US2022380459A1PendingUtilityA1

Multi-specific binding proteins that bind cd33, nkg2d, and cd16, and methods of use

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Assignee: DRAGONFLY THERAPEUTICS INCPriority: Feb 20, 2018Filed: May 3, 2022Published: Dec 1, 2022
Est. expiryFeb 20, 2038(~11.6 yrs left)· nominal 20-yr term from priority
Inventors:Gregory P. ChangAnn F. CheungAsya GrinbergDhruv Kam SethiWilliam HaneyBianka PrinzBradley M. LundeRonnie WeiDaniel FallonSteven O'Neil
C07K 2317/73C07K 2317/92C07K 2317/622C07K 2317/77C07K 2317/31C07K 16/2851A61P 35/00C07K 2317/33C07K 16/283A61K 2039/505C07K 16/2803C07K 2317/55C07K 2317/34C07K 2317/565C07K 2317/526C07K 2317/70C07K 2317/522
71
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Claims

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.

Claims

exact text as granted — not AI-modified
1 - 46 . (canceled) 
     
     
         47 . A protein comprising:
 (a) a first antigen-binding site comprising a Fab fragment that binds NKG2D, wherein the Fab fragment comprises the amino acid sequence of SEQ ID NO:114, the amino acid sequence of SEQ ID NO:115, the amino acid sequence of SEQ ID NO:116, the amino acid sequence of SEQ ID NO:111, the amino acid sequence of SEQ ID NO:112, and the amino acid sequences of SEQ ID NO:113;   (b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds CD33, wherein the scFv comprises the amino acid sequence of SEQ ID NO:48, the amino acid sequence of SEQ ID NO:49, the amino acid sequence of SEQ ID NO:50, the amino acid sequence of SEQ ID NO:45, the amino acid sequence of SEQ ID NO:46, and the amino acid sequences of SEQ ID NO:47; and   (c) an antibody Fc domain or a portion thereof sufficient to bind CD16.   
     
     
         48 . The protein according to  claim 47 , wherein the C-terminus of the Fab fragment is linked to the antibody Fc domain or a portion thereof sufficient to bind CD16. 
     
     
         49 . The protein according to  claim 47 , wherein the first antigen-binding site that binds NKG2D comprises a heavy chain variable domain amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:87 and a light chain variable domain amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:88. 
     
     
         50 . The protein according to  claim 47 , wherein the light chain variable domain of the scFv of the second antigen-binding site is positioned at the N-terminus of the heavy chain variable domain of the scFv. 
     
     
         51 . The protein according to  claim 50 , wherein the light chain variable domain of the scFv of the second antigen-binding site is linked to the heavy chain variable domain of the scFv via a linker, and wherein the linker amino acid sequence comprises the amino acid sequence of (GlyGlyGlyGlySer) 4  ((G4S) 4 ) (SEQ ID NO:186). 
     
     
         52 . The protein according to  claim 47 , wherein a disulfide bridge is formed between the heavy chain variable domain of the scFv and the light chain variable domain of the scFv. 
     
     
         53 . The protein according to  claim 47 , wherein the C-terminus of the heavy chain variable domain of the scFv of the second antigen-binding site is linked to the N-terminus of the antibody Fc domain or a portion thereof. 
     
     
         54 . The protein according to  claim 53 , wherein the C-terminus of the heavy chain variable domain of the scFv of the second antigen-binding site is linked to the N-terminus of the antibody Fc domain or a portion thereof via a hinge comprising Ala-Ser or Gly-Ala-Ser. 
     
     
         55 . The protein according to  claim 47 , wherein the scFv of the second 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:9 and a light chain variable domain amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:10. 
     
     
         56 . The protein according to  claim 47 , wherein the scFv of the second antigen-binding site comprises the amino acid sequence of SEQ ID NO:188. 
     
     
         57 . The protein according to  claim 47 , wherein the antibody Fc domain sufficient to bind CD16 comprises a hinge region and a CH2 domain of a human IgG1 antibody. 
     
     
         58 . The protein according to  claim 47 , 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. 
     
     
         59 . The protein according to  claim 47 , 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 from the Fc domain of a human IgG1 at one or more positions that is Q347, Y349, T350, L351, 5354, E356, E357, K360, Q362, 5364, T366, L368, K370, N390, K392, T394, D399, 5400, D401, F405, Y407, K409, T411, or K439, numbered according to the Kabat numbering. 
     
