of NK cells including inducing NK cell hyperactivity and / or ability to induce lysis of target cells by (activated / hyperactive) NK cells.

[0203] In one embodiment, the multispecific antigen binding protein described herein causes an increase in at least one NK cell activity selected from CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cytotoxicity, whereby preferably the increase is at least 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20, 50 or 100 fold greater compared to the increase achieved at the same effector:target cell ratio using the same NK cells and target cells that have not been contacted with the multispecific antigen binding protein.

[0204] In one embodiment the multispecific antigen binding protein described herein causes an increase in at least one NK cell activity selected from CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cytotoxicity, whereby preferably the increase is at least 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20, 50 or 100 fold greater compared to the increase achieved at the same effector:target cell ratio using the same NK cells and target cells contacted (under otherwise identical conditions) with a reference antigen binding protein or a reference feeder cell line.

[0205] In one embodiment, the reference antigen binding protein is a conventional human IgG1 monoclonal antibody that binds to the same NK cell activating receptor, preferably binds to the same epitope, and more preferably has the same NK cell activating receptor-specific antigen binding region as the multispecific antigen binding protein.

[0206] In one embodiment, the reference antigen binding protein is a (multispecific) antigen binding protein that comprises at least one antigen-binding region that binds to the same NK cell activating receptor as the multispecific antigen binding protein, preferably binds to the same epitope, more preferably has the same NK cell activating receptor-specific antigen-binding region, and comprises at least one NK cell-activating cytokine other than an IL21R agonist, preferably at least one NK cell-activating cytokine other than an IL21R agonist and a 4-1BB agonist. The NK cell-activating cytokine other than at least one of an IL21R agonist and a 4-1BB agonist may be an IL-15 receptor agonist, such as IL15, for example, a human modified IL-15 crosslinker as described in U.S. Patent Application Publication No. 2018282386 and Vallera et al. (2016, Clin Cancer Res.; 22 (14): 3440-3450).

[0207] In one embodiment, the reference antigen binding protein is an NK cell engager, such as those described in WO 2016 / 207278, WO 2018 / 148445, WO 2018 / 152518, WO 2019195409, US Patent Application Publication No. 2018282386, Vallera et al. (2016, supra) and Demaria et al. (2021, supra). An example of a (multispecific) reference antigen binding protein is AVC-006, as described in the Examples herein, which comprises, for example, one HER2-binding region and one NKG2D-binding region.

[0208] In one embodiment, the reference feeder cell line is an (irradiated) K562 feeder cell line (FC21 feeder cells) modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand, as described by Denman et al. (2012, supra).

[0209] Assays for detecting the expression of NK activation markers or detecting NK cell cytotoxicity or detecting NK cell activation and cytotoxicity assays (e.g., short-term and long-term cytotoxicity assays) are described in the Examples herein and, 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; Nolte-'t Hoen et al., (2007) Blood 109: 670-673); as described in WO 2016 / 207278 and WO 2018 / 148445.

[0210] In one embodiment, the multispecific antigen binding proteins described herein are capable of inducing hyperactivity (or a hyperactive phenotype) in an NK cell or population of NK cells. A hyperactive NK cell phenotype is herein understood as a phenotype having one or more of the phenotypic characteristics obtained by expanding NK cells obtained from a donor ex vivo by co-culturing the donor with irradiated K562 feeder cells (FC21 feeder cells) modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand, as described by Denman et al. (2012, supra).Thus, in one embodiment, ex vivo expansion of donor NK cells by co-culture (e.g., for 7, 14, or 21 days) with a multispecific antigen binding protein described herein produces a population of NK cells having one or more (or preferably all) characteristics selected from the group of: a) the fold increase in the number of expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or greater than the fold increase in the number of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells. b) the telomere length of the expanded NK cells is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55% compared to the telomere length of fresh NK cells, and preferably the percent increase in telomere length of the expanded NK cells compared to the telomere length of fresh NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or greater than the percent increase in telomere length of the expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells. 5.0-fold; c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46, and CD16 on the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0-fold higher than the expression level on NK cells obtained during ex vivo expansion in the presence of FC21 feeder cells; d) the secretion of at least one cytokine of TNF-α, IFN-γ, and IL-6 by the expanded NK cells is e) the cytokine secretion by the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times greater than that obtained upon ex vivo expansion in the presence of FC21 feeder cells; f) the cytotoxicity of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times greater than that of the NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells.Protocols for ex vivo expansion of donor NK cells and assays for determining fold expansion, telomere length increase, expression levels of NK cell activating receptors, cytokine secretion, and cytotoxicity (e.g., short-term or long-term cytotoxicity assays) are described in Denman et al. (2012, supra) and in the Examples herein.

[0211] Pharmaceutical Composition In a further aspect, the present invention relates to a pharmaceutical composition comprising a multispecific antigen-binding protein as described herein and a pharmaceutically acceptable carrier (excipient). The pharmaceutically acceptable carrier, such as an adjuvant or vehicle, is for administering the polypeptide to a subject. The pharmaceutical composition may be used in the treatment methods described below by administering an effective amount of the composition to a subject in need thereof. The term "subject" as used herein refers to all animals classified as mammals, including, but not limited to, primates and humans. The subject is preferably male or female of any age or race.

[0212] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the pharmaceutical carriers described in the "Handbook of Pharmaceutical Excipients," Rowe et al., eds., 7 thedition, 2012, www.pharmpress.com), and is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, etc., and absorption delaying agents, etc., compatible with the present invention. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentenol; benzoyl perfluorooctyl benzoate; benzoic acid; benzoyl perfluorooctyl benzoate ... ethanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn 2+- Protein complexing agents and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0213] Supplementary active compounds can also be incorporated into the pharmaceutical compositions of the present invention. Thus, in certain embodiments, the pharmaceutical compositions of the present invention can contain two or more active compounds necessary for the particular indication being treated, preferably active compounds with complementary activities that do not adversely affect each other. For example, it may be desirable to additionally provide a chemotherapeutic agent, cytokine, analgesic, thrombolytic agent, or immunomodulator, such as an immunosuppressant or immunostimulator. The effective amount of such other active agent will depend, among other things, on the amount of the polypeptide of the present invention present in the pharmaceutical composition, the type of disease or disorder, or treatment, etc.

[0214] In one embodiment, the polypeptides of the present invention are formulated using carriers that protect the compound from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems, such as liposomes. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Liposomal suspensions, including targeted liposomes, can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 or WO 2010 / 095940.

[0215] The route of administration of the polypeptides of the present invention can be parenteral. As used herein, the term "parenteral" includes intravenous, intraarterial, intralymphatic, intraperitoneal, intramuscular, or subcutaneous administration. Parenteral administration in intravenous or intramuscular dosage forms is preferred. "Systemic administration" refers to intravenous, intraperitoneal, and intramuscular administration. The amount of polypeptide required for therapeutic or prophylactic effect will, of course, vary depending on the polypeptide selected, the nature and severity of the condition being treated, and the patient. Furthermore, the polypeptide may be suitably administered by pulse infusion, e.g., with declining doses of the polypeptide. Preferably, administration is given by injection, most preferably intravenous, intramuscular, or subcutaneous injection, depending in part on whether the administration is brief or chronic.

[0216] Thus, in certain embodiments, the pharmaceutical compositions of the present invention may be in a form suitable for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in an appropriate unit dosage form. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CremophorEM (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and other pharmaceutically acceptable polyols, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0217] Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0218] Sterile injectable solutions can be prepared by incorporating the required amount of active compound (for example, the polypeptide of the present invention) into a suitable solvent with one or a combination of the ingredients listed above as needed, followed by filtration sterilization.Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients listed above.For the preparation of sterile powders for sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which allows the powder of the active ingredient and any additional desired ingredients to be obtained from its solution that has previously been sterile-filtered.

[0219] In certain embodiments, the pharmaceutical compositions are administered via intravenous (IV), intramuscular (IM), or subcutaneous (SC) routes. Suitable excipients, such as bulking agents, buffers, or surfactants, can be used. The above-described formulations will be prepared using standard methods for preparing parenterally administrable compositions, which are well known in the art and described in more detail in various sources, including, for example, "Remington: The Science and Practice of Pharmacy" (Ed. Allen, LV 22nd edition, 2012, www.pharmpress.com).

[0220] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate pharmaceutical compositions, i.e., parenteral compositions, in dosage unit form.Dosage unit form as used herein refers to a physically discrete unit suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound (polypeptide of the present invention) calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.The specifications of the dosage unit form of the present invention are determined and directly depend on the inherent characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of the technology of compounding such active compound for the treatment of individuals.

[0221] Generally, for the prevention and / or treatment of the diseases and disorders mentioned herein, and depending on the specific disease or condition being treated and its severity, the potency of the specific polypeptide of the invention used, the specific route of administration, and the specific pharmaceutical formulation or composition used, the polypeptides of the invention are generally administered continuously (e.g., by infusion) in the range of 0.001 to 1,000 mg / kg body weight / day, preferably about 0.01 to about 100 mg / kg body weight / day, and most preferably about 0.05 to 10 mg / kg body weight / day, e.g., about 1, 10, 100, or 1,000 mg / kg body weight / day, either as a single daily dose or in multiple divided doses throughout the day. A clinician will generally be able to determine an appropriate daily dose depending on the factors mentioned herein. It will also be apparent that in certain cases, a clinician may choose to deviate from these amounts, for example, based on the factors mentioned above and their professional judgment. The pharmaceutical compositions can be included in a container, pack, or dispenser along with instructions for administration.

