Interleukin-21 mutein/ anti-pd-1 antibody conjugates

Abstract

Provided herein are IL-21 muteins and fusion proteins comprising the same for use in methods of treating a disease. Related conjugates, nucleic acids, vectors, host cells, pharmaceutical compositions and kits are also provided herein. Methods of making the IL-21 muteins and fusion proteins comprising the same, as well as methods of treating a subject in need thereof, are provided by the present disclosure. Further provided are PD-1 antigen-binding proteins.

1 .- 51 . (canceled) 
     
     
         52 . A nucleic acid comprising a nucleotide sequence encoding an IL-21 mutein, wherein the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 2, 
       
         
           
                 
               
                   (SEQ ID NO: 2) 
                 
                   
                     QGQDX HMXXM XXXXX XVDXL KNXVN DLVPE FLPAP EDVET 
                   
                 
                     
                 
                   
                     NCEWS AFSCF QKAQL KSANT GNNEX XIXXX XXXLX XXXXX 
                   
                 
                     
                 
                   
                     TNAGR RQKHR LTCPS CDSYE KKPPK EFLXX FXXLL XXMXX 
                   
                 
                     
                 
                     QHXSS RTHGS EDS , 
                 
             
                
                
                
                
                
                
                
                
               
            
           
         
         wherein “X” represents any amino acid, and 
       
       and wherein said IL-21 mutein amino acid sequence differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by at least 1 amino acid. 
     
     
         53 . A vector comprising the nucleic acid of  claim 52 . 
     
     
         54 . A host cell comprising the nucleic acid of  claim 52 . 
     
     
         55 . A kit comprising a nucleic acid of  claim 52  and a container. 
     
     
         56 . (canceled) 
     
     
         57 . A method of making an IL-21 mutein, the method comprising:
 culturing the host cell of  claim 54  so as to express the IL-21 mutein; and   harvesting the expressed IL-21 mutein.   
     
     
         58 . (canceled) 
     
     
         59 . (canceled) 
     
     
         60 . The nucleic acid of  claim 52 , wherein the IL-21 mutein comprises an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by no more than 7 amino acids, or by 3, 4, 5, or 6 amino acids, optionally, by 1 or 2 amino acids. 
     
     
         61 . The nucleic acid of  claim 52 , wherein the difference(s) between the amino acid sequence of the IL-21 mutein and the amino acid sequence of SEQ ID NO: 1 occur(s) within (A) amino acids 10 −15 , inclusive, or amino acids 105-123, inclusive, of SEQ ID NO: 2, optionally, wherein the difference(s) occur(s) at amino acids 11, 14, 15, 109, 110, 112, 113, 116, 119, 120, and/or 123 of SEQ ID NO: 2 or (B) amino acids 5-25, inclusive, or amino acids 65-80, inclusive, of SEQ ID NO: 2, optionally, wherein the difference(s) occur(s) at amino acids 5, 8, 9, 12, 13, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, and/or 80 of SEQ ID NO: 2. 
     
     
         62 . The nucleic acid of  claim 60 , wherein the IL-21 mutein comprises an amino acid sequence with one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), optionally, wherein the amino acid substitution occurs at position 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123 of SEQ ID NO: 1, or at position 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123 of SEQ ID NO: 1. 
     
     
         63 . The nucleic acid of  claim 52 , wherein the IL-21 mutein comprises an amino acid substitution:
 a. at position 5, 8, 9, 12, 14, 15, 65, 66, 69, 70, 72, 73, 75, 76, 77, 80, 116, or 119 of SEQ ID NO: 1, wherein the substitute amino acid is an aliphatic amino acid   b. at position 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, 110, 112, 116, 117, 119, 120, or 123 of SEQ ID NO: 1, wherein the substitute amino acid is an acidic amino acid;   c. at position 5, 9, 73, 76, 109, 113, or 116 of SEQ ID NO: 1, wherein the substitute amino acid is a basic amino acid;   d. at position 5, 8, 9, 70, or 76 of SEQ ID NO: 1, wherein the substitute amino acid is an aromatic amino acid;   e. at position 5, 8, 9, 12, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, wherein the substitute amino acid is an amino acid comprising a side chain amide;   f. at position 5, 8, 9, 11, 12, 14, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, wherein the substitute amino acid is a non-aromatic amino acid comprising a side chain hydroxyl;   g. at position 65, 66, 69, 70, 72, 73, 75, 76, 77, or 80 of SEQ ID NO: 1, wherein the substitute amino acid is an imino acid;   h. at position 5, 9, 15, 76, 116, or 119 of SEQ ID NO: 1, wherein the substitute amino acid is an amino acid comprising a sulfur-containing side chain; or   i. or a combination thereof.   
     
