Multispecific antigen binding proteins for tumor-targeting of NK cells and use thereof

{
  "publication_number": "US2026103539A1",
  "country": "US",
  "kind": "A1",
  "title": "Multispecific antigen binding proteins for tumor-targeting of NK cells and use thereof",
  "abstract": "The present invention relates to multispecific antigen binding proteins that comprise antigen-binding regions specific for a tumor-associated antigen (TAA) and NK cell-activating cytokines. The NK cell-activating cytokine preferably is at least one of an interleukin 21 receptor (IL21R) agonist and a 4-1BB agonist. The multispecific antigen binding protein can further comprise an antigen-binding region that has affinity for a surface antigen expressed on NK cells, e.g CD16A. The multispecific antigen binding proteins of the invention specifically redirect and activate NK cells to lyse targeted tumor cells. The invention further relates to the use of such multispecific antigen binding proteins in the treatment of cancer, preferably a cancer expressing the TAA.",
  "claims": [
    "1. A multispecific antigen binding protein comprising: a) a first antigen-binding region that specifically binds a tumor associated antigen (TAA); b) a second antigen-binding region that has affinity for a surface antigen expressed on natural killer (NK) cells, wherein the second antigen-binding region comprises or consists of an immunoglobulin Fc region; and, c) at least two NK cell-activating cytokines comprising: i) an interleukin 21 receptor (IL21R) agonist; and, ii) a 4-1BB agonist, wherein the IL21R agonist comprises or consist of an IL21 polypeptide or an agonistic antigen-binding region that specifically binds IL21R, and wherein the 4-1BB agonist comprises or consist of at least one 4-1BB ligand (4-1BBL) extracellular domain (ECD) or at least one agonistic antigen-binding region that specifically binds 4-1BB. 2. A multispecific antigen binding protein according claim 1, wherein the IL21 polypeptide comprises an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 38 and having IL21R agonist activity, and wherein the 4-1BBL ECD comprises an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 37 and having 4-1BB agonist activity. 3. A multispecific antigen binding protein according claim 1, wherein the TAA is selected from the group consisting of: Her2 (ErbB2/Neu), Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1), Crypto, CD2, CD4, CD20, CD30, CD19, CD38, CD40, CD47, Glycoprotein NMB, CanAg, CD22 (Siglec2), CD33 (Siglec3), CD79, CD123, CD138, CD171, CTLA-4 (CD152), PD1, PSCA, L1-CAM, EpCAM, PSMA (prostate specific membrane antigen), BCMA, TROP2, STEAP1, CD52, CD56, CD80, CD70, E-selectin, EphB2, EPHA4, Melanotransferrin, Mud 6, TMEFF2, Killer 1g-Like Receptor, Killer 1g-Like Receptor 3DL2 (KIR3DL2), B7.1, B7.2, B7-H3, B7-H4, B7-H6, PD-L1, IL-6 receptor, IL-1 accessory Protein, MAGE, MART-1/Melan-A, gp100, MICA, MICB, adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017-1A/GA733, protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), NaPi2b, TYRP1, nectin-4, a UL16-binding protein (ULBP), a RAET1 protein, carcinoembryonic antigen (CEA), CEACAM5, etv6, aml1, prostate specific antigen (PSA), T-cell receptor/CD3-zeta chain, MAGE-A3, a GAGE-tumor antigen, anti-Müllerian hormone Type II receptor, delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), DR5, NTRKR1 (EC 2.7.10.1), SLAMF7, TRAILR1, TRAILR2, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC1, MUC1-C, VEGF, VEGFR2, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor (EGFR/ERBB1), HER-3/ERBB3, HER-4/ERBB4, a heterodimeric receptor comprised of at least one HER subunit, gastrin releasing peptide receptor antigen, cMET, CA125, integrin receptors, a5β3 integrins, a5β1 integrins, αllbβ3-integrins, PDGF alpha receptor, PDGF beta receptor, sVE-cadherin, IL-8 receptor, hCG, IL-6 receptor, IL-1 accessory protein, CSF1R, α-fetoprotein, mesothelin (MSLN), Isoform 2 of Claudin-18 (Claudin 18.2, CLDN18), folate receptor alpha (FRα, FOLR1), tissue factor (TF, CD142), P-cadherin, E-cadherin, α-catenin, β-catenin and γ-catenin, Plexin-A1, TNFRSF10B, AXL, EDNRB, OLR1, ADAM12, PLAUR, CCR4, CCR6, p120ctn, PRAME, NY-ESO-1, cdc27, CDCP1, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, a GM2 ganglioside, a GD2 ganglioside, a human papillomavirus protein, imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-I, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 CT-7, c-erbB-2, FcRL5/FcRH5, Flt3, muci6, muci7, mmp9, FAP, Lewis-Y, EGFRvIII, GPC3, GPRC5D, gpA33, 5T4, SSTR2, CD73, CD25, CD45, and CD133, and wherein preferably, the multispecific antigen binding protein comprises a third antigen-binding region that specifically binds a TAA and wherein the first and third antigen-binding regions can bind the same TAA or at least two different TAAs. 4. A multispecific antigen binding protein according to claim 1, wherein the Fc region is a dimeric Fc region that binds to CD16A and, preferably activates the NK cell. 5. A multispecific antigen binding protein according to claim 1, wherein the 4-1BB agonist comprises or consist 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 polypeptide linkers. 