[0222] therapeutic use In another aspect, there is provided a multispecific antigen binding protein as described herein for use as a medicament. In one embodiment, the multispecific antigen binding is used when the protein as described herein is used as an active ingredient, component or substance of a medicament.

[0223] In one aspect, the invention relates to the use of a multispecific antigen-binding protein as described herein for the manufacture of a medicament, such as a pharmaceutical formulation comprising the multispecific antigen-binding protein as an active ingredient, for the treatment, prevention or diagnosis of a disease in a subject in need thereof.

[0224] In one aspect, the invention relates to a multispecific antigen-binding protein as described herein or a pharmaceutical formulation comprising a multispecific antigen-binding protein as an active ingredient, for use in the treatment, prevention or diagnosis of a disease in a subject in need thereof.

[0225] In one aspect, the invention relates to a method of treating a disease in a subject in need thereof, comprising administering to the subject an (effective amount of) a multispecific antigen-binding protein or a pharmaceutical formulation comprising a multispecific antigen-binding protein as an active ingredient as described herein.

[0226] The disease to be treated, prevented or diagnosed using the multispecific antigen-binding protein may be cancer, an infectious disease, an inflammatory disease or an autoimmune disease.

[0227] In one embodiment, the disease to be treated, prevented or diagnosed using the multispecific antigen binding protein is cancer, such as those cancers described below.

[0228] In one embodiment, the present invention relates to a method of enhancing the anti-tumor activity of NK cells in a subject, the method comprising administering to the subject a multispecific antigen-binding protein as described herein or a pharmaceutical formulation comprising a multispecific antigen-binding protein as an active ingredient. In one embodiment, the subject has cancer, such as the cancers described below.

[0229] In one embodiment, the present invention relates to a method for expanding and / or inducing hyperactive NK cells in a subject, the method comprising administering to the subject a multispecific antigen-binding protein as described herein or a pharmaceutical formulation comprising a multispecific antigen-binding protein as an active ingredient. The fold expansion and hyperactivity are preferably as described herein above. In one embodiment, the subject has cancer, such as a cancer described below.

[0230] Subjects with cancer often have a lower number of NK cells and / or exhausted NK cells. Therefore, the multispecific antigen-binding proteins of the present invention can be advantageously used to increase the number of NK cells and / or induce NK cell hyperactivity in subjects suffering from cancer. Further benefits of NK cell hyperactivity induced by the multispecific antigen-binding proteins of the present invention include increased secretion of cytokines such as TNF-α, IFN-γ and IL-6, which help to form an adaptive immune response involving DCs and T cells. Indeed, NK cells are capable of secreting CD8 + It has been reported that DC subsets specialized in cross-expressing tumor antigens to T cells are recruited to the tumor microenvironment, and anti-tumor CD8 + These findings suggest a critical role for NK cells in enhancing T cell responses (Bottcher et al., Cell, 2018. 172: 1022-1037; and Barry et al., Nat. Med. 2018. 24: 1178-1191). The contribution of NK cells to orchestrating antitumor T cell responses has also been experimentally confirmed in mice, demonstrating that in addition to their direct effector function, NK cells can promote T cell responses and long-lasting immune control of tumors (Bonavita et al., Immunity 2020. 53: 1215-1229).

[0231] In one embodiment, the cancer to be treated, prevented or diagnosed using the multispecific antigen binding protein is a cancer selected from the group consisting of: carcinomas, such as carcinomas of the bladder, head and neck, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid and skin, such as squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myeloid leukemia and promyelocytic leukemia; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma, and teratocarcinoma; hematopoietic tumors of the lymphoid lineage, such as T-cell and B-cell tumors, including, but not limited to, T-cell disorders such as T-cell prolymphocytic leukemia (T-PLL), including the small cell and encephalomyeloma types; large granular lymphocyte leukemia (LGL), preferably of the T-cell type; Sézary syndrome (SS); adult T-cell leukemia-lymphoma (ATLL); a / d T-NHL hepatosplenic lymphoma; peripheral / retrothymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angioimmunoblastic T-cell lymphoma; angiocentric (nasal) T-cell lymphoma; undifferentiated (Ki 1+) large cell lymphoma; intestinal T-cell lymphoma; T-lymphoblastic; lymphoma / leukemia (T-Lbly / T-ALL).

[0232] In one embodiment, the multispecific antigen binding proteins described herein can be used as monotherapy (i.e., without other therapeutic agents), hi another embodiment, the multispecific antigen binding proteins described herein can be used in combination therapies.

[0233] In one embodiment, the multispecific antigen-binding proteins described herein are used in combination with another immunotherapy, such as a cellular immunotherapy. Thus, the multispecific antigen-binding proteins can be used in combination with the adoptive transfer of immune cells, including the adoptive transfer of T cells, e.g., CAR T cells, or NK cells. The NK cells can, for example, be enriched or expanded by methods known in the art, or can be ex vivo NK cells as described herein.

[0234] In one embodiment, a multispecific antigen-binding protein as described herein may be used in combination therapy with one or more other therapeutic agents. The additional therapeutic agent or agents are typically administered in amounts and with a treatment regimen typically used for that agent in monotherapy for the particular disease or condition being treated. Such therapeutic agents, when used in the treatment of cancer, include, but are not limited to, anti-cancer agents and chemotherapeutic agents. Exemplary therapeutic agents that may be used as part of a combination therapy in the treatment of cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinib acetate, ketanserin, doxifluridine, etretinate, isothionein, These include streptozocin, nimustine, vindesine, flutamide, drogenil, butosin, carmofur, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, iprosulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon alpha, interferon-2 alpha, interferon-beta, interferon-gamma, colony-stimulating factor-1, colony-stimulating factor-2, denileukin diftitox, interleukin-2, and luteinizing hormone-releasing factor.

[0235] A further class of agents that may be used as part of a combination therapy in the treatment of cancer are immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of: (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. Still other agents that may be used as part of a combination therapy in treating cancer are cytotoxic monoclonal antibodies against TAAs, including cytotoxic monoclonal antibodies against TAAs known in the art, such as trastuzumab (against HER2), pertuzumab (against HER2), rituximab (against CD20), tositumomab (against CD20), ibritumomab (against CD20), obinutuzumab (against CD20), ofatumumab (against CD20), alemtuzumab (against CD52), blinatumomab (against CD19), nebilizumab (against CD19), tabacitamab (against CD19), daratumumab (against CD38), isatuximab (against CD38), polatuzumab (against CD79b), dinutuximab (against GD2), ), naxitamab (against GD2), bevacizumab (against VEGF-A), elotuzumab (against SLAMF7), enfortumab (against nectin-4), sacituzumab (against TROP2), mogamulizumab (against CCR4), ipilimumab (against CTLA-4), tremelimumab (against CTLA-4), durvalumab (against PD-L1), pidilizumab (against PD-1), pembrolizumab (against PD-1), nivolumab (against PD-1), cemiplimab (against PD-1), avelumab (against PD-L1), cetuximab (against EGFR), necitumumab (against EGFR), panitumumab (against EGFR), olalatuzumab (against PDGFRα), and ramucirumab (against VEGFR2).

[0236] In some embodiments, administration of the multispecific antigen binding protein and another therapeutic agent can elicit additive or synergistic effects on immune and / or therapeutic efficacy.

[0237] In one embodiment, the multispecific antigen-binding proteins described herein are used as at least one of neoadjuvant and adjuvant therapy in addition to primary therapy, including, for example, surgery and / or radiation therapy. As neoadjuvant therapy, the multispecific antigen-binding protein is administered before primary treatment, for example, to help reduce tumor size (e.g., micrometastatic disease), kill spread cancer cells, and / or reduce the risk of tumor cell spread after surgery. As adjuvant therapy, the multispecific antigen-binding protein is administered after primary treatment, for example, to treat minimal residual disease (destroy remaining cancer cells). The use of multispecific antigen-binding proteins as neoadjuvant and / or adjuvant therapy reduces the recurrence rate. In neoadjuvant and / or adjuvant therapy, the multispecific antigen-binding protein can be used as a monotherapy or in combination therapy, as described above.

[0238] Ex vivo method In a further aspect, the present invention relates to a method in which the multispecific antigen-binding proteins described herein are used for the ex vivo (in vitro) treatment of NK cells or a population of NK cells. The method may be for at least one of expanding, pre-activating, activating, enhancing cytotoxicity and / or cytokine production and inducing a hyperactive phenotype as defined above. The method comprises at least the step of contacting NK cells or a population thereof with the multispecific antigen-binding protein or a composition comprising the multispecific antigen-binding protein described herein.

[0239] NK cells or NK cell populations for ex vivo treatment can be enriched from peripheral blood mononuclear cells (PBMCs). Methods for enriching and ex vivo treating NK cells from PBMCs are described, for example, in Denman et al. (PLoS One. 2012; 7(1): e30264) and U.S. Patent Application Publication No. 2020 / 0061115. For example, NK cells enriched from PBMCs can be cultured at 0.1 x 10 cells / mL in SCGM (CellGenix, Portsmouth, NH) supplemented with 10% FBS, 2 mM Glutamax, 100 U / mL IL-2 (Peprotech, Rocky Hill, NJ), and 1, 2, 5, 10, 20, 50, 100, 200, 500, or 1000 μg / mL of one or more multispecific antigen-binding proteins described herein. 6 The cells can be seeded at 100 NK cells / mL. The medium containing supplements can be changed every 2–3 days from day 5 onwards.

