Anti-cd73 antibodies and methods of use thereof

Abstract

The instant disclosure provides antibodies that specifically bind to CD73 (e.g., human CD73) and antagonize CD73 function. Also provided are anti-CD73 antibodies that further comprise a TGFβ-binding moiety or a VEGF-binding moiety. The instant disclosure additionally provides pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject using these antibodies.

What is claimed: 
     
         1 . An isolated polynucleotide encoding a heavy chain variable region or a light chain variable region of an antibody, wherein the heavy chain variable region comprises complementarity determining regions (CDRs) CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein:
 (a) CDRH1 comprises the amino acid sequence of X 1 X 2 WX 3 X 4 (SEQ ID NO: 13), wherein
 X 1  is S or N; 
 X 2  is S or Y; 
 X 3  is I or M; and 
 X 4  is N or H; 
   (b) CDRH2 comprises the amino acid sequence of X 1 IYPRX 2 X 3 DTNYX 4 X 5 KFKX 6  (SEQ ID NO: 14), wherein
 X 1  is R or T; 
 X 2  is N, A, or S; 
 X 3  is G or S; 
 X 4  is N, A, or S; 
 X 5  is G or Q; and 
 X 6  is D or G; 
   (c) CDRH3 comprises the amino acid sequence of LLDYSMDY (SEQ ID NO: 7);   (d) CDRL1 comprises the amino acid sequence of RASQDISX 1 X 2 LN (SEQ ID NO: 16), wherein
 X 1  is N or I; and 
 X 2  is Y or S; 
   (e) CDRL2 comprises the amino acid sequence of YTSRLHS (SEQ ID NO: 10); and   (f) CDRL3 comprises the amino acid sequence of QQGNTLPXT (SEQ ID NO: 17), wherein:
 X is L or W. 
   
     
     
         2 . The isolated polynucleotide of  claim 1 , wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 2, 4, 7, 8, 10, and 12; 2, 5, 7, 8, 10, and 12; 1, 3, 7, 8, 10, and 11; 2, 4, 7, 8, 10, and 11; 2, 4, 7, 9, 10, and 12; 2, 6, 7, 8, 10, and 12; 2, 5, 7, 9, 10, and 12; or 2, 6, 7, 9, 10, and 12. 
     
     
         3 . The isolated polynucleotide of  claim 2 , wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 2, 5, 7, 8, 10, and 12. 
     
     
         4 . The isolated polynucleotide of  claim 1 , wherein:
 (a) the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-21 and 23-30;   (b) the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-35 and 37-40; and/or   (c) the heavy chain variable region and the light chain variable region, respectively, comprise the amino acid sequences set forth in SEQ ID NOs: 24 and 37; 27 and 37; 19 and 33; 20 and 34; 21 and 35; 23 and 37; 23 and 38; 24 and 38; 25 and 37; 26 and 37; 28 and 37; 25 and 38; 26 and 38; 27 and 38; 28 and 38; 29 and 37; 30 and 37; 23 and 39; or 23 and 40.   
     
     
         5 . The isolated polynucleotide of  claim 4 , wherein the heavy chain variable region and the light chain variable region, respectively, comprise the amino acid sequences set forth in SEQ ID NOs: 27 and 37. 
     
     
         6 . The isolated polynucleotide of  claim 1 , wherein the antibody comprises:
 (a) (i) a heavy chain constant region selected from the group consisting of human IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 ,
 (ii) an IgG 1  heavy chain constant region comprising an N297A mutation, numbered according to the EU numbering system, 
 (iii) a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-58, or 
 (iv) a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 81, 82, 85, 86, 115, 117, 119, 121, and 127; 
   (b) (i) a light chain constant region selected from the group consisting of human kappa light chain and human lambda light chain,
 (ii) a light chain constant region comprising the amino acid sequence of SEQ ID NO: 93, or 
 (iii) a light chain comprising the amino acid sequence of SEQ ID NO: 97; or 
   (c) a heavy chain and a light chain comprising the amino acid sequences selected from the group consisting of SEQ ID NOs: 81 and 97; 82 and 97; 85 and 97; 86 and 97; 115 and 97; 117 and 97; 119 and 97; 121 and 97; and 127 and 97, respectively.   
     