     
         64 . The nucleic acid of  claim 62 , wherein the IL-21 mutein comprises an amino acid substitution with an amino acid at the position according to TABLE A: 
       
         
           
                 
                 
               
                   TABLE A 
                 
                     
                 
                   Amino Acid position 
                   Amino Acid 
                 
                   of SEQ ID NO: 1 
                   (in single letter code) 
                 
                     
                 
                     
                 
                 
                 
               
                   5 
                   A, D, E, G, H, I, K, L, M, 
                 
                     
                   N, Q, S, T, V, or Y 
                 
                   8 
                   A, D, E, N, S, T, V, or Y 
                 
                   9 
                   A, D, E, G, H, I, K, L, M, 
                 
                     
                   N, Q, S, T, V, or Y 
                 
                   11 
                   D or S 
                 
                   12 
                   A, D, E, N, S, T, or V 
                 
                   13 
                   D 
                 
                   14 
                   A, D, or S 
                 
                   15 
                   A, E, I, M, N, Q, S, T, or V 
                 
                   16 
                   D or E 
                 
                   19 
                   D 
                 
                   23 
                   D 
                 
                   65 
                   D, G, or P 
                 
                   66 
                   D, G, or P 
                 
                   68 
                   Q 
                 
                   69 
                   D, G, or P 
                 
                   70 
                   E, G, P, or Y 
                 
                   71 
                   L 
                 
                   72 
                   D, G, or P 
                 
                   73 
                   A, D, E, G, H, I, N, P, Q, S,  
                 
                     
                   or V 
                 
                   75 
                   D, G, or P 
                 
                   76 
                   A, D, E, G, H, I, K, L, M, 
                 
                     
                   N, P, Q, S, T, V, or Y 
                 
                   77 
                   D, G, or P 
                 
                   78 
                   D 
                 
                   79 
                   D 
                 
                   80 
                   G, or P 
                 
                   109 
                   K 
                 
                   110 
                   D 
                 
                   112 
                   D 
                 
                   113 
                   K 
                 
                   116 
                   A, D, E, I, K, L, M, N, S, T,  
                 
                     
                   or V 
                 
                   117 
                   D 
                 
                   119 
                   A, D, E, M, N, Q, S, or T 
                 
                   120 
                   D 
                 
                   123 
                   D 
                 
                     
                 
             
                
                
                
                
                
               
               
                
               
            
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         65 . The nucleic acid of  claim 52 , wherein the IL-21 mutein comprises an amino acid sequence of any one of SEQ ID NOs: 3-21, 23-56, 58-112, 114-198, 249-254, and 283, or an amino acid sequence of any one of SEQ ID NOs: 199 to 208, 210 to 222, 224 to 248, and 255. 
     
     
         66 . The nucleic acid of  claim 60 , wherein the IL-21 mutein comprises an amino acid sequence with two amino acid substitutions relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), wherein the amino acid substitution occurs at two of positions 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123 of SEQ ID NO: 1. 
     
     
         67 . The nucleic acid of  claim 66 , wherein the amino acid substitutions occur at two of positions 5, 9, 15, 70, 71, 72, 73, and 76 of SEQ ID NO: 1, or at two of positions 5, 9, 73, and 76 or SEQ ID NO: 1, optionally, wherein one of the substitutions occurs at position 76 of SEQ ID NO: 1. 
     
     
         68 . The nucleic acid of  claim 66 , wherein the IL-21 mutein comprises an amino acid substitution at position 5, 9, or 73 of SEQ ID NO: 1, wherein the substitute amino acid is an aliphatic amino acid or acidic amino acid, optionally, wherein the aliphatic amino acid is alanine or wherein the acidic amino acid is glutamic acid. 
     
     
         69 . The nucleic acid of  claim 66 , wherein the IL-21 mutein comprises an amino acid substitution at position 5 of SEQ ID NO: 1, wherein the substitute amino acid is an amino acid with a side chain amide, optionally, glutamine. 
     
     
         70 . The nucleic acid of  claim 52 , wherein the IL-21 mutein comprises an amino acid sequence of SEQ ID NO: 244.