[0240] It is understood that the duration of contact between the NK cells and the multispecific antigen-binding proteins described herein (i.e., the period of time during which cytotoxicity and / or cytokine production is expanded, pre-activated, activated, enhanced, or a hyperfunctional phenotype is induced) can be any length of time necessary to achieve the desired phenotype of the NK cells. For example, contact can be for as little as 1 minute or for 7 days (e.g., culturing NK cells in the presence of a multispecific antigen-binding protein described herein for 7 days). In one embodiment of the method, the NK cells are contacted with the multispecific antigen-binding protein for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours. In one embodiment of this method, the NK cells are contacted with the multispecific antigen-binding protein for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 days.

[0241] In one embodiment, the ex vivo treated (expanded) NK cells have one or more characteristics selected from the following: a) a fold increase in the number of expanded NK cells of at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times the fold increase in the number of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells; b) the telomere length of the expanded NK cells is at least 10, ... a 5, 20, 25, 30, 35, 40, 45, 50 or 55% increase in telomere length of the expanded NK cells compared to the telomere length of fresh NK cells, preferably at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the increase in telomere length of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells; c) NKG2D, NKp30, NKp44 on the expanded NK cells; the expression level of at least one NK cell activating receptor selected from NKp46 and CD16 is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times the expression level on NK cells obtained during ex vivo expansion in the presence of FC21 feeder cells; d) the secretion of at least one cytokine of TNF-α, IFN-γ, and IL-6 by the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times the expression level on NK cells obtained during ex vivo expansion in the presence of FC21 feeder cells; e) the cytotoxicity of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the cytokine secretion by the NK cells obtained upon in vivo expansion; and f) the cytotoxicity of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the cytotoxicity of the NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells.

[0242] In a still further aspect, the present invention relates to a method for treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of NK cells obtained by the above-described method for ex vivo treatment of NK cells or a population of NK cells. Once a sufficient number of NK cells with the desired (hyperactive) phenotype have been expanded by ex vivo treatment, the NK cells may be administered to a subject in need thereof.

[0243] In one embodiment, the method of treatment comprises the administration of ex vivo treated NK cells in combination with a multispecific antigen binding protein described herein or a pharmaceutical formulation comprising a multispecific antigen binding protein as an active ingredient.

[0244] In one embodiment, the method of treatment involves administering ex vivo treated NK cells in combination with another NK cell engager, such as those described in WO 2016 / 207278, WO 2018 / 148445, WO 2018 / 152518, WO 2019195409, U.S. Patent Application Publication No. 2018282386, Vallera et al. (2016, supra) and Demaria et al. (2021, supra), or a multispecific antigen-binding protein described in a co-pending application by the same applicant bearing reference number P6110749EP. One example of another NK cell engager is AVC-006, described in the Examples herein, which contains, for example, one HER2-binding region and one NKG2D-binding region. In a further embodiment, the ex vivo treated NK cells can be used in combination with other engagers and multispecific antigen-binding proteins described herein.

[0245] The disease to be treated may be cancer, an infectious disease, an inflammatory disease or an autoimmune disease, as described above. Preferably, the disease to be treated is cancer, as described above.

[0246] In one embodiment, the ex vivo treated NKs are autologous to the subject. In another embodiment, the ex vivo treated NKs are allogeneic, e.g., derived from donor PBMCs.

[0247] Nucleic acids, host cells and methods for producing multispecific antigen-binding proteins In one aspect, the present invention relates to a nucleic acid molecule comprising one or more nucleotide sequences encoding the polypeptide chains of a multispecific antigen-binding protein as described herein. The nucleotide sequences encoding such polypeptide chains preferably encode a signal peptide operably linked to the polypeptide chain. The nucleic acid molecule comprising one or more nucleotide sequences encoding the polypeptide chains further preferably comprises regulatory elements for (or that facilitate) expression of the polypeptide chain in a suitable host cell, wherein the regulatory elements are operably linked to the nucleotide sequences.

[0248] In one aspect, the present invention relates to a host cell comprising a nucleic acid molecule comprising one or more nucleotide sequences encoding the polypeptide chains of a multispecific antigen-binding protein described herein. In one embodiment, the host cell is an isolated cell or a cultured cell. Among the host cells that can be used are prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms, such as Escherichia coli or Bacillus. Suitable yeast cells include Saccharomyces cerevisiae and Pichia pastoris. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include monkey kidney cell lines COS-1, COS-7, L cells, HEK 293 cells, C127 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, HeLa cells, BHK cell lines such as BHK21, BSC-1, Hep G2, 653, SP2 / 0, and the CVI / EBNA cell line derived from the African green monkey kidney cell line CVI described by McMahan et al. (1991, EMBO J. 10:2821). The host cell can be any suitable species or organism capable of producing N-linked glycosylated polypeptides, for example, a mammalian host cell capable of producing human or rodent IgG-type N-linked glycosylation. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985). Host cells containing a nucleic acid molecule of the invention can be cultured under conditions that promote expression of the polypeptide.

[0249] Thus, in another aspect, the present invention relates to a method for producing a multispecific antigen-binding protein as described herein. The method preferably comprises culturing a host cell as described above such that one or more nucleotide sequences are expressed and the multispecific antigen-binding protein is produced. The method preferably comprises culturing a host cell comprising one or more nucleotide sequences encoding the polypeptide chains of the multispecific antigen-binding protein. The host cell is preferably cultured under conditions that promote expression of one or more polypeptide chains. The method may further comprise recovering the multispecific antigen-binding protein. The multispecific antigen-binding protein can be recovered by conventional protein purification procedures including, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, size-exclusion chromatography, or affinity chromatography using, for example, streptavidin / biotin (see, e.g., Low et al., 2007, J. Chromatography B, 848: 48-63; Shukla et al., 2007, J. Chromatography B, 848: 28-39).

[0250] In a further aspect, the present invention relates to a method for producing a pharmaceutical composition comprising a multispecific antigen-binding protein as described herein, said method comprising the steps of a) producing the multispecific antigen-binding protein by a method as defined above, and b) formulating the multispecific antigen-binding protein with a pharmaceutically acceptable carrier as defined above to obtain the pharmaceutical composition.

[0251] The present invention has been described with reference to several exemplary embodiments shown in the drawings and examples. Modifications and alternative implementations of several parts or elements are possible and fall within the scope of protection defined in the appended claims. [Brief explanation of the drawings]

[0252] Text description of the illustration image022.gif. [Figure 1]Schematic diagram of multispecific antigen-binding proteins and control proteins AVC-009 to AVC-015 (see Example 1 for sequence details). AVC-009 to AVC-010 are heterodimers. AVC-011 to AVC-015 are homodimers. NKp46Fab = Fab fragment specific for NKp46; TrasFc = trastuzumab Fc region; 41BBLT = 4-1BB ligand trimer; 41BBLM = 4-1BB ligand monomer; CD16- = L234A / L235A modification in the Fc region to reduce binding to CD16. [Figure 2] Ex vivo expansion of donor NK cells stimulated with multispecific antigen-binding proteins AVC-009 (AVC9), AVC-011 (AVC11), or AVC-014 (AVC14). FC21 is the reference expansion method using irradiated FC21 feeder cells. [Figure 3] Cytotoxicity of NK cells stimulated for one week with multispecific antigen-binding proteins AVC-009 (AVC9), AVC-011 (AVC11), or AVC-014 (AVC14) or with FC21 feeder cells as a reference against MDA-MB-231 target cells. All three, AVC-009, AVC-011, and AVC-014, induce strong cytotoxicity against MDA-MB-231 target cells. The cytotoxicity of NK cells expanded in the presence of AVC-009 or AVC-014 is not significantly different from the cytotoxicity of NK cells stimulated with the reference FC21 feeder cells. [Figure 4] Figure 4 shows the viSNE analysis described in Example 4.3 using 30 markers, showing the population distribution of cells from different expansion methods. AVC11, AVC14, AVC9, and FC21 are after one week of expansion as described in Example 3.5 in the presence of multispecific antigen-binding proteins AVC-009, AVC-011, or AVC-014, or FC21 feeder cells, respectively. Color mapping indicates expression of CD56 or Ki67, used to identify NK cells. Compared to the control condition of non-expanded NK cells, all four expansion methods result in enrichment of highly CD56-expressing NK cells with very similar phenotypes. [Figure 5] Up- or down-regulation of NK cell surface markers shown after one week of expansion of NK cells as described in Example 3.5 in the presence of multispecific antigen-binding proteins AVC-009, AVC-011, or AVC-014, respectively, or FC21 feeder cells (FC21). Fold change (Log2) is compared to the control state of non-expanded NK cells. [Figure 6] Comparison of NK cell proliferation induced by culturing the cells described in Example 3.5 in the presence of the multispecific antigen-binding proteins AVC9 (IL-21 and 4-1BBL), AVC81 (IL-21) or AVC82 (4-1BBL). DETAILED DESCRIPTION OF THE INVENTION

[0253] Example Example 1 Preparation of multispecific antigen-binding proteins Coding sequences were generated for multispecific antigen binding proteins, including proteins AVC-009 to AVC-015, shown generally in Figure 1, and control proteins, and the amino acid sequences are shown in Tables 1.1.1 and 1.1.2.

