Multi-specific antigen binding proteins for stimulating NK cells and uses thereof

Abstract

The present invention relates to multispecific antigen binding proteins comprising a natural killer (NK) cell activating cytokine and a region having an affinity for a surface antigen expressed on an NK cell, 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 proteins can further comprise antigen binding regions that specifically bind to NK cell activated receptors, such as NKp46, NKp44, NKp30, NKG2D, DNAM1, and CD16A. The multispecific antigen binding proteins of the present invention can be used for expanding and activating NK cells ex vivo or in vivo. The 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] The present invention relates to the field of medicine, 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 Art

[0002] Cancer immunotherapies are revolutionizing the treatment of cancer. Cancer immunotherapies are desirable because they are highly specific and can promote tumor destruction by inducing the patient's own immune system to recognize and eliminate tumor cells. Recent advances have focused on generating or releasing tumor antigen-specific T cell responses. They are 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 outstanding breakthroughs, clinical benefits remain limited to subsets of patients and certain tumor types, highlighting the need for alternative strategies.

[0003] One such alternative is to utilize the anti-tumor activity of natural killer (NK) cells. NK cells are components of the innate immune system and account for approximately 15% of circulating lymphocytes. NK cells infiltrate almost all tissues, and are initially characterized by their ability to effectively kill tumor cells without the need for prior sensitization. NK cells provide an effective immune surveillance mechanism by which undesirable cells, such as tumor cells or virally infected cells, can be eliminated. The biological characteristics 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 identify and kill cells that cannot express "self" MHC / HLA antigens by activating specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express stress ligands of NK activation receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit immune responses. Activated NK cells kill target cells in a manner similar to cytotoxic T cells (i.e., via cell lytic granules containing perforin and granzymes and via death receptor pathways). Activated NK cells also secrete inflammatory cytokines that promote other leukocytes to be recruited to target tissues, such as IFN-γ and chemokines. NK cells respond to signals through a variety of activating receptors and inhibitory receptors on their surfaces. For example, when NK cells encounter healthy self cells, their activity is inhibited by activating killer cell immunoglobulin-like receptors (KIR). Alternatively, when NK cells encounter foreign cells or cancer cells, they are activated via their activating receptors (e.g., NKG2D, NCR, DNAM1). NK cells are also activated via CD16 receptors on their surfaces through the constant regions of some immunoglobulins. The overall sensitivity of NK cells to activation depends on the sum of stimulating signals and inhibitory signals.

[0004] Strategies based on the recruitment of cytotoxic NK cells are currently being developed. Given the absence 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. In addition, unlike chimeric antigen receptor (CAR) T cells, the administration of allogeneic NK cells engineered by CAR is not associated with the occurrence of neurotoxicity, cytokine release syndrome (CRS), or graft-versus-host disease, and CAR-NK cell infusions do not increase circulating inflammatory cytokine concentrations above baseline levels. NK cell-based immunotherapy may be less likely to cause these adverse events because the cytokine profiles produced by these cells are different from those secreted by T cells. In addition, the frequency of NK cells is only about one-tenth of that of T cells, and this lower frequency ensures a more moderate release of cytokines, limiting the risk of CRS in patients treated with NK cell-targeted multispecific antibodies.

[0005] 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 targeted tumor cells and increase NK cell effector functions against tumor cells. A variety of NKCEs currently under development for clinical application are reviewed by Demaria et al. (Eur. J. Immunol. [European Journal of Immunology] 2021.51: 1934-1942).

[0006] There remains a need in the art for improved multispecific antigen-binding proteins with novel additional functionalities for inducing NK cells, particularly those that offer therapeutic advantages over existing NKCEs. Summary of the invention

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

[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 as described herein comprises an IL21R agonist, which is an IL21 polypeptide 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: 38, and preferably has an 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 mutant protein that is modified to reduce or enhance affinity for IL21R relative to the corresponding wild-type IL21 polypeptide. For example, an IL21 mutant protein having reduced affinity for IL21R relative to a corresponding wild-type IL21 polypeptide can be an IL21 mutant protein having a mutation in one or more amino acids 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. In one embodiment, the multispecific antigen-binding protein is a multispecific antigen-binding protein having an IL21R agonist titer greater than one.

[0009] In one embodiment, the multispecific antigen-binding protein is such 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 as described herein comprises a 4-1BB agonist, the 4-1BB agonist comprising 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 with SEQ ID NO: 37, and preferably having 4-1BB agonist activity as defined herein, and / or preferably having affinity for 4-1BB as defined herein. In one embodiment, the 4-1BBL ECD is a mutant protein, which is modified to reduce or enhance affinity to 4-1BB relative to the corresponding wild-type 4-1BBL ECD. In one embodiment, the 4-1BB agonist comprises or consists of a fusion protein, which comprises three 4-1BBL ECD monomers fused together in a single polypeptide chain, and wherein optionally, the three 4-1BBL ECD monomers are connected by a polypeptide linker. In one embodiment, the multispecific antigen-binding protein is such a multispecific antigen-binding protein having a 4-1BB agonist titer 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 that further comprises a 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 such a multispecific antigen-binding protein, wherein the region with affinity for surface antigens 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 such an Fc region that is modified to reduce or enhance affinity for CD16A relative to the corresponding wild-type Fc region. In one embodiment, the Fc region is such an Fc region that is modified to reduce or enhance NK cell activation by binding to CD16A relative to the 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, more preferably, the immunoglobulin variable region comprises or consists of a Fab or an immunoglobulin single variable domain (ISVD). In one embodiment, the antigen-binding region is a human or humanized antigen-binding region.

[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 can bind to the same NK cell-activating receptor, or they can bind 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 a multispecific antigen-binding protein wherein the NK cell activating receptor (which is 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 wherein 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) CDR-H1 (SEQ ID NO:24), CDR-H2 (SEQ ID NO:25), and CDR-H3 (SEQ ID NO:26) sequences as contained in SEQ ID NO:1, and CDR-L1 (SEQ ID NO:27), CDR-L2 (SEQ ID NO:28), and CDR-L3 (SEQ ID NO:29) sequences as contained in SEQ ID NO:2; and b) CDR-H1, CDR-H2, and CDR-H3 sequences as contained in SEQ ID NO:3, and CDR-L1, CDR-L2, and CDR-L3 sequences as contained in SEQ ID NO:20. Preferably, the multispecific antigen-binding protein is a protein wherein the antigen-binding region comprises a variable light chain (V L ) domain and variable heavy chain (V H ) domain combination: a) V as contained in SEQ ID NO: 1 H Sequence, and V as contained in SEQ ID NO:2 L Sequence; and b) V as contained in SEQ ID NO: 3 H Sequence, and V as contained in SEQ ID NO:20 L sequence.

[0017] In one embodiment, the multispecific antigen-binding protein is such a multispecific antigen-binding protein, wherein at least one NK cell activating cytokine is conjugated to the region with affinity for surface antigens expressed on NK cells. 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 with affinity for surface antigens expressed on NK cells, and wherein optionally, the NK cell activating cytokine is connected to the polypeptide chain of the region with affinity for surface antigens expressed on NK cells by 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 with affinity for surface antigens expressed on NK cells; iv) optionally a second flexible linker; and v) a second NK cell activating cytokine; and wherein preferably, the first NK cell activating cytokine and the second NK cell activating cytokine are different NK cell activating cytokines. Preferably, the at least one antigen binding region that specifically binds to the NK cell activating receptor is conjugated to the region with affinity for surface antigens expressed on NK cells. Preferably, at least one polypeptide chain in the at least one antigen binding region that specifically binds to the NK cell activating receptor forms a single polypeptide chain with at least one polypeptide chain in the region that has affinity for the surface antigen expressed on the NK cell. Preferably, the single polypeptide chain comprises, in order from N-terminus to C-terminus: i) at least one polypeptide chain in the at least one antigen binding region that specifically binds to the NK cell activating receptor; ii) optionally a flexible linker; and iii) the region that has affinity for the surface antigen expressed on the NK cell. In one embodiment, the region that has affinity for the surface antigen expressed on the NK cell is a dimeric immunoglobulin Fc region, wherein each of the two polypeptide chains of the dimeric Fc region is connected to a CH1 domain, and each CH1 domain is connected to an immunoglobulin variable region that specifically binds to the NK cell activating receptor. Preferably, 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. 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 a 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 the at least one antigen-binding region that specifically binds to the NK cell activating receptor, or to the region that has affinity for the surface antigen expressed on the NK cell. Preferably, at least one of these NK cell activating cytokines forms a single polypeptide chain with at least one of the following: i) at least one polypeptide chain in the at least one antigen-binding region that specifically binds to the NK cell activating receptor; and ii) at least one polypeptide chain in the region that has affinity for the surface antigen expressed on the NK cell; wherein optionally, a flexible linker is present between the agonist and the at least one polypeptide chain in the region defined in i) or ii). In one embodiment, at least one of these NK cell activating cytokines forms a single polypeptide chain with at least one of the following: i) a light chain in at least one of the two Fabs that specifically bind to the NK cell activating receptor; and ii) at least one of the two Fc chains in the Fc region of the dimeric immunoglobulin; wherein optionally, a flexible linker is present between the agonist and the light chain defined in i) or the Fc chain defined in ii). Preferably, at least one of these NK cell activating cytokines is fused to at least one of the following: i) the N-terminus of the light chain in 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 in 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 in 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 in at least one of the two Fc chains in the Fc region of the dimeric immunoglobulin, optionally through a flexible linker. In one embodiment, at least one of these NK cell activating cytokines is present on at least one side or both sides of the immunoglobulin structure.

[0019] In one embodiment, the multispecific antigen-binding protein is a multispecific antigen-binding protein, wherein the multispecific antigen-binding protein is a heterodimer with respect to at least one of: i) these antigen-binding regions that specifically bind to NK cell activating receptors; and ii) at least one fused NK cell activating cytokine, and wherein the dimeric Fc region comprises different first and second polypeptide chains, and the first polypeptide chain and the second polypeptide chain comprise a knob-into-hole modification that promotes the association of the first polypeptide chain and the second polypeptide chain of the Fc region.

[0020] In one embodiment, the multispecific antigen-binding protein is a multispecific antigen-binding protein having at least one biological activity selected from the group consisting of: a) the multispecific antigen-binding protein causes an increase in at least one NK cell activity selected from the group consisting of CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation, and NK cell toxicity, wherein preferably, the increase is at least 0.1-fold greater than the increase achieved with the same NK cells and target cells not contacted with the multispecific antigen-binding protein at the same effector cell:target cell ratio; and b) the multispecific antigen-binding protein causes an increase in at least one NK cell activity selected from the group consisting of CD107a degranulation, CD107 or CD69 expression, IFNy production, NK proliferation, and NK cell toxicity, wherein preferably, the increase is at least 0.1-fold greater than the increase achieved with the same NK cells and target cells 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.

[0021] In one embodiment, the multispecific antigen-binding protein is a multispecific antigen-binding protein wherein ex vivo expansion of donor NK cells by co-culturing with a multispecific antigen-binding protein as described herein produces an expanded NK cell population having one or more characteristics selected from the following: a) the expanded NK cells have an expansion fold 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 that of the expanded NK cells obtained by ex vivo expansion via co-culturing with irradiated K562 feeder cells; The feeder cells are 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, 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, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45 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 the expanded NK cells obtained by ex vivo expansion via co-culture with irradiated FC21 feeder cells; d) the expression level of at least one cytokine selected from TNF-α, IFN-γ and IL-6 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 the expanded NK cells obtained by ex vivo expansion via co-culture with irradiated FC21 feeder cells; The secretion of the 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 expanded by NK cells expanded ex vivo via co-culture with irradiated FC21 feeder cells; and e) the cytotoxicity of these 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 NK cells expanded ex vivo via co-culture with irradiated FC21 feeder cells.

[0022] In one embodiment, an expanded NK cell population having one or more characteristics selected from the following is generated by co-culturing with a multispecific antigen-binding protein as described herein and by ex vivo expansion of donor NK cells performed with irradiated tumor cells or co-stimulation with tumor cells: a) the expanded NK cells have an expansion fold 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 times that of the expanded NK 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, 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 that of the expanded NK cells; c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on these 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; d) the TNF- The secretion of at least one cytokine among α, IFN-γ and IL-6 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; and e) the cytotoxicity of these 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.

[0023] In a second aspect, the 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 relates to an ex vivo method for expanding NK cells, the method comprising the step of contacting NK cells with a multispecific antigen-binding protein as described herein or with a pharmaceutical composition comprising the protein, wherein preferably, the expanded NK cells have one or more characteristics selected from the following: a) the expansion fold 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 expansion fold of the expanded NK cells obtained by ex vivo expansion via co-culture with irradiated K562 feeder cells; 562 feeder cells are 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, 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, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45 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 the expanded NK cells obtained by ex vivo expansion via co-culture with irradiated FC21 feeder cells; d) at least one cytokine selected from TNF-α, IFN-γ and IL-6 on the expanded NK cells is at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times the expression level on the expanded NK cells The secretion of the 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 by expanded NK cells obtained by ex vivo expansion via co-culture with irradiated FC21 feeder cells; and e) the cytotoxicity of these 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 expanded NK cells obtained by ex vivo expansion via co-culture with irradiated FC21 feeder cells.

[0025] In addition, the present invention relates to an ex vivo method for expanding NK cells, the method comprising the step of contacting NK cells with a multispecific antigen-binding protein as described herein or with a pharmaceutical composition comprising the protein and with irradiated tumor cells or with tumor cells, wherein preferably, the expanded NK cells have one or more characteristics selected from the following: a) the expansion fold 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; 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, 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 .02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0 times; c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on these 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; d) the T cells of these expanded NK cells The secretion of at least one cytokine among NF-α, IFN-γ and IL-6 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; and e) the cytotoxicity of these 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.In one embodiment, the multispecific antigen-binding protein causes an increase in Glut1, Glut3, CD71 and / or CD98 expression, mitochondrial mass, glycolytic rate, the ratio of glycolytic rate to oxidative phosphorylation rate, metabolic fuel flexibility between glucose, glutamine and / or fatty acids, wherein preferably, the increase is at least 0.05 times the increase achieved with the same effector cell:target cell ratio, with the same NK cells and target cells not contacted with the multispecific antigen-binding protein; b) the multispecific antigen-binding protein causes an increase in at least one NK cell activity selected from the following: Glut1, Glut3, CD71 or CD98 expression, mitochondrial mass, glycolytic rate, the ratio of glycolytic rate to oxidative phosphorylation rate, metabolic fuel flexibility between glucose, glutamine and fatty acids, wherein preferably, the increase is at least 0.05 times the increase achieved with the same effector cell:target cell ratio, with the same NK cells and target cells contacted with a conventional human IgG1 monoclonal antibody having the same TAA-specific antigen-binding region as the multispecific antigen-binding protein.

[0026] In one embodiment, the ex vivo expansion of donor NK cells by co-culture with a multispecific antigen-binding protein as described herein produces an expanded NK cell population having one or more characteristics selected from the group consisting of: a) mitochondrial mass or expression of at least one nutrient transporter of Glut1, Glut3, CD71, or CD98 is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.06, 0.07, 0.08, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 or 20 times the glycolytic rate of NK cells expanded ex vivo 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) a glycolytic rate that 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 the glycolytic rate of NK cells expanded ex vivo by co-culture with irradiated K562 feeder cells modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells); to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells); c) a ratio of glycolysis rate to oxidative phosphorylation (OxPhos) rate that 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 the ratio of glycolysis rate to OxPhos rate of expanded NK cells obtained by ex vivo expansion via co-culture with irradiated K562 feeder cells modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells); 1) and 4-1BB ligand (FC21 feeder cells); 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 via co-culture with irradiated K562 feeder cells, which are 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 in the above method, optionally in combination with the 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 in the above method, optionally in combination with the multispecific antigen-binding protein, for use in treating 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 treating cancer, wherein the multispecific antigen-binding protein or the composition is used in combination with adoptive transfer of immune cells, wherein preferably, the immune cells are selected from T cells and NK cells.

[0029] In a sixth aspect, the present invention relates to a method for enhancing the anti-tumor activity of NK cells in a subject, the method comprising the following steps: administering to the subject a multispecific antigen-binding protein as described herein, a pharmaceutical composition comprising the protein, an ex vivo expanded NK cell obtained in the above method, optionally in combination with the multispecific antigen-binding protein, or a combination of the multispecific antigen-binding protein and an immune cell selected from T cells and NK cells. In one embodiment of the method, the subject suffers from cancer. In one embodiment, the present invention relates to the multispecific antigen-binding protein, the composition comprising the protein, or the ex vivo expanded NK cell, optionally in combination with the multispecific antigen-binding protein, for the above purposes, wherein at least one of the following: a) the multispecific antigen-binding protein and / or the ex vivo expanded NK cell is administered as a neoadjuvant therapy before a primary therapy comprising at least one of surgery and radiotherapy for the cancer; and b) the multispecific antigen-binding protein and / or the ex vivo expanded NK cell is administered as an adjuvant therapy after a primary therapy comprising at least one of surgery and radiotherapy for the cancer.

[0030] In a seventh aspect, the invention relates to a nucleic acid molecule comprising one or more nucleotide sequences encoding a polypeptide chain of a multispecific antigen-binding protein as described herein, Preferably, the nucleic acid molecule is a nucleic acid molecule wherein the one or more nucleotide sequences are operably linked to a regulatory sequence 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 for producing a multispecific antigen-binding protein as described herein, the method 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. DETAILED DESCRIPTION

[0033] definition

[0034] Various terms related to the methods, compositions, purposes and other aspects of the present invention are used throughout the specification and claims. Unless otherwise indicated, these terms should have the common meaning in the field to which the present invention belongs. Other specifically defined terms will be interpreted 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, preferred materials and methods are described herein.

[0035] "A," "an," and "the": These singular terms include plural referents unless the context clearly dictates otherwise. Thus, the indefinite article "a" or "an" generally means "at least one." Thus, for example, reference to "a cell" includes combinations of two or more cells, and so forth.

[0036] "About" and "approximately": When referring to measurable values ​​(such as amounts, durations, etc.), these terms are intended to cover variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% relative to the specified value, as long as such variations are suitable for performing the disclosed methods. In addition, amounts, ratios, and other numerical values ​​are sometimes presented herein in range format. It should be understood that this range format is used for convenience and brevity, and should be flexibly understood to include the numerical values ​​explicitly specified as range limits, and also include all individual numerical values ​​or subranges covered within the range, as if each numerical value and subrange was explicitly specified. For example, a ratio within the range of about 1 to about 200 should be understood to include the explicitly listed limits of about 1 and about 200, and also include individual ratios, such as about 2, about 3, and about 4, and subranges such as about 10 to about 50, about 20 to about 100.

[0037] “And / or”: The term “and / or” refers to situations where one or more of the stated situations may occur alone or in combination with at least one of the stated situations, up to all of the stated situations.

[0038] "Comprising": This term is interpreted as inclusive and open-ended, rather than exclusive. Specifically, this term and its variations mean including the specified features, steps or components. These terms should not be interpreted as excluding the presence of other features, steps or components.

[0039] “Exemplary”: This term means “serving as an example, instance, or illustration” and should not be interpreted to exclude other configurations disclosed herein.

[0040] As used herein, "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer also refers to a malignant neoplasm.

[0041] As used herein, "in combination with" is intended to refer to all forms of administration that provide a first drug together with an additional (second, third) drug. These drugs can be administered simultaneously, separately or sequentially and in any order. The drugs administered in combination are biologically active in the subject to which the drugs are delivered.

[0042] As used herein, "simultaneous" administration refers to the administration of more than one drug at the same time, but not necessarily via the same route of administration or in the form of a combined formulation. For example, one drug may be provided orally, while another drug may be provided intravenously during a patient's visit to a hospital. Separately includes administering the drugs in separate forms and / or at separate time points, but again not necessarily via the same route of administration. Sequentially refers to administering a second drug immediately or after a certain time after the administration of a first drug.

[0043] "Compositions," "products," or "combinations" as used herein that are useful in the methods of the present disclosure include those suitable for various routes of administration, including, but not limited to, intravenous, subcutaneous, intradermal, subdermal, 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 invention typically comprise a drug (alone or in combination) and one or more suitable pharmaceutically acceptable excipients.

[0044] As used herein, "effective amount" means the amount of a medicament required to improve the symptoms of a disease relative to an untreated patient. The effective amount of one or more active agents used to practice the present invention for therapeutic treatment of cancer varies depending on the mode of administration, the age, weight and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount. Therefore, in the context of the present disclosure, when it comes to administering a drug that is "effective" for a disease or condition, this indicates that administration in a clinically appropriate manner will produce a beneficial effect on at least a statistically significant proportion of patients, such as improvement of symptoms, cure, alleviation of at least one disease sign or symptom, extension of life span, improvement of quality of life, or other positive effects generally recognized by doctors familiar with treating that particular type of disease or condition.

[0045] As used herein, the phrase "NK cell" refers to a subpopulation of lymphocytes involved in innate immunity. NK cells can be identified by virtue of certain features and biological properties such as: expression of specific surface antigens (including CD56 and / or NKp46) of human NK cells; absence of α / β or γ / δTCR complexes on the cell surface; ability to recognize and kill cells that cannot express "self" MHC / HLA antigens by activating specific cell lysis mechanisms; ability to kill tumor cells or other diseased cells that express ligands for NK activation receptors; and the ability to release protein molecules (called cytokines) that stimulate or inhibit immune responses. Using methods well known in the art, any of these features and activities can be used to identify NK cells. The term NK cell will also cover any subpopulation of NK cells. In the context of this article, "active" NK cells refer to biologically active NK cells, including NK cells with 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 separation from blood samples, cell separation, tissue or cell collection, etc.). Useful protocols for assays involving NK cells can be found in Natural Killer Cells Protocols (2000, ed. by Campbell KS and Colonna M). Humana Press, pp. 219-238).

