# US 10,501,551 B2 · Multivalent and multispecific 41BB-binding fusion proteins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,501,551 B2 - Document ID: US10501551B2 - Status: Granted - Kind: B2 - Application number: 15/404,016 - Filed: 2017-01-11 - Published: 2017-07-13 - Granted: 2019-12-10 - Earliest claimed: 2016-01-11 - Assignees: Inhibrx, Inc. - Applicants: Inhibrx, Inc. - Inventors: Brendan P. Eckelman; John C. Timmer; Chelsie Hata; Kyle S. Jones; Abrahim Hussain; Amir S. Razai; Bryan Becklund; Rajay Pandit; Mike Kaplan; Lucas Rascon; Quinn Deveraux - Web page: https://patentgrep.com/patent/US10501551B2 ## CPC - C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95} - A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] - A61P 35/00: Antineoplastic agents - A61P 37/04: Immunostimulants - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - C07K 16/2827: {against B7 molecules, e.g. CD80, CD86} - C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered - C07K 2317/31: multispecific - C07K 2317/33: Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity - C07K 2317/35: Valency - C07K 2317/52: Constant or Fc region; Isotype - C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody® - C07K 2317/75: Agonist effect on antigen - C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding - C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value - C07K 2319/00: Fusion polypeptide - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS ## Abstract This invention relates generally to molecules that specifically engage 41BB, a member of the TNF receptor superfamily (TNFRSF). More specifically, this invention relates to multivalent and multispecific molecules that bind at least 41BB. ## Claims ### Claim 1 An isolated polypeptide comprising at least one VHH domain that binds PDL1 and at least one VHH domain that binds 41BB, wherein each VHH domain that binds PDL1 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 105, a CDR2 comprising the amino acid sequence of SEQ ID NO: 106, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 107; and wherein each VHH domain that binds 41BB comprises a CDR1, a CDR2, and a CDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 26, 27 and 28; or SEQ ID NOs: 26, 61, and 28; or SEQ ID NOs: 26, 63, and 28; or SEQ ID NOs: 26, 71, and 28; or SEQ ID NOs: 26, 73, and 28; or SEQ ID NOs: 26, 75, and 28; or SEQ ID NOs: 26, 77, and 28; or SEQ ID NOs: 26, 79, and 28; or SEQ ID NOs: 65, 61, and 28; or SEQ ID NOs: 67, 61, and 28; or SEQ ID NOs: 69, 61, and 28. ### Claim 2 (depends on 1) The isolated polypeptide of claim 1, wherein at least one VHH domain that binds PDL1 is humanized. ### Claim 3 (depends on 1) The isolated polypeptide of claim 1, wherein at least one VHH domain that binds PDL1 comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 104 and 119-124. ### Claim 4 (depends on 1) The isolated polypeptide of claim 1, wherein at least one VHH domain that binds PDL1 comprises an amino acid sequence selected from SEQ ID NOs: 104 and 119-124. ### Claim 5 (depends on 1) The isolated polypeptide of claim 1, wherein at least one VHH domain that binds PDL1 comprises the amino acid sequence of SEQ ID NO: 124. ### Claim 6 (depends on 1) The isolated polypeptide of claim 1, wherein at least one VHH domain that binds 41BB is humanized. ### Claim 7 (depends on 1) The isolated polypeptide of claim 1, wherein at least one VHH domain that binds 41BB comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 25, 59, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 81, 82, and 83. ### Claim 8 (depends on 1) The isolated polypeptide of claim 1, wherein at least one VHH domain that binds 41BB comprises an amino acid sequence selected from SEQ ID NOs: 25, 59, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 81, 82, and 83. ### Claim 9 (depends on 1) The isolated polypeptide of claim 1, wherein at least one VHH domain that binds 41BB comprises the amino acid sequence of SEQ ID NO: 60. ### Claim 10 (depends on 1) The isolated polypeptide of claim 1, wherein at least one VHH domain that binds 41BB comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28. ### Claim 11 (depends on 1) The isolated polypeptide of claim 1, wherein each VHH domain that binds 41BB comprises the amino acid sequence of SEQ ID NO: 60. ### Claim 12 (depends on 1) The isolated polypeptide of claim 1, wherein the polypeptide comprises one VHH domain that binds 41BB, wherein the VHH domain that binds 41BB comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28. ### Claim 13 (depends on 12) The isolated polypeptide of claim 12, wherein the VH H domain that binds 41BB is humanized. ### Claim 14 (depends on 12) The isolated polypeptide of claim 12, wherein the VHH domain that binds 41BB comprises the amino acid sequence of SEQ ID NO: 60. ### Claim 15 (depends on 1) The isolated polypeptide of claim 1, wherein the isolated polypeptide comprises an Fc region. ### Claim 16 (depends on 15) The isolated polypeptide of claim 15, wherein the Fc region comprises an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NOs: 1-6. ### Claim 17 (depends on 15) The isolated polypeptide of claim 15, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NOs: 1-6. ### Claim 18 (depends on 15) The isolated polypeptide of claim 15, which forms a homodimer under physiological conditions. ### Claim 19 (depends on 15) The isolated polypeptide of claim 15, wherein the polypeptide has the structure VHH-Linker-VHH-Linker-Hinge-Fc. ### Claim 20 (depends on 19) The isolated polypeptide of claim 19, wherein each linker is, independently, 5-20 amino acids in length. ### Claim 21 (depends on 19) The isolated polypeptide of claim 19, wherein the hinge comprises a sequence selected from SEQ ID NOs: 7-9. ### Claim 22 (depends on 1) The isolated polypeptide of claim 1, wherein each VHH domain that binds 41BB comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28. ### Claim 23 (depends on 22) The isolated polypeptide of claim 22, wherein the isolated polypeptide comprises two VHH domains that bind PDL1. ### Claim 24 (depends on 23) The isolated polypeptide of claim 23, wherein the isolated polypeptide comprises two VHH domains that bind 41BB. ### Claim 25 (depends on 22) The isolated polypeptide of claim 22, wherein the isolated polypeptide comprises two VHH domains that bind 41BB. ### Claim 26 (depends on 1) The isolated polypeptide of claim 1, wherein the isolated polypeptide comprises two VHH domains that bind PDL1. ### Claim 27 (depends on 26) The isolated polypeptide of claim 26, wherein the isolated polypeptide comprises two VHH domains that bind 41BB. ### Claim 28 (depends on 1) The isolated polypeptide of claim 1, wherein the isolated polypeptide comprises two VHH domains that bind 41BB. ### Claim 29 (depends on 1) The isolated polypeptide of claim 1, wherein each VI-11-1 domain of the isolated polypeptide is humanized. ### Claim 30 (depends on 1) The isolated polypeptide of claim 1, wherein the PDL1 is human PDL1, and wherein the 41BB is human 41BB. ### Claim 31 (depends on 1) The isolated polypeptide of claim 1, wherein the polypeptide comprises one VHH domain that binds PDL1. ### Claim 32 (depends on 31) The isolated polypeptide of claim 31, wherein the VHH domain that binds PDL1 is humanized. ### Claim 33 (depends on 31) The isolated polypeptide of claim 31, wherein the VHH domain that binds PDL1 comprises the amino acid sequence of SEQ ID NO: 124. ### Claim 34 (depends on 33) The isolated polypeptide of claim 33, wherein the polypeptide comprises one VHH domain that binds 41BB, wherein the VHH domain that binds 41BB comprises the amino acid sequence of SEQ ID NO: 60. ### Claim 35 (depends on 34) The isolated polypeptide of claim 34, wherein the isolated polypeptide comprises an Fc region. ### Claim 36 (depends on 35) The isolated polypeptide of claim 35, wherein the Fc region comprises an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NOs: 1-6. ### Claim 37 (depends on 35) The isolated polypeptide of claim 35, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NOs: 1-6. ### Claim 38 (depends on 35) The isolated polypeptide of claim 35, wherein the polypeptide has the structure