[0254] Expression constructs for expression of coding sequences in the HEK293-E-253 cell line were prepared using standard molecular biology materials and techniques. Briefly, coding sequences were generated by direct synthesis and / or PCR. PCR was performed using PrimeSTAR MAX DNA polymerase (Takara, #R045A), and PCR products were purified from 1% agarose gels using a NucleoSpin Gel and PCR Clean-Up Kit (Macherey-Nagel, #740609.250). Once purified, PCR products were quantified prior to the In-Fusion ligation reaction, which was performed as described in the manufacturer's protocol (ClonTech, #ST0345). Plasmids were obtained after miniprep preparation. Plasmids were then sequenced for sequence confirmation before transfection into the HEK293-E-253 cell line.

[0255] All buffers used were made with Versylene (endotoxin-free and sterile) water. Endotoxin was removed from the device by incubation with 0.1 M NaOH for at least 16 hours. Line clearance was performed at the beginning and end of production.

[0256] HEK293E-253 cells were transfected with endotoxin-free plasmid DNA using rPEx® technology. Six days after transfection, conditioned medium containing the recombinant protein was harvested by centrifugation and filtration through a 0.22 μm bottle-top filter. 100 μl samples were stored at 4°C. To avoid binding of nonspecific proteins or cell fragments to the MabSelect PrismA resin, samples were conditioned with 40 mL of 5 M NaCl per L of medium, increasing the NaCl concentration by 0.2 M.

[0257] A HiScreen Fibro PrismA column was equilibrated in 20 mM Tris, 150 mM NaCl, pH 7.8. The recombinant protein in the conditioned medium was loaded onto the column using a Teledyne ASX-560 autosampler. Nonspecifically bound proteins were removed by washing the column with 20 mM Tris, 1 M NaCl, pH 7.8 and 20 mM citrate, 150 mM NaCl, pH 5.0. Bound recombinant protein was eluted with a 12 CV block gradient to 20 mM citrate, 150 mM NaCl, pH 3.5 and a 6 CV block gradient to 20 mM citrate, 150 mM NaCl, pH 3.0. The eluate was neutralized directly in-line by mixing 62% of 1 M Tris, pH 8.0, at pH 7 in a 1.0 / 0.2 ratio, and 12.5 mL fractions were collected. Between injections, the HiScreen Fibro PrismA column was regenerated using 5 CV of 0.5 M NaOH, 2 CV of 1 M Tris pH 8.0, and equilibrated in 20 mM Tris 150 mM NaCl pH 7.8. The Teledyne autosampler was washed with 15 mL of 0.1 M NaOH and equilibrated in PBS.

[0258] The HiTrap Fibro PrismA pool was concentrated to 2-3 mL / injection on a Superdex 200 16 / 40 column using an Amicon 30 kDa spin filter. Aggregates in the concentrated pool were removed by filtration through a 0.22 μm syringe filter. The concentrated sample was stored at 4°C before undergoing gel filtration.

[0259] The recombinant protein products were analyzed by Labchip capillary electrophoresis and LAL assay.

[0260] [Table 5]

[0261] [Table 6]

[0262] [Table 7]

[0263] [Table 8]

[0264] [Table 9]

[0265] [Table 10]

[0266] [Table 11]

[0267] [Table 12]

[0268] [Table 13]

[0269] [Table 14]

[0270] [Table 15]

[0271] [Table 16]

[0272] [Table 17]

[0273] Example 2 Biochemical Characterization of Multispecific Antigen-Binding Proteins 2.1. Expression level and molecular weight The expression levels and molecular weights of multispecific antigen-binding proteins are determined by SDS-PAGE and size-exclusion chromatography (SEC) using Coomassie blue-stained 4%-15% SDS-PAGE under both reducing and non-reducing conditions (Zhang et al. Clin Cancer Res, 2007; 13 (9): 2758-2767).

[0274] Size exclusion chromatography was performed using a Yarra SEC-3000 column (Phenomenex, 00H-4513-K0) and a Waters 2695 HPLC (Waters Corporation) in a 0.1 M NaHPO / NaHPO, 0.1 M NaSO, pH 6.7 mobile phase at a flow rate of 0.5 mL / min. Thyroglobulin (669 kDa, Sr 8.5 nm), β-amylase (200 kDa, Sr 5.4 nm), bovine serum albumin (67 kDa, Sr 3.55 nm), carbonic anhydrase (29 kDa, Sr 2.35 nm), and FLAG peptide (1 kDa) served as standard proteins.

[0275] 2.2 Stability The stability of the multispecific antigen-binding protein is determined by repeating the SDS-PAGE and SEC described above on samples of the multispecific antigen-binding protein stored at room temperature for 1, 3 and 7 days.

[0276] The stability of the multispecific antigen-binding protein in plasma is determined by incubating the protein at 200 nM in 50% human plasma at 37° C. for 1, 3 and 7 days.

[0277] Samples are frozen at -20°C immediately after preparation (day 0) or after each incubation period. Intact protein levels are determined using SDS-PAGE and SEC as described above.

[0278] 2.3 Affinity for IL21R The binding affinity between IL-21R and multispecific antigen-binding proteins was analyzed using a Biacore T200 instrument at 25°C and a flow rate of 50 μl / min. Anti-human Fc antibodies were covalently immobilized on a CM5 sensor chip to capture the multispecific antigen-binding proteins in flow cells fc2 and fc4, using fc1 and fc3 as references. IL-21R was introduced at concentrations of 0.74 nM, 2.22 nM, 6.67 nM, 20 nM, and 60 nM, prepared by a series of 1:3 serial dilutions. The assay included a 180-second association phase and a 1,200-second dissociation phase after the highest concentration exposure. The assay buffer contained 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.05% Tween 20, and 3 mM EDTA. Regeneration was performed under standard conditions to remove complexes from all surfaces.

[0279] 2.4 Affinity for 4-1BB Interactions between multispecific antigen-binding proteins and 4-1BB receptors were analyzed using a Biacore T200 instrument at 25°C with a flow rate of 50 μl / min. A CM5 sensor chip from the Human Antibody Capture Kit (Cytiva) was used, and the analysis buffer consisted of 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.05% Tween 20, and 3 mM EDTA. Anti-human Fc antibodies were covalently immobilized on the CM5 sensor chip to capture the multispecific antigen-binding proteins on flow cells fc2 and fc4, while fc1 and fc3 served as references. Human 4-1BB was introduced at concentrations of 5 nM, 15 nM, 45 nM, 135 nM, and 405 nM following a series of 1:3 serial dilutions. The assay protocol included a 120-second association phase and a 600-second dissociation phase following the introduction of the highest concentration sample. After interaction analysis, regeneration was performed under standard conditions to effectively remove the complexes from all surfaces.

[0280] Recombinant biotinylated human, cynomolgus monkey, and mouse 4-1BB Fc(kih) fusion molecules (see Example 3 of WO 2016 / 075278) are directly coupled onto the SA chip using standard coupling instructions (Biacore, Freiburg / Germany). The immobilization level is approximately 30 RU. Polyspecific antigen binding or control, ranging in concentration from 0.39 nM to 200 nM, is passed through the flow cell for 120 seconds at a flow rate of 30 μL / min. Dissociation is monitored for 180 seconds. Bulk refractive index differences are corrected by subtracting the response obtained with a reference empty flow cell.

[0281] For affinity measurements, direct coupling of approximately 7200 resonance units (RU) of anti-human Fc-specific antibodies was performed on a CM5 chip at pH 5.0 using a standard amine coupling kit (GF Healthcare). 50 nM of 4-1BBL-containing multispecific antigen-binding protein or control was captured on flow cell 2 at a flow rate of 30 μL / min for 60 seconds. A dilution series (1.95 to 1000 nM) of human 4-1BB-avi-His (see Example 3 of WO 2016 / 075278) was flowed through both flow cells at 30 μL / min for 180 seconds to record the association phase. The dissociation phase was monitored for 180 seconds and initiated by switching from the sample solution to HBS-EP. The chip surface was regenerated after each cycle using a double injection of 10 mM glycine-HCl pH 2.1 for 60 seconds. Bulk refractive index differences were corrected by subtracting the response obtained on reference flow cell 1. For the interaction between 4-lBBL-containing multispecific antigen-binding proteins and hu4-lBB avi His, affinity constants are derived from the rate constants by fitting to 1:1 Langmuir binding curves using Biaeval software (GF Healthcare).

[0282] 2.5 Affinity for NKp46 The affinity of the multispecific antigen-binding protein for NKp46 was determined by SPR essentially as described in Gauthier et al., 2019, Cell 177, 1701-1713.

[0283] Example 3 Functional in vitro characterization of multispecific antigen-binding proteins 3.1 Binding to NK cells Binding of the multispecific antigen-binding proteins to NK cells is demonstrated using flow cytometry and competitive inhibition with unlabeled competing antibodies, essentially as described by Fellermeier et al. (2016, supra). Assays are performed in triplicate on NK cells from four different donors. Mean fluorescence intensity is plotted against dilutions of the multispecific antigen-binding proteins to determine the EC50 and EC90 concentrations for optimal effective engagement.