[0046] "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 sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as determined by the match between strings of such sequences, as the case may be. The "similarity" between two amino acid sequences is determined by comparing the amino acid sequence of a polypeptide and its conservative amino acid substitutes with the sequence of a second polypeptide. "Identity" and "similarity" can be easily calculated by known methods. The term "sequence identity" or "sequence similarity" means that two (poly) peptides or two nucleotide sequences (when optimally aligned, preferably aligned over the entire length (at least the shortest sequence in the comparison), and maximizing the number of matches and minimizing the number of gaps, such as by using default parameters for programs ClustalW (1.83), GAP or BESTFIT) share at least a certain percentage of sequence identity, as defined elsewhere herein. GAP uses the Needleman and Wengsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Typically, the GAP default parameters are used, with gap creation penalty = 50 (nucleotides) / 8 (proteins), and gap extension penalty = 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 [Proceedings of the National Academy of Sciences of the United States of America] 89, 915-919). The 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 alignments and percent sequence identity scores can be determined as follows: using a computer program such as the GCG Wisconsin software package version 10.3 available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752, USA; or using open source software such as the program "needle" (using the global Needleman-Wunsch algorithm) or "water" (using the local Smith-Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as GAP above, or using the default settings (both for "needle" and "water", and both for protein and DNA alignments, the default gap opening penalty is 10.0, and the default gap extension penalty is 0.5; for proteins, the default scoring matrix is ​​Blosum62; and for DNA, the default scoring matrix is ​​DNAFull).When the sequences are of significantly different overall length, local alignments, such as those using the Smith-Waterman algorithm, are preferred. Alternatively, percent similarity or identity can be determined by searching public databases using algorithms such as FASTA, BLAST, and the like.

[0047] Optionally, when determining the degree of amino acid similarity, the skilled person may also consider so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. The following table gives examples of classes of amino acid residues used for conservative substitutions.

[0048]

[0049] Alternative conservative amino acid residue substitution categories.

[0050] 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

[0051] Physical and functional classification of amino acid residues for their substitutability.

[0052]

[0053] The term "agent" generally refers to any entity that is not normally present or is not present at the level at which it is 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: a polynucleotide, a polypeptide, a small molecule, a (multi-specific) antigen binding protein, such as an antibody and a functional fragment thereof.

[0054] The term "antigen binding domain" or "antigen binding region" refers to the portion of an antigen binding protein that is capable of specifically binding to an antigen or epitope. In one embodiment, the antigen binding region is an immunoglobulin-derived antigen binding region, such as an antibody light chain variable region (V L ) and antibody heavy chain variable region (V H ) both. 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 from a single domain antibody, which consists only of heavy chains and lacks light chains, such as are known, for example, from camelids, where the antigen binding site is present on and formed by a single variable domain (also called "immunoglobulin single variable domain" or "ISVD"). Examples of such ISVDs include camelid heavy chain antibodies (V HH) (also known as nanobodies), domain antibodies (dAbs), and single domains derived from shark antibodies (IgNAR domains). In other embodiments, the antigen binding region comprises a non-immunoglobulin domain that can specifically bind to an antigen or epitope, such as darpin; affilin; antikalin, etc.

[0055] The term "antibody" herein is used in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments and derivatives, as long as they exhibit the desired biological activity and / or immunological activity. Various techniques related to the production of antibodies are provided in, for example, Harlow et al. Antibodies: A Laboratory Manual [Antibodies: Laboratory Manual], Cold Spring Harbor Laboratory Press [Cold Spring Harbor Laboratory Press], Cold Spring Harbor, New York, (1988). Antibodies can be human and / or humanized. A "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody containing minimal sequence derived from a non-human antibody.

[0056] The terms "full-length antibody", "complete antibody" and "whole antibody" are used interchangeably herein to refer to antibodies having a structure that is substantially similar to a natural antibody structure. "Native antibody" refers to naturally occurring immunoglobulin molecules with various structures. For example, natural IgG class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two light chains and two heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3), also referred to as a heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a light chain constant domain (CL), also referred to as a light chain constant region. The heavy chains of antibodies 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 chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domains of the antibodies.

[0057] "Antibody fragments" include a portion of a full-length antibody, such as its antigen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab), Fv (typically the V fragment of a single arm of an antibody), H and V L domain), single-chain Fv (scFv), dsFv, Fd fragment (typically V H and CH1 domains) and dAbs (typically V H domain) fragment; V H 、V L 、V HH and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies and kappa antibodies (see, e.g., 111 et al. Protein Eng 1997; 10: 949-57); camel IgG; IgNAR; and multispecific antibody fragments formed by antibody fragments and one or more isolated CDRs or functional paratopes, wherein 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 certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, e.g., Plückthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 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 comprising salvage receptor binding epitope residues and having increased in vivo half-life, 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 [Nature Medicine] 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med [Nature Medicine] 9, 129-134 (2003). Various types of antibody fragments have been described or reviewed, e.g., in Heiliger and Hudson, Nat Biotechnol [Nature Biotechnology] 2005; 23, 1126-1136; WO 2005 / 040219, US 20050238646, and US 20020161201. Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, CHO, E. coli, or phage), as described herein.

[0058] As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone (including any eukaryotic clone, prokaryotic clone or phage clone), rather than a method for producing it. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombinant and phage display techniques or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma technology, which includes those known in the art and taught in, for example, the following documents: Harlow and Lane, "Antibodies: A Laboratory Manual [antibodies: laboratory manual], "Cold Spring Harbor Laboratory Press [Cold Spring Harbor Laboratory Press], New York (1988); Hammerling et al., in: "Monoclonal Antibodies and T-Cell Hybridomas [monoclonal antibodies and T cell hybridomas], "Elsevier [Elsevier], New York (1981), pp. 563-681 (both of which are incorporated herein by reference in their entirety).

[0059] As used herein, the term "monospecific" antibody means that the antibody portion of the multispecific antigen-binding protein as described herein has one or more antigen binding sites, each antigen binding site binding the same epitope of the same antigen. The term "bispecific" means that the antibody portion of the multispecific antigen-binding protein as described herein has at least two antigen binding sites, which can specifically bind to at least two different antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which has specificity for different antigenic determinants. In certain embodiments, a bispecific antigen binding molecule can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0060] The term "valence" or "valency" as used in this application indicates that there are a specified number of binding sites in an antigen binding molecule. Thus, the terms "divalent", "tetravalent" and "hexavalent" indicate that there are two binding sites, four binding sites and six binding sites, respectively, in an antigen binding molecule.

[0061] Antibodies immunoreactive with a particular antigen can be produced by recombinant methods, such as selection of a recombinant antibody library in a phage or similar vector (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 an animal 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 an antigen. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and the splenocytes are isolated. The splenocytes are then fused with 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 to select for cells that secrete antibodies capable of binding the antigen. Ascites fluid, which typically contains high levels of antibodies, can be generated by intraperitoneally inoculating mice with positive hybridoma clones.

[0062] Typically, an immunoglobulin has a heavy chain and a light chain. Each heavy chain and light chain contains a constant region and a variable region (these regions are also referred to as "domains"). The light chain variable region and the heavy chain variable region contain four "framework" regions interrupted by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). The sequences of the framework regions of different light or heavy chains are relatively conservative within species. The framework region of an antibody (which is the combined framework region of the constituent light and heavy chains) is used to locate and align the CDRs in three-dimensional space.

[0063] The term "hypervariable region" as used herein refers to the amino acid residues in an antibody that are responsible for antigen binding. The hypervariable region typically comprises amino acid residues from a "complementarity determining region" or "CDR" (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 [National Institutes of Health], Bethesda, Maryland, USA) and / or those residues from the "hypervariable loop" (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 [Journal of Molecular Biology] 1987; 196: 901-917). Typically, the numbering of amino acid residues in this region is by the method of Kabat et al., supra. Phrases such as "Kabat position", "variable domain residue numbering as in Kabat", and "according to Kabat" refer herein to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to shortening of a FR or CDR of a variable domain or insertions in a FR or CDR of a variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of CDR H2 (residue 52a according to Kabat) and an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues for a given antibody may be determined by aligning the antibody sequence with a region of homology to a "standard" Kabat numbering sequence.

[0064] As used herein, the term "framework" or "FR" residues refers to the region of an antibody variable domain excluding those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into continuous regions (FR1, FR2, FR3, and FR4) separated by CDRs.

[0065] The term "constant region" as defined herein refers to an antibody-derived constant region encoded by one of a light chain immunoglobulin constant region gene or a heavy chain immunoglobulin constant region gene. As used herein, "constant light chain" or "light chain constant region" means an antibody region encoded by a κ (Ck) or λ (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, wherein numbering is performed according to the EU index (Kabat et al., 1991, supra).

[0066] As used herein, the term "constant heavy chain" or "heavy chain constant region" refers to the region of an antibody encoded by the μ, δ, γ, α or ε gene, thereby defining the isotype of the antibody as IgM, IgD, IgG, IgA or IgE, respectively. For a full-length IgG antibody, the constant heavy chain as defined herein refers to the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, thus comprising positions 118-447, wherein numbering is performed according to the EU index.

[0067] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each of which contains a heavy chain variable domain and a light chain variable domain, and also contains 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 "Fab region" refers to an antibody fragment that contains a light chain fragment containing the VL domain and the constant domain (CL) of the light chain and the VH domain and the first constant domain (CH1) of the heavy chain. Fab can refer to this region alone, or this region in the context of a polypeptide, a multispecific antigen-binding protein or an antigen-binding region, or this region in any other embodiment as outlined herein. The difference between the Fab' fragment and the Fab fragment is that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. Fab'-SH is a Fab' fragment in which one or more cysteine ​​residues of the constant domain carry a free thiol group. Pepsin treatment produces a F(ab')2 fragment with two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0068] As used herein, the term "single-chain Fv" or "scFv" refers to a V fragment comprising an antibody. H and V L The Fv polypeptide is further comprised of a V domain and a V domain. H With V LThe polypeptide linker between the domains enables the scFv to form the desired structure for antigen binding. Methods for producing scFv are well known in the art. For a review of methods for producing scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994).

[0069] "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, for example, 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 selected from the group consisting of: CTLA-4 (Evibody), lipocalin (Anticalin), protein A-derived molecules such as the Z domain of protein A (affine), A domain (Avimer / Maxibody), serum transferrin (trans-body); designed ankyrin repeat protein (DARPin), variable domain of antibody light chain or heavy chain (single domain antibody, sdAb), variable domain of antibody heavy chain (nanoantibody, aVH), V NAR fragment, fibronectin (AdNectin), C-type lectin domain (tetranectin); variable domain of neoantigen receptor β-lactamase (V NAR fragments), human γ-crystallin or ubiquitin (Affilin molecules); Kunitz-type domains of human protease inhibitors, microbodies (such as proteins from the knottin family), peptide aptamers and fibronectin (adnectin).

[0070] CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is a + CD28 family receptor expressed on T cells. Its extracellular domain has a variable domain-like Ig fold. The loop corresponding to the CDR of the antibody can be replaced by a heterologous sequence to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also called Evibodies (e.g., US7166697B1). Evibodies are roughly the same size as the isolated variable regions of antibodies (e.g., domain antibodies). For further details, see Journal of Immunological Methods [Journal of Immunological Methods] 248 (1-2), 31-45 (2001).

[0071] Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bile acids, retinoids, and lipids. They have a rigid beta-sheet secondary structure with multiple loops at the open end of the cone-shaped structure that can be engineered to bind different target antigens. Anticalins are between 160-180 amino acids in size and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482:337-350 (2000); US 7250297 B1; and US 20070224633.

[0072] Affibodies are scaffolds derived from protein A of Staphylococcus aureus that can be engineered to bind to antigens. The domain consists of a three-helix bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details, see Protein Eng. Des. Sel. [Protein Engineering Design and Selection] 17, 455-462 (2004); and EP 1641818 A1.

[0073] Avidin is a multidomain protein derived from the A-domain scaffold family. The native domain of approximately 35 amino acids adopts a defined disulfide-bonded structure. Diversity is generated by shuffling the natural variation 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).

[0074] Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind different target antigens by inserting peptide sequences into permitted surface loops. Examples of engineered transferrin scaffolds include Trans-body. For further details, see J.Biol.Chem [Journal of Biological Chemistry] 274, 24066-24073 (1999).

[0075] Designed ankyrin repeat proteins (DARPins) are derived from ankyrin, 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 α helices and a β turn. They can be engineered to bind to different target antigens by randomizing the residues in the first α helix and β turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details, see J. Mol. Biol. [Journal of Molecular Biology] 332, 489-503 (2003); PNAS [Proceedings of the National Academy of Sciences of the United States of America] 100 (4), 1700-1705 (2003); and J. Mol. Biol. [Journal of Molecular Biology] 369, 1015-1028 (2007); and US 20040132028A1.

[0076] Single domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first single variable domain is derived from the variable domain of an antibody heavy chain from a camelid (nanobody or V H H fragment). In addition, the term single variable domain antibody includes the autonomous human heavy chain variable domain (aVH) or the VH derived from sharks. NAR Fragment.

[0077] Fibronectin is a scaffold that can be engineered to bind to an antigen. Adnectin consists of a skeleton of the natural amino acid sequence of the 10th domain of 15 repeating units of type III human fibronectin (FN3). The three loops at one end of the β sandwich can be engineered to enable Adnectin to specifically recognize the therapeutic target of interest. For further details, see Protein Eng. Des. Sel. [Protein Engineering Design and Selection] 18, 435-444 (2005); US20080139791; WO2005056764; and US 6818418 B1.

[0078] Peptide aptamers are combinatorial recognition molecules consisting of a constant scaffold protein, typically thioredoxin (TrxA), containing a constrained variable peptide loop inserted at the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005).

[0079] Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length (which contain 3-4 cysteine ​​bridges) - examples of microproteins include KalataBI and conotoxins and knottin. Microproteins have loops that can be engineered to include up to 25 amino acids without affecting the overall folding of the microprotein. For further details on engineered knottin domains, see WO 2008098796.

[0080] As used herein, the term "Fv" or "Fv fragment" or "Fv region" refers to a single antibody V fragment. H and V L Domain polypeptides.

[0081] As used herein, the term "Fc" or "Fc region" refers to a polypeptide comprising a constant region other than the first constant region immunoglobulin domain of an antibody. Fc can refer to this region alone, or to this region in the context of an Fc polypeptide, as described below. As used herein, "Fc polypeptide" or "polypeptide from Fc" means a polypeptide comprising all or part of an Fc region. Fc polypeptides herein include, but are not limited to, antibodies, Fc fusions, and Fc fragments. In addition, the Fc region according to the present invention includes a variant containing at least one modification that changes (enhances or reduces) Fc-related effector functions. In addition, the Fc region according to the present invention includes chimeric Fc regions that include different parts or domains of different Fc regions, which are derived, for example, from antibodies of different isotypes or species. Therefore, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and is the last three constant region immunoglobulin domains of IgE and IgM, as well as the flexible hinge at the N-terminus of these domains. For IgA and IgM, Fc can include a J chain. For IgG, 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 generally defined as comprising residues C226, P230 or A231 to its carboxyl terminus, wherein numbering is performed according to the EU index. The "CH2 domain" in the human IgG Fc region generally extends from the amino acid residue at about position 231 to the amino acid residue at about position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein can be a native sequence CH2 domain or a variant CH2 domain." CH3 domain" is included in the residue segment at the C-terminus of the CH2 domain in the Fc region (i.e., from the amino acid residue at about position 341 of IgG to the amino acid residue at about position 447). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "knob" in one chain thereof and a corresponding introduced "cavity" in the other chain thereof; see U.S. Pat. No. 5,821,333, which is expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two different antibody heavy chains as described herein. In one embodiment, the 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 may or may not be present.Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is performed according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Maryland, 1991 (also known as the EU index).

[0082] The "knob-in-hole" technique is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., ProtEng [Protein Engineering] 9, 617-621 (1996); and Carter, J Immunol Meth [Journal of Immunological Methods] 248, 7-15 (2001). In general, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protrusion can be positioned in the cavity to promote heterodimer formation and hinder homodimer formation. The protrusion is constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory cavity of the same or similar size as the protrusion is created in the interface of the second polypeptide. The protrusions and cavities can be prepared by altering the nucleic acid encoding the polypeptide (e.g., by site-specific mutagenesis) or by 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 another specific embodiment, the subunit of the Fc region comprising the knob modification additionally comprises the amino acid substitution S354C, and the subunit of the Fc region comprising the hole modification additionally comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). Numbering is according to the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Maryland, 1991.

[0083] "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 and variants with alterations that produce substitutions, additions or deletions, but these alterations do not substantially reduce the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular toxicity). For example, one or more amino acids may be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantially losing biological function. Such variants may be selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, JU et al., Science 247: 1306-10 (1990)).

[0084] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with 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, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0085] "Activation of Fc receptors" is such an Fc receptor that triggers a signaling event that stimulates cells carrying receptors to perform effector functions after engaging with the Fc region of an antibody. Activation of Fc receptors includes FcγRIIIa (CD16A), FcyRI (CD64), FcyRIIa (CD32) and FcaRI (CD89). Specific activation of Fc receptors is human FcγRIIIa (see UniProt accession number P08637, version 141), also referred to as CD16 or CD16A. 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, while CD16B is connected to the plasma membrane via a GPI anchor, and is mainly expressed by neutrophils. Therefore, unless otherwise indicated, when CD16 is mentioned herein in the context of expression on NK cells, CD16A is generally meant.

[0086] As used herein, "variable region" refers to the region of an antibody that consists essentially of V L (including Vκ and Vλ) and / or V H One or more Ig domains encoded by any of the genes that constitute the light chain (including κ and λ) and heavy chain immunoglobulin genetic loci, respectively. The light chain variable region or the heavy chain variable region (V L or VH ) comprises four conserved framework regions (FR) and three hypervariable regions (HVR). See, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.

[0087] 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 a structurally defined loop ("hypervariable loop"). Typically, a natural four-chain antibody comprises six HVRs; three in VH (H1, H2, H3), and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and / or from "complementarity determining regions" (CDRs), which have the highest sequence variability and / or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. [Journal of Molecular Biology] 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, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 31-35B of H1, amino acid residues 50-65 of H2, and amino acid residues 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Maryland (1991)). Hypervariable regions (HVRs) are also called complementarity determining regions (CDRs), and these terms are used interchangeably herein to refer to the portions of the variable regions that form the antigen binding region. This particular region has been described by Kabat et al., U. S. Department 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 to each other. However, the application of either definition to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. Appropriate amino acid residues covering the CDRs as defined by each of the references cited above are shown in Table A below for comparison. The exact residue number covering a particular CDR will vary depending on the sequence and size of the CDR.Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a particular CDR.

[0088] Table A .CDR Definition 1

[0089]

[0090]

[0091] Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. One of ordinary skill in the art can clearly assign this "Kabat numbering" system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system shown in Kabat et al., USDept.ofHealth and Human Services [U.S. Department of Health and Human Services], "Sequence of Proteins of Immunological Interest [Sequence of Proteins with Immunological Significance]" (1983). Unless otherwise stated, the numbering of specific amino acid residue positions in the variable region of an antibody is based on the Kabat numbering system.

[0092] In addition to the CDR1 in VH, CDRs generally include amino acid residues that form hypervariable loops. CDRs also include "specificity determining residues" or "SDRs", which are residues that contact antigens. SDRs are included in regions called brief CDRs or a-CDRs in CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDRL2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) appear 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. [Frontiers of Biological Sciences] 13: 1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0093] As used herein, in the context of antigen binding molecules (e.g., antibodies), the term "affinity maturation" 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 antigen bound by the reference antibody, preferably to the same epitope; and has a higher affinity for the antigen than the affinity of the reference antigen binding molecule for the antigen. Affinity maturation generally involves modification of one or more amino acid residues in one or more CDRs of the antigen binding molecule. Typically, affinity-matured antigen binding molecules bind to the same epitope as the initial reference antigen binding molecule.

[0094] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as 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.

[0095] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking antibodies 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.

[0096] The term "specific binding" refers to the number of different types of antigens or antigenic determinants to which 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. Avidity (determined by the equilibrium constant (K) for the dissociation of an antigen from an antigen binding protein) D Affinity is a measure of the strength of binding between an antigenic determinant and an antigen binding site on an antigen binding protein. Alternatively, affinity can also be expressed as an affinity constant (K A ), which is 1 / K D Affinity can be determined in a manner known per se according to a specific combination of antigen binding protein and target antigen. Avidity is understood herein to refer to the strength of binding of a target molecule to multiple binding sites through a larger complex of a binding agent, i.e. the binding strength of a multivalent binding. Avidity is related to both the affinity and the valence (i.e. the number of binding sites present on the antigen binding protein) between an antigenic determinant and its antigen binding site on the antigen binding protein. On the other hand, affinity refers to a simple monovalent receptor ligand system.

[0097] Typically, the antigen binding region or region of a multispecific antigen binding protein of the invention that has affinity for a surface antigen expressed on NK cells will be about 10 -6 Up to 10 -12M or less, and preferably 10 -8 Up to 10 -12 M or less 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 -9 M, such as at least 10 -10 , 10 -11 , 10 -12 M or greater binding affinity binds to its target molecule (antigen). Greater than 10 -4 Any K D Values ​​are generally considered to indicate nonspecific binding. Thus, an antigen binding region that "specifically binds" an antigen is one that is no more than 10 -4 M of K D The antigen binding domain of the multispecific antigen binding protein of the invention that binds to the antigen, as can be determined as described below. Preferably, the antigen binding region or region having affinity for a surface antigen expressed on NK cells of the multispecific antigen binding protein of the invention will bind to the target molecule with an affinity of less than 800, 400, 200, 100, 50, 10 or 5 nM, more preferably less than 1 nM, such as less than 500, 200, 100, 50, 10 or 5 pM. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the 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 illustrative examples are described below.

