Antigen-binding molecule having regulated conjugation between heavy-chain and light-chain

Abstract

It was found that association between CH1 and CL can be suppressed by substituting amino acids that exist on the interface between CH1 and CL with electrically-charged amino acids, and that formation of heterogeneous molecules is enabled more efficiently than by introducing knobs into holes mutations into CH3 domain.

1 . An antigen-binding molecule in which association of the heavy chain and light chain is regulated, wherein
 one set or two or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) to (c) below in the heavy chain and light chain in the antigen-binding molecule are amino acid residues that mutually repel electrically:   
       (a) the amino acid residue comprised in the heavy chain constant region (CH1) at position 147 as indicated by EU numbering, and the amino acid residue comprised in the light chain constant region (CL) at position 180 as indicated by EU numbering; 
       (b) the amino acid residue comprised in CH1 at position 147 as indicated by EU numbering, and the amino acid residue comprised in CL at position 131 as indicated by EU numbering; and, 
       (c) the amino acid residue comprised in CH1 at position 175 as indicated by EU numbering, and the amino acid residue comprised in CL at position 160 as indicated by EU numbering. 
     
     
         2 . The antigen-binding molecule of  claim 1 , further wherein amino acid residues in the set of amino acid residues shown in (d) below are amino acid residues that mutually repel electrically: 
       (d) the amino acid residue comprised in CH1 at position 213 as indicated by EU numbering, and the amino acid residue comprised in CL at position 123 as indicated by EU numbering. 
     
     
         3 . The antigen-binding molecule of  claim 1  or  2 , wherein the amino acid residues that mutually repel electrically are selected from amino acid residues comprised in either set of (X) and (Y) below:
 (X) glutamic acid (E) or aspartic acid (D); and 
 (Y) lysine (K), arginine (R) or histidine (H). 
 
     
     
         4 . The antigen-binding molecule of any one of  claims 1  to  3 , further wherein two or more amino acid residues forming an interface between the heavy chain variable region and light chain variable region are amino acid residues that mutually repel electrically. 
     
     
         5 . The antigen-binding molecule of  claim 4 , wherein the amino acid residues that mutually repel electrically are one set or two sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) or (b):
 (a) the amino acid residue comprised in the heavy chain variable region at position 39 as indicated by Kabat numbering and the amino acid residue comprised in the light chain variable region at position 38 as indicated by Kabat numbering; or   (b) the amino acid residue comprised in the heavy chain variable region at position 45 as indicated by Kabat numbering, and the amino acid residue comprised in the light chain variable region at position 44 as indicated by Kabat numbering.   
     
     
         6 . The antigen-binding molecule of  claim 4  or  5 , wherein the amino acid residues that mutually repel electrically are selected from the amino acid residues comprised in either set of (X) and (Y) below:
 (X) glutamic acid (E) or aspartic acid (D); and 
 (Y) lysine (K), arginine (R), or histidine (H). 
 
     
     
         7 . An antigen-binding molecule in which association of the heavy chain and light chain is regulated, wherein
 one set or two or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) to (c) below in the associating heavy chain and light chain in the antigen-binding molecule are amino acid residues that do not mutually repel electrically:   
       (a) the amino acid residue comprised in the heavy chain constant region (CH1) at position 147 as indicated by EU numbering, and the amino acid residue comprised in the light chain constant region (CL) at position 180 as indicated by EU numbering; 
       (b) the amino acid residue comprised in CH1 at position 147 as indicated by EU numbering, and the amino acid residue comprised in CL at position 131 as indicated by EU numbering; and, 
       (c) the amino acid residue comprised in CH1 at position 175 as indicated by EU numbering, and the amino acid residue comprised in CL at position 160 as indicated by EU numbering. 
     
     
         8 . The antigen-binding molecule of  claim 7 , further wherein amino acid residues of the set of amino acid residues shown in (d) below are amino acid residues that do not mutually repel electrically: 
       (d) the amino acid residue comprised in CH1 at position 213 as indicated by EU numbering, and the amino acid residue comprised in CL at position 123 as indicated by EU numbering. 
     
     
         9 . The antigen-binding molecule of  claim 7  or  8 , wherein the amino acid residues that do not mutually repel electrically are amino acid residues selected from each of two sets selected from the group consisting of (X) to (Z) below, and wherein the two sets are selected from among the combinations of (X) and (Y), (X) and (Z), (Y) and (Z), and (Z) and (Z):
 (X) glutamic acid (E) or aspartic acid (D); 
 (Y) lysine (K), arginine (R) or histidine (H); and 
 (Z) alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), or valine (V). 
 
