Bispecific antibody exhibiting increased alternative fviii-cofactor-function activity

Abstract

The present invention provides light chain amino acid substitutions that improve the FVIII cofactor function-substituting activity of ACE910 (Emicizumab), novel light chains showing FVIII cofactor function-substituting activity, and heavy chain amino acid substitutions that improve the FVIII cofactor function-substituting activity of novel light chain-containing bispecific antibodies.

1 . A polypeptide comprising an antibody light chain variable domain, wherein the polypeptide comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47, in which one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, I29, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and197, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine. 
     
     
         2 . A bispecitic antibody which recognizes FIX and/or FIXa, and FX, wherein the antibody comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47, in which one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, 129, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and197, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine. 
     
     
         3 . A bispecific antibody which recognizes FIX and/or FIXa, and FX, wherein a first polypeptide and a third polypeptide form a pair and a second polypeptide and a fourth polypeptide form a pair, wherein the first polypeptide comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 45 and the second polypeptide comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 46, wherein either the third polypeptide or the fourth polypeptide comprises the antibody light chain variable domain amino acid sequence of SEQ If) NO: 47 and the other polypeptide comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47, and wherein in the other polypeptide one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, 129, E30, R31, Q32, L33, A34, Q50A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and 197, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine. 
     
     
         4 . A polypeptide comprising an antibody heavy chain variable domain, wherein the polypeptide comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 45, in which one or more amino acids selected from the group consisting of Y31, Y32, D33, 134, Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine. 
     
     
         5 . A bispecific antibody which recognizes FIX and/or FIXa, and FX, wherein the antibody comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or comprises an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 45, in which one or more amino acids selected from the group consisting of Y31, Y32, D33, 134. Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine. 
     
     
         6 . A polypeptide comprising an antibody heavy chain variable domain, wherein the polypeptide comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 46, in which one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, I51, N52, T52a, R53, S54, G55, G56, S57, 158, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine. 
     
     
         7 . A bispecific antibody which recognizes FIX and/or FIXa, and FX, wherein the antibody comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or comprises an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 46, in which one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, I51, N52, T52a, R53, S54, G55, G56, S57, I58, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, numbered according to the Kabat numbering system, have been substituted with an arbitrary amino acids other than cysteine. 
     
     
         8 . A bispecific antibody which recognizes FIX and/or FIXa, and FX, wherein a first polypeptide and a third polypeptide form a pair and a second polypeptide and a fourth polypeptide form a pair, wherein the second polypeptide comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 46, the third polypeptide comprises the antibody light chain amino acid sequence of SEQ ID NO: 42, and the fourth polypeptide comprises the antibody light chain amino acid sequence of SEQ ID NO: 44, and the first polypeptide comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 45, and wherein in the first polypeptide one or more amino acids selected from the group consisting of Y31, Y32, D33, I34, Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine. 
     
     
         9 . A bispecific antibody which recognizes FIX and/or FIXa, and FX, wherein a first polypeptide and a third polypeptide form a pair and a second polypeptide and a fourth polypeptide form a pair, wherein the first polypeptide comprises the antibody heavy chain amino acid sequence of SEQ ID NO: 45, the third polypeptide comprises the antibody light chain amino acid sequence of SEQ ID NO: 43, and the fourth polypeptide comprises the antibody light chain amino acid sequence of SEQ ID NO: 44, and the second polypeptide comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ NOs: 4, 5, and 6, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 46, and wherein in the second polypeptide one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, 151, T52a, N52, R53, S54, G55, G56, S57, 158, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine. 
     
