Bispecific binding proteins

Abstract

The present disclosure provides bispecific proteins that bind to two antigens, as well as their compositions, uses, and methods of making.

1 . A bispecific binding protein capable of binding CD40 and capable of binding mesothelin, comprising two polypeptides of formula (II):
   scFv X -H-Fc-L-scFv Y   (II),
   wherein
 L is a polypeptide linker, 
 H is a hinge region, 
 Fc comprises CH2 and CH3 regions of an immunoglobulin, 
 scFv X  and scFv Y  are each independently a single chain variable fragment, 
 scFv X  specifically binds a first antigen, 
 scFv Y  specifically binds a second antigen, and 
 one of the two antigens is CD40 and the other is mesothelin. 
   
     
     
         2 . The bispecific binding protein of  claim 1 , wherein scFv X - has the structure of formula (V) or (VI):
   V H   X -L X -V L   X -  (V),
     V L   X -L X -V H   X -  (VI),
   wherein V H   X  is a variable heavy chain, V L   X  is a variable light chain, and L X  is a polypeptide linker.   
     
     
         3 . The bispecific binding protein of  claim 1 , wherein -scFv Y  has the structure of formula (VII) or (VIII):
   -V L   Y -L Y -V H   Y   (VII),
     -V H   Y -L Y -V L   Y   (VIII),
   wherein V H   Y  is a variable heavy chain, V L   Y  is a variable light chain, and L Y  is a polypeptide linker.   
     
     
         4 . The bispecific binding protein of  claim 1 , wherein L has an amino acid sequence comprising any one of SEQ ID NOS:251 and 253-262. 
     
     
         5 . The bispecific binding protein of  claim 2 , wherein L X  and L Y  each has an amino acid sequence comprising any one of SEQ ID NOS:301-306. 
     
     
         6 . The bispecific binding protein of  claim 1 , wherein the scFv that binds CD40 comprises a V H  having a CDR-H1 according to SEQ ID NO:25, a CDR-H2 according to SEQ ID NO:26, and a CDR-H3 according to SEQ ID NO:27, and a V L  having a CDR-L1 according to SEQ ID NO:55, a CDR-L2 according to SEQ ID NO:56, and a CDR-L3 according to SEQ ID NO:57. 
     
     
         7 . (canceled) 
     
     
         8 . The bispecific binding protein of  claim 1 , wherein the scFv that binds CD40 comprises a V H  according to SEQ ID NO: 17 and a V L  according to SEQ ID NO:44. 
     
     
         9 .- 11 . (canceled) 
     
     
         12 . The bispecific binding protein of  claim 1 , wherein the scFv that binds mesothelin comprises a V H  having a CDR-H1 according to SEQ ID NO:241, a CDR-H2 according to SEQ ID NO:242, and a CDR-H3 according to SEQ ID NO:243, and a V L  having a CDR-L1 according to SEQ ID NO:244, a CDR-L2 according to SEQ ID NO:245, and a CDR-L3 according to SEQ ID NO:246. 
     
     
         13 . (canceled) 
     
     
         14 . The bispecific binding protein of  claim 1 , wherein the scFv that binds mesothelin comprises a V H  according to SEQ ID NO:120 and a V L  according to SEQ ID NO:137. 
     
     
         15 . The bispecific binding protein of  claim 1 , wherein the scFv that binds mesothelin comprises a V H  according to SEQ ID NO:120 and a V L  according to SEQ ID NO:141. 
     
     
         16 .- 18 . (canceled) 
     
     
         19 . The bispecific binding protein of  claim 1 , wherein the two polypeptides each comprise the amino acid sequence of SEQ ID NO:406. 
     
     
         20 . The bispecific binding protein of  claim 1 , wherein the two polypeptides each comprise the amino acid sequence of SEQ ID NO:407. 
     
     
         21 . The bispecific binding protein of  claim 1 , wherein the two polypeptides each comprise the amino acid sequence of SEQ ID NO:408. 
     