     
     
         10 . The antigen-binding molecule of any one of  claims 7  to  9 , wherein the amino acid residues that do not mutually repel electrically are the amino acid residue comprised in CH1 at position 175 as indicated by EU numbering which is lysine (K); and the amino acid residues comprised in CL at position 180, position 131, and position 160 as indicated by EU numbering which are all glutamic acid (E). 
     
     
         11 . The antigen-binding molecule of any one of  claims 7  to  9 , wherein the amino acid residues that do not mutually repel electrically are the amino acid residues comprised in CH1 at position 147 and position 175 as indicated by EU numbering which are glutamic acid (E); and the amino acid residues comprised in CL at position 180, position 131, and position 160 as indicated by EU numbering which are all lysine (K). 
     
     
         12 . The antigen-binding molecule of  claim 11 , further wherein the amino acid residue comprised in CH1 at position 213 as indicated by EU numbering is glutamic acid (E), and the amino acid residue comprised in CL at position 123 as indicated by EU numbering is lysine (K). 
     
     
         13 . The antigen-binding molecule of any one of  claims 7  to  12 , wherein two or more amino acid residues forming the interface between the heavy chain variable region and light chain variable region are amino acid residues that do not mutually repel electrically. 
     
     
         14 . The antigen-binding molecule of  claim 13 , wherein the amino acid residues that do not mutually repel electrically are one set or two sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) or (b) below:
 (a) the amino acid residue comprised in the heavy chain variable region at position 39 as indicated by Kabat numbering, and the amino acid residue comprised in the light chain variable region at position 38 as indicated by Kabat numbering; or   (b) the amino acid residue comprised in the heavy chain variable region at position 45 as indicated by Kabat numbering, and the amino acid residue comprised in the light chain variable region at position 44 as indicated by Kabat numbering.   
     
     
         15 . The antigen-binding molecule of  claim 13  or  14 , wherein the amino acid residues that do not mutually repel electrically are amino acid residues selected from each of two sets selected from the group consisting of (X) to (Z) below, and wherein the two sets are selected from among the combinations of (X) and (Y), (X) and (Z), (Y) and (Z), and (Z) and (Z):
 (X) glutamic acid (E) or aspartic acid (D); 
 (Y) lysine (K), arginine (R) or histidine (H); and 
 (Z) alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), or valine (V). 
 
     
     
         16 . The antigen-binding molecule of any one of  claims 1  to  15 , wherein the antigen-binding molecule is a bispecific antibody. 
     
     
         17 . A method for producing an antigen-binding molecule in which association of the heavy chain and light chain is regulated, comprising steps of (1) to (3) below:
 (1) modifying nucleic acids encoding the heavy chain constant region (CH1) and the light chain constant region (CL) so that one set or two or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) to (c) below mutually repel electrically:
 (a) the amino acid residue comprised in CH1 at position 147 as indicated by EU numbering, and the amino acid residue comprised in CL at position 180 as indicated by EU numbering; 
 (b) the amino acid residue comprised in CH1 at position 147 as indicated by EU numbering, and the amino acid residue comprised in CL at position 131 as indicated by EU numbering; and, 
 (c) the amino acid residue comprised in CH1 at position 175 as indicated by EU numbering, and the amino acid residue comprised in CL at position 160 as indicated by EU numbering, 
   (2) introducing the modified nucleic acids into a host cell and culturing the host cell so that it expresses the nucleic acids, and   (3) collecting the antigen-binding molecule from a culture of the host cell.   
     
     
         18 . The method for producing an antigen-binding molecule of  claim 17 , further comprising in step (1), modifying nucleic acids so that the amino acid residues in the set of amino acid residues shown in (d) below mutually repel electrically:
 (d) the amino acid residue comprised in CH1 at position 213 as indicated by EU numbering, and the amino acid residue comprised in CL at position 123 as indicated by EU numbering.   
     
     
         19 . The method for producing an antigen-binding molecule of  claim 17  or  18 , comprising in step (1), modifying nucleic acids so that the amino acid residues that mutually repel electrically are selected from among amino acid residues comprised in either group of (X) and (Y) below:
 (X) glutamic acid (E) or aspartic acid (D); and 
 (Y) lysine (K), arginine (R), or histidine (H). 
 
