US2022089760A1PendingUtilityA1

Multi-specific binding proteins that bind bcma, nkg2d and cd16, and methods of use

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Assignee: DRAGONFLY THERAPEUTICS INCPriority: Aug 8, 2018Filed: Dec 6, 2021Published: Mar 24, 2022
Est. expiryAug 8, 2038(~12.1 yrs left)· nominal 20-yr term from priority
Inventors:Mitchell BigelowGregory P. ChangAnn F. CheungAsya GrinbergWilliam HaneyNicolai WagtmannBradley M. LundeBianka PrinzRonnie WeiDaniel FallonSteven O'Neil
C07K 16/283A61P 35/00C07K 16/2878C07K 16/2851C07K 2317/73A61K 2039/505C07K 2317/92C07K 2317/31C07K 2317/55C07K 2317/53C07K 2317/624C07K 2317/622C07K 2317/565
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Claims

Abstract

Multi-specific binding proteins that bind to and kill human cancer cells are described, as well as pharmaceutical compositions and therapeutic methods useful for the treatment of cancer. The cancer can be B-cell maturation antigen (BCMA)-expressing cancer. The multi-specific binding proteins provided herein exhibit high potency and maximum lysis of target cells compared to anti-BCMA monoclonal antibodies.

Claims

exact text as granted — not AI-modified
1 - 57 . (canceled) 
     
     
         58 . A protein comprising:
 (a) a first antigen-binding site comprising a single-chain variable fragment (scFv) that binds NKG2D and comprises the amino acid sequence of SEQ ID NO:190, the amino acid sequence of SEQ ID NO:96, the amino acid sequence of SEQ ID NO:191, the amino acid sequence of SEQ ID NO:99, the amino acid sequence of SEQ ID NO:100, and the amino acid sequences of SEQ ID NO:101;   (b) a second antigen-binding site that binds B-cell maturation antigen (BCMA) and comprises the amino acid sequence of SEQ ID NO:149, the amino acid sequence of SEQ ID NO:150, the amino acid sequence of SEQ ID NO:151, the amino acid sequence of SEQ ID NO:153, the amino acid sequence of SEQ ID NO:154, and the amino acid sequences of SEQ ID NO:155;   (c) an additional antigen-binding site that binds BCMA and comprises the amino acid sequence of SEQ ID NO:149, the amino acid sequence of SEQ ID NO:150, the amino acid sequence of SEQ ID NO:151, the amino acid sequence of SEQ ID NO:153, the amino acid sequence of SEQ ID NO:154, and the amino acid sequences of SEQ ID NO:155; and   (d) an antibody Fc domain or a portion thereof sufficient to bind CD16.   
     
     
         59 . The protein according to  claim 58 , wherein the light chain variable domain of the scFv of the first antigen-binding site is positioned at the N-terminus of the heavy chain variable domain of the scFv. 
     
     
         60 . The protein according to  claim 59 , wherein the light chain variable domain of the scFv of the first antigen-binding site is linked to the heavy chain variable domain of the scFv via a linker, wherein the linker amino acid sequence comprises the amino acid sequence of (GlyGlyGlyGlySer) n  ((G4S) n ) (SEQ ID NO:198), and wherein n is an integer between 1-10. 
     
     
         61 . The protein according to  claim 60 , wherein the linker amino acid sequence comprises the amino acid sequence of (GlyGlyGlyGlySer) 4  ((G4S) 4 ) (SEQ ID NO:164). 
     
     
         62 . The protein according to  claim 58 , wherein a disulfide bridge is formed between the heavy chain variable domain of the scFv and the light chain variable domain of the scFv. 
     
     
         63 . The protein according to  claim 62 , wherein the disulfide bridge is formed between C44 of the heavy chain variable domain of the scFv and C100 of the light chain variable domain of the scFv. 
     
     
         64 . The protein according to  claim 58 , wherein the N-terminus of the light chain variable domain of the scFv of the first antigen-binding site is linked to the C-terminus of the antibody Fc domain or a portion thereof. 
     
     
         65 . The protein according to  claim 64 , wherein the N-terminus of the light chain variable domain of the scFv of the first antigen-binding site is linked to the C-terminus of the antibody Fc domain or a portion thereof via a linker, and wherein the linker amino acid sequence comprises the amino acid sequence of SEQ ID NO:168. 
     
     
         66 . The protein according to  claim 58 , wherein the scFv of the first antigen-binding site comprises a heavy chain variable domain amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:94 and a light chain variable domain amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:98. 
     
     
         67 . The protein according to  claim 58 , wherein the scFv of the first antigen-binding site comprises the amino acid sequence of SEQ ID NO:161. 
     
     
         68 . The protein according to  claim 58 , wherein the second and additional antigen-binding sites that bind BCMA are Fab fragments. 
     
     
         69 . The protein according to  claim 58 , wherein the C-terminus of each of the second and additional antigen-binding sites is linked to the antibody Fc domain or a portion thereof sufficient to bind CD16 via a hinge. 
     
     
         70 . The protein according to  claim 69 , wherein the hinge comprises Ala-Ser. 
     
     
         71 . The protein according to  claim 58 , wherein the second and additional antigen-binding sites that bind BCMA each comprises a heavy chain variable domain amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:148 and a light chain variable domain amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:152. 
     
     
         72 . The protein according to  claim 58 , wherein the antibody Fc domain sufficient to bind CD16 comprises hinge and CH2 domains of a human IgG1 antibody. 
     