     
         22 . (canceled) 
     
     
         23 . The bispecific binding protein of  claim 1 , wherein the two polypeptides each comprise the amino acid sequence of SEQ ID NO:419. 
     
     
         24 . The bispecific binding protein of  claim 1 , wherein the two polypeptides each comprise the amino acid sequence of SEQ ID NO:420. 
     
     
         25 . (canceled) 
     
     
         26 . A pharmaceutical composition comprising a bispecific binding protein of any  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         27 . A method of treating a cancer, comprising administering to a patient in need thereof a bispecific binding protein of  claim 1 , optionally wherein the cancer is a solid tumor. 
     
     
         28 . A bispecific binding protein capable of binding 4-1BB and capable of binding prostate-specific membrane antigen, comprising two polypeptides of formula (II):
   scFv X -H-Fc-L-scFv Y   (II),
   wherein
 L is a polypeptide linker, 
 H is a hinge region, 
 Fc comprises CH2 and CH3 regions of an immunoglobulin, 
 scFv X  and scFv Y  are each independently a single chain variable fragment, 
 scFv X  specifically binds a first antigen, 
 scFv Y  specifically binds a second antigen, and 
 one of the two antigens is 4-1BB and the other is prostate-specific membrane antigen. 
   
     
     
         29 .- 32 . (canceled) 
     
     
         33 . The bispecific binding protein of  claim 28 , wherein the scFv that binds 4-1BB comprises a V H  having a CDR-H1 according to SEQ ID NO:280, a CDR-H2 according to SEQ ID NO:281, and a CDR-H3 according to SEQ ID NO:282, and a V L  having a CDR-L1 according to SEQ ID NO:283, a CDR-L2 according to SEQ ID NO:284, and a CDR-L3 according to SEQ ID NO:285. 
     
     
         34 . (canceled) 
     
     
         35 . The bispecific binding protein of  claim 33 , wherein the scFv that binds 4-1BB comprises a V H  according to SEQ ID NO:69 and a V L  according to SEQ ID NO:89. 
     
     
         36 . The bispecific binding protein of  claim 33 , wherein the scFv that binds 4-1BB comprises a V H  according to SEQ ID NO:71 and a V 1  according to SEQ ID NO:94. 
     
     
         37 . The bispecific binding protein of  claim 28 , wherein the scFv that binds prostate-specific membrane antigen comprises a V H  according to any one of SEQ ID NOS:191-197 and a V L  according to any one of SEQ ID NOS:201-207. 
     
     
         38 .- 40 . (canceled) 
     
     
         41 . The bispecific binding protein of  claim 28 , wherein the two polypeptides each comprise the amino acid sequence of SEQ ID NO:447. 
     
     
         42 . (canceled) 
     
     
         43 . A pharmaceutical composition comprising a bispecific binding protein of  claim 28 , and a pharmaceutically acceptable carrier. 
     
     
         44 . A method of treating a cancer, comprising administering to a patient in need thereof a bispecific binding protein of  claim 28 , optionally wherein the cancer is a solid tumor. 
     
     
         45 . A bispecific binding protein capable of binding CD40 and capable of binding mesothelin, comprising two polypeptides of formula (III):
   Fab-H-Fc-L-scFv Y   (III),
   wherein
 L is a polypeptide linker, 
 H is a hinge region, 
 Fc comprises CH2 and CH3 regions of an immunoglobulin, 
 Fab is a fragment antigen-binding region, 
 scFv Y  is a single chain variable fragment, 
 Fab specifically binds a first antigen, 
 scFv Y  specifically binds a second antigen, and 
 the first antigen is mesothelin and the second antigen is CD40. 
   
     
     
         46 .- 48 . (canceled) 
     
     
         49 . The bispecific binding protein of  claim 45 , wherein the scFv that binds CD40 comprises a Vii having a CDR-H1 according to SEQ ID NO:25, a CDR-H2 according to SEQ ID NO:26, and a CDR-H3 according to SEQ ID NO:27, and a V L  having a CDR-L1 according to SEQ ID NO:55, a CDR-L2 according to SEQ ID NO:56, and a CDR-L3 according to SEQ ID NO:57. 
     