[0098] “K D ” or “K D The "value" can be determined by using an ELISA as described in the Examples herein or by using a BIAcore TM -2000 or BIAcore TMThe surface plasmon resonance assay of the BIAcore-3000 (BIAcore, Inc., Piscataway, NJ) was measured at 25°C with an immobilized antigen CM5 chip at approximately 10-50 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore) were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5 μg / ml (approximately 0.2 μM) with 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. After the injection of the antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of antibodies or Fabs (0.78 nM to 500 nM) were injected into PBS with 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25 μl / min. The association rates (k) were calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. on ) and dissociation rate (k off ). The equilibrium dissociation constant (K D ) is calculated as the ratio k off / k on See, for example, Chen, Y., et al., (1999) J. Mol Biol [Journal of Molecular Biology] 293: 865-881. If the on-rate exceeds 10 as measured by surface plasmon resonance, 6 M -1 S -1 , the binding rate can be determined by using a fluorescence quenching technique that measures 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 the presence of increasing concentrations of antigen in PBS (pH 7.2) at 25°C, as measured in a spectrometer with a stirred cuvette (such as a spectrophotometer equipped with a choke (Aviv Instruments) or a 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic).

[0099] The term "humanized antibody" or "humanized immunoglobulin" refers to an immunoglobulin comprising a human framework, at least one and preferably all complementary determining regions (CDRs) from a non-human antibody, and any constant regions present therein are substantially identical to human immunoglobulin constant regions, i.e., at least about 85%, at least 90%, and at least 95% identical. Thus, all parts of a humanized immunoglobulin (except possible CDRs) are substantially identical to corresponding parts of one or more natural human immunoglobulin sequences. Typically, framework residues in the human framework region will be substituted with corresponding residues from a CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, such as by modeling the interactions of the CDRs with the framework residues to identify framework residues that are important for antigen binding, and by 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; 6,180,370 (each of which is incorporated by reference in its entirety). Antibodies can be humanized using a variety of techniques known in the art, including, for example, 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 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332), all of which are hereby incorporated by reference in their entirety.

[0100] One type of antigen binding region for use in the present invention comprises an immunoglobulin single variable domain (ISVD) having an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring single variable domain but which has been "humanized", i.e., by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring single variable domain sequence with those in the V sequences of a conventional 4-chain antibody from a human. HOne or more substitutions in the amino acid residues occurring at one or more corresponding positions in the domain. This can be done in a manner known per se that will be clear to the skilled person, for example based on the prior art on humanization (including, for example, Jones et al. (Nature [Nature] 321: 522-525, 1986); Riechmann et al. (Nature [Nature] 332: 323-329, 1988); Presta (Curr. Op. Struct. Biol. [Current Structural Biology Opinion] 2: 593-596, 1992); Vaswani and Hamilton (Ann. Allergy, Asthma and Immunol. [Allergy, Asthma and Immunology Annals], 1: 105-115); 1998); Harris (Biochem. Soc. Transactions, 23:1035-1038, 1995); Hurle and Gross (Curr. Op. Biotech., 5:428-433, 1994)) and those involving V H The humanization of H is carried out according to the specific prior art (e.g. Vincke et al. (2009, J. Biol. Chem. [Journal of Biological Chemistry] 284: 3273-3284)). Again, it should be noted that such humanized single variable domains of the present invention can be obtained in any suitable manner known per se and are therefore not strictly limited to polypeptides that have been obtained using polypeptides comprising naturally occurring single variable domains as starting material.

[0101] "Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain is generally composed of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0102] For the purposes of this paper, "acceptor human framework" is such a framework, the framework comprises the amino acid sequence of the light chain variable domain (VL) framework or the heavy chain variable domain (VH) framework derived from the human immunoglobulin framework or the people's common framework, as defined below. The acceptor human framework "derived from" the human immunoglobulin framework or the people's common framework can comprise its identical amino acid sequence, or it can contain amino acid sequence changes. In certain 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 certain embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the people's common framework sequence.

[0103] As an alternative to humanization, human antibodies can be produced. "Human antibodies" means antibodies that contain human light chains and heavy chains and constant regions completely produced by any known standard method. For example, transgenic animals (e.g., mice) are available, and these transgenic animals can produce a complete library of human antibodies in the absence of endogenous immunoglobulin production after immunization. For example, homozygous deletions of antibody heavy chain joining region PH genes in chimeric and germline mutant mice have been described, resulting in complete inhibition of endogenous antibody production. Transferring human germline immunoglobulin gene arrays to such germline mutant mice will result in the production of human antibodies after immunization. See, for example, Jakobovits et al., Proc. Nat. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States], 90: 2551 (1993); Jakobovits et al., Nature [Nature], 362: 255-258 (1993). Alternatively, phage display technology (McCafferty et al., Nature [Nature] 348: 552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from donors. According to this technology, antibody V domain genes are cloned in-frame into major or minor coat protein genes of filamentous phage (such as M13 or fd) and displayed as functional antibody fragments on the surface of phage particles. Because filamentous particles contain single-stranded DNA copies of the phage genome, selection based on the functional properties of antibodies also results in the selection of genes encoding antibodies that exhibit those properties. Therefore, phages mimic some properties of B cells. Phage display can be performed in a variety of forms; for their review, see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology [Current Structural Biology Viewpoint] 3: 564-571 (1993). Human antibodies can also be produced by B cells activated in vitro or SCID mice reconstructed with human cells of the immune system. Once human antibodies are obtained, their encoding DNA sequences can be isolated, the sequences are cloned and introduced into a suitable expression system (i.e., cell lines, preferably from mammals), which then express the encoding DNA sequences and release them into a culture medium from which the antibodies can be isolated.

[0104] As used herein, the term "tumor-associated antigen" (TAA) means any antigen, including but not limited to proteins, glycoproteins, gangliosides, carbohydrates, lipids associated with cancer. Such antigens may be expressed on malignant cells or in the tumor microenvironment, such as on tumor-associated blood vessels, extracellular matrix, mesenchymal matrix, or immune infiltrates. Explicitly included in the term TAA are homologs of wild-type TAAs that differ from wild-type TAAs due to tumor-specific mutations (which may be patient-specific or shared) and result in altered amino acid sequences, so-called neoantigens.

[0105] "Nucleic acid construct" or "nucleic acid vector" should be understood herein to mean an artificial nucleic acid molecule produced using recombinant DNA technology. Therefore, the term "nucleic acid construct" does not include naturally occurring nucleic acid molecules, although nucleic acid constructs may include (a part of) naturally occurring nucleic acid molecules. The term "expression vector" or "expression construct" refers to a nucleic acid molecule that can realize the 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 include regulatory sequence elements that are operably connected to the nucleotide sequence to be expressed to affect the expression of the nucleotide sequence. Such regulatory elements typically include at least a suitable transcriptional regulatory sequence and optionally a 3' transcription termination signal. Other elements that are necessary or helpful in realizing expression may also be present, such as expression enhancer elements. The expression vector will be introduced into a suitable host cell, and the expression of the coding sequence in the in vitro cell culture of the host cell can be realized. The expression vector will be suitable for replication in the host cell or organism of the present invention, and the expression construct will generally be integrated into the genome of the host cell so that it is maintained. The technology for introducing nucleic acid into cells is well established in the art, and any suitable technology can be adopted according to the specific circumstances.For eukaryotic cells, suitable technology can include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other viruses (such as adenovirus, AAV, slow virus or vaccinia virus).For microorganisms (such as, bacteria) cells, suitable technology can include calcium chloride conversion, electroporation and transfection using bacteriophage.The nucleic acid introduced can be on an extrachromosomal vector in the cell, or the nucleic acid can be integrated into the genome of the host cell.According to standard techniques, integration can be promoted by including a sequence that promotes recombination with the genome in nucleic acid or a vector.Can express nucleic acid to produce encoded fusion protein after introduction.In certain embodiments, host cell (it can include actual transformed cells, although more likely these cells will be the offspring of transformed cells) can be cultured in vitro under the conditions for expressing nucleic acid, so that the fusion protein polypeptide encoded is produced, when using an inducible promoter, expression may need to activate the inducible promoter.

[0106] As used herein, the term "promoter" or "transcription regulatory sequence" refers to a nucleic acid fragment that plays a role in controlling the transcription of one or more coding sequences and is located upstream relative to the transcription direction of the transcription start site of the coding sequence, and is structurally identified by the presence of a binding site for a DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequence, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art that acts directly or indirectly to 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, such as by the application of a chemical inducer.

[0107] The term "selectable marker" is a term familiar to those of ordinary skill 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, although it is primarily used to refer to visible markers, such as green fluorescent protein (GFP). Selectable markers can be dominant or recessive or bidirectional.

[0108] As used herein, the term "operably linked" refers to the connection of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a transcription regulatory sequence affects the transcription of a coding sequence, the transcription regulatory sequence is operably linked to a coding sequence. Operably linked means that the DNA sequences being linked are typically continuous, and in the case of two protein coding regions, continuous and in reading frame.

[0109] The terms "protein" or "polypeptide" are used interchangeably and refer to a molecule composed of a chain of amino acids without regard to a particular mode of action, size, 3-dimensional structure or origin.

[0110] The term "signal peptide" (sometimes referred to as signal sequence) is a short peptide (usually 16-30 amino acids long) that is present at the N-terminus of most newly synthesized proteins destined to enter 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 into the secretory pathway, i.e., the ER) to produce a free signal peptide and mature protein. Signal peptides are extremely heterogeneous, and many prokaryotic and eukaryotic signal peptides are functionally interchangeable even between different species, however the efficiency of protein secretion may depend on the signal peptide. Suitable signal peptides are generally known in the art, for example from et al. (2004 J. Mol. Biol. 338: 1027-1036) and von Heijne (1985, J Mol Biol. 184 (1): 99-105).

[0111] The term "gene" refers to a DNA fragment comprising a region (transcribed region) operably linked to a suitable regulatory region (e.g., a promoter) that is transcribed into an RNA molecule (e.g., mRNA) in a cell. A gene typically comprises several operably linked segments, such as a promoter, a 5' leader sequence, a coding region, and a 3' non-translated sequence (3' end) comprising a polyadenylation site. "Gene expression" refers to a process in which a DNA region operably linked to a suitable regulatory region (particularly a promoter) is transcribed into RNA that has biological activity, i.e., can be translated into a biologically active protein or peptide.

[0112] 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 should be understood to mean that in nature, the nucleic acid or polypeptide molecule is produced by a host cell or organism of the same species (preferably the same variety or strain). If homologous to the host cell, the nucleic acid sequence encoding the polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and (if applicable) another (heterologous) secretion signal sequence and / or terminator sequence different from its natural environment. It should be understood that regulatory sequences, signal sequences, terminator sequences, etc. can also be homologous to the host cell. When used to indicate the correlation of two nucleic acid sequences, the term "homologous" means that a single-stranded nucleic acid sequence can hybridize with a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a variety of factors, including the amount of identity between the sequences and hybridization conditions (such as temperature and salt concentration discussed later).

[0113] The term "heterologous" when used with respect to nucleic acids (DNA or RNA) or proteins refers to such nucleic acids or proteins that are not naturally present as part of an organism, cell, genome or DNA or RNA sequence in which it is present, or that are found in one or more positions in a cell or genome or DNA or RNA sequence different from the one in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cells into which they are introduced, but have been obtained or synthesized or recombinantly produced from another cell. Typically, although not necessarily, such nucleic acid encoding is not a protein normally produced by a cell that transcribes or expresses DNA. Similarly, exogenous RNA encoding is not normally expressed in a cell in which exogenous RNA is present. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Those skilled in the art will recognize that any nucleic acid or protein that is exogenous or foreign to the cell expressing it is covered by the term heterologous nucleic acids or proteins herein. The term heterologous is also applicable to non-natural combinations of nucleic acids or amino acid sequences, i.e., combinations in which at least two of the combined sequences are foreign to each other.

[0114] DETAILED DESCRIPTION OF THE INVENTION

[0115] The present invention arises in part from the observation that a multispecific antigen-binding protein comprising a NK cell-activating cytokine that triggers at least one of the interleukin 21 receptor and 4-1BB and comprising a region having affinity for a surface antigen expressed on NK cells, such as an Fc region, is able to induce hyperfunctionality in NK cells, including proliferation of NK cells, resistance to the tumor microenvironment, enhanced ability of NK cells to mediate target cell lysis, and prolonged duration of these abilities ( Figure 1 Such capabilities may be even further enhanced if the multispecific antigen binding protein comprises at least one antigen binding region that specifically binds to an NK cell activating receptor.

[0116] Multispecific antigen binding proteins

[0117] In the first aspect, the present invention relates to multispecific antigen binding proteins. In one embodiment, the multispecific antigen binding protein comprises a) NK cell activation cytokines; and ii) 4-1BB agonists; and b) regions with affinity for surface antigens expressed on natural killer (NK) cells. NK cell activation cytokines are preferably at least one of the following: i) interleukin 21 receptor (IL21R) agonists; and ii) 4-1BB agonists. The region with affinity for NK cell surface antigens allows multispecific antigen binding proteins to bind to NK cells, after which one or more NK cell activation cytokines can induce a superfunctional phenotype in NK cells. In one embodiment, the multispecific antigen binding protein further comprises c) at least one antigen binding region that specifically binds to NK cell activation receptors.

[0118] 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 NK cells; and c) at least one antigen binding region that specifically binds to an NK cell activating receptor; an NK cell activating receptor.

[0119] NK cell activating cytokines

[0120] Therefore, the multispecific antigen binding protein as described herein includes at least one NK cell activation cytokine. In one embodiment, the NK cell activation cytokine is at least one of the following: i) interleukin 21 receptor (IL21R) agonist; and ii) 4-1BB agonist. In one embodiment, the multispecific antigen binding protein at least includes an IL21R agonist. In one embodiment, the multispecific antigen binding protein at least includes a 4-1BB agonist. In addition, in one embodiment, the multispecific antigen binding protein at least includes both an IL21R agonist and a 4-1BB agonist. In a further embodiment, in addition to at least one of an IL21R agonist and a 4-1BB agonist, the multispecific antigen binding protein as described herein may also include additional NK cell activation cytokines. Such additional NK cell activation cytokines may be selected from the group consisting of: IL15 receptor agonist, IL2 receptor agonist, IL12 receptor agonist, and IL18 receptor agonist, as further described below.

[0121] Thus, in one embodiment, a multispecific antigen-binding protein as described herein comprises at least an interleukin 21 receptor (IL21R) agonist.

[0122] Interleukin 21 (IL21) is a protein encoded by the IL21 gene (Entrez gene ID: 59067) in humans. IL21 is a cytokine that has an effective regulatory effect 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 human IL21 precursor (including its signal sequence) is described in NCBI accession number NP_001193935 and NP_068575, and its disclosure is 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 effect on target cells through the IL-21 receptor (IL21R) expressed on the surface of T cells, B cells and NK cells. IL21R is structurally similar to the receptors of other type I cytokines (like IL-2R or IL-15), and needs to dimerize with common gamma chain (γc) in order to combine IL-21.IL21R is encoded by IL21R gene (Entrez gene ID: 50615) in people.The amino acid sequence of people IL21R is described in NCBI accession numbers NP_068570, NP_851564 and NP_851565, and its disclosure is incorporated into this paper by quoting.

[0123] As used herein, "IL21R agonist" is an agent with "agonist" activity at the IL21 receptor, which means an agent that can cause or increase "IL21R signaling". "IL21R signaling" refers to the ability of IL21R to activate or transduce intracellular signaling pathways when, for example, expressed on the surface of T cells, B cells and NK cells and triggered by its natural ligand IL21. "Natural ligand IL21" is understood herein to include the amino acid sequence as indicated above or the human wild-type IL21 consisting thereof. When combined with IL-21, the IL-21 receptor works through the Jak / STAT pathway, utilizing Jak1 and Jak3 and STAT3 homodimers to activate its target genes. IL21R agonist activity (i.e., changes in IL21R signaling activity) can be measured, for example, by the following means: a determination designed to measure changes in IL21R signaling pathways (e.g., by monitoring the phosphorylation of signal transduction components), a determination for measuring the association of certain signal transduction components with other proteins or intracellular structures (or changes in the biochemical activity of components such as kinases), or a determination designed to measure reporter gene expression under the control of IL21R sensitive promoters and enhancers, or indirectly by downstream effects mediated by IL21R (e.g., activation of specific cell lysis mechanisms in NK cells). Suitable cell-based determinations of the biological activity of IL21R agonists are described, for example, in Maurer et al. (mAbs. [monoclonal antibodies] 2012; 4 (1): 69-83.), wherein both human IL21R and STAT response type luciferase reporter genes are transfected into mouse pre-B cell lines. IL21R agonist activity can be determined by using this cell line to measure STAT3 phosphorylation level and / or by detecting luciferase luminescence using beads conjugated with anti-pSTAT3 antibodies after the cell line is contacted with the IL21R agonist. The natural ligand IL21 can be used as a positive control in the determination of 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 (such as a multispecific antigen binding protein comprising an IL21R agonist as described herein).

[0124] In one embodiment, the multispecific antigen-binding protein as described herein comprises an IL21R agonist having reduced IL21R agonist activity compared to human wild-type IL21. In one embodiment, the IL21R agonist activity of the IL21R agonist is 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 200, 1 / 500 or 1 / 1000 of the IL21R agonist activity of human wild-type IL21.

[0125] In one embodiment, a multispecific antigen-binding protein as described herein comprises an IL21R agonist having enhanced IL21R agonist activity compared to human wild-type IL21. In one embodiment, the IL21R agonist activity of the IL21R agonist is 2, 5, 10, 20, 50, 100, 200, 500, or 1000 times greater than the IL21R agonist activity of human wild-type IL21.

[0126] In one embodiment, the multispecific antigen-binding protein as described herein comprises an IL21R agonist, which is an IL21 polypeptide 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:38, and preferably has IL21R agonist activity as defined above, and / or preferably has affinity for IL21R as defined below.

[0127] In one embodiment, a multispecific antigen-binding protein as described herein comprises an IL21R agonist with reduced or enhanced affinity for IL21R compared to human wild-type IL21. The affinity of an IL21R agonist for IL21R can be determined using methods generally known in the art, such as surface plasmon resonance.

[0128] In one embodiment, a multispecific antigen-binding protein as described herein comprises an IL21R agonist having reduced affinity for IL21R compared to human wild-type IL21. In one embodiment, the affinity of the IL21R agonist for IL21R is 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 200, 1 / 500, 1 / 1000, 1 / 10000 or 1 / 100000 of the affinity of human wild-type IL21 for IL21R.

[0129] In one embodiment, a multispecific antigen-binding protein as described herein comprises an IL21R agonist having an 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 that of human wild-type IL21 for IL21R.

[0130] In one embodiment, the multispecific antigen-binding protein as described herein includes an IL21R agonist, which is IL21 or a fragment thereof having IL21R agonist activity. Preferably, the IL21R agonist is human IL21 or a fragment thereof having IL21R agonist activity. In one embodiment, the IL21R agonist is an IL21 mutant protein having a reduced affinity for IL21R compared to human wild-type IL21. The IL21 mutant protein having a reduced affinity for IL21R compared to human wild-type IL21 is described in Shen et al. (Front Immunol. [Immunology Frontier] 2020; 11: 832). Thus, in one embodiment, the IL21R agonist is an IL21 mutant protein having a mutation (i.e., an amino acid substitution, deletion or insertion) of one or more amino acids 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 refer to positions in SEQ ID NO: 38 or corresponding positions in IL-21 allelic variants). Preferably, the IL21R agonist is an IL21 mutein comprising one or more amino acid substitutions 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, K73E, K75P, K77P, Q12E, R5E, R65P, R76E, R9E, S70P, V69P, I8G, K73G, Q12N, R5G, R76G, R9G, S70Y, I8N, K73H, Q12S, R5H , R76H, R9H, I8S, K73I, Q12T, R5I, R76I, R9I, K73N, Q12V, R5K, R76K, R9K, K73 P, R5L, R76L, R9L, K73Q, R5M, R76M, R9M, K73S, R5N, R76N, R9N, K73V, R5Q, R76 P, R9Q, R5S, R76Q, R9S, R5T, R76S, R9T, R5V, R76T, R9V, R5Y, R76V, R9Y and R76Y.

[0131] In one embodiment, IL21R agonist is the IL21 mutant protein with affinity to human IL21R-Fc between 0.028 and 0.099nM in Table 2 of Shen et al. (2020; The same). In one embodiment, IL21R agonist is the IL21 mutant protein with affinity to human IL21R-Fc between 0.10 and 0.29nM in Table 2 of Shen et al. (2020; The same). In one embodiment, IL21R agonist is the IL21 mutant protein with affinity to human IL21R-Fc between 0.30 and 0.99nM in Table 2 of Shen et al. (2020; The same). In one embodiment, IL21R agonist is the IL21 mutant protein with affinity to human IL21R-Fc between 1.0 and 2.9nM in Table 2 of Shen et al. (2020; The same). In one embodiment, the IL21R agonist is an IL21 mutant protein in Table 2 of Shen et al. (2020; supra) with an affinity for human IL21R-Fc > 2.9 nM.

[0132] In one embodiment, a multispecific antigen-binding protein as described herein comprises an IL21R agonist, which 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 above.

[0133] In one embodiment, the multispecific antigen-binding protein as described herein comprises more than one IL21R agonist as described above. Therefore, in one embodiment, the multispecific antigen-binding protein has an IL21R agonist titer greater than one. The IL21R agonist titer 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 higher.

[0134] In one embodiment, a multispecific antigen-binding protein as described herein comprises at least a 4-1BB agonist.

