# US 12,215,157 B2 · Multi-specific binding proteins that bind CD33, NKG2D, and CD16, and methods of use > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,215,157 B2 - Document ID: US12215157B2 - Status: Granted - Kind: B2 - Application number: 16/971,104 - Filed: 2019-02-20 - Published: 2021-04-08 - Granted: 2025-02-04 - Earliest claimed: 2018-02-20 - Assignees: Dragonfly Therapeutics, Inc. - Applicants: Dragonfly Therapeutics, Inc. - Inventors: Gregory P. Chang; Ann F. Cheung; Asya Grinberg; Dhruv Kam Sethi; William Haney; Bianka Prinz; Bradley M. Lunde; Ronnie Wei; Daniel Fallon; Steven O'Neil - Web page: https://patentgrep.com/patent/US12215157B2 ## CPC - C07K 16/2803: {against the immunoglobulin superfamily} - C07K 16/283: {against Fc-receptors, e.g. CD16, CD32, CD64 (CD23 C07K16/2851)} - A61P 35/00: Antineoplastic agents - C07K 16/2851: {against the lectin superfamily, e.g. CD23, CD72} - C07K 2317/31: multispecific - C07K 2317/33: Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity - C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues - C07K 2317/522: CH1 domain - C07K 2317/526: CH3 domain - C07K 2317/55: Fab or Fab' - C07K 2317/565: Complementarity determining region [CDR] - C07K 2317/622: Single chain antibody (scFv) - C07K 2317/70: characterized by effect upon binding to a cell or to an antigen - C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation - C07K 2317/77: Internalization into the cell - C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value - 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 Multi-specific binding proteins that bind to and kill human cancer cells expressing CD33 (Siglec-3) are described, as well as pharmaceutical compositions and therapeutic methods useful for the treatment of CD33 expressing cancer. The invention relates to multi-specific binding proteins that bind to human cancer cells expressing CD33 and exhibit high potency and maximum lysis of target cells compared to anti-CD33 monoclonal antibodies. The multi-specific binding proteins comprise a CD33-binding domain, an NKG2D-binding domain and a CD16-binding domain. ## Claims ### Claim 1 A protein comprising: (a) a first antigen-binding site that binds human NKG2D; (b) a second antigen-binding site that binds human CD33; and (c) an antibody Fc domain that binds human CD16 or a portion thereof that binds human CD16, wherein the first antigen-binding site that binds human NKG2D comprises a Fab fragment, and wherein the Fab fragment comprises a light chain complementarity-determining region 1 (CDR1) sequence comprising the amino acid sequence of SEQ ID NO:114, a light chain complementarity-determining region 2 (CDR2) sequence comprising the amino acid sequence of SEQ ID NO:115, a light chain complementarity-determining region 3 (CDR3) sequence comprising the amino acid sequence of SEQ ID NO: 116, a heavy chain complementarity-determining region 1 (CDR1) sequence comprising the amino acid sequence of SEQ ID NO:111, a heavy chain complementarity-determining region 2 (CDR2) sequence comprising the amino acid sequence of SEQ ID NO:112, and a heavy chain complementarity-determining region 3 (CDR3) sequence comprising the amino acid sequence of SEQ ID NO:113; and wherein the second antigen-binding site that binds human CD33 comprises a single-chain variable fragment (scFv), and wherein the scFv comprises a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:48, a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:49, a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:50, a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:45, a heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:46, and a heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:47. ### Claim 2 (depends on 1) The protein of claim 1, wherein the antibody Fc domain that binds human CD16 comprises an amino acid sequence at least 90% identical to the amino acid sequence of a human IgG1 constant region without CHI domain, and wherein the amino acid sequence contains one or more mutations to enable heterodimerization. ### Claim 3 (depends on 1) The protein of claim 1, wherein the protein comprises (a) a first polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 187; (b) a second polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:189; and (c) a third polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:190. ### Claim 4 (depends on 3) The protein of claim 3, wherein the first polypeptide is linked to the second polypeptide via heterodimerization and at least one disulfide bond, and wherein the second polypeptide is linked to the third polypeptide via at least one disulfide bond. ### Claim 5 (depends on 1) The protein of claim 1, wherein the scFv of the second antigen-binding site is linked to the antibody Fc domain that binds human CD16 or a portion thereof that binds human CD16, via a hinge comprising Ala-Ser or Gly-Ala-Ser. ### Claim 6 (depends on 1) The protein of claim 1, wherein the first antigen-binding site that binds human NKG2D comprises a heavy chain variable region amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:87 and a light chain variable region amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:88. ### Claim 7 (depends on 1) The protein of claim 1, wherein the second antigen-binding site that binds human CD33 comprises a heavy chain variable region amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:9 and a light chain variable region amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:10. ### Claim 8 (depends on 1) The protein of claim 1, wherein the scFv of the second antigen-binding site that binds human CD33 comprises the amino acid