VHH-Linker-VHH-Linker-Hinge-Fc. ### Claim 39 (depends on 38) The isolated polypeptide of claim 38, wherein each linker is, independently, 5-20 amino acids in length. ### Claim 40 (depends on 38) The isolated polypeptide of claim 38, wherein the hinge comprises a sequence selected from SEQ ID NOs: 7-9. ### Claim 41 (depends on 38) The isolated polypeptide of claim 38, which forms a homodimer under physiological conditions. ### Claim 42 (depends on 35) The isolated polypeptide of claim 35, which forms a homodimer under physiological conditions. ### Claim 43 (depends on 31) The isolated polypeptide of claim 31, wherein the polypeptide comprises one VHH domain that binds 41BB, wherein the VHH domain that binds 41BB comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28. ### Claim 44 (depends on 43) The isolated polypeptide of claim 43, wherein the PDL1 is human PDL1, and wherein the 41BB is human 41BB. ### Claim 45 (depends on 43) A method of treating cancer comprising administering to a subject with cancer a polypeptide of claim 43. ### Claim 46 (depends on 45) The method of claim 45, wherein the cancer is selected from carcinoma, sarcoma, lymphoma, and leukemia. ### Claim 47 (depends on 43) A method of increasing T cell activation and/or proliferation comprising contacting T cells with a polypeptide of claim 43. ### Claim 48 (depends on 47) The method of claim 47, wherein the T cells are CD4+ T cells and/or CD8+ T cells. ### Claim 49 (depends on 1) A method of treating cancer comprising administering to a subject with cancer a polypeptide of claim 1. ### Claim 50 (depends on 49) The method of claim 49, wherein the cancer is selected from carcinoma, sarcoma, lymphoma, and leukemia. ### Claim 51 (depends on 1) A method of increasing T cell activation and/or proliferation comprising contacting T cells with a polypeptide of claim 1. ### Claim 52 (depends on 51) The method of claim 51, wherein the T cells are CD4+ T cells and/or CD8+ T cells. ### Claim 53 An isolated polypeptide comprising a first VHH domain (VHH1) that binds PDL1 and a second VHH domain (VHH2) that binds 41BB, wherein VHH1 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 105, a CDR2 comprising the amino acid sequence of SEQ ID NO: 106, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 107; and VHH2 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28; and wherein the polypeptide has the structure VHH1-Linker-VHH2-Linker-Hinge-Fc. ### Claim 54 (depends on 53) The isolated polypeptide of claim 53, wherein the first VHH domain comprises the amino acid sequence of SEQ ID NO: 124 and the second VHH domain comprises the amino acid sequence of SEQ ID NO: 60, and wherein the Fc region comprises an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NOs: 1-6. ### Claim 55 (depends on 54) The isolated polypeptide of claim 54, wherein the Fc region comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 2. ### Claim 56 (depends on 54) The isolated polypeptide of claim 54, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 2. ### Claim 57 (depends on 53) The isolated polypeptide of claim 53, wherein the polypeptide comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 449. ### Claim 58 (depends on 53) The isolated polypeptide of claim 53, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 449. ### Claim 59 (depends on 58) The isolated polypeptide of claim 58, which forms a dimer at physiological conditions. ### Claim 60 (depends on 53) The isolated polypeptide of claim 53, wherein the PDL1 is human PDL1, and wherein the 41BB is human 41BB. ### Claim 61 (depends on 53) A method of treating cancer comprising administering to a subject with cancer a polypeptide of claim 53. ### Claim 62 (depends on 61) The method of claim 61, wherein the cancer is selected from carcinoma, sarcoma, lymphoma, and leukemia. ### Claim 63 (depends on 53) A method of increasing T cell activation and/or proliferation comprising contacting T cells with a polypeptide of claim 53. ### Claim 64 (depends on 63) The method of claim 63, wherein the T cells are CD4+ T cells and/or CD8+ T cells.