[0284] 3.2 Short-term NK cytotoxicity The short-term (4-hour) NK cytotoxicity of multispecific antigen-binding proteins was determined using a calcein-acetoxymethyl release assay (Somanchi et al., 2011, J Vis Exp., 2: 2540; Lee et al., 2010, Methods Mol. Biol., 651: 61-77). Target cells (MDA-MB-231, K562, Karpas 299, SK-N-AS, and A253) were loaded with 0.025 μM calcein-AM and incubated at 37°C for 1 hour with gentle resuspension every 10–15 minutes. After this time, target cells were washed twice with medium, and 10,000 target cells were seeded into a 96-well plate. NK cells were purified by negative selection using RosetteSep (In: Methods in Molecular Biology, Ex Vivo Expansion of Human NK Cells Using K562 Engineered to Express Membrane Bound IL21 Srinivas S. Somanchi and Dean A. Lee DOI 10.1007 / 978-1-4939-3684-7) from four normal donor buffy coats, as described in Example 3.5 below, and expanded ex vivo with the multispecific antigen-binding proteins AVC-009, AVC-011, or AVC-014 (at saturating concentrations). As a control, NK cells were stimulated with irradiated FC21 feeder cells at a ratio of 1:2 (NK:FC21). Assays were performed in triplicate. The cytotoxicity of NK cells expanded ex vivo for one week in the presence of multispecific antigen-binding proteins (AVC-009, AVC-011, or AVC-014) was compared with the cytotoxicity of NK cells expanded ex vivo in the presence of irradiated FC21 feeder cells. NK cells were harvested from the expansions and added to target cells at an E:T ratio of 2:1 and co-cultured at 37°C for 4 hours. In addition, two control conditions were also established: 1. Target cells only - This sample was used to quantify the spontaneous release of calcein-AM from tumor cells. 2. Maximum release - This sample was treated with Triton X-100 to kill and permeabilize the target cells and used to quantify the maximum possible release of calcein-AM.

[0285] After this incubation, the supernatant was transferred to a new plate and the fluorescence was measured in a plate reader using an excitation filter of 485 nm and an emission filter of 530 nm. Specific lysis was calculated using the following formula:

number

[0286] The results are shown in Figure 3 and demonstrate that all three multispecific antigen-binding proteins, AVC-009, AVC-011, and AVC-014, induce strong NK cell cytotoxicity against MDA-MB-231 target cells. Furthermore, the cytotoxicity of NK cells expanded in the presence of AVC-009 or AVC-014 is not significantly different from that of NK cells stimulated with FC21 feeder cells. Similar data were obtained using K562, Karpas 299, SK-N-AS, and A253 as target cells (data not shown).

[0287] 3.3 Long-term NK cytotoxicity To assess sequential killing, the long-term NK cytotoxicity of the multispecific antigen-binding proteins was determined using the xCelligence assay (Naeimi Kararoudi et al., 2022, Cell Reports Methods 2, 100236 June 20, 2022). The hematological target cells were K562 and Karpas 299 (T-cell NHL). The solid tumor target cell lines were SK-N-AS (neuroblastoma) and A253 (salivary gland carcinoma). NK cells were purified by negative selection using RosetteSep (see above) from four normal donor buffy coats and co-cultured with the multispecific antigen-binding proteins at a low E:T ratio (0.5:1) in the presence or absence of the multispecific antigen-binding proteins (at saturating concentrations) or with anti-NKp46 antibodies of SEQ ID NOs: 1 and 2. Assays were performed in triplicate. The cytotoxicity of fresh NK cells (described above) is compared to the cytotoxicity of i) NK cells expanded ex vivo in the presence of the multispecific antigen-binding protein, and ii) NK cells expanded ex vivo in the presence of irradiated FC21 feeder cells. NK cells are expanded ex vivo as described in 3.5 below.

[0288] 3.4 NK cell proliferation assay NK cells are labeled with CellTrace Violet Cell Proliferation Dye and expanded in the presence or absence of multispecific antigen-binding protein (at saturating concentrations or at three low, medium, and high dose levels, e.g., 25, 50, and 100%). Assays are performed in triplicate. After 7 days, cells are harvested and stained for viability with Live / Dead stain and viable CD3+ cells are counted. - CD56 + NK cell populations are stained for surface markers to gate. Proliferation is plotted against tumor cell and booster dilutions to identify EC50 and EC90 concentrations for optimal effective concentrations.

[0289] 3.5 Ex vivo expansion of NK cells A protocol for ex vivo expansion of donor NK cells is described in Denman et al. (2012, supra).

[0290] Briefly, NK cells were isolated from the buffy coats of eight healthy donors using RosetteSep enrichment and Ficoll (GE HealthCare, Piscataway, NJ). On day 0, NK cells were stimulated as follows: 1.0 × 10 6 NK cells were incubated with 25 nM of each of the multispecific antigen-binding proteins AVC-009, AVC-011, or AVC-014. As a control, NK cells were incubated at 1 x 10 6 The cells were stimulated with irradiated (100 cGy) FC21 feeder cells at a ratio of 1:2 (NK:FC21) in NK cell medium at 0.2 × 10 NK / mL. Cells were then cultured in AIM V™ medium (12055091, Gibco, Thermo Scientific) supplemented with CTS™ immune cell serum replacement (A2596101, Gibco, Thermo Scientific) and 50 IU / mL recombinant human IL-2 (Proleukin, Novartis Vaccines and Diagnostics, Inc.). 6 Cells were seeded at a density of 1000 cells / mL.

[0291] Every other day of expansion, count NK cells using a Nexcelom Cellometer and AOPI staining solution. Determine total cell number. Add AIM V medium supplemented with CTS immune cell serum replacement to culture cells at a concentration of 0.3 x 10 6 Maintain a concentration of NK cells / mL of medium close to 50 IU / mL of human IL-2 for each condition / donor.

[0292] Figure 2 shows that each of the multispecific antigen-binding proteins AVC-009 or AVC-011 and AVC-014 can induce ex vivo expansion of NK cells. FC21 is the reference expansion scheme.

[0293] Figure 6 shows a graph comparing the different rates of NK cell proliferation induced by AVC9 (IL-21 and 4-1BBL), AVC81 (IL-21), and AVC82 (4-1BBL). As can be seen, after 2 weeks of proliferation, cells were counted and proliferation induced by AVC9 (IL-21 and 4-1BBL) is superior to both AVC81 (IL-21) and AVC82 (4-1BBL).

[0294] Example 4 Phenotypic Characterization of NK Cells 4.1 Cytokine secretion by expanded NK cells NK cells were cultured for 7 days in the absence and presence of saturating concentrations of multispecific antigen-binding proteins. Supernatants were collected, and the concentrations of TNF-α, IFN-γ, IL-2, and IL-6 were simultaneously determined using microparticle-based cytokine capture on a Cytometric Based Array kit. Concentrations were calculated from mean fluorescence intensity based on the standard curve and formula provided with the kit (Denman et al. PLoS ONE, 2012, supra). Cytokine secretion from fresh NK cells (described above) was compared to cytokine secretion by i) NK cells expanded ex vivo in the presence of multispecific antigen-binding proteins and ii) NK cells expanded ex vivo in the presence of irradiated FC21 feeder cells. NK cells were expanded ex vivo as described in 3.5 above.

[0295] 4.2 Transcriptome analysis of expanded NK cells Gene expression in NK cells stimulated with multispecific antigen-binding proteins was assayed using the nCounter platform (nanoString Technologies, Inc., Seattle, WA; Geiss et al. 2008, Nat Biotechnol 26: 317-325). Purified NK cells from four donors were stimulated with either multispecific antigen-binding proteins or FC21 feeder cells (Denman et al., pLoS ONE, 2012, supra) in parallel growth for one week. Total RNA was purified from each sample and assessed for expression of 96 genes (Denman et al., pLoS ONE, 2012, supra). Gene expression was normalized to LDH (average 6,076 copies detected from 100 ng of loaded mRNA) and plotted as mean + SEM. Genes with borderline detection (fewer than 10 normalized transcripts detected) were excluded from further analysis. Genes with a 2-fold or greater difference in mean expression between cultures containing the multispecific antigen binding protein and FC21 are then identified.

[0296] 4.3 Analysis of NK cell phenotype by multiparameter mass cytometry NK cell phenotype is identified by multiparameter mass cytometry. NK cells were harvested at the beginning of expansion (baseline) and after one week of expansion in the presence of multispecific antigen-binding proteins AVC-009, AVC-011, or AVC-014 or FC21 feeder cells (see Example 3.5) and incubated with the antibodies listed in Table 4.3.1. Following this, cells were fixed with 2% formaldehyde in PBS and stored in this solution until harvest.

[0297] [Table 18]

[0298] Immediately prior to acquisition, samples were washed in Cell Staining Media (CSM; PBS + 0.5% FBS) and resuspended in a 1:20 dilution of EQ™ Four Element Calibration Beads (Fluidigm) in CSM at a concentration of 1 million cells / mL, as previously described (Rahman et al., 2016, Cytometry A, 89: 601-607).

[0299] After acquisition on a Helios mass cytometer (Fluidigm), the resulting FCS files were normalized using the normalization tool developed by Finck et al. and analyzed in Cyto-bank (www.cytobank.org) (Finck et al., 2013, Cytometry A, 83 (5): 483-94; Kotecha et al., Curr Protoc Cytom. 2010 Jul; Chapter 10: Unit10.17).

[0300] Once the singlet gate was established, cells were identified using markers (listed in Table 4.3.1) and analyzed using viSNE, a high-dimensional single-cell data visualization tool based on the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm (see Amir et al., 2013, Nat. Biotechnol. 31 (6): 545-552).