     
         60 . The protein according to  claim 47 , wherein the antibody Fc domain sufficient to bind CD16 is an Fc domain of a human IgG1 comprising substitutions for forming a heterodimer. 
     
     
         61 . The protein according to  claim 47 , 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. 
     
     
         62 . The protein according to  claim 47 , 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. 
     
     
         63 . The protein according to  claim 47 , wherein the protein comprises:
 (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:189;   (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:187; 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, wherein the third polypeptide is linked to the first polypeptide via a disulfide bond.   
     
     
         64 . The protein according to  claim 47 , wherein the first antigen-binding site comprising a Fab fragment that binds NKG2D, the second antigen-binding site comprising a single-chain variable fragment (scFv) that binds CD33 and the antibody Fc domain or a portion thereof sufficient to bind CD16 are linked as represented in  FIG.  19   . 
     
     
         65 . The protein according to  claim 47 , wherein the first antigen-binding site comprising a Fab fragment that binds NKG2D, the second antigen-binding site comprising a single-chain variable fragment (scFv) that binds CD33 and the antibody Fc domain or a portion thereof sufficient to bind CD16 are linked as represented in  FIG.  44   . 
     
     
         66 . A method of producing the protein according to  claim 47 , 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 second polypeptide comprising the amino acid sequence of SEQ ID NO:189, a second nucleic acid encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO:187, and a third nucleic acid encoding a third polypeptide comprising the amino acid sequence of SEQ ID NO:190; and   (b) isolating and purifying the protein.   
     
     
         67 . A method of treating CD33-expressing cancer comprising administering a therapeutically effective amount of the protein according to  claim 47  to a patient in need thereof. 
     
     
         68 . The method according to  claim 67 , wherein the CD33-expressing cancer is selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), myeloid blast crisis of CIVIL, acute lymphoblastic leukemia (ALL), acute lymphoblastic lymphoma, myeloproliferative neoplasms (MPNs), lymphoma, non-Hodgkin lymphomas, and classical Hodgkin lymphoma. 
     
     
         69 . A protein comprising:
 (a) a first antigen-binding site comprising a Fab fragment that binds NKG2D, wherein the Fab fragment comprises the amino acid sequence of SEQ ID NO:114, the amino acid sequence of SEQ ID NO:115, the amino acid sequence of SEQ ID NO:116, the amino acid sequence of SEQ ID NO:192, the amino acid sequence of SEQ ID NO:112, and the amino acid sequences of SEQ ID NO:193;   (b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds CD33, wherein the scFv comprises the amino acid sequence of SEQ ID NO:48, the amino acid sequence of SEQ ID NO:49, the amino acid sequence of SEQ ID NO:50, the amino acid sequence of SEQ ID NO:181, the amino acid sequence of SEQ ID NO:46, and the amino acid sequences of SEQ ID NO:182; and   (c) an antibody Fc domain or a portion thereof sufficient to bind CD16.   
     
     
         70 . A pharmaceutical composition comprising a protein and a pharmaceutically acceptable carrier, wherein the protein comprises:
 (a) a first antigen-binding site comprising a Fab fragment that binds NKG2D, wherein the Fab fragment comprises the amino acid sequence of SEQ ID NO:114, the amino acid sequence of SEQ ID NO:115, the amino acid sequence of SEQ ID NO:116, the amino acid sequence of SEQ ID NO:111, the amino acid sequence of SEQ ID NO:112, and the amino acid sequences of SEQ ID NO:113;   (b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds CD33, wherein the scFv comprises the amino acid sequence of SEQ ID NO:48, the amino acid sequence of SEQ ID NO:49, the amino acid sequence of SEQ ID NO:50, the amino acid sequence of SEQ ID NO:45, the amino acid sequence of SEQ ID NO:46, and the amino acid sequences of SEQ ID NO:47; and   (c) an antibody Fc domain or a portion thereof sufficient to bind CD16.   
     
     
         71 . A nucleic acid encoding:
 (a) a polypeptide comprising the amino acid sequence of SEQ ID NO:189;   (b) a polypeptide comprising the amino acid sequence of SEQ ID NO:187; or   (c) a polypeptide comprising the amino acid sequence of SEQ ID NO:190.   
     
     
         72 . The nucleic acid of  claim 71 , wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:189. 
     
     
         73 . The nucleic acid of  claim 71 , wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:187. 
     
     
         74 . The nucleic acid of  claim 71 , wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:190.

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