[0135] 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, and its disclosure is incorporated herein by reference. 4-1BB is referred to as a co-stimulatory immune checkpoint molecule. 4-1BB is expressed by activated T cells of both CD4+ and CD8+ lineages, and is expressed on activated NK cells. It is known that NK cells with increased 4-1BB expression are highly active to target cells (e.g., tumor cells) expressing 4-1BB ligands. 4-1BB ligand (4-1BBL) (also referred to as TNFSF9 or CD137L) is a protein encoded by the TNFSF9 gene (Entrez gene ID: 8744) in humans. The amino acid sequence of human 4-1BBL is described in NCBI accession number NP_003802, and its disclosure is incorporated herein by reference.4-1BB / 4-1BBL complex is composed of three monomer 4-1BBs combined with trimer 4-1BBL.Each 4-1BB monomer is combined with two 4-1BBLs via a cysteine-rich domain (CRD).The interaction between 4-1BB and the second 4-1BBL is required for stabilizing its interaction.

[0136] As used herein, "4-1BB agonist" is an agent with "agonist" activity at 4-1BB, which means that it can cause or increase the agent of "4-1BB signaling". "4-1BB signaling" refers to the ability of 4-1BB to activate or transduce intracellular signaling pathways when expressed on the surface of T cells, B cells and NK cells and triggered by its natural ligand 4-1BBL, for example. "Natural 4-1BB ligands" are understood herein as the extracellular domain (ECD) of human wild-type 4-1BBL, which includes the amino acid sequence (i.e., SEQ ID NO:37) of people 4-1BBL from position 71 to 254 or consisting of it. Therefore, 4-1BBL extracellular domain (ECD) is understood herein as polypeptide, which includes the amino acid sequence from position 71 to 254 of people 4-1BBL or its fragment with 4-1BB agonist activity or consisting of the amino acid sequence or its fragment.

[0137] 4-1BB agonist activity (i.e., changes in 4-1BB signaling activity) can be measured, for example, by an assay designed to measure changes in the 4-1BB signaling pathway (e.g., by monitoring phosphorylation of signal transduction components), an assay for measuring the association of certain signal transduction components with other proteins or intracellular structures (or changes in the biochemical activity of components such as kinases), or indirectly by downstream effects mediated by 4-1BB (e.g., production of specific cytokines). Suitable cell-based assays for the in vitro biological activity of 4-1BB agonists are described, for example, in Zhang et al. (Clin Cancer Res, 2007; 13(9): 2758-2767), using measurements of IL-2 production from spleen cells aseptically removed from BALB / c mice in microtiter plates pre-coated with anti-CD3 monoclonal antibodies (145-11C clone). Other suitable cell-based assays for in vitro biological activity of 4-1BB agonists are described in Example 6 of WO2016 / 075278 (see, e.g., Example 6.1). A natural 4-1BB ligand, a 4-1BBL ECD trimer as described by Fellermeier et al. (Oncoimmunol. 2016, 5(11): e1238540) (e.g., a 4-1BBL ECD trimer comprising the amino acid sequence of SEQ ID NO: 36), or an anti-CD137 agonist antibody (such as antibody 2A, Epstein et al., Tumor necrosis imaging and treatment of solid tumors. In: VP Torchilin, ed., Handbook of targeted delivery of imaging agents, Vol. 16, Boca Raton: CRC Press; 1995. p. 259) can be used as a positive control in the determination of 4-1BB agonist activity, and can also be used as a reference for the amount of 4-1BB agonist activity of a given non-natural 4-1BB agonist (such as a multispecific antigen-binding protein comprising a 4-1BB agonist as described herein).

[0138] In one embodiment, the multispecific antigen-binding protein as described herein comprises a 4-1BB agonist with reduced 4-1BB agonist activity compared to human wild-type 4-1BBL or anti-4-1BB agonist antibody 2A. In one embodiment, the 4-1BB agonist activity of the 4-1BB agonist is 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 200, 1 / 500 or 1 / 1000 of the 4-1BB agonist activity of the ECD of human wild-type 4-1BBL or anti-4-1BB agonist antibody 2A.

[0139] In one embodiment, the multispecific antigen-binding protein as described herein comprises a 4-1BB agonist having enhanced 4-1BB agonist activity compared to human wild-type 4-1BBL. In one embodiment, the 4-1BB agonist activity of the 4-1BB agonist is 2, 5, 10, 20, 50, 100, 200, 500 or 1000 times that of the ECD of human wild-type 4-1BBL or the 4-1BB agonist activity of anti-4-1BB agonist antibody 2A.

[0140] In one embodiment, the multispecific antigen-binding protein as described herein comprises a 4-1BB agonist comprising 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 above, and / or preferably has affinity for 4-1BB as defined below.

[0141] In one embodiment, a multispecific antigen-binding protein as described herein comprises a 4-1BB agonist with reduced or enhanced affinity for 4-1BB compared to the ECD of human wild-type 4-1BBL. The affinity of a 4-1BB agonist to 4-1BB can be determined using methods generally known in the art, such as surface plasmon resonance.

[0142] In one embodiment, the multispecific antigen-binding protein as described herein comprises a 4-1BB agonist having a reduced affinity for 4-1BB compared to human wild-type 4-1BBL. In one embodiment, the affinity of the 4-1BB agonist to 4-1BB is 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 200, 1 / 500, 1 / 1000 or 1 / 10000 of the affinity of the ECD of human wild-type 4-1BBL or anti-4-1BB agonist antibody 2A to 4-1BB.

[0143] In one embodiment, the multispecific antigen-binding protein as described herein comprises a 4-1BB agonist having an enhanced affinity for 4-1BB compared to human wild-type 4-1BBL. In one embodiment, the affinity of the 4-1BB agonist to 4-1BB is 2, 5, 10, 20, 50, 100, 200, 500 or 1000 times that of the ECD of human wild-type 4-1BBL or the affinity of anti-4-1BB agonist antibody 2A to 4-1BB.

[0144] In one embodiment, the multispecific antigen-binding protein as described herein comprises a 4-1BB agonist, which comprises an ECD of 4-1BBL or a fragment thereof having 4-1BB agonist activity or is composed of the ECD or a fragment thereof. 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 mutant protein of the ECD of 4-1BBL having a reduced affinity for 4-1BB compared to the ECD of human wild-type 4-1BBL or anti-4-1BB agonist antibody 2A.

[0145] In one embodiment, a multispecific antigen-binding protein as described herein comprises a 4-1BB agonist comprising or consisting of a fusion protein comprising three 4-1BBL ECD monomers fused together in a single polypeptide chain, as described in, for example, Fellermeier et al. (2016, supra). In one embodiment, three 4-1BBL ECD monomers are connected by a polypeptide linker. In one embodiment, three 4-1BBL ECD monomers are connected by a polypeptide linker selected from the group consisting of: (GGGGS) 4, GGGSGGG, GGSGGGGSGG and G, wherein (GGGGS) 4 is preferred. Other suitable one or more flexible polypeptide linkers are described below. In one embodiment, a multispecific antigen-binding protein as described herein comprises a 4-1BB agonist comprising or consisting of a fusion protein comprising three 4-1BBL ECD monomers fused together in a single polypeptide chain, for example, comprising an amino acid sequence of SEQ ID NO: 36.

[0146] In one embodiment, the multispecific antigen-binding protein as described herein comprises a 4-1BB agonist, which comprises three 4-1BBL ECD monomers present in more than one polypeptide chain of the multispecific antigen-binding protein. For example, two 4-1BBL ECD monomers can be fused together in a single polypeptide chain, optionally connected together by 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, as described in, for example, WO 2016 / 075278. The first polypeptide chain and the second polypeptide chain of the multispecific antigen-binding protein can be chains comprising heavy chains and light chains, respectively, or vice versa, wherein preferably, 4-1BBL ECD is fused to the N-terminus of the variable domain. Alternatively, the first polypeptide chain and the second polypeptide chain of the multispecific antigen-binding protein can be two chains comprising two heavy chains, wherein preferably, 4-1BBL ECD is fused to the C-terminus of the constant domain.

[0147] In one embodiment, the multispecific antigen-binding protein as described herein includes a 4-1BB agonist, which includes an antigen binding region that specifically binds to 4-1BB and has 4-1BB agonistic activity. Antibodies for 4-1BB are, for example, described in WO2005035584, WO2006088464, and US2006188439.

[0148] In one embodiment, the multispecific antigen-binding protein as described herein comprises more than one 4-1BB agonist as described above. Therefore, in one embodiment, the multispecific antigen-binding protein has a 4-1BB agonist titer higher than one. The 4-1BB agonist titer 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 higher.

[0149] In preferred embodiments, the multispecific antigen-binding protein as described herein comprises both at least one IL21R agonist as described above and at least one 4-1BB agonist as described above.

[0150] In a further embodiment, in addition to at least one of an IL21R agonist and a 4-1BB agonist, a multispecific antigen-binding protein as described herein may also include additional NK cell-activating cytokines selected from the group consisting of: IL15 receptor agonists, type I interferon (IFN-1) agonists, IL2 receptor agonists, IL12 receptor agonists, and IL18 receptor agonists. In one embodiment, the IL15 receptor agonist is an IL15 polypeptide or an agonistic antigen binding region that specifically binds to an IL15 receptor. In one embodiment, the IL2 receptor agonist is an IL2 polypeptide or an agonistic antigen binding region that specifically binds to an IL2 receptor. In one embodiment, the IL12 receptor agonist is an IL12 polypeptide or an agonistic antigen binding region that specifically binds to an IL12 receptor. In one embodiment, the IL18 receptor agonist is an IL18 polypeptide or an agonistic antigen binding region that specifically binds to an IL18 receptor.

[0151] Region with affinity for surface antigens expressed on NK cells

[0152] In one embodiment, a multispecific antigen-binding protein as described herein can further comprise a region having affinity for a surface antigen expressed on NK cells. Thus, the presence of such a region having affinity for a surface antigen expressed on NK cells in a multispecific antigen-binding protein is optional.

[0153] In one embodiment of the multispecific antigen-binding protein, the region with affinity for surface antigens expressed on NK cells comprises an immunoglobulin Fc region or at least a portion thereof or consists of an immunoglobulin Fc region or at least a portion thereof, which binds to a 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 the CH2 and CH3 domains. In one embodiment, the immunoglobulin Fc region comprises at least one of the CH2 and CH3 domains and the hinge region. In one embodiment, the immunoglobulin Fc region comprises or consists of a hinge region and CH2 and CH3 domains. In one embodiment, the immunoglobulin Fc region is a dimer Fc region or at least a portion thereof that binds to CD16A.

[0154] In one embodiment, the Fc region or portion thereof that binds CD16A is a wild-type region or portion thereof.

[0155] In one embodiment, the Fc region or part thereof in conjunction with CD16A can be modified to enhance or reduce its binding affinity to CD16A. In the Fc region, CD16A binding is mediated by the hinge region and the CH2 domain. For example, in human IgG1, the interaction with CD16 is mainly concentrated in the amino acid residues D265-E269, N297-T299, A327-I332, L234-S239 and carbohydrate residues N-acetyl-D-glucosamine in the CH2 domain (see, Sondermann et al., 2000Nature [Natural], 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 mutations can be designed based on the known three-dimensional structure of the interaction.

[0156] Therefore, in one embodiment, in the case where the multispecific antigen-binding protein is intended to have an increased affinity for CD16A, the Fc region or portion thereof that binds CD16A may include modifications to increase affinity for CD16A. Therefore, the Fc region or portion thereof that binds CD16A may include one or more amino acid modifications (e.g., amino acid substitutions, deletions, insertions) that increase binding to (human) CD16A and optionally another receptor (such as FcRn). Typical modifications include a modified constant region of human IgG1 origin that includes at least one amino acid modification (e.g., substitution, deletion, insertion) and / or an altered glycosylation type (e.g., low fucosylation). The modification may, for example, increase the binding of the Fc region to FcγRIIIa (CD16A) on NK cells. Examples of modifications are provided in US10,577,419, the disclosure of which is incorporated herein by reference. Specific mutations (in the IgG1 Fc region) that enhance FcyRIIIa (CD16A) binding include E333A, S239D / I332E, and S239D / A330L / I332E.

[0157] In one embodiment, the multispecific antigen-binding protein comprises an Fc region, or portion thereof, that binds CD16A, which Fc region, or portion thereof, 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 an enhanced binding affinity for (human) CD16A relative to a molecule comprising the wild-type Fc region, optionally wherein 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), optionally wherein the variant Fc region comprises a substitution at residues S239 and I332, e.g., S239D and I332E substitutions (Kabat EU numbering).

[0158] In one embodiment, the multispecific antigen-binding protein comprises an Fc region or portion thereof that binds CD16A, the Fc region or portion thereof comprising an altered glycosylation pattern that increases binding affinity to (human) CD16A. Such carbohydrate modifications can be accomplished, for example, by expressing a nucleic acid encoding a multispecific protein in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery are known in the art and can be used as host cells in which to express recombinant antibodies, thereby producing antibodies with altered glycosylation. See, e.g., Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1 and European Patent Nos.: EP 1,176,195; WO 06 / 133148; WO 03 / 035835; WO 99 / 54342, each of which is incorporated herein by reference in its entirety. In one embodiment, the multispecific antigen-binding protein comprises one or more hypofucosylated constant regions. Such a multispecific antigen-binding protein may or may not comprise amino acid changes and / or may be expressed or synthesized or processed under conditions that result in hypofucosylation. In one embodiment, in a composition comprising a multispecific antigen-binding protein described herein, at least 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, or substantially all of the multispecific antigen-binding proteins have a constant region 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 contain N-linked glycans that comprise a core carbohydrate structure with fucose. The core carbohydrate will preferably be a sugar chain at Asn297.

[0159] In one embodiment, the multispecific antigen-binding protein comprising an Fc region, or a portion thereof, that binds CD16A that is modified to have increased binding affinity for CD16A has a binding affinity for human CD16A that is at least 1, 2, or 3 logs greater than the binding affinity of a conventional or wild-type human IgG1 antibody, e.g., as assessed by surface plasmon resonance.

[0160] In another embodiment, in the case where the multispecific antigen-binding protein is intended to have a reduced affinity for CD16A, the CH2 and / or CH3 domains, Fc regions or portions thereof that bind to CD16A may include modifications that reduce the affinity for CD16A. For example, a CH2 mutation at residue N297 (Kabat numbering) in a dimeric Fc region protein may eliminate CD16A binding. Other modifications that reduce or eliminate binding to CD16A in the Fc region include L234A / L235A, also referred to as "LALA" modifications, as exemplified herein. Modifications to reduce or eliminate the binding to CD16A in the Fc region may be used for 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. It is expected that cross-linking of NK cells with NK cells or other immune cells will reduce the therapeutic efficacy of NK cell engagement. Most importantly, cross-linking of NK cells with one or more NK cells or other immune cells through bivalent or multivalent interactions with FcRy or in combination with a second immune cell antigen (e.g., NKp46, NKG2D, NKp30, or SLAMF7) may result in immune cell activation. This may result in the induction of target cell-driven fratricide or immune cell killing (e.g., NK-NK cell lysis), ultimately leading to effective NK cell depletion in vivo, as previously described for CD16-directed murine IgG antibodies (3G8), CD38-directed antibodies daratumumab, and other approaches (Choi et al. 2008 Immunology 124(2)215-22; DOI:10.1111 / 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 [Clinical Cancer Research], 24(16): 4006-4017; DOI: 10.1158 / 1078-0432.CCR-17-3117; His et al. 2008; Nakamura 2013 PNAS [Proceedings of the National Academy of Sciences of the United States of America]; 110(23) 9421-9426; DOI: 10.1073 / pnas.1300140110; Breman et al. 2018 Front Immunol [Frontiers in Immunology], 12(9) 2940; DOI: 10.3389 / fimmu.2018.02940).

[0161] Those skilled in the art will appreciate that other configurations for modifying the Fc region can be implemented. For example, it is shown that substitution of human IgG1 or IgG2 residues at positions 233-236 and substitution of IgG4 residues at positions 327, 330 and 331 greatly reduce binding to Fcγ receptors, and therefore reduce ADCC and CDC. In addition, Idusogie et al. (2000) J.Immunol. [Journal of Immunology] 164 (8): 4178-84 demonstrate that alanine substitutions at different positions (including K322) significantly reduce complement activation.

[0162] In one embodiment, the multispecific antigen-binding protein comprising an Fc region, or a portion thereof, that binds CD16A that is modified to have a decreased binding affinity for CD16A has a binding affinity for human CD16A that is at least 1, 2, or 3 logs less than the binding affinity of a conventional or wild-type human IgG1 antibody, e.g., as assessed by surface plasmon resonance.

[0163] In one embodiment, the multispecific antigen-binding protein comprises an Fc region having an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity with the Fc region of at least one of SEQ ID NOs.: 1, 3, 11-19 and 23, and preferably has one or more of the above structural and / or functional characteristics.

[0164] Specific binding to the antigen binding region of NK cell activating receptor

[0165] 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.

[0166] 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 an affinity body based on the Z domain of Staphylococcus protein A, an engineered Kunitz domain, a single antibody or adnectin based on the 10th extracellular domain of human fibronectin III, anticalins derived from lipocalin, DARPins (designed ankyrin repeat domains), Affilins, multimerized LDLR-A molecules, avidin polymers, or cysteine-rich knottin peptides. See, e.g., Gebauer and Skerra (2009) Current Opinion in Chemical Biology 13:245-255, the disclosure of which is incorporated herein by reference. In alternative embodiments, the antigen binding region that specifically binds to an NK cell activating receptor is a ligand, such as a peptide ligand, that specifically binds to and preferably activates an NK cell activating receptor.

[0167] In preferred embodiments, the antigen binding region used in the multispecific antigen binding proteins described herein comprises or consists of an immunoglobulin variable region. Such immunoglobulin variable regions may comprise or consist of variable domains, which are typically derived from, for example, associated V domains found on two polypeptide chains (such as present in Fab). L and V H Alternatively, the immunoglobulin variable region may comprise a single chain antigen binding domain (such as scFv, V H Domain, V L domain) or an immunoglobulin single variable domain (ISVD) (such as a dAb, a V-NAR domain or a V H The immunoglobulin variable region to be used in the multispecific antigen-binding proteins described herein may be a human or humanized immunoglobulin variable region or an immunoglobulin single variable domain as defined above.

[0168] In one embodiment of the multispecific antigen-binding proteins described herein, the antigen binding region that specifically binds to the NK cell activation receptor is an agonistic antigen binding region that activates the NK cell receptor. As used herein, an antigen binding region with "agonist" activity at the NK cell activation receptor is an agent that can cause or increase "signaling by the NK cell activation receptor". "Signaling by the NK cell activation receptor" refers to the ability of the NK cell activation receptor to activate or transduce intracellular signaling pathways. Changes in the signaling activity of the NK cell activation receptor can be measured, for example, by the following means: an assay designed to measure changes in the NK cell activation receptor signaling pathway (e.g., by monitoring the phosphorylation of signal transduction components), an assay for measuring the association of certain signal transduction components with other proteins or intracellular structures (or changes in the biochemical activity of components such as kinases), or an assay designed to measure the expression of reporter genes under the control of NK cell activation receptor sensitive promoters and enhancers, or indirectly by downstream effects mediated by NK cell activation receptor polypeptides (e.g., activation of specific cytolytic mechanisms in NK cells). Reporter genes can be naturally occurring genes (e.g., monitoring cytokine production), or they can be genes artificially introduced into cells. Other genes can be placed under the control of such regulatory elements and thus used to report the level of NK cell activating receptor signaling activity.

[0169] In one embodiment, the antigen binding region that specifically binds to the 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 the NK cell activating receptor comprises or consists of at least one immunoglobulin variable region. More preferably, the antigen binding region that specifically binds to the NK cell activating receptor comprises or consists of a Fab that specifically binds to the NK cell activating receptor or an immunoglobulin single variable domain (ISVD) that specifically binds to the NK cell activating receptor. In one embodiment, the antigen binding region that specifically binds to the NK cell activating receptor is no more than 10 -4 M of K D The value binds to the antigen binding region of the NK cell activating receptor, such as can be determined as described above.

[0170] In one embodiment, the antigen binding region that specifically binds to the NK cell activating receptor comprises or consists of a human or humanized immunoglobulin variable region or an immunoglobulin single variable domain as defined above.

[0171] In one embodiment, the multispecific antigen-binding protein as described herein comprises two antigen-binding regions that specifically bind to NK cell activating receptors. In a multispecific antigen-binding protein comprising two antigen-binding regions that specifically bind to NK cell activating receptors, the two antigen-binding regions can bind to the same NK cell activating receptor, or they can bind 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 NK cell activating receptors, the two antigen-binding regions are the same. Therefore, for two antigen-binding regions that specifically bind to NK cell activating receptors, the multispecific antigen-binding protein as described herein can be a homodimeric antigen-binding protein or a heterodimeric antigen-binding protein.

[0172] In one embodiment, the multispecific antigen-binding protein as described herein comprises 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, among which NKp46, NKp30, NKG2D, CD16A and SLAMF7 are preferred.

[0173] In one embodiment, the multispecific antigen-binding protein as described herein comprises an antigen-binding region that specifically binds to an NK cell activation receptor, which is a natural cytotoxicity receptor (NCR). Natural cytotoxicity receptors are type 1 transmembrane proteins of the immunoglobulin superfamily that mediate NK killing and the release of IFNγ after stimulation. They bind viral ligands (such as hemagglutinin and hemagglutinin neuraminidase), some bacterial ligands, and cell ligands (such as PCNA) associated with tumor growth. Natural cytotoxicity receptors include NKp46, NKp44, and NKp30. In one embodiment, the multispecific antigen-binding protein as described herein comprises an antigen-binding region that specifically binds to an NK cell activation receptor, which is an NCR selected from the group consisting of: NKp46, NKp44, and NKp30.

[0174] "NKp46" refers to a protein or polypeptide encoded by the Ncr1 gene or by a cDNA prepared from this gene. NKp46 is also named NCR1, CD335 (differentiation group, NKP46, NK-p46 and LY94. The term NKp46 polypeptide encompasses any naturally occurring isoform, allele, ortholog or variant (e.g., a NKp46 polypeptide that is 90%, 95%, 98% or 99% identical to SEQ ID NO:50 or a continuous sequence of at least 20, 30, 50, 100 or 200 amino acid residues thereof). The sequence of 304 amino acid residues of human NKp46 (isoform a) is shown in SEQ ID NO:50 (which corresponds to NCBI accession number NP_004820, the disclosure of which is incorporated herein by reference). The human NKp46 mRNA sequence is described in NCBI accession number NM_004829, the disclosure of which is incorporated herein by reference.

