Antibodies with modified isoelectric points

Abstract

The invention relates generally to compositions and methods for altering the isoelectric point of an antibody, and in some cases, resulting in improved plasma pharmacokinetics, e.g. increased serum half-life in vivo.

1 . An antibody comprising a variant heavy chain constant domain having the formula: 
       
         
           
                 
               
                   A-X 119 -T-K-G-P-S-V-F-P-L-A-P-X 131 -S-X 133 -S-T-S-X 137 - 
                 
                     
                 
                   X 138 -T-A-A-L-G-C-L-V-K-D-Y-F-P-E-P-V-T-V-S-W-N-S- 
                 
                     
                 
                   G-A-L-X 164 -S-G-V-H-T-F-P-A-V-L-Q-S-S-G-L-Y-S-L-S- 
                 
                     
                 
                   S-V-V-T-V-P-S-S-X 192 -X 193 -G-T-X 196 -T-Y-X 199 -C-N-V- 
                 
                     
                 
                   X 203 -H-X 205 -P-S-X 208 -T-X 210 -V-D-K-X 214 -V-E-X 217 -K-X 219 - 
                 
                     
                 
                   X 220 -X 221 -X 222 -X 223 -X 224 -X 225 -C-P-P-C-P-A-P-X 233 -X 234 - 
                 
                     
                 
                   X 235 -X 236 -G-P-S-V-F-L-F-P-P-K-P-K-D-T-L-M-I-S-R-T-P- 
                 
                     
                 
                   E-V-T-C-V-V-V-D-V-S-H-E-D-P-E-V-X 274 -F-N-W-Y-V-D-G- 
                 
                     
                 
                   V-E-V-H-N-A-K-T-K-P-R-E-E-Q-X 296 -N-S-T-X 300 -R-V-V-S- 
                 
                     
                 
                   V-L-T-V-X 309 -H-Q-D-W-L-N-G-K-E-Y-X 320 -C-X 322 -V-S-N- 
                 
                     
                 
                   X 326 -X 327 -L-P-A-P-I-E-X 334 -T-I-S-K-X 339 -K-G-Q-P-R-E-P- 
                 
                     
                 
                   Q-V-Y-T-L-P-P-S-X 355 -E-E-M-T-K-N-Q-V-S-L-T-C-L-V-K- 
                 
                     
                 
                   G-F-Y-P-S-D-I-A-V-E-W-E-S-X 384 -G-Q-P-E-N-N-Y-X 392 -T- 
                 
                     
                 
                   T-P-P-X 397 -L-D-S-D-G-S-F-F-L-Y-S-K-L-T-V-D-K-S-R-W- 
                 
                     
                 
                   Q-X 419 -G-N-V-F-S-C-S-V-X 428 -H-E-A-L-H-X 434 -H-Y-T-Q-K- 
                 
                     
                 
                   S-L-S-L-S-P-G-X-X-X-X 447 , 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein X 119  is selected from the group consisting of S and E; 
         wherein X 131  is selected from the group consisting of S and C; 
         wherein X 133  is selected from the group consisting of K, R, E, and Q; 
         wherein X 137  is selected from the group consisting of G and E; 
         wherein X 138  is selected from the group consisting of G and S; 
         wherein X 164  is selected from the group consisting of T and E; 
         wherein X 192  is selected from the group consisting of S and N; 
         wherein X 193  is selected from the group consisting of L and F; 
         wherein X 196  is selected from the group consisting of Q and K; 
         wherein X 199  is selected from the group consisting of I and T; 
         wherein X 203  is selected from the group consisting of N and D; 
         wherein X 205  is selected from the group consisting of K, E, and Q; 
         wherein X 208  is selected from the group consisting of N and D; 
         wherein X 210  is selected from the group consisting of K, E, and Q; 
         wherein X 214  is selected from the group consisting of K and T; 
         wherein X 217  is selected from the group consisting of P and R; 
         wherein X 219  is selected from the group consisting of S and C; 
         wherein X 220  is selected from the group consisting of C, PLG, and G; 
         wherein X 221  is selected from the group consisting of D and a deletion; 
         wherein X 222  is selected from the group consisting of K, V, and T; 
         wherein X 223  is selected from the group consisting of T and a deletion; 
         wherein X 224  is selected from the group consisting of H and E; 
         wherein X 225  is selected from the group consisting of T and a deletion; 
         wherein X 233  is selected from the group consisting of E and P; 
         wherein X 234  is selected from the group consisting of L and V; 
         wherein X 235  is selected from the group consisting of L, A, and a deletion; 
         wherein X 236  is selected from the group consisting of G, A, and a deletion; 
         wherein X 274  is selected from the group consisting of K, Q, and E; 
         wherein X 296  is selected from the group consisting of Y and F; 
         wherein X 300  is selected from the group consisting of Y and F; 
         wherein X 309  is selected from the group consisting of L and V; 
         wherein X 320  is selected from the group consisting of K and E; 
         wherein X 322  is selected from the group consisting of K and E; 
         wherein X 326  is selected from the group consisting of K and E; 
         wherein X 327  is selected from the group consisting of A and G; 
         wherein X 334  is selected from the group consisting of K and E; 
         wherein X 339  is selected from the group consisting of A and T; 
         wherein X 355  is selected from the group consisting of R, Q, and E; 
         wherein X 384  is selected from the group consisting of N and S; 
         wherein X 392  is selected from the group consisting of K, N, and E; 
         wherein X 397  is selected from the group consisting of V and M; 
         wherein X 419  is selected from the group consisting of Q and E; 
         wherein X 428  is selected from the group consisting of M and L; 
         wherein X 434  is selected from the group consisting of N and S; and 
         wherein X 447  is selected from the group consisting of K, DEDE, and a deletion; 
       