[0301] The results in Figures 4 and 5 show that the phenotype of NK cells stimulated and activated by AVC-009, AVC-011, or AVC-014 during expansion closely matches the previously described hyperactive phenotype of NK cells stimulated and activated by the reference FC21 feeder cells (Denman et al. (2012, supra). The main difference between the phenotype induced by the multispecific antigen-binding proteins and FC21 feeder cells is the increase in Ki67 and perforin expression (see also Figure 5).

[0302] Example 5 In vivo characterization of multispecific antigen-binding proteins In vivo PK and biodistribution of the multispecific antigen-binding proteins are performed using a whole body clearance study in BALB / c mice with radiolabeled fusion proteins as described by Zhang et al. (Clin Cancer Res, 2007, supra).

[0303] Embodiments: 1. A multispecific antigen-binding protein comprising: a) an NK cell-activating cytokine, i) interleukin 21 receptor (IL21R) agonists, and ii) 4-1BB agonists and an NK cell-activating cytokine, which is at least one of b) a region that has affinity for a surface antigen expressed on natural killer (NK) cells; 1. A multispecific antigen-binding protein comprising: 2. The multispecific antigen-binding protein of embodiment 1, wherein the IL21R agonist comprises or consists of an agonistic antigen-binding region that specifically binds to an IL21 polypeptide or IL21R. 3. The multispecific antigen-binding protein of embodiment 2, wherein the IL21 polypeptide comprises an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 38, and preferably has at least one of IL21R agonist activity and affinity for IL21R. 4. The multispecific antigen-binding protein of embodiment 2 or 3, wherein the IL21 polypeptide is an IL21 mutein that has been modified to have reduced or enhanced affinity for IL21R compared to the corresponding wild-type IL21 polypeptide. 5. The multispecific antigen binding protein of embodiment 4, wherein the IL21 mutein has reduced affinity for IL21R compared to a corresponding wild-type IL21 polypeptide, and wherein the IL21 mutein comprises a mutation in one or more amino acids selected from the group consisting of 116, 166, 18, K72, K73, K75, K77, L13, P78, Q12, Q19, R5, R65, R76, R9, S70, S80, V69 and Y23. 6. The multispecific antigen-binding protein of any one of the preceding embodiments, wherein the multispecific antigen-binding protein has an IL21R agonist valency of greater than 1. 7. The multispecific antigen-binding protein of any one of the preceding embodiments, wherein the 4-1BB agonist comprises or consists of at least one 4-1BB ligand (4-1BBL) extracellular domain (ECD) or at least one agonistic antigen-binding region that specifically binds to 4-1BB. 8. The multispecific antigen binding protein of embodiment 6, wherein the 4-1BBL ECD comprises an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 37, and preferably has at least one of 4-1BB agonist activity and affinity for 4-1BB. 9. The multispecific antigen-binding protein of embodiment 7 or 8, wherein the 4-1BBL ECD is a mutein that has been engineered to decrease or enhance affinity for 4-1BB compared to the corresponding wild-type 4-1BBL ECD. 10. The multispecific antigen-binding protein of any one of embodiments 7-9, wherein the 4-1BB agonist comprises or consists of a fusion protein comprising three 4-1BBL ECD monomers fused together in a single polypeptide chain, optionally wherein the three 4-1BBL ECD monomers are linked by a polypeptide linker. 11. The multispecific antigen-binding protein of any one of embodiments 7 to 10, having a 4-1BB agonist titer of greater than 1.1. 12. The multispecific antigen-binding protein of any one of the preceding embodiments, comprising an IL21R agonist and a 4-1BB agonist. 13. The multispecific antigen-binding protein of any one of the preceding embodiments, further comprising an NK cell-activating cytokine selected from an IL15 receptor agonist, a Type I interferon (IFN-1) agonist, an IL2 receptor agonist, an IL12 receptor agonist and an IL18 receptor agonist. 14. The multispecific antigen-binding protein of any one of the preceding embodiments, wherein the region with affinity for a surface antigen expressed on an NK cell comprises or consists of an immunoglobulin Fc region. 15. The multispecific antigen-binding protein of embodiment 14, wherein the Fc region is a dimeric Fc region. 16. The multispecific antigen-binding protein of embodiment 14 or 15, wherein the Fc region binds to CD16A. 17. The multispecific antigen-binding protein of embodiment 16, wherein the immunoglobulin Fc region has been modified to reduce or enhance affinity for CD16A compared to the corresponding wild-type Fc region. 18. The multispecific antigen-binding protein is c) at least one antigen-binding region that specifically binds to an NK cell-activating receptor 10. The multispecific antigen-binding protein of any one of the preceding embodiments, further comprising: 19. The multispecific antigen-binding protein of embodiment 18, wherein the antigen-binding region that specifically binds to the NK cell activating receptor is an agonistic antigen-binding region that activates the NK cell receptor. 20. The multispecific antigen-binding protein of any one of embodiments 18-19, wherein the antigen-binding region comprises at least one immunoglobulin variable region. 21. The multispecific antigen-binding protein of embodiment 20, wherein the immunoglobulin variable region comprises or consists of a Fab or an immunoglobulin single chain variable domain (ISVD). 22. The multispecific antigen-binding protein of any one of embodiments 18 to 21, wherein the antigen-binding region is a human or humanized antigen-binding region. 23. The multispecific antigen-binding protein of any one of embodiments 18 to 22, comprising two antigen-binding regions that specifically bind to an NK cell activating receptor. 24. The multispecific antigen-binding protein of embodiment 23, wherein the two antigen-binding regions bind to the same NK cell activating receptor or at least two different NK cell activating receptors. 25. The multispecific antigen-binding protein of embodiment 24, wherein the two antigen-binding regions are identical. 26. The multispecific antigen-binding protein of any one of embodiments 18 to 25, wherein the NK cell activating receptor is selected from the group consisting of NKp46, NKp30, NKG2D, CD16A, SLAMF7, NKp44, CD94-NKG2C / E, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR2DS2, KIR2DL4, KIR3DS1, CD160, NKp80, DNAM1, 2B4, CD59, PD-L1, Tim3, CRACC, 4-BB, OX40, CRTAM, CD27, PSGL1, CD96, CD100, CEACAM1 and NTB-A. 27. The antigen-binding region is a) the CDR-H1 (SEQ ID NO: 24), CDR-H2 (SEQ ID NO: 25), and CDR-H3 (SEQ ID NO: 26) sequences contained in SEQ ID NO: 1 and the CDR-L1 (SEQ ID NO: 27), CDR-L2 (SEQ ID NO: 28), and CDR-L3 (SEQ ID NO: 29) sequences contained in SEQ ID NO: 2, and b) the CDR-H1, CDR-H2, and CDR-H3 sequences contained in SEQ ID NO: 3 and the CDR-L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 20 27. The multispecific antigen-binding protein of embodiment 26, comprising a combination of complementarity determining regions (CDRs) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of: 28. The antigen-binding region is a) V contained in SEQ ID NO: 1 H Sequence and V contained in SEQ ID NO:2L and b) V contained in SEQ ID NO: 3 H V contained in the sequence and SEQ ID NO: 20 L array A variable weight (V) selected from the group consisting of L ) and variable weight (V H 28. The multispecific antigen-binding protein of embodiment 27, comprising a combination of .ALPHA.-, ... 29. The multispecific antigen-binding protein of any one of the preceding embodiments, wherein at least one NK cell-activating cytokine is conjugated to a region having affinity for a surface antigen expressed on an NK cell. 30. The multispecific antigen-binding protein of embodiment 29, wherein at least one NK cell-activating cytokine forms a single polypeptide chain with at least one polypeptide chain of the region with affinity for a surface antigen expressed on NK cells, and optionally the NK cell-activating cytokine is linked to the polypeptide chain of the region with affinity for a surface antigen expressed on NK cells via a flexible linker. 31. The multispecific antigen-binding protein of embodiment 30, wherein the single polypeptide chain comprises, in order from N-terminus to C-terminus: i) a first NK cell-activating cytokine, ii) optionally a first flexible linker, iii) a polypeptide chain of a region having affinity for a surface antigen expressed on an NK cell, iv) optionally a second flexible linker, and v) a second NK cell-activating cytokine, preferably wherein the first and second NK cell-activating cytokines are different NK cell-activating cytokines. 32. The multispecific antigen-binding protein of any one of embodiments 18 to 30, wherein at least one antigen-binding region that specifically binds to an NK cell activating receptor is conjugated to a region with affinity for a surface antigen expressed on NK cells. 33. The multispecific antigen-binding protein of embodiment 32, wherein at least one polypeptide chain in at least one antigen-binding region that specifically binds to an NK cell-activating receptor forms a single polypeptide chain with at least one polypeptide chain of a region having affinity for a surface antigen expressed on NK cells. 34. The multispecific antigen-binding protein of embodiment 33, wherein the single polypeptide chain comprises, in order from N-terminus to C-terminus: i) at least one polypeptide chain in at least one antigen-binding region that specifically binds to an NK cell activating receptor; ii) optionally, a flexible linker; iii) a region with affinity for a surface antigen expressed on NK cells. 35. The multispecific antigen-binding protein of embodiment 33 or 34, wherein the region having affinity for a surface antigen expressed on NK cells is a dimeric immunoglobulin Fc region, each of the two polypeptide chains of the dimeric Fc region being linked to a CH1 domain, and each of the CH1 domains being linked to an immunoglobulin variable region that specifically binds to an NK cell activating receptor. 36. The multispecific antigen-binding protein of embodiment 35, wherein the two immunoglobulin variable regions bind to the same NK cell-activating receptor or wherein the two immunoglobulin variable regions each bind to a different NK cell-activating receptor. 37. The multispecific antigen-binding protein of embodiment 35 or 36, comprising a dimeric immunoglobulin Fc region, wherein each of the two Fc polypeptide chains is operably linked to a Fab that specifically binds to an NK cell activating receptor. 