[0175] "NKp44" refers to a protein or polypeptide encoded by the Ncr2 gene or by a cDNA prepared from this gene. NKp44 is also named NCR2, CD336 (differentiation group 336), NKP44, NK-p44, LY95 and dJ149M18.1. The term NKp44 polypeptide encompasses any naturally occurring isoform, allele, ortholog or variant (e.g., a NKp44 polypeptide that is 90%, 95%, 98% or 99% identical to SEQ ID NO:51 or a continuous sequence of at least 20, 30, 50, 100 or 200 amino acid residues thereof). The sequence of 276 amino acid residues of human NKp46 is shown in SEQ ID NO:51 (which corresponds to NCBI accession number NP_004819, the disclosure of which is incorporated herein by reference). The human NKp46 mRNA sequence is described in NCBI accession number NM_004828, the disclosure of which is incorporated herein by reference.

[0176] "NKp30" refers to a protein or polypeptide encoded by the Ncr3 gene or by a cDNA prepared from this gene. NKp30 is also named NCR3 and CD337 (differentiation group 337). The term NKp30 polypeptide encompasses any naturally occurring isoform, allele, ortholog or variant (e.g., a NKp30 polypeptide that is 90%, 95%, 98% or 99% identical to a continuous sequence of SEQ ID NO:52 or at least 20, 30, 50, 100 or 200 amino acid residues thereof). The sequence of 201 amino acid residues of human NKp30 is shown below in SEQ ID NO:52 (which corresponds to NCBI accession number NP_667341, the disclosure of which is incorporated herein by reference). The human NKp30 mRNA sequence is described in NCBI accession number NM_147130, the disclosure of which is incorporated herein by reference.

[0177] NKG2D is an activating receptor (transmembrane protein) belonging to the NKG2 family of C-type lectin-like receptors. NKG2D is encoded by the KLRK1 gene in humans. NKG2D recognizes the self-protein induced by the MIC and RAET1 / ULBP families, which appears on the surface of stress cells, malignant transformed cells and infected cells." NKG2D" refers to a protein or polypeptide encoded by the KLRK1 gene or by a cDNA prepared from this gene. NKG2D is also named KLRK1, CD314 (differentiation group 314), D12S2489E, KLR, NKG2-D, natural killer cell group 2D, killer cell lectin-like receptor K1, killer cell lectin-like receptor K1. The term NKG2D polypeptide encompasses any naturally occurring isoform, allele, ortholog or variant (e.g., a 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 sequence of 216 amino acid residues of human NKG2D is shown 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 described in NCBI Accession No. NM_007360, the disclosure of which is incorporated herein by reference.

[0178] DNAM-1 is a glycoprotein of about 65kDa expressed especially on the surface of NK cells. It is a member of the immunoglobulin superfamily containing 2 Ig-like domains of V-set. DNAM-1 mediates cell adhesion to other cells with its ligands CD112 and CD155, and cross-linking of DNAM-1 with antibodies causes cell activation. "DNAM-1" refers to a protein or polypeptide encoded by the KLRK1 gene or by a cDNA prepared from this gene. DNAM-1 is also named CD226 (differentiation group 226), DNAM-1, DNAM1, PTA1 and TLiSA1. The term DNAM-1 polypeptide encompasses any naturally occurring isoform, allele, ortholog or variant (e.g., a DNAM-1 polypeptide identical to SEQ ID NO:54 or a continuous sequence of at least 20, 30, 50, 100 or 200 amino acid residues thereof, 90%, 95%, 98% or 99%). The sequence of 336 amino acid residues of human DNAM-1 is shown in SEQ ID NO:54 (which corresponds to NCBI Accession No. NP_006557, the disclosure of which is incorporated herein by reference). The human DNAM-1 mRNA sequence is described in NCBI Accession No. NM_006566, the disclosure of which is incorporated herein by reference.

[0179] As indicated above, CD16A is an immunoglobulin gamma Fc region receptor (FcγRIIIa), which is expressed on NK cells, and NK cells recognize IgG bound to the surface of target cells infected with pathogens or expressing TAAs through it. The term CD16A polypeptide encompasses any naturally occurring isoform, allele, ortholog or variant (e.g., a CD16A polypeptide identical to SEQ ID NO: 55 or a continuous sequence of at least 20, 30, 50, 100 or 200 amino acid residues thereof, 90%, 95%, 98% or 99%). The sequence of 254 amino acid residues of human CD16A is shown in SEQ ID NO: 55 (which corresponds to UniProt accession number P08637, the disclosure of which is incorporated herein by reference).

[0180] "SLAMF7" is a protein encoded by the human SLAMF7 gene in humans. Isoform 1 SLAMF7 mediates NK cell activation through an ERK-mediated pathway regulated by SH2D1A-independent extracellular signals. SLAMF7 is also named CD319 (differentiation group 319), 19A, CRACC and CS1. The term SLAMF7 polypeptide encompasses any naturally occurring isoform, allele, ortholog or variant (e.g., a SLAMF7 polypeptide identical to SEQ ID NO:56 or a continuous sequence of at least 20, 30, 50, 100 or 200 amino acid residues thereof of 90%, 95%, 98% or 99%). The sequence of 335 amino acid residues of human SLAMF7 is shown in SEQID NO:56 (which corresponds to UniProt accession number Q9NQ25-1, the disclosure of which is incorporated herein by reference).

[0181] In one embodiment, the multispecific antigen-binding protein as described herein comprises at least one antigen-binding region 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 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 at least a variable light chain (V L ) domain and variable heavy chain (V H) domain sequence, which is obtained / can be obtained from a monoclonal antibody against an NK cell activating receptor as known in the art. Many examples of monoclonal antibodies against NK cell activating receptors are described in the art. Anti-NKp46 monoclonal antibodies are described in WO 2011 / 086179, WO 2016 / 209021 and Gauthier et al. (2019, Cell [Cell] 177, 1701-1713) or WO 2016 / 207278, such as NKp46-1, NKp46-2, NKp46-3, NKp46-4, NKp46-6 or NKp46-9. 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, which include the heavy chain sequences and light chain sequences of SEQ ID NO.: 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 for DNAM-1 are described, for example, in WO 2013 / 140787. Examples of anti-SLAMF7 monoclonal antibodies include elotuzumab and other antibodies described in US2018208653. Monoclonal antibodies for 4-1BB (CD137) are described, for example, in WO 2005 / 035584, WO 2006 / 088464, and US2006188439. Monoclonal antibodies for OX40 are described, for example, in WO 2007 / 062245, US2010136030, US2019100596, WO2013 / 008171, and WO 2013 / 028231. Monoclonal antibodies for CD96 are described, for example, in WO 2019 / 091449. Monoclonal antibodies against CD160 are described, for example, in US2012003224 and US2013122006. Monoclonal antibodies against KIR2DS1-5 are described, for example, in WO 2016 / 031936.

[0182] Accordingly, in one embodiment, a multispecific antigen-binding protein as 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) CDR-H1 (SEQ ID NO:24), CDR-H2 (SEQ ID NO:25), and CDR-H3 (SEQ ID NO:26) sequences as contained in SEQ ID NO:1, and CDR-L1 (SEQ ID NO:27), CDR-L2 (SEQ ID NO:28), and CDR-L3 (SEQ ID NO:29) sequences as contained in SEQ ID NO:2; and b) CDR-H1, CDR-H2, and CDR-H3 sequences as contained in SEQ ID NO:3, and CDR-L1, CDR-L2, and CDR-L3 sequences as contained in SEQ ID NO:20.

[0183] In one embodiment, a multispecific antigen-binding protein as described herein comprises a variable heavy chain (V H ) domain and variable light chain (V L ) domain combination: a) V as contained in SEQ ID NO: 1 H Sequence, and V as contained in SEQ ID NO:2 L Sequence; and b) V as contained in SEQ ID NO: 3 H Sequence, and V as contained in SEQ ID NO: 20 L sequence.

[0184] In one embodiment, the multispecific antigen-binding protein as described herein comprises a combination of heavy chains 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.

[0185] Structure of multispecific antigen-binding proteins

[0186] In one embodiment, in a multispecific antigen-binding protein as described herein, at least the NK cell activating cytokine is conjugated to a region with affinity for surface antigens expressed on NK cells. The conjugation of cytokines to regions should be understood to mean that they are covalently linked to each other. Cytokines can be cross-linked with regional chemistry using a cross-linking agent for connecting two protein molecules, as is well known in the art. There are a variety of commercially available cross-linking agents for preparing protein or peptide bioconjugates. Many of these cross-linking agents allow biomolecules to be homo-conjugated or hetero-conjugated to dimers through free amines or sulfhydryl groups in protein side chains. Other cross-linking methods involve coupling to hydrazide moieties through carbohydrate groups. In order to cross-link NK cell activating cytokines to NK cell binding regions, it is preferred that a cross-linking agent with heterofunctional specificity is used. In one embodiment, the cross-linking agent comprises a flexible spacer to provide two entities with flexibility or freedom of movement relative to each other.

[0187] However, in one embodiment, at least one NK cell activating cytokine is conjugated to a region having affinity for a surface antigen expressed on NK cells by being included in a single polypeptide chain. Since the region having affinity for a surface antigen expressed on NK cells can contain two polypeptide chains, such as a dimer Fc region of an antibody, in one embodiment, at least one polypeptide chain of a region having affinity for a surface antigen expressed on NK cells forms a single polypeptide chain with at least one NK cell activating cytokine. In one embodiment, the NK cell activating cytokine is connected to a polypeptide chain of a region having affinity for a surface antigen expressed on NK cells by a flexible linker. In one embodiment, wherein a single polypeptide chain comprises more than one NK cell activating cytokine, wherein optionally, these NK cell activating cytokines are connected to each other or to a polypeptide chain of a region having affinity for a surface antigen expressed on NK cells by a flexible linker. The flexible linker can be an immunoglobulin hinge region or can be a linker as described below.

[0188] Therefore, in one embodiment, a multispecific antigen-binding protein as described herein comprises a single polypeptide chain, which 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 having an affinity region for surface antigens expressed on NK cells; iv) optionally a second flexible linker; and v) a second NK cell activating cytokine; and wherein preferably, the first NK cell activating cytokine and the second NK cell activating cytokine are different NK cell activating cytokines. The first NK cell activating cytokine and the second NK cell activating cytokine are such NK cell activating cytokines, which can be selected from IL21R agonists and 4-1BB agonists as defined above.

[0189] In one embodiment, the multispecific antigen-binding protein as described herein further comprises at least one antigen-binding region that specifically binds to an NK cell activating receptor. In one embodiment, 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 NK cells. Preferably, the antigen-binding region that specifically binds to an NK cell activating receptor is conjugated to at least one polypeptide chain in a region that has affinity for a surface antigen expressed on NK cells. As described above, the conjugation of the two regions is understood to mean that they are covalently linked to each other, which can be chemically cross-linked to each other using a cross-linking agent for linking two protein molecules, as is well known in the art.

[0190] However, in one embodiment, at least one antigen binding region that specifically binds to an NK cell activating receptor is conjugated with a region having affinity for a surface antigen expressed on NK cells by being contained in a single polypeptide chain. H and V L Chain, in one embodiment, at least one polypeptide chain in the antigen binding region that specifically binds to the NK cell activating receptor forms a single polypeptide chain with at least one polypeptide chain in the region that has affinity for the surface antigen expressed on the NK cell. Similarly, since the region that has affinity for the surface antigen expressed on the NK cell can also contain two polypeptide chains, such as the dimer Fc region of the antibody, in one embodiment, at least one polypeptide chain in the region that has affinity for the surface antigen expressed on the NK cell forms a single polypeptide chain with at least one polypeptide chain in the antigen binding region that specifically binds to the NK cell activating receptor.

[0191] Thus, in one embodiment, a multispecific antigen-binding protein as described herein comprises 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) a region (at least one polypeptide chain) that has affinity for a surface antigen expressed on NK cells. The flexible linker may be an immunoglobulin hinge region or may be a linker as described below.

[0192] In one embodiment, the region having affinity for the surface antigen expressed on NK cells is a dimeric immunoglobulin Fc region, wherein each of the two polypeptide chains of the Fc region is connected to a CH1 domain, and each CH1 domain is connected to an immunoglobulin variable region that specifically binds to an NK cell activating receptor. The dimeric immunoglobulin Fc region is preferably a dimer of the Fc region that binds to CD16A as described above. The immunoglobulin variable region can be a scFv, V H Domain, V Ldomain or immunoglobulin single variable domain (ISVD) (such as a dAb, V-NAR domain or V H In one embodiment, the immunoglobulin variable region connected to the CH1 domain is a V domain connected to a Cκ or Cλ domain. L Domain-paired V H In this embodiment, preferably, V H and V L The domains together specifically bind to NK cell activating receptors.

[0193] In one embodiment, 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. With regard to the specificity for NK cell activating receptors, the multispecific antigen-binding proteins as described herein can therefore be homodimeric, with two identical immunoglobulin variable regions that bind to the same NK cell activating receptor. Alternatively, the multispecific antigen-binding proteins as described herein can therefore be heterodimeric with regard to the specificity for NK cell activating receptors, wherein each of the two immunoglobulin variable regions each binds to a different NK cell activating receptor. In an embodiment, wherein the multispecific antigen-binding protein is bispecific relative to the NK cell activating receptor, preferably, one of the two immunoglobulin variable regions is an immunoglobulin single variable domain, and the other immunoglobulin variable region is not. Next, the assembly of heterodimeric antibody heavy chains can be completed by expressing two different antibody heavy chain sequences in the same cell, which can result in the assembly of homodimers of each antibody heavy chain and the assembly of heterodimers. Promoting the preferential assembly of heterodimers can be accomplished by incorporating different mutations in the CH3 domain of each antibody heavy chain constant region, as shown in US 13 / 494,870, US16 / 028850, US11 / 533,709, US12 / 875,015, US13 / 289,934, US 14 / 773,418, US12 / 811,207, US13 / 866,756, US14 / 647,480, US14 / 830,336 and WO 2019 / 195409. For example, mutations can be made in the CH3 domain based on human IgG1 and incorporating different amino acid substitution pairs within the first polypeptide and the second polypeptide, which allow the two chains to selectively heterodimerize with each other. For example, the CH3 domain comprises amino acid substitutions wherein the CH3 domain interface of the antibody Fc region is mutated to produce altered charge polarity at the Fc dimer interface such that co-expression of electrostatically matched Fc chains supports favorable attractive interactions, thereby promoting the desired Fc heterodimer formation, while unfavorable repulsive charge interactions inhibit the unwanted Fc homodimer formation.

[0194] In one embodiment, a "knob-in-hole" approach is used in which the CH3 domain interface of the antibody Fc region is mutated such that the antibody preferentially forms heterodimers (further including attached light chains). These mutations produce altered charge polarity at the Fc dimer interface such that co-expression of electrostatically matched Fc chains supports favorable attractive interactions, thereby promoting the desired Fc heterodimer formation, while unfavorable repulsive charge interactions inhibit unwanted Fc homodimer formation. For example, one heavy chain comprises a T366W substitution, and a second heavy chain comprises 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 WO2009 / 089004, the disclosures of which are incorporated herein by reference. In another approach, one heavy chain comprises a F405L substitution and a second heavy chain comprises a K409R substitution, see, e.g., Labrijn et al. (2013) Proc. Natl. Acad. Sci. USA, 110, pp. 5145-5150. In another approach, one heavy chain comprises a T350V, L351Y, F405A, and Y407V substitution and a second heavy chain comprises a T350V, T366S, K392L, and T394W substitution, see, e.g., Von Kreudenstein et al., (2013) mAbs 5: 646-654. In another approach, one heavy chain comprises both K409D and K392D substitutions, and the second heavy chain comprises both D399K and E356K substitutions, see, e.g., Gunasekaran et al., (2010) J. Biol. Chem. 285: 19637-19646. In another approach, one heavy chain comprises D221E, P228E, and L368E substitutions, and the second heavy chain comprises D221R, P228R, and K409R substitutions, see, e.g., Strop et al., (2012) J. Mol. Biol. 420: 204-219. In another approach, one heavy chain comprises S364H and F405A substitutions, and a second heavy chain comprises Y349T and T394F substitutions, see, e.g., Moore et al., (2011) mAbs [monoclonal antibodies] 3: 546-557. In another approach, one heavy chain comprises H435R substitution, and a second heavy chain optionally may or may not comprise a substitution, see, e.g., U.S. Pat. No. 8,586,713.When such heteromultimeric antibodies have an Fc region derived from human IgG2 or IgG4, the Fc region of these antibodies can be engineered to contain amino acid modifications that allow CD16 binding. In some embodiments, the antibody can comprise mammalian antibody-type N-linked glycosylation at residue N297 (Kabat EU numbering).

[0195] In a preferred embodiment, the multispecific antigen-binding protein as described herein comprises a dimeric immunoglobulin Fc region, which is a dimer of the Fc region that binds CD16A as 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. In addition to the presence of one or more NK cell activating cytokines, the multispecific antigen-binding protein comprising such a dimeric Fc linked to two Fabs thus forms an immunoglobulin structure, such as a conventional IgG immunoglobulin.

[0196] Therefore, the multispecific antigen-binding protein as described herein comprising an antigen-binding region that specifically binds to an NK cell activation receptor further comprises at least one NK cell activation cytokine. In one embodiment, at least one of the NK cell activation cytokines is conjugated to at least one antigen-binding region that specifically binds to an NK cell activation receptor, or to a region that has affinity for surface antigens expressed on NK cells. As indicated above, the conjugation of two protein entities should be understood to mean that they are covalently linked to each other, which can be accomplished by chemical cross-linking using a cross-linking agent for connecting two protein molecules, which can include a flexible spacer as is well known in the art.

[0197] However, in one embodiment, at least one NK cell activating cytokine forms a single polypeptide chain with at least one 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 in a region that has affinity for a surface antigen expressed on NK cells. In one embodiment, a flexible linker (as described below) is present between the agonist and the region defined in i) or ii).

[0198] In one embodiment, at least one NK cell activating cytokine forms a single polypeptide chain with at least one of the following: i) a light chain in at least one of the two Fabs that specifically bind to the NK cell activating receptor; and ii) at least one of the two Fc chains in the Fc region of the dimeric immunoglobulin. In one embodiment, a flexible linker (as described below) is present between the agonist and the light chain defined in i) or the Fc chain defined in ii).

[0199] In one embodiment, at least one NK cell activating cytokine is fused to at least one of the following: i) the N-terminus of the light chain in 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 in 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 in 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 in at least one of the two Fc chains in the Fc region of the dimeric immunoglobulin, optionally through a flexible linker, wherein the flexible linker can be as described below.

[0200] In one embodiment, wherein the multispecific antigen-binding protein includes a (same or different) dimer antigen-binding protein as described above, the dimer can include at least one NK cell activation cytokine only on one of the two monomers in the dimer, or the dimer can include at least one NK cell activation cytokine on each of the two monomers in the dimer (i.e., both).Therefore, in one embodiment, wherein the multispecific antigen-binding protein includes an immunoglobulin structure, at least one of the NK cell activation cytokines can be present in at least one side or both sides of the immunoglobulin structure.In an embodiment in which at least one NK cell activation cytokine is present in each of the two monomers in the dimer or immunoglobulin structure, the multispecific antigen-binding protein can, for example, include an IL21R agonist on two monomers, include a 4-1BB agonist on two monomers, or include an IL21R agonist on a first monomer and include a 4-1BB agonist on a second monomer. It will be appreciated that when the multispecific antigen-binding protein comprises a heterodimeric heavy chain, the "knob-in-hole" technique as described above may be applied, wherein the CH3 domain of the first chain is modified to have a "protrusion" ("knob"), and the second chain is modified to have a corresponding "cavity" ("hole").

[0201] Thus, in one embodiment, the multispecific antigen-binding protein as described herein 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 fused NK cell activating cytokine, and wherein the dimeric Fc region comprises a different first polypeptide chain and a second polypeptide chain, which comprises a knob-in-hole structure modification that promotes the association of the first polypeptide chain and the second polypeptide chain of the Fc region.

[0202] Suitable linker amino acid sequences for connecting various functional domains and regions in multispecific antigen-binding proteins as described herein are known in the art (e.g., from Chen et al., 2013, Adv Drug Deliv Rev. [Advanced Drug Delivery Review] 65 (10): 1357-1369). Linker amino acid sequences can be rigid, but are generally flexible. Flexible linkers are generally applied when the connected domains require a certain degree of mobility or interaction. They are generally composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows mobility of the connected 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, and thus reduce adverse interactions between the linker and the protein portion. Preferred flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues ("GS" linkers). Examples of preferred (and widely used) flexible linkers have (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 to maintain necessary 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 amino acids or polar amino acids (such as Gly and Ser), but may 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 flexible linkers KESGSVSSEQLAQFRSLD (SEQ ID NO:34) and EGKSSGSGSESKST (SEQ ID NO:35), which have been applied to the construction of biologically active scFv.

[0203] In one embodiment, the multispecific antigen-binding protein as described herein is a multispecific antigen-binding protein as exemplified herein, such as, for example, 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 chain (V H ) domain and variable light chain (V L ) domain was replaced by the variable heavy chain (V H ) domain and variable light chain (VL ) domain replacement.