sequence of SEQ ID NO:188. ### Claim 9 (depends on 1) The protein of claim 1, wherein the antibody Fc domain that binds human CD16 or a portion thereof that binds human CD16 comprises an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgGI antibody according to EU numbering. ### Claim 10 (depends on 1) The protein of claim 1, wherein the antibody Fc domain that binds human CD16 is an Fc domain of a human IgGI comprising Y349C, K360E and K409W substitutions according to EU numbering. ### Claim 11 (depends on 1) The protein of claim 1, wherein the antibody Fc domain that binds human CD16 is an Fc domain of a human IgG1 comprising S354C, Q347R, D399V, and F405T substitutions according to EU numbering. ### Claim 12 A protein comprising: (a) a first antigen-binding site that binds human NKG2D; (b) a second antigen-binding site that binds human CD33; and (c) an antibody Fc domain that binds human CD16 or a portion thereof that binds human CD16, wherein the first antigen-binding site that binds human NKG2D comprises a Fab fragment, and wherein the Fab fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:87 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:88; and wherein the second antigen-binding site that binds human CD33 comprises a single-chain variable fragment (scFv), and wherein the scFv comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:10, wherein the heavy chain variable region of the scFv comprises one amino acid substitution relative to the amino acid sequence of SEQ ID NO:9 located in a framework region of the heavy chain variable region of the scFv, and wherein the light chain variable region of the scFv comprises one amino acid substitution relative to the amino acid sequence of SEQ ID NO: 10 located in a framework region of the light chain variable region of the scFv; and wherein the antibody Fc domain that binds human CD16 or a portion thereof that binds human CD16 comprises an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody according to EU numbering. ### Claim 13 A protein comprising: (a) a first antigen-binding site that binds human NKG2D; (b) a second antigen-binding site that binds human CD33; and (c) an antibody Fc domain that binds human CD16 or a portion thereof that binds human CD16, wherein the first antigen-binding site that binds human NKG2D comprises a Fab fragment, and wherein the Fab fragment comprises a light chain complementarity-determining region 1 (CDR1) sequence comprising the amino acid sequence of SEQ ID NO:114, a light chain complementarity-determining region 2 (CDR2) sequence comprising the amino acid sequence of SEQ ID NO:115, a light chain complementarity-determining region 3 (CDR3) sequence comprising the amino acid sequence of SEQ ID NO:116, a heavy chain complementarity-determining region 1 (CDR1) sequence comprising the amino acid sequence of SEQ ID NO:192, a heavy chain complementarity-determining region 2 (CDR2) sequence comprising the amino acid sequence of SEQ ID NO:112, and a heavy chain complementarity-determining region 3 (CDR3) sequence comprising the amino acid sequence of SEQ ID NO:193; and wherein the second antigen-binding site that binds human CD33 comprises a single-chain variable fragment (scFv), and wherein the scFv comprises a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:48, a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:49, a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:50, a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:181, a heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:46, and a heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 182. ### Claim 14 (depends on 13) The protein of claim 13, wherein the protein comprises (a) a first polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:187; (b) a second polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:189; and (c) a third polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:190. ### Claim 15 (depends on 13) The protein of claim 13, wherein the first antigen-binding site that binds human NKG2D comprises a heavy chain variable region amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:87 and a light chain variable region amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:88. ### Claim 16 (depends on 13) The protein of claim 13, wherein the second antigen-binding site that binds human CD33 comprises a heavy chain variable region amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:9 and a light chain variable region amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:10. ### Claim 17 (depends on 13) The protein of claim 13, wherein the antibody Fc domain that binds human CD16 or a portion thereof that binds human CD16 comprises an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgGI antibody according to EU numbering. ### Claim 18 (depends on 13) The protein of claim 13, wherein the antibody Fc domain that binds human CD16 is an Fc domain of a human IgGI comprising Y349C, K360E and K409W substitutions according to EU numbering. ### Claim 19 (depends on 13) The protein of claim 13, wherein the antibody Fc domain that binds human CD16 is an Fc domain of a human IgG1 comprising S354C, Q347R, D399V, and F405T substitutions according to EU numbering. ### Claim 20 A protein comprising: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:187; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:189; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:190, wherein the first polypeptide is linked to the second polypeptide via heterodimerization and at least one disulfide bond, and wherein the second polypeptide is linked to the third polypeptide via at least one