     
         7 . The isolated polynucleotide of  claim 1 , wherein the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 85; and a light chain comprising the amino acid sequence of SEQ ID NO: 97. 
     
     
         8 . The isolated polynucleotide of  claim 1 , wherein the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 115, and a light chain comprising the amino acid sequence of SEQ ID NO: 97. 
     
     
         9 . The isolated polynucleotide of  claim 1 , wherein the polynucleotide further encodes a TGFβ-binding moiety selected from the group consisting of:
 (i) an extracellular domain of a human TGFβ receptor; and 
 (ii) an antibody that specifically binds to TGFβ. 
 
     
     
         10 . The isolated polynucleotide of  claim 9 , wherein the extracellular domain of the human TGFβ receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 109-112. 
     
     
         11 . The isolated polynucleotide of  claim 9 , wherein the extracellular domain of the human TGFβ receptor comprises the amino acid sequence of SEQ ID NO: 111. 
     
     
         12 . The isolated polynucleotide of  claim 9 , wherein the polynucleotide further encodes a peptide linker comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 103-108. 
     
     
         13 . The isolated polynucleotide of  claim 1 , wherein the antibody comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 117, 119, and 121, and/or a light chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 97. 
     
     
         14 . The isolated polynucleotide of  claim 1 , wherein the polynucleotide further encodes a VEGF-binding moiety selected from the group consisting of:
 (i) an extracellular domain of a human VEGF receptor; and   (ii) an antibody that specifically binds to VEGF.   
     
     
         15 . The isolated polynucleotide of  claim 14 , wherein the extracellular domain of the human VEGF receptor comprises the amino acid sequence of SEQ ID NO: 122. 
     
     
         16 . The isolated polynucleotide of  claim 14 , wherein the polynucleotide further encodes a peptide linker comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 103-108. 
     
     
         17 . The isolated polynucleotide of  claim 1 , wherein the antibody comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 127, and/or a light chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 97. 
     
     
         18 . A vector comprising:
 (a) the polynucleotide of  claim 1 ;   (b) the polynucleotide of  claim 1  and a second polynucleotide encoding an extracellular domain of a human TGFβ receptor; or   (c) the polynucleotide of  claim 1  and a second polynucleotide encoding an extracellular domain of a human VEGF receptor.   
     
     
         19 . A recombinant host cell comprising the polynucleotide of  claim 1 . 
     
     
         20 . A recombinant host cell comprising a first polynucleotide encoding a heavy chain variable region or a heavy chain of an antibody and a second polynucleotide encoding a light chain variable region or a light chain of the antibody, wherein the antibody comprises: a heavy chain variable region comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region of SEQ ID NO: 27; and a light chain variable region comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region of SEQ ID NO: 37. 
     
     
         21 . The recombinant host cell of  claim 20 , wherein:
 (a) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 2, 5, 7, 8, 10, and 12;   (b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 27 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 37;   (c) the heavy chain comprises the amino acid sequence of SEQ ID NO: 85 or 115; and   the light chain comprises the amino acid sequence of SEQ ID NO: 97; and/or   (d) the recombinant host cell further comprises a polynucleotide encoding an extracellular domain of a human TGFβ or VEGF receptor.   
     
     
         22 . The recombinant host cell of  claim 20 , wherein the antibody comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 117, 119, 121, and 127, and/or a light chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 97. 
     
     
         23 . A lipid nanoparticle comprising the polynucleotide of  claim 1 . 
     
     
         24 . A method of producing an antibody, the method comprising culturing the host cell of  claim 19  under suitable conditions so that the polynucleotide is expressed and the antibody is produced. 
     
     
         25 . A method of producing an antibody, the method comprising culturing the host cell of  claim 20  under suitable conditions so that the polynucleotide is expressed and the antibody is produced.