[0204] In one embodiment, the multispecific antigen-binding protein as described herein is a multispecific antigen-binding protein comprising the following: a) a first heavy chain of a monoclonal antibody against an NK cell activating receptor as described above, wherein preferably, the first NK cell activating cytokine is optionally fused to the C-terminus of the first heavy chain through a flexible linker; b) a second heavy chain of a monoclonal antibody against an NK cell activating receptor, wherein preferably, the second NK cell activating cytokine is optionally fused to the C-terminus of the second heavy chain through a flexible linker; c) a first light chain and a second light chain of a monoclonal antibody against an NK cell activating receptor as described above, wherein optionally, the first NK cell activating cytokine or the second NK cell activating cytokine is optionally fused to the C-terminus of the light chain through a flexible linker, wherein preferably, the amino acid sequence of the Fc region of the first heavy chain and the second heavy chain comprises a knob-and-hole structure modification that promotes the association of the first heavy chain and the second heavy chain. In one embodiment, the first NK cell activating cytokine is a 4-1BB agonist as described above, preferably comprising a fusion protein of 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 activation cytokine is an IL21R agonist as described above, preferably an IL21R polypeptide as described above. In one embodiment, the monoclonal antibody for NK cell activation receptor is a monoclonal antibody for NKp46 as described herein. In one embodiment, the Fc region in the heavy chain comprises a modification of the affinity of the reduction to CD16A as described above, such as "LALA" modification.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] In one embodiment, the multispecific antigen-binding protein as described herein is a multispecific antigen-binding protein comprising the following: the first polypeptide chain and the second polypeptide chain of the Fc region as described above, wherein preferably, the first NK cell activation cytokine is optionally fused to the N-terminus of at least one polypeptide chain of the Fc region through a flexible linker, and preferably, the second NK cell activation cytokine is optionally fused to the C-terminus of at least one polypeptide chain of the Fc region through a flexible linker. In one embodiment, the first NK cell activation cytokine is an IL21R agonist as described above, preferably an IL21R polypeptide as described above. In one embodiment, the second NK cell activation cytokine is a 4-1BB agonist as described above, preferably comprising a fusion protein of 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 in the heavy chain includes a modification that reduces affinity for CD16A as described above, such as a "LALA" modification. In one embodiment, the multispecific antigen-binding protein (AVC-014) comprises: a) a first polypeptide chain and a second polypeptide chain 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) a first polypeptide chain and a second polypeptide chain comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 40.

[0211] Biological activity of multispecific antigen binding proteins

[0212] The multispecific antigen-binding proteins described herein can have one or more biological activities, including, for example, antigen binding, binding to NK cells, the ability to direct NK cells to target cells, the ability to activate NK cells (including inducing hyperfunctionality of NK cells), and / or the ability to cause target cells to be lysed by (activated / hyperfunctional) NK cells.

[0213] In one embodiment, the multispecific antigen-binding protein as described herein causes an increase in at least one NK cell activity selected from the group consisting of CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation, and NK cell cytotoxicity, wherein preferably, the increase is at least 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20, 50, or 100-fold the increase achieved with the same effector cell:target cell ratio, with the same NK cells and target cells not contacted with the multispecific antigen-binding protein.

[0214] In one embodiment, the multispecific antigen-binding protein as described herein causes an increase in at least one NK cell activity selected from the group consisting of: CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation, and NK cytotoxicity, wherein preferably, the increase is at least 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20, 50, or 100-fold the increase achieved with the same effector cell:target cell ratio, with the same NK cells and target cells contacted with a reference antigen-binding protein or a reference feeder cell line (under otherwise identical conditions).

[0215] 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 one or more NK cell activating receptor-specific antigen binding regions as the multispecific antigen binding protein.

[0216] In one embodiment, the reference antigen binding protein is a (multi-specific) antigen binding protein, which includes binding to the same NK cell activation receptor, preferably binding to the same epitope, more preferably having at least one antigen binding region of one or more NK cell activation receptor specific antigen binding regions identical to the multi-specific antigen binding protein, and includes at least one NK cell activation cytokine other than IL21R agonist, preferably at least one NK cell activation cytokine other than IL21R agonist and 4-1BB agonist. NK cell activation cytokine other than at least one of IL21R agonist and 4-1BB agonist can be IL-15 receptor agonist, such as IL15, for example, modified human IL-15 cross-linking agent, such as US 2018282386 and Vallera et al. (2016, Clin Cancer Res. [Clinical Cancer Research]; 22 (14): 3440-3450) described in.

[0217] In one embodiment, the reference antigen binding protein is an NK cell engager, such as described in WO 2016 / 207278, WO 2018 / 148445, WO 2018 / 152518, WO 2019195409, US2018282386, Vallera et al. (2016, supra) and Demaria et al. (2021, supra). An example of a (multispecific) reference antigen binding protein is AVC-006, such as described in the Examples herein, which comprises a HER2 binding region and a NKG2D binding region.

[0218] In one embodiment, the reference feeder cell line is the (irradiated) K562 feeder cell line that has been modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells) as described in Denman et al. (2012, supra).

[0219] Assays for detecting expression of NK activation markers or detecting NK cytotoxicity or for detecting NK cell activation and cytotoxicity assays (e.g., short-term cytotoxicity assays and long-term cytotoxicity assays) are described in the Examples herein and, for example, in Pessino et al., J. Exp. Med [Journal of Experimental Medicine], 1998, 188(5):953-960; Sivori et al., Eur J Immunol [European Journal of Immunology], 1999. 29:1656-1666; Brando et al., (2005) J. Leukoc. Biol. [Journal of White Cell Biology] 78:359-371; El-Sherbiny et al., (2007) Cancer Research [Cancer Research] 67(18):8444-9; Nolte-'t Hoen et al., (2007) Blood [Blood] 109:670-673; WO 2016 / 207278 and WO2018 / 148445.

[0220] In one embodiment, multispecific antigen-binding proteins as described herein have the ability to induce superfunctionality (or superfunctional phenotype) in NK cells or NK cell colonies.Superfunctional NK cell phenotype is understood herein as a phenotype with one or more phenotypic characteristics, which is obtained by amplifying in vitro via NK cells obtained from donors and irradiated K562 feeder cells co-cultured, these irradiated K562 feeder cells are modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligands (FC21 feeder cells), as described in Denman et al. (2012, supra).Thus, in one embodiment, the ex vivo expansion of donor NK cells by co-culturing (e.g., for 7, 14, or 21 days) with a multispecific antigen-binding protein as described herein produces a population of NK cells having one or more (or preferably all) characteristics selected from the following group: a) the expansion fold 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 expansion fold of the expanded NK cells obtained by ex vivo expansion via 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, 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, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0 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 NK cells obtained after ex vivo expansion in the presence of FC21 feeder cells; d) the secretion of at least one cytokine among TNF-α, IFN-γ and IL-6 by the expanded NK cells is at least 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 after ex vivo expansion in the presence of FC21 feeder cells; 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 secretion of cytokines of NK cells obtained after ex vivo expansion in the case of FC21 feeder cells; and e) the cytotoxicity of the amplified 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 NK cells obtained after ex vivo expansion in the presence of FC21 feeder cells. The scheme for ex vivo expansion of donor NK cells and the determination of expansion times, telomere length increase, expression level of NK cell activation receptors, cytokine secretion and cytotoxicity (e.g., short-term cytotoxicity assay or long-term cytotoxicity assay) are described in Denman et al. (2012, supra) and Examples herein.

[0221] Pharmaceutical composition

[0222] In another aspect, the invention relates to a pharmaceutical composition comprising a multispecific antigen-binding protein as described herein and a pharmaceutically acceptable carrier (excipient). A pharmaceutically acceptable carrier (such as an adjuvant or vehicle) is used to administer the polypeptide to a subject. The pharmaceutical composition can be used in the treatment methods described below by administering an effective amount of the composition to a subject in need thereof. As used herein, the term "subject" refers to all animals classified as mammals, and includes, but is not limited to, primates and humans. The subject is preferably a male or female human of any age or race.

[0223] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (see, e.g., "Handbook of Pharmaceutical Excipients", Rowe et al., ed., 7th ed., 2012, www.pharmpress.com ). The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, their use in the composition is contemplated. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and isocyanate. 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 complex); and / or nonionic surfactants such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG).

[0224] Supplementary active compounds may also be incorporated into the pharmaceutical compositions of the present invention. Thus, in particular embodiments, the pharmaceutical compositions of the present invention may also contain more than one active compound necessary for the particular indication being treated, preferably those active compounds having complementary activities that do not adversely affect each other. For example, it may be desirable to further provide a chemotherapeutic agent, a cytokine, an analgesic, a thrombolytic agent, or an immunomodulator (e.g., an immunosuppressant or an immunostimulant). The effective amount of such other active agents depends, inter alia, on the amount of the polypeptide of the present invention present in the pharmaceutical composition, the type or treatment of the disease or disorder, etc.

[0225] In one embodiment, the polypeptide of the present invention is prepared together with a carrier (such as a controlled release formulation, including implants and microencapsulated delivery systems, such as liposomes) that will protect the compound from rapid elimination from the body. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The method for preparing such a formulation will be clear 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 US 4,522,811 or WO 2010 / 095940).

[0226] The administration route of the polypeptide of the present invention can be parenteral. As used herein, the term "parenteral" includes intravenous, intraarterial, intralymphatic, intraperitoneal, intramuscular or subcutaneous. The intravenous or intramuscular form of parenteral administration is preferred. "Systemic administration" means intravenous, intraperitoneal and intramuscular administration. The amount of polypeptide required for the therapeutic effect or preventive effect will certainly vary with the selected polypeptide, the nature and severity of the disease being treated, and the patient. In addition, the polypeptide can be appropriately administered by pulse infusion (for example, with a decreasing dose of polypeptide). Preferably, administration is performed by injection, most preferably intravenous, intramuscular or subcutaneous injection, depending in part on whether the administration is short-lived or long-term.

[0227] Therefore, in a particular embodiment, the pharmaceutical composition of the present invention can be in a form suitable for parenteral administration, such as a sterile solution, suspension or lyophilized product in a suitable unit dosage form. Pharmaceutical compositions suitable for injection include sterile aqueous solutions (in the case of water-soluble) or dispersants and sterile powders for the temporary preparation of sterile injectable solutions or dispersants. For intravenous administration, suitable carriers include physiological saline, antibacterial water, CremophorEM (BASF, Parsippany, New Jersey) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under manufacturing and storage conditions and must be preserved to prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol (such as glycerol, propylene glycol, liquid polyethylene glycol) and a suitable mixture thereof. Suitable fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersant, and by using a surfactant. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. 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.

[0228] 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.

[0229] Sterile injectable solutions can be prepared in the following manner: the required amount of active compound (e.g., polypeptide of the present invention) is incorporated into an appropriate solvent with one or a combination of ingredients listed above (as required), followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying, which produce powders of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0230] In certain embodiments, the pharmaceutical composition is administered via intravenous (IV), intramuscular (IM) or subcutaneous (SC) routes. Suitable excipients such as bulking agents, buffers or surfactants may be used. The formulations mentioned will be prepared using standard methods for preparing parenterally administrable compositions, as is well known in the art and described in more detail in various sources, including, for example, "Remington: The Science and Practice of Pharmacy" (Editor Allen, LV 22nd edition, 2012, www.pharmpress.com ).

[0231] Formulating pharmaceutical compositions (i.e., parenteral compositions) in dosage unit form is particularly advantageous for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suitable as single dosages for subjects to be treated; each unit contains a predetermined quantity of active compound (polypeptide of the present invention) calculated to produce the desired therapeutic effect associated with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are dictated by and directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the limitations inherent in the art of formulating such active compounds for treating individuals.

[0232] Typically, in order to prevent and / or treat the diseases and disorders mentioned herein, and depending on the specific disease or condition to be treated and its severity, the effectiveness of the specific polypeptide of the present invention to be used, the specific route of administration used and the specific pharmaceutical formulation or composition, the polypeptide of the present invention will typically range from 0.001 to 1,000 mg / kg body weight / day, preferably from about 0.01 to about 100 mg / kg body weight / day, more preferably from about 0.05 to 10 mg / kg body weight / day (such as about 1, 10, 100 or 1000 micrograms / kg body weight / day) continuously (e.g., by infusion) with a single daily dose or in multiple divided doses during the day. Clinicians will generally be able to determine the appropriate daily dose based on the factors mentioned herein. It is also clear that in certain cases, clinicians can choose to deviate from these amounts, for example, based on the factors cited above and his expert judgment. The pharmaceutical composition can be included in a container, package or dispenser together with the instructions for use.

[0233] Therapeutic Uses

[0234] In another aspect, a multispecific antigen-binding protein as described herein is provided for use as a medicament. In one embodiment, a multispecific antigen-binding protein as described herein is used as an active ingredient, component or substance in a medicament.

[0235] In one aspect, the invention relates to the use of a multispecific antigen-binding protein as described herein for the manufacture of a medicament (e.g., a pharmaceutical formulation comprising a multispecific antigen-binding protein as an active ingredient) for treating, preventing, or diagnosing a disease in a subject in need thereof.

[0236] In one aspect, the invention relates to a multispecific antigen-binding protein as described herein or a pharmaceutical formulation comprising the multispecific antigen-binding protein as an active ingredient for use in treating, preventing, or diagnosing a disease in a subject in need thereof.

[0237] In one aspect, the present invention relates to a method for treating a disease in a subject in need thereof, wherein the method comprises the step of administering to the subject (an effective amount of) a multispecific antigen-binding protein as described herein or a pharmaceutical formulation comprising the multispecific antigen-binding protein as an active ingredient.

[0238] The disease to be treated, prevented or diagnosed using the multispecific antigen binding protein can be cancer, an infectious disease, an inflammatory disease or an autoimmune disease.

[0239] In one embodiment, the disease to be treated, prevented or diagnosed using the multispecific antigen-binding protein is cancer, such as a cancer described below.

[0240] In one embodiment, the present invention relates to a method for 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 or a pharmaceutical formulation comprising a multispecific antigen-binding protein as an active ingredient. In one embodiment, the subject suffers from cancer, such as a cancer as described below.

[0241] In one embodiment, the present invention relates to a method for amplifying and / or inducing super-functional NK cells in a subject, the method comprising administering to the subject a multispecific antigen-binding protein as described herein or a pharmaceutical preparation comprising a multispecific antigen-binding protein as an active ingredient. The amplification multiple and super-functionality are preferably as described above. In one embodiment, the subject suffers from cancer, such as a cancer as described below.

[0242] Subjects with cancer often exhibit low numbers of NK cells and / or NK cell exhaustion. Therefore, the multispecific antigen-binding proteins of the present invention can be advantageously used to expand the number of NK cells and / or induce super functionality of NK cells in subjects with cancer. Additional advantages of the super functionality of NK cells as induced by the multispecific antigen-binding proteins of the present invention include that they increase the secretion of cytokines (such as TNF-α, IFN-γ and IL-6), which helps to form an adaptive immune response involving DCs and T cells. Indeed, it has been reported that NK cells promote the cross-presentation of tumor antigens specifically to CD8+ DC subsets of T cells are recruited to the tumor microenvironment, suggesting that NK cells play a role in enhancing anti-tumor CD8 + NK cells play a key role in T cell responses (Bottcher et al., Cell, 2018.172:1022-1037; and Barry et al., Nat. Med. [Nature Medicine] 2018.24:1178-1191). The contribution of NK cells to the coordination of anti-tumor T cell responses has also been confirmed experimentally in mice, demonstrating that in addition to their direct effector functions, NK cells can also promote T cell responses and persistent immune control of tumors (Bonavita et al., Immunity [Immunity] 2020.53:1215-1229).

[0243] 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, including bladder cancer, head and neck cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, and skin cancer, including squamous cell carcinoma; hematopoietic tumors of the lymphoid lineage, including leukemias, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma, and Burkitt lymphoma; hematopoietic tumors of the myeloid lineage, including acute myeloid leukemia and chronic myeloid leukemia and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; sarcomas; other tumors, including neuroblastoma and glioma; central and peripheral nervous system tumors, including astrocytomas, neuroblastomas, gliomas and schwannomas; tumors of mesenchymal origin, including fibrosarcomas, rhabdomyosarcomas and osteosarcomas; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma and teratoma, hematopoietic tumors of the lymphoid lineage (e.g., T-cell and B-cell tumors, including but not limited to T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including small cell and brain-shaped cell types; large granular lymphocytic leukemia (LGL), preferably T-cell type; Sezary syndrome (SS); adult T-cell leukemia lymphoma (ATLL); a / d T-NHL hepatosplenic lymphoma; peripheral / postthymic T-cell lymphoma (polymorphic and immunoblastic subtypes); angioimmunoblastic T-cell lymphoma; angiocentric (nasal) T-cell lymphoma; anaplastic (Ki 1+) large cell lymphoma; intestinal T-cell lymphoma; T-lymphoblastic; and lymphoma / leukemia (T-Lbly / T-ALL)).

[0244] In one embodiment, the multispecific antigen-binding proteins described herein can be used as a monotherapy (ie, without other therapeutic agents). In another embodiment, the multispecific antigen-binding proteins described herein can be used in combination therapy.

[0245] In one embodiment, a multispecific antigen-binding protein as described herein is used in combination with another immunotherapy (e.g., cellular immunotherapy). Therefore, a multispecific antigen-binding protein can be used in combination with adoptive transfer of immune cells (including adoptive transfer of T cells (e.g., CAR T cells) or NK cells). NK cells can be enriched or amplified, for example, by methods known in the art, or can be ex vivo NK cells as described herein.

[0246] In one embodiment, a multispecific antigen-binding protein as described herein can be used in combination therapy with one or more other therapeutic agents. One or more additional therapeutic agents will generally be administered in an amount and treatment regimen typically used for that agent in a monotherapy for the particular disease or condition being treated. When used to treat cancer, such therapeutic agents include, but are not limited to, anticancer agents and chemotherapeutic agents. Exemplary therapeutic agents that can be used as part of a combination therapy to treat cancer include, for example, radiation, mitomycin, tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine, dibromomannitol, methotrexate, doxorubicin, carboquinone, pentostatin, diamine nitrazepam, netastatin, cetrorelix, letrozole, raltitrexed, daunomycin, fadrozole, fotemustine, thymalfasin, sobuzosine, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesanaline, aminoglutethimide, amsacrine, proglumide, eliprolamide, ketanserin, doxifluridine, etretinate, ester, isotretinoin, streptozotocin, nimustine, vindesine, flutamide, drogenil, butocin, carmofur, razoxane, sizolan, carboplatin, dibromodulan, tegafur, ifosfamide, prednimustine, sapellose, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, melastane, cyclothiocarb, formestane, interferon-alpha, interferon-2alpha, interferon-beta, interferon-gamma, colony stimulating factor-1, colony stimulating factor-2, denileukin diftitox, interleukin-2, and luteinizing hormone releasing factor.

[0247] Another class of agents that can be used as part of a combination therapy for treating cancer is an immune checkpoint inhibitor. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of the following: (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. Yet another agent that can be used as part of a combination therapy for treating cancer is a cytotoxic monoclonal antibody against TAA, including cytotoxic monoclonal antibodies against TAA known in the art, such as: trastuzumab (for HER2), pertuzumab (for HER2), rituximab (for CD20), tositumomab (for CD20), ibritumomab tiuxetan (for CD20), obinutuzumab (for CD20), ofatumumab (for CD20), alemtuzumab (for C D52), blinatumomab (targeting CD19), inebilizumab (targeting CD19), tafasitamab (targeting CD19), daratumumab (targeting CD38), isatuximab (targeting CD38), polatuzumab (targeting CD79b), dinutuximab (targeting GD2), Naxitamab (targeting GD2), bevacizumab (targeting VEGF-A), elotuzumab (targeting SLAMF7), enfortumab (targeting connexin 4), sacituzumab (targeting TROP2), mogamulizumab (targeting CCR4), ipilimumab (targeting CTLA-4), tremelimumab (targeting CTLA-4), durvalumab (targeting PD1), pidilizumab pidilizumab (targeting PD-1), pembrolizumab (targeting PD-1), nivolumab (targeting PD-1), cemiplimab (targeting PD-1), avelumab (targeting PD-L1), cetuximab (targeting EGFR), necituzumab (targeting EGFR), panitumumab (targeting EGFR), olaratumab (targeting PDGFRα), and ramucirumab (targeting VEGFR2).

[0248] In some embodiments, administration of a multispecific antigen-binding protein and other therapeutic agents may result in additive or synergistic effects on immunity and / or on therapeutic efficacy.

[0249] In one embodiment, a multispecific antigen-binding protein as described herein is used as at least one of a neoadjuvant therapy and an adjuvant therapy as a supplement to a primary therapy including, for example, surgery and / or radiotherapy. As a neoadjuvant therapy, a multispecific antigen-binding protein is administered prior to the primary therapy, for example, to help reduce the size of the tumor (so that less extensive surgery and / or radiotherapy is required), kill diffused cancer cells (e.g., micrometastatic disease), and / or reduce the risk of postoperative tumor cell spread. As an adjuvant therapy, a multispecific antigen-binding protein is administered after the primary therapy, for example, to treat minimal residual disease (destroy remaining cancer cells). Using a multispecific antigen-binding protein as a neoadjuvant therapy and / or adjuvant therapy reduces the recurrence rate. In a neoadjuvant therapy and / or adjuvant therapy, a multispecific antigen-binding protein can be used as a monotherapy or in a combination therapy, as described above.

[0250] In vitro methods

[0251] In another aspect, the present invention relates to a method in which a multispecific antigen-binding protein as described herein is used to treat NK cells or NK cell populations ex vivo (in vitro). The method can be a method for amplifying, preactivating, activating, enhancing cytotoxicity and / or cytokine production, and inducing at least one of a hyperfunctional phenotype, as defined above. These methods include at least the step of contacting NK cells or their populations with a multispecific antigen-binding protein as described herein or with a composition comprising a multispecific antigen-binding protein.

[0252] 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. [Public Library of Science Comprehensive] 2012; 7(1): e30264) and US2020 / 0061115. For example, NK cells enriched from PBMCs can be concentrated at 0.1 x 10 6 NK cells / mL are seeded 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, 1000 μg / mL of one or more multispecific antigen-binding proteins as described herein. After day 5, the medium containing the supplements can be refreshed every 2-3 days.