38. The multispecific antigen-binding protein of any one of embodiments 18 to 30, wherein at least one NK cell-activating cytokine is conjugated to at least one antigen-binding region that specifically binds to an NK cell-activating receptor or a region with affinity for a surface antigen expressed on an NK cell. 39. At least one of the NK cell-activating cytokines is i) at least one polypeptide chain in at least one antigen-binding region that specifically binds to an NK cell activating receptor; and ii) at least one polypeptide chain in a region having affinity for a surface antigen expressed on NK cells a single polypeptide chain with at least one of wherein optionally a flexible linker is present between the agonist and at least one polypeptide chain in the region defined in i) or ii). 40. At least one of the NK cell-activating cytokines is i) at least one light chain of two Fabs that specifically bind to an NK cell activating receptor, and ii) a single polypeptide chain with at least one of the two Fc chains in a dimeric immunoglobulin Fc region and optionally a flexible linker is present between the agonist and the light chain as defined in i) or the Fc chain as defined in ii). 41. At least one of the NK cell-activating cytokines is i) the N-terminus of the light chain of at least one of the two Fabs, optionally through a flexible linker, that specifically binds to an NK cell activating receptor; ii) the C-terminus of the light chain of at least one of the two Fabs, optionally through a flexible linker, that specifically binds to an NK cell activating receptor; iii) the N-terminus of the heavy chain of at least one of the two Fabs that specifically binds to an NK cell activating receptor, and iv) the C-terminus of at least one heavy chain of the two Fc chains in the dimeric immunoglobulin Fc region, optionally through a flexible linker 41. The multispecific antigen-binding protein of embodiment 40, fused to at least one of: 42. The multispecific antigen-binding protein of embodiment 40 or 41, wherein at least one of the NK cell-activating cytokines is present on at least one or both sides of the immunoglobulin structure. 43. The multispecific antigen-binding protein of any one of embodiments 35 to 42, which is a heterodimer with respect to at least one of i) an antigen-binding region that specifically binds to an NK cell-activating receptor, and ii) at least one fusion NK cell-activating cytokine, wherein the dimeric Fc region comprises distinct first and second polypeptide chains, the distinct first and second polypeptide chains comprising knob-into-hole modifications that facilitate association of the first and second polypeptide chains of the Fc region. 44. a) the multispecific antigen binding protein causes an increase in at least one NK cell activity selected from CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cytotoxicity, whereby preferably the increase is at least 0.1 fold greater compared to the increase achieved at the same effector:target cell ratio using the same NK cells and target cells that have not been contacted with the multispecific antigen binding protein; and b) the multispecific antigen-binding protein causes an increase in at least one NK cell activity selected from CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cytotoxicity, whereby preferably the increase is at least 0.1-fold greater than that achieved at the same effector:target cell ratio using the same NK cells and target cells that have been contacted with a conventional human IgG1 monoclonal antibody having the same NK cell activating receptor-specific antigen-binding region as the multispecific antigen. 10. The multispecific antigen-binding protein of any one of the preceding embodiments, having at least one biological activity selected from: 45. Ex vivo expansion of donor NK cells by co-culturing with a multispecific antigen-binding protein described herein results in expanded NK cells, a) the fold increase in expanded NK cells is at least 0.001-fold, 0.002-fold, 0.005-fold, 0.01-fold, 0.02-fold, 0.05-fold, 0.1-fold, 0.2-fold, 0.5-fold, 1.0-fold, 2.0-fold, or 5.0-fold greater than the fold increase in expanded NK cells obtained by ex vivo expansion by co-culture with irradiated K562 feeder cells modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells); b) the telomere length of the expanded NK cells is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55% compared to the telomere length of fresh NK cells, and preferably the percentage increase in telomere length of the expanded NK cells compared to the telomere length of fresh NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times the percentage increase in telomere length of NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells; c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the expression level on expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells; d) secretion of at least one of the cytokines TNF-α, IFN-γ, and IL-6 by the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times greater than the cytokine secretion by expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells; and e) the cytotoxicity of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times the cytotoxicity of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells 10. The multispecific antigen-binding protein of any one of the preceding embodiments, wherein the multispecific antigen-binding protein generates a population of expanded NK cells having one or more characteristics selected from: 46. ​​A pharmaceutical composition comprising a multispecific antigen-binding protein according to any one of the preceding embodiments and a pharmaceutically acceptable carrier. 47. An ex vivo method for expanding NK cells, comprising contacting NK cells with a multispecific antigen-binding protein according to any one of embodiments 1 to 45 or the pharmaceutical composition according to embodiment 46, wherein preferably the expanded NK cells are a) the fold increase in the number of expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times the fold increase in the number of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated K562 feeder cells modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells); b) the telomere length of the expanded NK cells is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55% compared to the telomere length of fresh NK cells, and preferably the percentage increase in telomere length of the expanded NK cells compared to the telomere length of fresh NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times the percentage increase in telomere length of NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells; c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the expression level on expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells; d) secretion of at least one of the cytokines TNF-α, IFN-γ and IL-6 by the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times greater than the cytokine secretion by expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells; e) the cytotoxicity of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times the cytotoxicity of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells A method having one or more features selected from the following: 48. A multispecific antigen-binding protein according to any one of embodiments 1 to 45, a composition according to embodiment 46 or ex vivo expanded NK cells obtained by the method according to embodiment 47, optionally in combination with the multispecific antigen-binding protein, for use as a medicament. 49. A multispecific antigen-binding protein according to any one of embodiments 1 to 45, a composition according to embodiment 46 or ex vivo expanded NK cells obtained by the method according to embodiment 47, for use in treating cancer. 50. A multispecific antigen-binding protein according to any one of embodiments 1 to 45, or a composition according to embodiment 46, for use in the treatment of cancer, used in combination with adoptive transfer of immune cells, preferably the immune cells being selected from T cells and NK cells. 48. A multispecific antigen-binding protein according to any one of embodiments 1 to 45, a composition according to embodiment 46, for use according to claim 49 or 50, a) the multispecific antigen binding protein is administered as neoadjuvant therapy prior to a primary therapy comprising at least one of surgery and radiation therapy for the cancer; and b) the multispecific antigen-binding protein is administered as an adjuvant therapy following primary therapy comprising at least one of surgery and radiation therapy for the cancer; a multispecific antigen-binding protein or composition, wherein the multispecific antigen-binding protein or composition is at least one of: 51. A method of enhancing the anti-tumor activity of NK cells in a subject, comprising the step of administering to the subject a multispecific antigen-binding protein according to any one of embodiments 1 to 45, a composition according to embodiment 46, or ex vivo expanded NK cells obtained by the method according to embodiment 47, optionally in combination with the multispecific antigen-binding protein, or a combination of the multispecific antigen-binding protein and immune cells selected from T cells and NK cells. 52. The method according to 51, wherein the subject has cancer. 53. a) the multispecific antigen-binding protein is administered to the subject as neoadjuvant therapy prior to a primary therapy comprising at least one of surgery and radiation therapy for the cancer; and b) the multispecific antigen-binding protein is administered to the subject as an adjuvant therapy following a primary therapy comprising at least one of surgery and radiation therapy for the cancer; 53. The method of claim 52, wherein the at least one of 54. A nucleic acid molecule comprising one or more nucleotide sequences encoding the polypeptide chains of a multispecific antigen-binding protein according to any one of embodiments 1 to 45. 55. The nucleic acid molecule of embodiment 55, wherein the one or more nucleotide sequences are operably linked to regulatory sequences for expression of one or more polypeptide chains in a host cell. 56. A host cell comprising a nucleic acid molecule according to embodiment 54 or 55. 57. A method of producing a multispecific antigen-binding protein according to any one of embodiments 1 to 45, comprising culturing a host cell according to embodiment 56 such that a nucleotide sequence of one of the nucleotides is expressed and the multispecific antigen-binding protein is produced. 58. The method of embodiment 57, comprising the steps of recovering the multispecific antigen-binding protein and optionally formulating the multispecific antigen-binding protein with a pharmaceutically acceptable carrier.