     
         10 . An antibody light chain, which comprises any one of the amino acid sequences selected from the following (a1) to (a6), (b1) to (b23), and (c1) to (c3):
 (a1) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 13 (QNK131);   (a2) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 14 (QNK284);   (a3) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 15 (QNK315);   (a4) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 16 (QNL182);   (a5) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 17 (QNL492);   (a6) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 18 (QNL576);   (b1) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 19 (JNK131);   (b2) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 20 (JNK163);   (b3) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 21 (JNK252);   (b4) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 22 (JNK263);   (b5) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 23 (JNK339);   (b6) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 24 (JNK348);   (b7) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 25 (JNK351);   (b8) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 26 (JNK:360);   (b9) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 27 (JNK378);   (b10) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 28 (JNK382);   (b11) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 29 (JNL036);   (b12) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 30 (JNL072);   (b13) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 31 (JNL095);   (b14) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 32 (JNL176);   (b15) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 33 (JNL208);   (b16) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 34 (JNL224);   (b17) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 35 (JNL260);   (b18) an antibody light chain comprising the amino acid sequence of SEQ NO: 36 (JNL056);   (b19) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 37 (JNL059);   (b20) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 38 (JNL226);   (b21) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 39 (JNL250);   (b22) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 40 (JNL263);   (b23) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 41 (JNL281);   (c1) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 42 (QAL187);   (c2) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 43 (QAL201); and   (c3) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 44 (JYL280).   
     
     
         11 . A bispecific antibody which recognizes FIX andlor FIXa, and FX, wherein a first polypeptide (d) and a third polypeptide (f) form a pair, a second polypeptide (e) and a fourth polypeptide (g) form a pair, and wherein each of the polypeptides is the following polypeptide:
 (d) the first polypeptide is (d1) or (d2):
 (d1) a polypeptide which comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ NOs: 1, 2, and 3, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 45; 
 (d2) a polypeptide which comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 45, in which one or more amino acids selected from the group consisting of Y31, Y32, D33, I34, Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, numbered according to the Kabat numbefing system, have been substituted with arbitrary amino acids other than cysteine; 
   (e) the second polypeptide is (e1) or (e2):
 (e1) a polypeptide which comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 46; 
 (e2) a polypeptide Which comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 46, in which one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, I51, 152a, N52, R53, S54, G55, G56, S57, I58, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine; 
   (f) the third polypeptide is (f1), (f2), or (f3):
 (f1) a polypeptide which comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47; 
 (f2) a polypeptide which comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47, in which one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, I29, E30, R31, Q32, I33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine; 
 (f3) the polypeptide of any one of (a1) to (a6) and (c1) to (c2) described in  claim 10 ; and 
   (g) the fourth polypeptide is (g1), (g2), or (g3):
 (g1) a polypeptide which comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47; 
 (g2) a polypeptide which comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47, in which one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, I29, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine; 
 (g3) the polypeptide of any one of (b1) to (b23), and (c3) described in  claim 10 . 
   
     
     
         12 . A method of producing a variant of Emicizumab, which comprises the following step (a):
 (a) performing one or more of the following substitutions (i) to (iii), wherein the numbering is according to the Kabat numbering system:
 (i) substitution of one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, 129, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and197, in an antibody light chain variable domain comprising the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively; 
 (ii) substitution of one or more amino acids selected. from the group consisting of Y31, Y32, D33, 134, Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, in an antibody heavy chain variable domain comprising the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ NOs: 1, 2, and 3, respectively; and 
 (iii) substitution of one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, I51, T52a, N52, R53, S54, G55, G56, S57, I58, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, in an antibody heavy chain variable domain comprising the heavy chain CDR 1, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. 
   
     
     
         13 . A method of isolating a variant of Emicizumab, which comprises the steps of (a) to (c) below:
 (a) producing a variant of Emicizumab by performing one or more of the following (i) to (iii), wherein the numbering is according to the Kabat numbering system:
 (i) substitution of one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, I29, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, in an antibody light chain variable domain comprising the light chain CDR I, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively; 
 (ii) substitution of one or more amino acids selected from the group consisting of Y31, Y32, D33, 134, Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, in an antibody heavy chain variable domain comprising the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; and 
 (iii) substitution of one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, I51, T52a, N52, R53, S54, G55, G56, S57, I58, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, in an antibody heavy chain variable domain comprising the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; 
   (b) measuring FVIII cofactor function-substituting activity of the variant produced in (a); and   (c) selecting a variant of Emicizumab having improved FVIII cofactor function-substituting activity compared to that of Emicizumab.