     
         50 .- 51 . (canceled) 
     
     
         52 . The bispecific binding protein of  claim 45 , wherein the scFv that binds CD40 comprises a Vii according to SEQ ID NO:22 and a V L  according to SEQ ID NO:48. 
     
     
         53 . The bispecific binding protein of  claim 45 , wherein the scFv that binds CD40 comprises a V H  according to SEQ ID NO:23 and a V L  according to SEQ ID NO:49. 
     
     
         54 . (canceled) 
     
     
         55 . The bispecific binding protein of  claim 45 , wherein the Fab that binds mesothelin comprises a V H  according to SEQ ID NO: 120 and a V L  according to SEQ ID NO:137. 
     
     
         56 . The bispecific binding protein of  claim 45 , wherein the Fab that binds mesothelin comprises a Vii according to SEQ ID NO:129 and a V L  according to SEQ ID NO:143. 
     
     
         57 . (canceled) 
     
     
         58 . The bispecific binding protein of  claim 45 , wherein the two polypeptides each comprise the heavy chain amino acid sequence of SEQ ID NO: 359 and the light chain amino acid sequence of SEQ ID NO: 371. 
     
     
         59 . The bispecific binding protein of  claim 45 , wherein the two polypeptides each comprise the heavy chain amino acid sequence of SEQ ID NO: 360 and the light chain amino acid sequence of SEQ ID NO: 371. 
     
     
         60 .- 61 . (canceled) 
     
     
         62 . A pharmaceutical composition comprising a bispecific binding protein of  claim 45 , and a pharmaceutically acceptable carrier. 
     
     
         63 . A method of treating a cancer, comprising administering to a patient in need thereof a bispecific binding protein of  claim 45 , optionally wherein the cancer is a solid tumor. 
     
     
         64 . A bispecific binding protein comprising two polypeptides of formula (I):
   X-H-Fc-L-scFv Y   (I),
   wherein
 X is scFv X  or a Fab region, 
 wherein X specifically binds a first antigen and scFv Y  specifically binds a second antigen, 
 H is a hinge region, 
 Fc comprises CH2 and CH3 regions of an immunoglobulin, 
 L is a polypeptide linker, 
 scFv X  and scFv Y  are each independently a single chain variable fragment, 
 one of the two antigens is an immunomodulatory protein and the other is a tumor antigen, and 
 the bispecific binding protein activates the immunomodulatory protein. 
   
     
     
         65 . (canceled) 
     
     
         66 . The bispecific binding protein of  claim 64 , wherein the immunomodulatory protein is selected from CD40, 4-1BB, TNFR2, ICOS, TRAILR1, and TRAILR2. 
     
     
         67 .- 68 . (canceled) 
     
     
         69 . The bispecific binding protein of  claim 64 , wherein the tumor antigen is selected from mesothelin, nectin-4, epidermal growth factor receptor, prostate-specific membrane antigen, and B7-H4. 
     
     
         70 . (canceled) 
     
     
         71 . The bispecific binding protein of  claim 64 , wherein the first antigen is an immunomodulatory protein and the second antigen is a tumor antigen. 
     
     
         72 . (canceled) 
     
     
         73 . A pharmaceutical composition comprising a bispecific binding protein of  claim 64 , and a pharmaceutically acceptable carrier. 
     
     
         74 . (canceled) 
     
     
         75 . A nucleic acid comprising a nucleotide sequence encoding a bispecific binding protein of  claim 1 . 
     
     
         76 .- 77 . (canceled) 
     
     
         78 . A eukaryotic host cell transformed with the vector comprising the nucleic acid of  claim 75 . 
     
     
         79 .- 80 . (canceled) 
     
     
         81 . A method of producing a bispecific binding protein, comprising: (a) culturing the host cell of  claim 78  and (b) recovering the protein. 
     
     
         82 . A method of activating the immune system, comprising administering to a patient in need thereof a bispecific binding protein of  claim 1 .