       wherein said variant heavy chain constant domain comprises at least 6 mutations as compared to SEQ ID NO: 2, said variant heavy chain constant domain is not SEQ ID NO: 3, and wherein said numbering is according to EU index. 
     
     
         2 . An antibody according to  claim 1  wherein said variant heavy chain constant domain comprises at least 10 mutations as compared to SEQ ID NO: 2. 
     
     
         3 . An antibody according to  claim 1  wherein said variant heavy chain constant domain comprises at least 15 mutations as compared to SEQ ID NO: 2. 
     
     
         4 . An antibody according to  claim 1 ,  2  or  3  further comprising a light chain constant domain. 
     
     
         5 . An antibody according to  claim 4  wherein said light chain constant domain is SEQ ID NO: 112. 
     
     
         6 . An antibody according to  claim 4  wherein said light chain constant domain is a variant light chain constant domain having the formula: 
       
         
           
                 
               
                   X 108 -T-V-A-A-P-S-V-F-I-F-P-P-S-D-E-X 124 -L-X 126- S-G-T- 
                 
                     
                 
                   A-S-V-V-C-L-L-N-X 138 -F-Y-P-R-E-A-X 145 -V-Q-W-K-V-D- 
                 
                     
                 
                   X 152 -A-L-Q-X 156 -G-N-S-Q-E-S-V-T-E-Q-D-S-X 169 -D-S-T- 
                 
                     
                 
                   Y-S-L-S-S-T-L-T-L-S-K-A-D-Y-E-K-H-K-V-Y-A-C-E-V- 
                 
                     
                 
                   T-H-X 199 -G-L-X 202 -S-P-V-T-X 207 -S-F-N-R-G-E-X 214 -X 215 - 
                 
                     
                 
                   X 216 -X 217 , 
                 
             
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein X 108  is selected from the group consisting of R and Q; 
         wherein X 124  is selected from the group consisting of Q and E; 
         wherein X 126  is selected from the group consisting of K, E, and Q; 
         wherein X 138  is selected from the group consisting of N and D; 
         wherein X 145  is selected from the group consisting of K, E, Q, and T; 
         wherein X 152  is selected from the group consisting of N and D; 
         wherein X 156  is selected from the group consisting of S and E; 
         wherein X 169  is selected from the group consisting of K, E, and Q; 
         wherein X 199  is selected from the group consisting of Q and E; 
         wherein X 202  is selected from the group consisting of S and E; and 
         wherein X 207  is selected from the group consisting of K and E; and 
         wherein X 214  is selected from the group consisting of C and CDEDE, 
       
       wherein said variant light chain constant domain comprises at least 2 mutations as compared SEQ ID NO: 112. 
     
     
         7 . An antibody according to  claim 7  wherein said antibody is selected from the group consisting of:
 a) an antibody comprising SEQ ID NO:52 in the heavy chain constant domain and SEQ ID NO:117 in the light chain constant domain; 
 b) an antibody comprising SEQ ID NO:98 in the heavy chain constant domain and SEQ ID NO:152 in the light chain constant domain; 
 c) an antibody comprising SEQ ID NO:66 in the heavy chain constant domain and SEQ ID NO:152 in the light chain constant domain; and 
 d) an antibody comprising SEQ ID NO:191; and 
 e) an antibody comprising SEQ ID NO:98 in the heavy chain constant domain and SEQ ID NO:150 in the light chain constant domain. 
 
     
     
         8 . A nucleic acid encoding the antibody of  claims 1  to  7 . 
     
     
         9 . A host cell containing the nucleic acid of  claim 8 . 
     
     
         10 . A method of producing the antibody of any of  claims 1  to  7  comprising culturing a host cell of  claim 9  and recovering said antibody from the cell culture.