disulfide bond. ### Claim 21 (depends on 12) The protein of claim 12, wherein the one amino acid substitution in the heavy chain variable region of the scFv of the second antigen-binding site and the one amino acid substitution in the light chain variable region of the scFv of the second antigen-binding site form a disulfide bond. ### Claim 22 (depends on 12) The protein of claim 12, wherein the one amino acid substitution in the heavy chain variable region of the scFv of the second antigen-binding site is G44C numbered according to Kabat numbering, and the one amino acid substitution in the light chain variable region of the scFv of the second antigen-binding site is G100C numbered according to Kabat numbering. ### Claim 23 (depends on 12) The protein of claim 12, wherein the scFv of the second antigen-binding site that binds human CD33 comprises the amino acid sequence of SEQ ID NO:188. ### Claim 24 (depends on 12) The protein of claim 12, wherein the protein comprises (a) a first polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:187; (b) a second polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 189; and (c) a third polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:190. ### Claim 25 (depends on 24) The protein of claim 24, wherein the first polypeptide is linked to the second polypeptide via heterodimerization and at least one disulfide bond, and wherein the second polypeptide is linked to the third polypeptide via at least one disulfide bond. ### Claim 26 (depends on 13) The protein of claim 13, wherein the antibody Fc domain that binds human CD16 comprises an amino acid sequence at least 90% identical to the amino acid sequence of a human IgG1 constant region without CHI domain, and wherein the amino acid sequence contains one or more mutations to enable heterodimerization. ### Claim 27 (depends on 13) The protein of claim 13, wherein the scFv of the second antigen-binding site that binds human CD33 comprises the amino acid sequence of SEQ ID NO: 188. ### Claim 28 (depends on 14) The protein of claim 14, wherein the first polypeptide is linked to the second polypeptide via heterodimerization and at least one disulfide bond, and wherein the second polypeptide is linked to the third polypeptide via at least one disulfide bond. ### Claim 29 (depends on 13) The protein of claim 13, wherein the scFv of the second antigen-binding site is linked to the antibody Fc domain that binds human CD16 or a portion thereof that binds human CD16, via a hinge comprising Ala-Ser or Gly-Ala-Ser. ### Claim 30 (depends on 12) The protein of claim 12, wherein the antibody Fc domain that binds human CD16 comprises an amino acid sequence at least 90% identical to the amino acid sequence of a human IgG1 constant region without CH1 domain, and wherein the amino acid sequence contains one or more mutations to enable heterodimerization. ### Claim 31 (depends on 12) The protein of claim 12, wherein the scFv of the second antigen-binding site is linked to the antibody Fc domain that binds human CD16 or a portion thereof that binds human CD16, via a hinge comprising Ala-Ser or Gly-Ala-Ser. ### Claim 32 (depends on 12) The protein of claim 12, wherein the antibody Fc domain that binds human CD16 or a portion thereof that binds human CD16 comprises an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody according to EU numbering. ### Claim 33 (depends on 12) The protein of claim 12, wherein the antibody Fc domain that binds human CD16 is an Fc domain of a human IgG1 comprising Y349C, K360E and K409W substitutions according to EU numbering. ### Claim 34 (depends on 12) The protein of claim 12, wherein the antibody Fc domain that binds human CD16 is an Fc domain of a human IgG1 comprising S354C, Q347R, D399V, and F405T substitutions according to EU numbering. ### Claim 35 (depends on 1) The protein of claim 1, wherein the antibody Fc domain that binds human CD16 comprises an amino acid sequence at least 95% identical to the amino acid sequence of a human IgG1 constant region without CHI domain, and wherein the amino acid sequence contains one or more mutations to enable heterodimerization. ### Claim 36 (depends on 12) The protein of claim 12, wherein the antibody Fc domain that binds human CD16 comprises an amino acid sequence at least 95% identical to the amino acid sequence of a human IgGI constant region without CHI domain, and wherein the amino acid sequence contains one or more mutations to enable heterodimerization. ### Claim 37 (depends on 13) The protein of claim 13, wherein the antibody Fc domain that binds human CD16 comprises an amino acid sequence at least 95% identical to the amino acid sequence of a human IgG1 constant region without CHI domain, and wherein the amino acid sequence contains one or more mutations to enable heterodimerization. ### Claim 38 (depends on 1) The protein of claim 1, wherein the antibody Fc domain that binds human CD16 or a portion thereof that binds human CD16 comprises an amino acid sequence at least 95% identical to amino acids 234-332 of a human IgGI antibody according to EU numbering. ### Claim 39 (depends on 12) The protein of claim 12, wherein the antibody Fc domain that binds human CD16 or a portion thereof that binds human CD16 comprises an amino acid sequence at least 95% identical to amino acids 234-332 of a human IgG1 antibody according to EU numbering. ### Claim 40 (depends on 13) The protein of claim 13, wherein the antibody Fc domain that binds human CD16 or a portion thereof that binds human CD16 comprises an amino acid sequence at least 95% identical to amino acids 234-332 of a human IgG1 antibody according to EU numbering.