[0253] It is understood that the duration of contact between the NK cells and the multispecific antigen-binding protein as described herein (i.e., the duration of pre-activation or activation (i.e., the duration of expansion, pre-activation, activation, enhancement of cytotoxicity and / or cytokine production, and induction of a hyperfunctional phenotype) can last for any length of time necessary to achieve the desired phenotype of the NK cells. For example, the contact can be as little as 1 minute or as much as 7 days (e.g., culturing the NK cells in the presence of a multispecific antigen-binding protein as 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, 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 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, 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.

[0254] In one embodiment, the ex vivo treated (expanded) NK cells have one or more characteristics selected from the following: a) the expansion fold 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 expansion fold of the expanded NK cells obtained by ex vivo expansion via co-culture with irradiated FC21 feeder cells; b) the telomere length of the expanded NK cells is increased by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 710%, 720%, %, 20%, 25%, 30%, 35%, 40%, 45%, 50% or 55%, 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 the expanded NK cells obtained by ex vivo expansion via co-culture with irradiated FC21 feeder cells; c) a gene selected from NKG2D on the expanded NK cells d) the secretion of at least one cytokine among 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 after ex vivo expansion in the presence of FC21 feeder cells; d) the secretion of at least one cytokine among TNF-α, IFN-γ and IL-6 by the expanded NK cells is at least 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 after ex vivo expansion in the presence of FC21 feeder cells. 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 NK cells obtained after ex vivo expansion; 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 that of the NK cells obtained after ex vivo expansion in the presence of FC21 feeder cells.

[0255] In yet another aspect, the present invention relates to a method for treating a disease in a subject in need thereof, wherein the method comprises administering to the subject (an effective amount of) the NK cells obtained in the above method for ex vivo treatment of NK cells or NK cell colonies. After a sufficient number of NK cells with a desired (superfunctional) phenotype have been amplified by ex vivo treatment, the NK cells can be administered to a subject in need thereof.

[0256] In one embodiment, the method of treatment comprises administering ex vivo treated NK cells in combination with a multispecific antigen-binding protein as described herein or a pharmaceutical formulation comprising a multispecific antigen-binding protein as an active ingredient.

[0257] In one embodiment, the method of treatment comprises administering the NK cells treated ex vivo in combination with another NK cell adapter (e.g., as described in WO 2016 / 207278, WO 2018 / 148445, WO 2018 / 152518, WO 2019195409, US2018282386, Vallera et al. (2016, supra) and Demaria et al. (2021, supra)), or in combination with a multispecific antigen-binding protein as described in a co-pending application belonging to the same applicant with reference number P6110749EP. An example of another NK cell adapter is AVC-006, for example, as described in the examples herein, which comprises a HER2 binding region and a NKG2D binding region. In further embodiments, the NK cells treated ex vivo can be used in combination with other adapters and in combination with a multispecific antigen-binding protein as described herein.

[0258] 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.

[0259] 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.

[0260] Nucleic acids, host cells, and methods for producing multispecific antigen-binding proteins

[0261] In one aspect, the invention relates to nucleic acid molecules comprising one or more nucleotide sequences encoding a polypeptide chain of a multispecific antigen-binding protein as described herein. The nucleotide sequence encoding such a polypeptide chain preferably encodes a signal peptide operably linked to the polypeptide chain. The nucleic acid molecule comprising one or more nucleotide sequences encoding the polypeptide chain further preferably comprises regulatory elements for (or beneficial for) expression of the polypeptide chain in an appropriate host cell, these regulatory elements being operably linked to the nucleotide sequence.

[0262] In one aspect, the invention relates to a host cell comprising a nucleic acid molecule comprising one or more nucleotide sequences encoding a polypeptide chain of a multispecific antigen-binding protein as described herein. In one embodiment, the host cell is an isolated cell or a cultured cell. Host cells that can be used include prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms, such as Escherichia coli or bacilli. 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 COS-1, COS-7 lines of monkey kidney cells (Gluzman et al., 1981, Cell 23: 175), L cells, HEK 293 cells, C127 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, HeLa cells, BHK cell lines (e.g., BHK21), BSC-1, Hep G2, 653, SP2 / 0, and CVI / EBNA cell lines derived from the African green monkey kidney cell line CVI, as described by McMahan et al. (1991, EMBO J. [Journal of the European Molecular Biology Association] 10: 2821). The host cell can be any suitable species or organism capable of producing N-linked glycosylated polypeptides, such as mammalian host cells capable of producing human or rodent IgG-type N-linked glycosylated polypeptides. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985). Host cells containing nucleic acid molecules of the invention can be cultured under conditions that promote expression of the polypeptide.

[0263] Thus, another aspect of the 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 the step of culturing a host cell comprising one or more nucleotide sequences encoding a polypeptide chain of the multispecific antigen-binding protein. The host cell is preferably cultured under conditions conducive to the expression of one or more polypeptide chains. The method may further comprise the step of recovering the multispecific antigen-binding protein. The multispecific antigen-binding protein may be recovered by conventional protein purification procedures including, for example, protein A-agarose, hydroxyapatite 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).

[0264] 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, the 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 a pharmaceutical composition.

[0265] The invention has been described above with reference to a number of exemplary embodiments as shown in the drawings and examples. Modifications and alternative embodiments of some parts or elements are possible and are included within the scope of protection as defined in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0266] Figure 1 Schematic depiction 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 modifications in the Fc region to reduce binding to CD16.

[0267] 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.

[0268] Figure 3 NK cells cytotoxicity against MDA-MB-231 target cells, which had been stimulated for one week with the multispecific antigen binding proteins AVC-009 (AVC9), AVC-011 (AVC11) or AVC-014 (AVC14) or FC21 feeder cells as reference. All three of AVC-009, AVC-011 and AVC-014 induced strong cytotoxicity against MDA-MB-231 target cells. The cytotoxicity of NK cells amplified in the presence of AVC-009 or AVC-014 was not significantly different from that of NK cells stimulated with reference FC21 feeder cells.

[0269] Figure 4 . Here is shown a viSNE analysis using 30 markers as described in Example 4.3, which shows the population distribution of cells from different expansion methods. AVC11, AVC14, AVC9 and FC21 are in the presence of multispecific antigen binding proteins AVC-009, AVC-011 or AVC-014 or FC21 feeder cells, respectively, after one week of expansion as described in Example 3.5. The color map indicates CD56 or Ki67 expression, which is used to identify NK cells. Compared to the control condition of NK cells that were not expanded, all 4 expansion methods resulted in the enrichment of NK cells with high expression of CD56 with very similar phenotypes.

[0270] Figure 5 . Upregulation or downregulation of NK cell surface markers as indicated after expansion of NK cells for one week 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 (FC21), respectively. The fold changes (Log2) were compared to control conditions of non-expanded NK cells.

[0271] Figure 6 : Comparison of proliferation of NK cells induced by culturing cells as 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).

[0272] Examples

[0273] Example 1 Preparation of multispecific antigen binding proteins

[0274] Generation of multispecific antigen-binding proteins and control proteins (including Figure 1 The coding sequences of the proteins AVC-009 to AVC-015 schematically depicted in (the amino acid sequences of which are depicted in Tables 1.1.1 and 1.1.2).

[0275] The expression construct for expressing the coding sequence in HEK293-E-253 cell line is prepared using standard molecular biology materials and techniques. In brief, the coding sequence is produced by direct synthesis and / or by PCR. PCR is carried out using PrimeSTAR MAXDNA polymerase (Takara Biotechnology Co., Ltd., #R045A), and the PCR product is purified from 1% agarose gel using NucleoSpin gel and PCR purification kit (Macherey-Nagel, #740609.250). After purification, the PCR product is quantitatively carried out, and then In-Fusion ligation reaction is carried out, which is carried out as described in the manufacturer's scheme (ClonTech, #ST0345). Plasmid is obtained after a small amount of preparation. Then plasmid is sequenced for sequence confirmation, and then it is transfected into the HEK293-E-253 cell line.

[0276] All buffers used were prepared with Versylene (endotoxin-free and sterile) water. Endotoxins were removed from the instrument by incubation with 0.1 M NaOH for at least 16 hours. Line cleanup was performed at the beginning and end of production.

[0277] use HEK293E-253 cells were transfected with endotoxin-free plasmid DNA using the ELISA technique. Six days after transfection, the conditioned medium containing the recombinant protein was harvested by centrifugation and filtered through a 0.22 μm bottle-top filter. 100 μl of the sample was stored at 4°C. To avoid nonspecific protein or cell fragment binding to the MabSelect PrismA resin, the sample was pretreated with 40 ml of 5 M NaCl / 1 L of culture medium. This resulted in a 0.2 M increase in NaCl concentration.

[0278] The HiScreen Fibro PrismA post is balanced in 20mM Tris 150mM NaCl pH 7.8. Teledyne ASX-560 automatic sampler is used to load the recombinant protein in the conditioned medium onto the post, and by washing the post with 20mMTris 1M NaCl pH 7.8 and 20mM citrate 150mM NaCl pH 5.0, the protein of non-specific binding is removed. The combined recombinant protein is eluted to 20mM citrate 150mM NaCl pH 3.5 by 12CV block gradient, and to 20mM citrate 150mM NaCl pH 3.0 by 6CV block gradient. By being mixed into 1M Tris pH 8.0 (for 62ot and 62zationn to pH 7) with 1.0 / 0.2 ratio, eluent is directly neutralized online, and 12.5ml fraction is collected. Between injections, the HiScreenFibro PrismA column was regenerated using 5CV 0.5M NaOH, 2CV 1M Tris pH 8.0 and equilibrated in 20mM Tris 150mM NaCl pH 7.8. The Teledyne autosampler was cleaned with 15ml 0.1M NaOH and equilibrated in PBS.

[0279] The HiTrap Fibro PrismA pool was concentrated to 2-3 ml per injection on a Superdex200 increase 16 / 40 column using an Amicon 30 kDa spin filter. Aggregates in the concentrated pool were removed by filtration on a 0.22 μm syringe filter. The concentrated sample was stored at 4 °C prior to gel filtration.

[0280] The recombinant protein products were analyzed by Labchip capillary electrophoresis and LAL assay.

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0297] Example 2 Biochemical Characterization of Multispecific Antigen Binding Proteins

[0298] 2.1. Expression level and molecular weight

[0299] The expression level and molecular weight of the multispecific antigen-binding protein were determined by SDS-PAGE and size exclusion chromatography (SEC). Coomassie blue-stained 4% to 15% SDS-PAGE was performed under both reducing and non-reducing conditions (Zhang et al. Clin Cancer Res, 2007; 13(9):2758-2767).

[0300] Size exclusion chromatography was performed using a Yarra SEC-3000 column (Phenomenex, 00H-4513-KO) and a Waters 2695 HPLC (Waters Corporation) in a 0.1 M Na2HPO4 / NaH2PO4, 0.1 M Na2SO4 (pH 6.7) mobile phase at a flow rate of 0.5 mL / min. Thyroglobulin (669 kDa, Sr 8.5 nm), b-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) were used as standard proteins.

[0301] 2.2 Stability

[0302] The stability of the multispecific antigen-binding protein was determined by repeating the above SDS-PAGE and SEC on samples of the multispecific antigen-binding protein stored at room temperature for 1, 3, and 7 days.

[0303] The stability of the multispecific antigen-binding protein in plasma was determined by incubating 200 nM of the protein in 50% human plasma at 37°C for 1, 3, and 7 days.

[0304] The samples were frozen at -20°C immediately after preparation (0d) or after the corresponding incubation period. The levels of intact protein were determined using SDS-PAGE and SEC as described above.

[0305] 2.3 Affinity for IL21R

[0306] Use Biacore T200 instrument to analyze the binding affinity between IL-21R and multi-specific antigen binding protein at 25 ℃ and 50 μ l / min flow rate.Anti-human Fc antibody is covalently fixed on CM5 sensor chip, to capture multi-specific antigen binding protein in flow pool fc2 and fc4, wherein fc1 and fc3 are used as reference.IL-21R is introduced with the concentration of 0.74nM, 2.22nM, 6.67nM, 20nM and 60nM prepared by a series of 1:3 serial dilutions.Measurement is included in the association phase of 180 seconds and the dissociation phase of 1200 seconds after the highest concentration exposure.Analysis buffer contains 10mM HEPES (pH 7.4), 150mM NaCl, 0.05% Tween 20 and 3mM EDTA.Regeneration is carried out under standard conditions to remove complex from all surfaces.

[0307] 2.4 Affinity for 4-1BB

[0308] The interaction between multi-specific antigen binding protein and 4-1BB receptor was analyzed using Biacore T200 instrument at 25 ° C with 50 μ l / min flow rate. Using the CM5 sensor chip from human antibody capture kit (Cytiva), and the analysis buffer is composed of 10mM HEPES (pH 7.4), 150mM NaCl, 0.05% Tween 20 and 3mMEDTA. Anti-human Fc antibody is covalently fixed to the CM5 sensor chip to capture the multi-specific antigen binding protein on the flow pool fc2 and fc4, and fc1 and fc3 are used as reference. After a series of 1: 3 serial dilutions, people 4-1BB is introduced with a concentration of 5nM, 15nM, 45nM, 135nM and 405nM. The determination scheme includes an association phase of 120 seconds and a dissociation phase of 600 seconds after the introduction of the highest concentration sample. After the interaction analysis, regeneration is performed under standard conditions to effectively remove the complex from all surfaces. Recombinant biotinylated human, cynomolgus monkey and mouse 4-1BB Fc (kih) fusion molecules (see Example 3 of WO 2016 / 075278) were directly coupled to the SA chip using standard coupling instructions (Bio-Aiqor, Freiburg / Germany). The fixed level was about 30RU. The multispecific antigen binding or control was flowed through the flow cell at a flow rate of 30 μL / min at a concentration ranging from 0.39 to 200 nM for 120 seconds. Dissociation was monitored for 180 seconds. The bulk refractive index difference was corrected by subtracting the response obtained on the reference empty flow cell.

[0309] To measure affinity, direct coupling of about 7200 resonance units (RU) of anti-human Fc-specific antibodies was performed on a CM5 chip using a standard amine coupling kit (GF Healthcare) at pH 5.0. 50 nM of multispecific antigen-binding proteins containing 4-1BBL or controls were captured at a flow rate of 30 μL / min on flow cell 2 for 60 seconds. Serial dilutions (1.95 to 1000 nM) of human 4-1BB-avi-His (see Example 3 of WO 2016 / 075278) were flowed through two flow cells at 30 μL / min for 180 seconds to record the association phase. The dissociation phase was monitored for 180 s and triggered by switching from the sample solution to HBS-EP. After each cycle, the chip surface was regenerated using a 60 s double injection of 10 mM glycine-HCl pH 2.1. The bulk refractive index difference was corrected by subtracting the response obtained on the reference flow cell 1. For the interaction between the multispecific antigen binding protein containing 4-1BBL and hu4-lBB avi His, affinity constants were derived from rate constants by fitting to a 1:1 Langmuir binding curve using Biaeval software (GF Medical).

[0310] 2.5 Affinity for NKp46

[0311] 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.

[0312] Example 3 Functional in vitro characterization of multispecific antigen-binding proteins

[0313] 3.1 Binding to NK cells

[0314] Essentially as described in Fellermeier (2016, supra), flow cytometry and competitive inhibition with unlabeled competing antibodies were used to demonstrate the binding of multispecific antigen-binding proteins to NK cells. NK cells from four different donors were assayed in triplicate. The mean fluorescence intensity was plotted against the dilution of the multispecific antigen-binding protein to determine the EC50 and EC90 concentrations for optimal effective engagement.

[0315] 3.2 Short-term NK cell cytotoxicity

[0316] Short-term (4 h) NK cytotoxicity of multispecific antigen binding proteins was determined using the calcein-acetoxymethyl release assay (Somanchi et al., 2011, J Vis Exp. [Journal of Visualized Experiments], 2:2540; Lee et al., 2010, Methods Mol. Biol. [Molecular Biology Methods], 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 h with gentle resuspension every 10-15 min. Thereafter, target cells were washed twice with culture medium and 10,000 target cells were seeded in 96-well plates. NK cells were purified from four normal donor buffy coats via 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) and expanded ex vivo in the presence of (saturating concentrations of) multispecific antigen-binding proteins AVC-009, AVC-011 or AVC-014 as described in Example 3.5 below. As a control, NK cells were stimulated with irradiated FC21 feeder cells at a ratio of 1:2 (NK:FC21). The assay was performed in triplicate. The cytotoxicity of NK cells expanded ex vivo for 1 week in the presence of multispecific antigen binding proteins (AVC-009, AVC-011, or AVC-014) was compared to that of NK cells expanded ex vivo in the presence of irradiated FC21 feeder cells. NK cells were removed from the expansion 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 set up:

[0317] 1. Target cells only - use this sample to quantify spontaneous release of Calcein-AM from tumor cells

[0318] 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

[0319] After this incubation, the supernatant was transferred to a new plate and fluorescence was measured on 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:

[0320]

[0321] The results are shown in Figure 3 In the present study, all three multispecific antigen binding proteins AVC-009, AVC-011 and AVC-014 were shown to induce strong cytotoxicity of NK cells against MDA-MB-231 target cells. In addition, the cytotoxicity of NK cells expanded in the presence of AVC-009 or AVC-014 was not even significantly different from that of NK cells stimulated by FC21 feeder cells. Similar data were obtained using K562, Karpas 299, SK-N-AS and A253 as target cells (data not shown).

[0322] 3.3 Long-term NK cell cytotoxicity

[0323] Long-term NK cytotoxicity of multispecific antigen binding proteins was determined using the xCelligence assay to assess serial killing (Naeimi Kararoudi et al., 2022, Cell Reports Methods 2, 100236 June 20, 2022). Hematological target cells were K562 and Karpas 299 (T cell NHL). Solid tumor target cell lines were SK-N-AS (neuroblastoma) and A253 (salivary gland carcinoma). NK cells were purified from four normal donor buffy coats via negative selection using RosetteSep (see above) and co-cultured with and without (saturating concentrations of) multispecific antigen binding proteins or with anti-NKp46 antibodies of SEQ ID NO.: 1 and 2 at a low E:T ratio (0.5: 1). The assay was performed in triplicate. The cytotoxicity of fresh NK cells (above) was compared to that of i) NK cells expanded ex vivo in the presence of multispecific antigen-binding protein 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 below.

[0324] 3.4 NK cell proliferation assay

[0325] NK cells were labeled with CellTrace Violet cell proliferation dye and expanded with and without (saturating concentration or three dose levels (low, medium and high, e.g. 25%, 50% and 100%)) the multispecific antigen binding protein. The assay was performed in triplicate. After 7 days, cells were harvested, stained with Live / Dead stain for viability, and stained for surface markers to gate live CD3 - CD56 + NK cell populations. Proliferation was plotted against tumor cells and booster dilutions to identify EC50 and EC90 concentrations to determine the optimal effective concentration.

[0326] 3.5 Ex vivo expansion of NK cells

[0327] The protocol for ex vivo expansion of donor NK cells is described in Denman et al. (2012, supra).

[0328] Briefly, NK cells were isolated from buffy coats of 8 healthy donors using RosetteSep enrichment and Ficoll (GE HealthCare, Piscataway, NJ). On day 0, NK cells were stimulated as follows: 1.0 x 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, 1x 10 6 NK cells were stimulated with irradiated (100 cGy) FC21 feeder cells at a ratio of 1:2 (NK:FC21) in NK cell culture medium containing 10 NK cells / mL. The cells were then cultured at 0.2 x 10 6 The cells were plated at a density of 10 cells / mL in a PBS solution supplemented with CTS TM Immune cell serum replacement (A2596101, Gibco, Thermo Scientific) and AIM V with 50 IU / mL recombinant human IL-2 (Proleukin, Novartis Vaccines and Diagnostics, Inc) TM culture medium (12055091, Gibco, Thermo Scientific).

[0329] Every other day of expansion, count NK cells using a Nexcelom Cellometer and AOPI staining solution. Determine the total cell count. Add AIM V medium supplemented with CTS Immune Cell Serum Replacement to maintain cells at approximately 0.3 x 10 650 IU / mL human IL-2 was added to each condition / donor.

[0330] Figure 2 It was shown that each of the multispecific antigen binding proteins AVC-009 or AVC-011 and AVC-014 was able to induce ex vivo expansion of NK cells. FC21 was the reference expansion method.

[0331] Figure 6 A graph showing the different rates of NK cell proliferation induced by AVC9 (IL-21 and 4-1BBL), AVC81 (IL-21) and AVC82 (4-1BBL) is shown. As can be observed, after 2 weeks of expansion, cells were counted and the expansion induced by AVC9 (IL-21 and 4-1BBL) was superior to both AVC81 (IL-21) and AVC82 (4-1BBL).

[0332] Example 4 Phenotypic Characterization of NK Cells

[0333] 4.1 Cytokine secretion by expanded NK cells

[0334] In the absence and presence of saturating concentrations of multispecific antigen binding proteins, NK cells were cultured for 7 days. 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. Based on the standard curve and formula provided by the kit, the concentration was calculated according to the mean fluorescence intensity (Denman et al. PLoS ONE [Public Library of Science Comprehensive], 2012, supra). The cytokine secretion of fresh NK cells (above) was compared with i) NK cells amplified in vitro in the presence of multispecific antigen binding proteins and ii) NK cells amplified in vitro in the presence of irradiated FC21 feeder cells. NK cells were amplified in vitro as described in 3.5 above.

[0335] 4.2 Transcriptome analysis of expanded NK cells

[0336] Gene expression in NK cells stimulated with multispecific antigen binding proteins was assessed 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 multispecific antigen binding proteins or FC21 feeder cells in parallel expansion for one week (Denman et al. PLoS ONE, 2012, supra). Total RNA was purified from each sample and the expression of 96 genes was assessed (Denman et al. PLoS ONE, 2012, supra). Gene expression was normalized to LDH (average 6,076 copies detected from 100 ng of load mRNA) and plotted as mean ± SEM. Genes with borderline detection (≤10 normalized transcripts detected) were excluded from further analysis. Genes with a > 2-fold difference in mean expression between cultures with the multispecific antigen binding protein and FC21 were then identified.