6. A multispecific antigen binding protein according to claim 1, wherein at least one polypeptide chain in the at least one of the first and third antigen-binding regions forms a single polypeptide chain with at least one polypeptide chain of the second antigen-binding region, wherein preferably, the single polypeptide chain comprises in an N- to C-terminal order: i) at least one polypeptide chain in the at least one of the first and third antigen-binding region; ii) optionally a flexible linker; and iii) the second antigen-binding region, wherein more preferably the second antigen-binding region is a dimeric Fc region, wherein each of the two polypeptide chains of the dimeric Fc region is linked to a CH1 domain, each of which CH1 domains is linked to an immunoglobulin-derived antigen-binding region that specifically binds a TAA, wherein most preferably, the protein comprises a dimeric Fc region, wherein each of the two Fc polypeptide chains is operably linked to a Fab that specifically binds a TAA. 7. A multispecific antigen binding protein according to claim 6, wherein at least one of the NK cell-activating cytokines, is conjugated to the at least one antigen-binding region that specifically binds a TAA, or to the second antigen-binding region, wherein preferably, at least one of the NK cell-activating cytokines forms a single polypeptide chain with at least one of: i) at least one polypeptide chain in at least one of the first and third antigen-binding regions; and, ii) at least one polypeptide chain in the second antigen-binding region; 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), wherein more preferably, wherein at least one of the NK cell-activating cytokines forms a single polypeptide chain with at least one of: i) a light chain in at least one of the two Fab's that specifically bind a TAA; and, ii) at least one of the two Fc chains in the dimeric Fc region; 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 more preferably, at least one of the NK cell-activating cytokines is fused to at least one of: i) the N-terminus of the light chain in at least one of the two Fab's that specifically bind a TAA, optionally through a flexible linker; ii) the C-terminus of the light chain in at least one of the two Fab's that specifically bind a TAA, optionally through a flexible linker; iii) the N-terminus of the heavy chain in at least one of the two Fab's that specifically bind a TAA; and, iv) the C-terminus of the heavy chain in at least one of the two Fc chains in the dimeric immunoglobulin Fc domain, optionally through a flexible linker wherein most preferably, at least one of the NK cell-activating cytokines is present on at least one or on both sides of the immunoglobulin structure. 8. A multispecific antigen binding protein according to claim 6, wherein the multispecific antigen binding protein is heterodimeric with respect to at least one of i) the first and third antigen-binding regions; and ii) at least one fused NK cell-activating cytokine, and wherein the dimeric Fc region comprises different first and a second polypeptide chains comprising knob-into-hole modifications promoting association of the first and the second polypeptide chains of the Fc region. 9. A multispecific antigen binding protein according to claim 1, wherein the multispecific antigen binding protein has at least one biological activity selected from: a) the multispecific antigen binding protein causes an increase in at least one NK cell activity selected from CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cell cytotoxicity, whereby preferably, the increase is at least a factor 0.1 higher as compared to the increase achieved with the same effector:target cell ratio, with the same NK cells and target cells that are not brought into contact with the multispecific antigen binding protein; and, b) the multispecific antigen binding protein causes an increase in at least one NK cell activity selected from CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cell cytotoxicity, whereby preferably, the increase is at least a factor 0.1 higher as compared to the increase achieved with the same effector:target cell ratio, with the same NK cells and target cells that are brought into contact with a conventional human IgG1 monoclonal antibody that has the same TAA-specific antigen-binding regions as the multispecific antigen binding protein. 10. A multispecific antigen binding protein according to claim 1, wherein ex vivo expansion of donor NK cells by co-culturing with the multispecific antigen binding protein, produces a population of expanded NK cells having one or more features selected from: a) the fold expansion of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 fold of the fold expansion of expanded NK cells obtained by ex vivo expansion by co-culturing 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% as compared to the telomere length of fresh NK cells, preferably, the percentage telomere length increase of the expanded NK cells as compared to the telomere length of fresh NK cells, is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 fold of the percentage telomere length increase of NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells; c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 fold of the expression level on expanded NK cells obtained by ex vivo expansion by co-culturing 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 fold of the secretion of the cytokine by expanded NK cells obtained by ex vivo expansion by co-culturing 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 fold of the cytotoxicity of expanded NK cells obtained by ex vivo expansion by co-culturing with irradiated FC21 feeder cells, wherein, preferably the NK cells are co-cultured with tumor cells expressing a TAA specifically bound by the multispecific antigen binding protein. 