Claims

1. 1. A multispecific antigen-binding protein comprising: a) at least two NK cell-activating cytokines, i) interleukin 21 receptor (IL21R) agonists, and ii) 4-1BB agonist and at least two NK cell-activating cytokines, including b) a region having affinity for a surface antigen expressed on natural killer (NK) cells, the region comprising or consisting of an immunoglobulin Fc region; Including, the IL21R agonist comprises or consists of an agonistic antigen-binding region that specifically binds to an IL21 polypeptide or IL21R; The 4-1BB agonist comprises or consists of at least one 4-1BB ligand (4-1BBL) extracellular domain (ECD) or at least one agonistic antigen-binding region that specifically binds to 4-1BB, preferably the 4-1BB agonist comprises or consists of a fusion protein comprising three 4-1BBL ECD monomers fused together in a single polypeptide chain, optionally the three 4-1BBL ECD monomers are linked by a polypeptide linker, a multispecific antigen-binding protein.

2. 2. The multispecific antigen-binding protein of claim 1, wherein said IL21 polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 38 and having IL21R agonist activity, and said 4-1BBL ECD comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 37 and having 4-1BB agonist activity.

3. 3. The multispecific antigen-binding protein of claim 1, wherein the Fc region is a dimeric Fc region that binds to CD16A and preferably activates the NK cells.

4. c) at least one antigen-binding region that specifically binds to an NK cell activating receptor; Preferably, the antigen-binding region that specifically binds to an NK cell activating receptor is an agonistic antigen-binding region that activates the NK cell receptor, and preferably, the NK cell activating receptor is selected from the group consisting of NKp46, NKp30, NKG2D, CD16A, SLAMF7, NKp44, CD94-NKG2C / E, KIR2DS1, KIR2DS3, KIR2DS4, and KIR2DS5.

4. The multispecific antigen-binding protein of any one of claims 1 to 3, which is selected from the group consisting of: 2DS5, KIR2DS2, KIR2DL4, KIR3DS1, CD160, NKp80, DNAM1, 2B4, CRACC, 4-BB, OX40, CRTAM, CD27, PSGL1, CD96, CD100, CEACAM1, CD59, PD-L1, Tim3 and NTB-A.

5. 5. The multispecific antigen-binding protein of any one of claims 1 to 4, wherein at least one NK cell-activating cytokine is conjugated to said region having affinity for a surface antigen expressed on an NK cell, preferably said at least one NK cell-activating cytokine forms a single polypeptide chain with at least one polypeptide chain of said region having affinity for a surface antigen expressed on an NK cell, optionally said NK cell-activating cytokine is linked to said polypeptide chain of said region having affinity for a surface antigen expressed on an NK cell through a flexible linker, more preferably said single polypeptide chain comprises, in order from N-terminus to C-terminus: i) a first NK cell-activating cytokine, ii) optionally a first flexible linker, iii) the polypeptide chain of said region having affinity for a surface antigen expressed on an NK cell, iv) optionally a second flexible linker, and v) a second NK cell-activating cytokine, preferably said first and second NK cell-activating cytokines are different NK cell-activating cytokines.

6. the at least one antigen-binding region that specifically binds to an NK cell activating receptor is conjugated to a region that has affinity for a surface antigen expressed on an NK cell; Preferably, at least one polypeptide chain in said at least one antigen-binding region that specifically binds to an NK cell activating receptor forms a single polypeptide chain with at least one polypeptide chain in said region that has affinity for a surface antigen expressed on an NK cell; More preferably, said single polypeptide chain comprises, in order from N-terminus to C-terminus: i) at least one polypeptide chain in said at least one antigen-binding region that specifically binds to an NK cell activating receptor; ii) optionally, a flexible linker; and iii) said region having affinity for a surface antigen expressed on an NK cell; Even more preferably, said region having affinity for a surface antigen expressed on an NK cell is a dimeric immunoglobulin Fc region, each of the two polypeptide chains of said dimeric Fc region being linked to a CH1 domain, each of said CH1 domains being linked to an immunoglobulin variable region that specifically binds to an NK cell activating receptor; 6. The multispecific antigen-binding protein of claim 5, wherein the protein comprises a dimeric immunoglobulin Fc region, each of two Fc polypeptide chains operably linked to a Fab that specifically binds to an NK cell activating receptor.

7. at least one NK cell-activating cytokine is conjugated to said at least one antigen-binding region that specifically binds to an NK cell-activating receptor or said region that has affinity for a surface antigen expressed on an NK cell; Preferably, at least one of said NK cell-activating cytokines is i) at least one polypeptide chain in said at least one antigen-binding region that specifically binds to an NK cell activating receptor; and ii) at least one polypeptide chain in said region having affinity for a surface antigen expressed on NK cells forming a single polypeptide chain with at least one of Optionally, a flexible linker is present between the agonist and the at least one polypeptide chain in the region defined in i) or ii); More preferably, at least one of said NK cell-activating cytokines is i) a light chain in at least one of the two Fabs that specifically binds to an NK cell activating receptor; and ii) at least one of the two Fc chains in the dimeric immunoglobulin Fc region; forming a single polypeptide chain with at least one of Optionally, a flexible linker is present between said agonist and said light chain defined in i) or said Fc chain defined in ii); Even more preferably, at least one of said NK cell-activating cytokines is i) the N-terminus of the light chain of at least one of the two Fabs, optionally through a flexible linker, that specifically binds to an NK cell activating receptor; ii) the C-terminus of the light chain of at least one of the two Fabs, optionally through a flexible linker, that specifically binds to an NK cell activating receptor; iii) the N-terminus of the heavy chain of at least one of the two Fabs that specifically binds to an NK cell activating receptor; and iv) the C-terminus of the heavy chain of at least one of the two Fc chains in the dimeric immunoglobulin Fc region, optionally through a flexible linker fused to at least one of 7. The multispecific antigen-binding protein of claim 5 or 6, wherein at least one of said NK cell-activating cytokines is present on at least one or both sides of the immunoglobulin structure.

8. 8. The multispecific antigen-binding protein of any one of claims 5 to 7, which is heterodimeric with respect to at least one of i) the antigen-binding region that specifically binds to an NK cell activating receptor, and ii) at least one fusion NK cell activating cytokine, wherein the dimeric Fc Region comprises distinct first and second polypeptide chains, the distinct first and second polypeptide chains comprising knob-into-hole modifications of the Fc Region that facilitate association of the first and second polypeptide chains.

9. a) said multispecific antigen binding protein causes an increase in at least one NK cell activity selected from CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cytotoxicity, whereby preferably said increase is at least 0.1 fold higher compared to the increase achieved at the same effector:target cell ratio using the same NK cells and target cells that have not been contacted with said multispecific antigen binding protein; and b) the multispecific antigen-binding protein causes an increase in at least one NK cell activity selected from CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cytotoxicity, whereby preferably said increase is at least 0.1 fold higher compared to the increase achieved at the same effector:target cell ratio using the same NK cells and target cells that have been contacted with a conventional human IgG1 monoclonal antibody having the same NK cell activating receptor-specific antigen-binding region as the multispecific antigen-binding protein.

9. The multispecific antigen-binding protein of claim 1, having at least one biological activity selected from the group consisting of:

10. The ex vivo expansion of donor NK cells by co-culture with the multispecific antigen-binding protein described herein comprises expanding NK cells, a) the fold expansion of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times greater than the fold expansion of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated K562 feeder cells modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells); b) the telomere length of said expanded NK cells is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55% compared to the telomere length of fresh NK cells, and preferably the increase in telomere length of said expanded NK cells compared to the telomere length of fresh NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the increase in telomere length of NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells; c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the expression level on expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells; d) secretion of at least one of the cytokines TNF-α, IFN-γ and IL-6 by said expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times greater than the secretion of said cytokine by expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells; and e) the cytotoxicity of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times the cytotoxicity of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells 10. The multispecific antigen-binding protein of any one of claims 1 to 9, which generates a population of expanded NK cells having one or more characteristics selected from:

11. 11. A pharmaceutical composition comprising the multispecific antigen-binding protein of any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. 12. An ex vivo method for expanding NK cells, comprising contacting NK cells with a multispecific antigen-binding protein according to any one of claims 1 to 10 or a composition according to claim 11, preferably wherein said expanded NK cells a) the fold expansion of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times greater than the fold expansion of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated K562 feeder cells modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells); b) the telomere length of said expanded NK cells is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55% compared to the telomere length of fresh NK cells, and preferably the increase in telomere length of said expanded NK cells compared to the telomere length of fresh NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the increase in telomere length of NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells; c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the expression level on expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells; d) secretion of at least one of the cytokines TNF-α, IFN-γ and IL-6 by said expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times greater than the secretion of said cytokine by expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells; and e) the cytotoxicity of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 times the cytotoxicity of expanded NK cells obtained by ex vivo expansion by co-culture with irradiated FC21 feeder cells An ex vivo method having one or more features selected from:

13. 14. A multispecific antigen-binding protein according to any one of claims 1 to 10, a composition according to claim 12 or ex vivo expanded NK cells obtained by the method of claim 13, optionally in combination with said multispecific antigen-binding protein, for use as a medicament, preferably for use in the treatment of cancer.

14. 13. The multispecific antigen-binding protein of any one of embodiments 1 to 10, or the composition of embodiment 12, for use in the treatment of cancer, used in combination with adoptive transfer of immune cells, preferably wherein said immune cells are selected from T cells and NK cells.

15. 15. Ex vivo expanded NK cells obtained by the method of claim 12, in combination with a multispecific antigen-binding protein according to any one of embodiments 1 to 10, a composition according to embodiment 11 or optionally said multispecific antigen-binding protein, for use according to claim 13 or 14. a) the multispecific antigen-binding protein and / or the ex vivo expanded NK cells are administered as neoadjuvant therapy prior to a primary therapy comprising at least one of surgery and radiation therapy for the cancer; and b) said multispecific antigen-binding protein and / or said ex vivo expanded NK cells are administered as an adjuvant therapy following a primary therapy comprising at least one of surgery and radiation therapy for said cancer. a multispecific antigen-binding protein, a composition, or ex vivo expanded NK cells,

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