[0337] 4.3 Analysis of NK cell phenotype by multiparameter mass cytometry

[0338] NK cell phenotype was identified by multiparameter mass cytometry. 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), NK cells were collected and incubated with the antibodies listed in Table 4.3.1. Thereafter, cells were fixed in PBS containing 2% formaldehyde and stored in this solution until collection.

[0339] Table 4.3.1 Antibodies conjugated to stable metal isotopes as used for mass cytometry All antibodies were purchased from Fluidigm (pre-conjugated) or conjugated using the X8 MaxPar conjugation kit according to the manufacturer's protocol.

[0340]

[0341]

[0342] Immediately before collection, samples were washed with cell staining medium (CSM; PBS + 0.5% FBS) and plated at a concentration of 1 million cells / mL at a 1:20 dilution of EQ TMFour-element calibration beads (Fluida) were resuspended in CSM as previously described (Rahman et al., 2016, Cytometry A, 89:601-607).

[0343] After acquisition on a Helios mass spectrometer (Fluda), the resulting FCS files were normalized using the normalization tool developed by Finck et al. and in Cyto-bank ( www.cytobank.org ) (Finck et al., 2013, Cytometry A, 83(5):483-94; Kotecha et al., Curr Protoc Cytom. [Flow Cytometry Laboratory Manual] July 2010; Chapter 10: Unit 10.17).

[0344] After establishing single-cell gating, cells were identified using markers (listed in Table 4.3.1) and analyzed using viSNE (a visualization tool for high-dimensional single-cell data based on the t-distributed stochastic neighbor embedding (t-SNE) algorithm) (see Amir et al., 2013, Nat. Biotechnol. 31(6):545-552).

[0345] Figure 4 and Figure 5 The results in show that the phenotype of NK cells stimulated and activated by AVC-009, AVC-011 or AVC-014 during expansion closely matches the hyperfunctional phenotype of NK cells stimulated and activated by reference FC21 feeder cells described previously (Denman et al. (2012, supra)). The main difference between the phenotypes induced by the multispecific antigen binding protein and FC21 feeder cells is the increase in Ki67 and perforin expression (see also Figure 5 ).

[0346] Example 5 In vivo characterization of multispecific antigen-binding proteins

[0347] In vivo PK and biodistribution of multispecific antigen-binding proteins were performed using systemic clearance studies of radiolabeled fusion proteins in BALB / c mice as described in Zhang et al. (Clin Cancer Res, 2007, supra).

[0348] Example:

[0349] 1. A multispecific antigen-binding protein, comprising:

[0350] a) NK cell activating cytokine, which is at least one of the following:

[0351] i) interleukin 21 receptor (IL21R) agonists; and

[0352] ii) 4-1BB agonists; and

[0353] b) The region with affinity for surface antigens expressed on natural killer (NK) cells.

[0354] 2. The multispecific antigen-binding protein of embodiment 1, wherein the IL21R agonist comprises or consists of an IL21 polypeptide or an agonistic antigen-binding region that specifically binds to IL21R.

[0355] 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.

[0356] 4. The multispecific antigen-binding protein of embodiment 2 or 3, wherein the IL21 polypeptide is an IL21 mutein that is modified to reduce or increase affinity for IL21R relative to the corresponding wild-type IL21 polypeptide.

[0357] 5. The multispecific antigen-binding protein of embodiment 4, wherein the IL21 mutant protein has a reduced affinity for IL21R relative to the corresponding wild-type IL21 polypeptide, and wherein the IL21 mutant protein has a mutation in one or more amino acids 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.

[0358] 6. The multispecific antigen-binding protein of any preceding embodiment, wherein the multispecific antigen-binding protein has an IL21R agonist titer greater than one.

[0359] 7. The multispecific antigen-binding protein according to 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.

[0360] 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.

[0361] 9. The multispecific antigen-binding protein of embodiment 7 or 8, wherein the 4-1BBL ECD is a mutant protein that is modified to reduce or increase affinity for 4-1BB relative to the corresponding wild-type 4-1BBL ECD.

[0362] 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, and wherein optionally, the three 4-1BBL ECD monomers are connected by a polypeptide linker.

[0363] 11. The multispecific antigen-binding protein of any one of embodiments 7-10, wherein the multispecific antigen-binding protein has a 4-1BB agonist titer greater than one.

[0364] 12. The multispecific antigen-binding protein of any preceding embodiment, wherein the multispecific antigen-binding protein comprises an IL21R agonist and a 4-1BB agonist.

[0365] 13. A multispecific antigen-binding protein according to any one of the preceding embodiments, wherein the multispecific antigen-binding protein further comprises an NK cell-activating cytokine selected from the following: an IL15 receptor agonist, a type I interferon (IFN-1) agonist, an IL2 receptor agonist, an IL12 receptor agonist, and an IL18 receptor agonist.

[0366] 14. The multispecific antigen-binding protein of any preceding embodiment, wherein the region having affinity for surface antigens expressed on NK cells comprises or consists of an immunoglobulin Fc region.

[0367] 15. The multispecific antigen-binding protein of embodiment 14, wherein the Fc region is a dimeric Fc region.

[0368] 16. The multispecific antigen-binding protein of embodiment 14 or 15, wherein the Fc region binds to CD16A.

[0369] 17. The multispecific antigen-binding protein of embodiment 16, wherein the immunoglobulin Fc region is modified to reduce or increase affinity for CD16A relative to a corresponding wild-type Fc region.

[0370] 18. The multispecific antigen-binding protein of any preceding embodiment, wherein the multispecific antigen-binding protein further comprises:

[0371] c) specifically binds to at least one antigen binding region of an NK cell activating receptor.

[0372] 19. A multispecific antigen-binding protein according to Example 18, wherein the antigen-binding region that specifically binds to the NK cell activating receptor is an agonist antigen-binding region that activates the NK cell receptor.

[0373] 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.

[0374] 21. The multispecific antigen-binding protein of embodiment 20, wherein the immunoglobulin variable region comprises or consists of a Fab or an immunoglobulin single variable domain (ISVD).

[0375] 22. The multispecific antigen-binding protein of any one of embodiments 18-21, wherein the antigen binding region is a human or humanized antigen binding region.

[0376] 23. A multispecific antigen-binding protein according to any one of embodiments 18-22, wherein the multispecific antigen-binding protein comprises two antigen-binding regions that specifically bind to an NK cell activating receptor.

[0377] 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.

[0378] 25. The multispecific antigen-binding protein of embodiment 24, wherein the two antigen-binding regions are identical.

[0379] 26. The multispecific antigen-binding protein of any one of embodiments 18-25, 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.

[0380] 27. The multispecific antigen-binding protein of embodiment 26, wherein 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:

[0381] a) CDR-H1 (SEQ ID NO:24), CDR-H2 (SEQ ID NO:25) and CDR-H3 (SEQ ID NO:26) sequences as contained in SEQ ID NO:1, and CDR-L1 (SEQ ID NO:27), CDR-L2 (SEQ ID NO:28) and CDR-L3 (SEQ ID NO:29) sequences as contained in SEQ ID NO:2; and b) CDR-H1, CDR-H2 and CDR-H3 sequences as contained in SEQ ID NO:3, and CDR-L1, CDR-L2 and CDR-L3 sequences as contained in SEQ ID NO:20.

[0382] 28. The multispecific antigen-binding protein of embodiment 27, wherein the antigen-binding region comprises a variable light chain (V L ) domain and variable heavy chain (V H ) domain combination:

[0383] a) V as contained in SEQ ID NO: 1 H Sequence, and V as contained in SEQ ID NO:2 L Sequence; and b) V as contained in SEQ ID NO: 3 H Sequence, and V as contained in SEQ ID NO:20 L sequence.

[0384] 29. A multispecific antigen-binding protein according to any of the preceding embodiments, wherein at least one NK cell activating cytokine is conjugated to the region having affinity for a surface antigen expressed on NK cells.

[0385] 30. A multispecific antigen-binding protein according to Example 29, wherein 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 the surface antigen expressed on NK cells, and wherein optionally, the NK cell activating cytokine is connected to the polypeptide chain of the region having affinity for the surface antigen expressed on NK cells via a flexible linker.

[0386] 31. A multi-specific antigen-binding protein according to 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 the region having affinity for a surface antigen expressed on NK cells; iv) optionally a second flexible linker; and v) a second NK cell activating cytokine; and wherein preferably, the first NK cell activating cytokine and the second NK cell activating cytokine are different NK cell activating cytokines.

[0387] 32. A multispecific antigen-binding protein according to any one of embodiments 18-30, wherein the at least one antigen-binding region that specifically binds to an NK cell activating receptor is conjugated to the region that has affinity for a surface antigen expressed on NK cells.

[0388] 33. A multispecific antigen-binding protein according to 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 in the region that has affinity for a surface antigen expressed on NK cells.

[0389] 34. A multispecific antigen-binding protein according to 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; and iii) the region that has affinity for surface antigens expressed on NK cells.

[0390] 35. A multispecific antigen-binding protein according to embodiment 33 or 34, wherein the region having affinity for surface antigens expressed on NK cells is a dimeric immunoglobulin Fc region, wherein each of the two polypeptide chains of the dimeric Fc region is connected to a CH1 domain, and each CH1 domain is connected to an immunoglobulin variable region that specifically binds to an NK cell activating receptor.

[0391] 36. A multispecific antigen-binding protein according to 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.

[0392] 37. A multispecific antigen-binding protein according to embodiment 35 or 36, wherein 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.

[0393] 38. A multispecific antigen-binding protein according to any one of embodiments 18-30, 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 to the region that has affinity for a surface antigen expressed on NK cells.

[0394] 39. The multispecific antigen-binding protein of embodiment 37, wherein at least one of the NK cell activating cytokines forms a single polypeptide chain with at least one of:

[0395] i) specifically binds to at least one polypeptide chain in the at least one antigen binding region of an NK cell activating receptor; and

[0396] ii) at least one polypeptide chain in the region that has affinity for a surface antigen expressed on NK cells;

[0397] Optionally, a flexible linker is present between the agonist and the at least one polypeptide chain in the region defined in i) or ii).

[0398] 40. The multispecific antigen-binding protein of embodiment 38 or 39, wherein at least one of the NK cell activating cytokines forms a single polypeptide chain with at least one of:

[0399] 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 Fc region of the dimeric immunoglobulin;

[0400] wherein optionally, a flexible linker is present between the agonist and the light chain defined in i) or the Fc chain defined in ii).

[0401] 41. The multispecific antigen-binding protein of embodiment 40, wherein at least one of the NK cell activating cytokines is fused to at least one of:

[0402] i) specifically binds to the N-terminus of the light chain in at least one of the two Fabs that specifically bind to a NK cell activating receptor, optionally through a flexible linker;

[0403] ii) the C-terminus of the light chain in at least one of the two Fabs that specifically binds to a NK cell activating receptor, optionally via a flexible linker;

[0404] iii) the N-terminus of the heavy chain in at least one of the two Fabs that specifically binds to a NK cell activating receptor; and

[0405] iv) the C-terminus of the heavy chain of at least one of the two Fc chains in the Fc region of the dimeric immunoglobulin, optionally via a flexible linker.

[0406] 42. A multispecific antigen-binding protein according to embodiment 40 or 41, wherein at least one of these NK cell activating cytokines is present on at least one side or both sides of the immunoglobulin structure.

[0407] 43. A multispecific antigen-binding protein according to any one of embodiments 35-42, wherein the multispecific antigen-binding protein is a heterodimer with respect to at least one of the following: i) these antigen-binding regions that specifically bind to NK cell activating receptors; and ii) at least one fused NK cell activating cytokine, and wherein the dimeric Fc region comprises different first and second polypeptide chains, and the first polypeptide chain and the second polypeptide chain comprise a knob-in-hole structure modification that promotes the association of the first polypeptide chain and the second polypeptide chain of the Fc region.

[0408] 44. The multispecific antigen-binding protein of any preceding embodiment, wherein the multispecific antigen-binding protein has at least one biological activity selected from:

[0409] a) the multispecific antigen-binding protein causes an increase in at least one NK cell activity selected from the group consisting of: CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation, and NK cytotoxicity, wherein preferably the increase is at least 0.1-fold the increase achieved with the same NK cells and target cells not contacted with the multispecific antigen-binding protein, at the same effector cell:target cell ratio; and

[0410] b) the multispecific antigen-binding protein causes an increase in at least one NK cell activity selected from the group consisting of: CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cell toxicity, wherein preferably, the increase is at least 0.1-fold the increase achieved with the same effector cell:target cell ratio, with the same NK cells and target cells 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.

[0411] 45. The multispecific antigen-binding protein of any preceding embodiment, wherein ex vivo expansion of donor NK cells by co-culturing with the multispecific antigen-binding protein as described herein produces an expanded NK cell population having one or more characteristics selected from the group consisting of:

[0412] a) the expanded NK cells have an expansion fold 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 times greater than the expansion fold of the expanded NK cells obtained by ex vivo expansion via co-culture with irradiated K562 feeder cells modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells);

[0413] 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, 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 after ex vivo expansion in the presence of FC21 feeder cells;

[0414] 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 via co-culture with irradiated FC21 feeder cells;

[0415] 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 higher than the secretion of the cytokine by expanded NK cells obtained by ex vivo expansion via co-culture with irradiated FC21 feeder cells; and

[0416] 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 via co-culture with irradiated FC21 feeder cells.

[0417] 46. ​​A pharmaceutical composition comprising the multispecific antigen-binding protein of any preceding embodiment and a pharmaceutically acceptable carrier.

[0418] 47. An ex vivo method for expanding NK cells, the method comprising the step of contacting NK cells with the multispecific antigen-binding protein of any one of embodiments 1-45 or with the composition of embodiment 46, and wherein preferably the expanded NK cells have one or more characteristics selected from the group consisting of:

[0419] a) the expanded NK cells have an expansion fold 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 times greater than the expansion fold of the expanded NK cells obtained by ex vivo expansion via co-culture with irradiated K562 feeder cells modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligand (FC21 feeder cells);

[0420] 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, 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 after ex vivo expansion in the presence of FC21 feeder cells;

[0421] 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 via co-culture with irradiated FC21 feeder cells;

[0422] 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 higher than the secretion of the cytokine by expanded NK cells obtained by ex vivo expansion via co-culture with irradiated FC21 feeder cells; and

[0423] 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 via co-culture with irradiated FC21 feeder cells.

[0424] 48. The multispecific antigen-binding protein according to any one of embodiments 1-45, the composition according to embodiment 46, or the ex vivo expanded NK cells obtained in the method according to embodiment 47, optionally in combination with the multispecific antigen-binding protein, for use as a medicament.

[0425] 49. The multispecific antigen-binding protein according to any one of Examples 1-45, the composition according to Example 46, or the ex vivo expanded NK cells obtained according to the method according to Example 47, optionally in combination with the multispecific antigen-binding protein, for use in treating cancer.

[0426] 50. The multispecific antigen-binding protein according to any one of Examples 1-45, the composition according to Example 46, for use in the treatment of cancer, wherein the multispecific antigen-binding protein or the composition is used in combination with adoptive transfer of immune cells, wherein preferably, these immune cells are selected from T cells and NK cells.

[0427] 48. The multispecific antigen-binding protein of any one of embodiments 1-45 for use according to claim 49 or 50, the composition of embodiment 46, wherein at least one of the following:

[0428] a) administering the multispecific antigen-binding protein as a neoadjuvant therapy prior to a primary treatment comprising at least one of surgery and radiation therapy for the cancer; and

[0429] b) administering the multispecific antigen-binding protein as adjuvant therapy following primary treatment comprising at least one of surgery and radiation therapy for the cancer.

[0430] 51. A method for enhancing the anti-tumor activity of NK cells in a subject, the method comprising the following steps: administering to the subject a multispecific antigen-binding protein according to any one of Examples 1-45, a composition according to Example 46, ex vivo expanded NK cells obtained in the method according to Example 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.

[0431] 52. The method of 51, wherein the subject suffers from cancer.

[0432] 53. The method of claim 52, wherein at least one of the following:

[0433] a) administering the multispecific antigen-binding protein to the subject as neoadjuvant therapy prior to primary treatment comprising at least one of surgery and radiation therapy for the cancer; and

[0434] b) administering the multispecific antigen-binding protein to the subject as adjuvant therapy following primary treatment comprising at least one of surgery and radiation therapy for the cancer.

[0435] 54. A nucleic acid molecule comprising one or more nucleotide sequences encoding a polypeptide chain of the multispecific antigen-binding protein of any one of embodiments 1-45.

[0436] 55. A nucleic acid molecule according to embodiment 54, wherein the one or more nucleotide sequences are operably linked to a regulatory sequence for expression of the one or more polypeptide chains in a host cell.

[0437] 56. A host cell comprising the nucleic acid molecule according to embodiment 54 or 55.

[0438] 57. A method for producing the multispecific antigen-binding protein of any one of embodiments 1-45, the method comprising culturing the host cell of embodiment 56 so that one or more nucleotide sequences are expressed and the multispecific antigen-binding protein is produced.

[0439] 58. The method according to embodiment 57 further comprises the following steps: recovering the multi-specific antigen-binding protein; and optionally, formulating the multi-specific antigen-binding protein with a pharmaceutically acceptable carrier.

Claims

1. A multispecific antigen-binding protein, comprising: a) at least two NK cell activating cytokines comprising: i) interleukin 21 receptor (IL21R) agonists; and ii) 4-1BB agonists; and b) a region having affinity for a surface antigen expressed on a natural killer (NK) cell, wherein the region comprises or consists of an immunoglobulin Fc region, wherein the IL21R agonist comprises or consists of an IL21 polypeptide or an agonistic antigen binding region that specifically binds to IL21R, And 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, wherein 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, and wherein optionally, the three 4-1BBL ECD monomers are connected by a polypeptide linker.

2. The multispecific antigen-binding protein of claim 1, wherein the IL21 polypeptide comprises an amino acid sequence that has at least 70% sequence identity to SEQ ID NO: 38 and has IL21R agonist activity, and wherein the 4-1BBLECD comprises an amino acid sequence that has at least 70% sequence identity to SEQ ID NO: 37 and has 4-1BB agonist activity. 3 . The multispecific antigen-binding protein according to claim 1 , wherein the Fc region is a dimeric Fc region that binds to CD16A and preferentially activates NK cells.

4. The multispecific antigen-binding protein of any one of the preceding claims, wherein the multispecific antigen-binding protein further comprises: c) specifically binds to at least one antigen binding region of an NK cell activating receptor, Preferably, the antigen binding region that specifically binds to the NK cell activating receptor is an agonist 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, 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.

5. A multispecific antigen-binding protein according to any of the preceding claims, wherein at least one NK cell activating cytokine is conjugated to the region having affinity for surface antigens expressed on NK cells, wherein preferably, 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 surface antigens expressed on NK cells, and wherein optionally, the NK cell activating cytokine is connected to the polypeptide chain of the region having affinity for surface antigens expressed on NK cells via a flexible linker, and wherein more 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 surface antigens expressed on NK cells; iv) optionally a second flexible linker; and v) a second NK cell activating cytokine; and wherein preferably, the first NK cell activating cytokine and the second NK cell activating cytokine are different NK cell activating cytokines.

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

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

8. A multispecific antigen-binding protein according to any one of claims 5-7, wherein the multispecific antigen-binding protein is a heterodimer with respect to at least one of the following: i) these antigen-binding regions that specifically bind to NK cell activating receptors; and ii) at least one fused NK cell activating cytokine, and wherein the dimeric Fc region comprises different first and second polypeptide chains, and the first polypeptide chain and the second polypeptide chain comprise a knob-in-hole structure modification that promotes the association of the first polypeptide chain and the second polypeptide chain of the Fc region.

9. The multispecific antigen-binding protein of any one of the preceding claims, wherein the multispecific antigen-binding protein has at least one biological activity selected from the group consisting of: a) the multispecific antigen-binding protein causes an increase in at least one NK cell activity selected from the group consisting of: CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation, and NK cytotoxicity, wherein preferably the increase is at least 0.1-fold the increase achieved with the same NK cells and target cells not contacted with the multispecific antigen-binding protein, at the same effector cell:target cell ratio; and b) the multispecific antigen-binding protein causes an increase in at least one NK cell activity selected from the group consisting of: CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cell toxicity, wherein preferably, the increase is at least 0.1-fold the increase achieved with the same effector cell:target cell ratio, with the same NK cells and target cells 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.

10. The multispecific antigen-binding protein of any of the preceding claims, wherein ex vivo expansion of donor NK cells by co-culturing with the multispecific antigen-binding protein as described herein produces an expanded NK cell population having one or more characteristics selected from the group consisting of: a) the expanded NK cells have an expansion fold 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 times greater than the expansion fold of the expanded NK cells obtained by ex vivo expansion via 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, 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 after 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 via 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 higher than the secretion of the cytokine by expanded NK cells obtained by ex vivo expansion via 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 that of the expanded NK cells obtained by ex vivo expansion via co-culture with irradiated FC21 feeder cells.

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

12. An ex vivo method for expanding NK cells, the method comprising the step of contacting NK cells with a multispecific antigen-binding protein according to any one of claims 1 to 10 or with a composition according to claim 11, and wherein preferably, the expanded NK cells have one or more characteristics selected from the group consisting of: a) the expanded NK cells have an expansion fold 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 times greater than the expansion fold of the expanded NK cells obtained by ex vivo expansion via 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, 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 after 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 via 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 higher than the secretion of the cytokine by expanded NK cells obtained by ex vivo expansion via 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 that of the expanded NK cells obtained by ex vivo expansion via co-culture with irradiated FC21 feeder cells.

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

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

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

Images