11. (canceled) 12. An ex vivo method for expansion of NK cells, the method comprising the step of contacting an NK cell with a multispecific antigen binding protein according to a claim 1, and wherein preferably, the expanded NK cells have one or more features selected from: a) the fold expansion of the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 fold of the fold expansion of expanded NK cells obtained by ex vivo expansion by co-culturing 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% as compared to the telomere length of fresh NK cells, preferably, the percentage telomere length increase of the expanded NK cells as compared to the telomere length of fresh NK cells, is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 fold of the percentage telomere length increase of NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells; c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on the expanded NK cells is at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 fold of the expression level on expanded NK cells obtained by ex vivo expansion by co-culturing 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 fold of the secretion of the cytokine by expanded NK cells obtained by ex vivo expansion by co-culturing 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 fold of the cytotoxicity of expanded NK cells obtained by ex vivo expansion by co-culturing with irradiated FC21 feeder cells, wherein, preferably the method comprising the further step of co-culturing the NK cells with tumor cells expressing a TAA specifically bound by the multispecific antigen binding protein. 13 - 15. (canceled) 16. A method for treating cancer in a subject in need thereof, the method comprising administering the multispecific antigen binding protein according to claim 1 to the subject. 17. The method according to claim 16, wherein the treatment is for a cancer comprising tumor cells expressing the TAA. 18. The method according to claim 16, wherein the multispecific antigen binding protein is used in combination with an adoptive transfer of immune cells, wherein preferably the immune cells are selected from T cells and NK cells. 19. The method according to claim 16, wherein at least one of: 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 radiation therapy of 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 radiation therapy of the cancer."
  ],
  "description_excerpt": "The present invention relates to the field of medicine, in particular to the fields of oncology, immunology and immunotherapy of tumors. Specifically, the invention relates to multispecific antigen binding proteins that specifically redirect and activate NK cells to lyse targeted tumor cells. The invention further relates to the use of such multispecific antigen binding proteins in the treatment of cancer.\n\nImmunotherapy of cancer is revolutionizing the treatment of cancers. Cancer immunotherapies are desirable because they are highly specific and can facilitate destruction of a tumor by inducing the recognition and elimination of tumor cells by the patient's own immune system. Recent advances have focused on generating or unleashing tumor antigen-specific T cell responses. They were based on the use of immune checkpoint inhibitors targeting inhibitory pathways, or bispecific T cell engagers and chimeric antigen receptor (CAR) T cells targeting a tumor antigen. Despite these outstanding breakthroughs, the clinical benefit has been limited to a subset of patients and certain tumor types, highlighting the need for alternative strategies.\n\nOne such alternative approach is to exploit the antitumor activity of natural killer (NK) cells. NK cells are a component of the innate immune system and make up approximately 15% of circulating lymphocytes. NK cells infiltrate virtually all tissues and were originally characterized by their ability to kill tumor cells effectively without the need for prior sensitization.",
  "cpc": [
    "C07K 16/30",
    "A61K 35/17",
    "A61K 40/11",
    "A61K 40/15",
    "A61P 35/00",
    "C07K 14/54",
    "C07K 14/70596",
    "C07K 19/00",
    "C07K 2317/31",
    "C07K 2317/52",
    "C07K 2317/55",
    "C12N 2502/30",
    "C12N 5/0646"
  ],
  "ipc": [
    "A61K 35/17",
    "A61K 40/11",
    "A61K 40/15",
    "A61P 35/00",
    "C07K 14/54",
    "C07K 14/705",
    "C07K 16/30",
    "C07K 19/00",
    "C12N 5/0783"
  ],
  "assignees": [
    "Avidicure Ip BV"
  ],
  "inventors": [
    "Robert Heinz Edward Friesen"
  ],
  "filing_date": "2023-09-15",
  "publication_date": "2026-04-16",
  "priority_date": "2022-09-15",
  "application_number": "US-202319111337-A",
  "family_id": "88068592",
  "cited_by_count": 0
}