     
         73 . The protein according to  claim 58 , wherein the antibody Fc domain sufficient to bind CD16 comprises an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody. 
     
     
         74 . The protein according to  claim 58 , wherein the antibody Fc domain sufficient to bind CD16 comprises an amino acid sequence at least 90% identical to the Fc domain of a human IgG1 and differs at one or more positions that is Q347, Y349, T350, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, or K439, numbered according to the Kabat numbering. 
     
     
         75 . The protein according to  claim 58 , wherein the antibody Fc domain sufficient to bind CD16 is an Fc domain of a human IgG1 comprising substitutions for forming a heterodimer. 
     
     
         76 . The protein according to  claim 58 , wherein the antibody Fc domain sufficient to bind CD16 is an Fc domain of a human IgG1 comprising Y349C, K360E and K409W substitutions, numbered according to the Kabat numbering. 
     
     
         77 . The protein according to  claim 58 , wherein the antibody Fc domain sufficient to bind CD16 is an Fc domain of a human IgG1 comprising S354C, Q347R, D399V, and F405T substitutions, numbered according to the Kabat numbering. 
     
     
         78 . The protein according to  claim 58 , wherein the protein comprises:
 (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:162;   (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:163; and   (c) a third and fourth polypeptides each comprising the amino acid sequence of SEQ ID NO:165,   wherein the first polypeptide is linked to the second polypeptide via heterodimerization and at least one disulfide bond, wherein the third polypeptide is linked to the first polypeptide via a disulfide bond, and wherein the fourth polypeptide is linked to the second polypeptide via a disulfide bond.   
     
     
         79 . A pharmaceutical composition comprising a protein and a pharmaceutically acceptable carrier, wherein the protein comprises:
 (a) a first antigen-binding site comprising a single-chain variable fragment (scFv) that binds NKG2D and comprises the amino acid sequence of SEQ ID NO:190, the amino acid sequence of SEQ ID NO:96, the amino acid sequence of SEQ ID NO:191, the amino acid sequence of SEQ ID NO:99, the amino acid sequence of SEQ ID NO:100, and the amino acid sequences of SEQ ID NO:101;   (b) a second antigen-binding site that binds B-cell maturation antigen (BCMA) and comprises the amino acid sequence of SEQ ID NO:149, the amino acid sequence of SEQ ID NO:150, the amino acid sequence of SEQ ID NO:151, the amino acid sequence of SEQ ID NO:153, the amino acid sequence of SEQ ID NO:154, and the amino acid sequences of SEQ ID NO:155;   (c) an additional antigen-binding site that binds BCMA and comprises the amino acid sequence of SEQ ID NO:149, the amino acid sequence of SEQ ID NO:150, the amino acid sequence of SEQ ID NO:151, the amino acid sequence of SEQ ID NO:153, the amino acid sequence of SEQ ID NO:154, and the amino acid sequences of SEQ ID NO:155; and   (d) an antibody Fc domain or a portion thereof sufficient to bind CD16.   
     
     
         80 . A nucleic acid encoding:
 (a) a polypeptide comprising the amino acid sequence of SEQ ID NO:162;   (b) a polypeptide comprising the amino acid sequence of SEQ ID NO:163; or   (c) a polypeptide comprising the amino acid sequence of SEQ ID NO:165.   
     
     
         81 . The nucleic acid of  claim 80 , wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:162. 
     
     
         82 . The nucleic acid of  claim 80 , wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:163. 
     
     
         83 . The nucleic acid of  claim 80 , wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:165. 
     
     
         84 . A method of producing the protein according to  claim 58 , wherein the method comprises:
 (a) culturing a host cell under conditions suitable for expression of the protein, wherein the host cell comprises a first nucleic acid encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO:162, a second nucleic acid encoding a second polypeptide comprising the amino acid sequence of SEQ ID NO:163, and a third nucleic acid encoding a third polypeptide comprising the amino acid sequence of SEQ ID NO:165; and   (b) isolating and purifying the protein.   
     
     
         85 . A method of treating BCMA-expressing cancer comprising administering a therapeutically effective amount of a protein to a patient in need thereof, wherein the protein comprises:
 (a) a first antigen-binding site comprising a single-chain variable fragment (scFv) that binds NKG2D and comprises the amino acid sequence of SEQ ID NO:190, the amino acid sequence of SEQ ID NO:96, the amino acid sequence of SEQ ID NO:191, the amino acid sequence of SEQ ID NO:99, the amino acid sequence of SEQ ID NO:100, and the amino acid sequences of SEQ ID NO:101;   (b) a second antigen-binding site that binds B-cell maturation antigen (BCMA) and comprises the amino acid sequence of SEQ ID NO:149, the amino acid sequence of SEQ ID NO:150, the amino acid sequence of SEQ ID NO:151, the amino acid sequence of SEQ ID NO:153, the amino acid sequence of SEQ ID NO:154, and the amino acid sequences of SEQ ID NO:155;   (c) an additional antigen-binding site that binds BCMA and comprises the amino acid sequence of SEQ ID NO:149, the amino acid sequence of SEQ ID NO:150, the amino acid sequence of SEQ ID NO:151, the amino acid sequence of SEQ ID NO:153, the amino acid sequence of SEQ ID NO:154, and the amino acid sequences of SEQ ID NO:155; and   (d) an antibody Fc domain or a portion thereof sufficient to bind CD16.   
     
     
         86 . The method according to  claim 85 , wherein the BCMA-expressing cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, B cell lymphomas, or acute myeloid leukemia.

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