     
     
         20 . The method for producing an antigen-binding molecule of any one of  claims 17  to  19 , further comprising in step (1), modifying nucleic acids so that two or more amino acid residues forming the interface between the heavy chain variable region and light chain variable region are amino acid residues that mutually repel electrically. 
     
     
         21 . The method for producing an antigen-binding molecule of  claim 20 , wherein the amino acid residues that mutually repel electrically are amino acid residues of any one set selected from the group consisting of the sets of amino acid residues shown in (a) or (b) below:
 (a) the amino acid residue comprised in the heavy chain variable region at position 39 as indicated by Kabat numbering, and the amino acid residue comprised in the light chain variable region at position 38 as indicated by Kabat numbering; or   (b) the amino acid residue comprised in the heavy chain variable region at position 45 as indicated by Kabat numbering, and the amino acid residue comprised in the light chain variable region at position 44 as indicated by Kabat numbering.   
     
     
         22 . The method for producing an antigen-binding molecule of  claim 20  or  21 , wherein the amino acid residues that mutually repel electrically are selected from amino acid residues comprised in either set of (X) and (Y) below:
 (X) glutamic acid (E) or aspartic acid (D); and 
 (Y) lysine (K), arginine (R), or histidine (H). 
 
     
     
         23 . A method for producing an antigen-binding molecule in which association of the heavy chain and light chain is regulated, comprising the following steps of (1) to (3):
 (1) modifying nucleic acids encoding a heavy chain constant region (CH1) and a light chain constant region (CL) which associate so that one set or two or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) to (c) below do not mutually repel electrically:
 (a) the amino acid residue comprised in the heavy chain constant region (CH1) at position 147 as indicated by EU numbering, and the amino acid residue comprised in the light chain constant region (CL) at position 180 as indicated by EU numbering; 
 (b) the amino acid residue comprised in CH1 at position 147 as indicated by EU numbering, and the amino acid residue comprised in CL at position 131 as indicated by EU numbering; and, 
 (c) the amino acid residue comprised in CH1 at position 175 as indicated by EU numbering, and the amino acid residue comprised in CL at position 160 as indicated by EU numbering, 
   (2) introducing the modified nucleic acids into a host cell and culturing the host cell so that it expresses the nucleic acids, and   (3) collecting the antigen-binding molecule from a culture of the host cell.   
     
     
         24 . The method for producing an antigen-binding molecule of  claim 23 , further comprising in step (1), modifying nucleic acids so that the amino acid residues in the set of amino acid residues shown in (d) below do not mutually repel electrically:
 (d) the amino acid residue comprised in CH1 at position 213 as indicated by EU numbering, and the amino acid residue comprised in CL at position 123 as indicated by EU numbering.   
     
     
         25 . The method for producing an antigen-binding molecule of  claim 23  or  24 , comprising in step (1), modifying the nucleic acids so that the amino acid residues that do not mutually repel electrically are amino acids residues selected from each of two sets selected from the group consisting of (X) to (Z) below, and wherein the two sets are selected from among the combinations of (X) and (Y), (X) and (Z), (Y) and (Z), and (Z) and (Z):
 (X) glutamic acid (E) or aspartic acid (D); 
 (Y) lysine (K), arginine (R), or histidine (H); and 
 (Z) alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), or valine (V). 
 
     
     
         26 . The method for producing an antigen-binding molecule of any one of  claims 23  to  25 , comprising in step (1), modifying nucleic acids so that the amino acid residues that do not mutually repel electrically are the amino acid residue comprised in CH1 at position 175 as indicated by EU numbering which is lysine (K), and the amino acid residues comprised in CL at position 180, position 131, and position 160 as indicated by EU numbering which are all glutamic acid (E). 
     
     
         27 . The method for producing an antigen-binding molecule of any one of  claims 23  to  25 , comprising in step (1), modifying nucleic acids so that the amino acid residues that do not mutually repel electrically are the amino acid residues comprised in CH1 at position 147 and position 175 as indicated by EU numbering which are glutamic acid (E), and the amino acid residues comprised in CL at position 180, position 131, and position 160 as indicated by EU numbering which are all lysine (K). 
     
     
         28 . The method for producing an antigen-binding molecule of  claim 27 , further comprising modifying nucleic acids so that the amino acid residue comprised in CH1 at position 213 as indicated by EU numbering is glutamic acid (E), and the amino acid residue comprised in CL at position 123 as indicated by EU numbering is lysine (K). 
     
     
         29 . The method for producing an antigen-binding molecule of any one of  claims 23  to  28 , further comprising in step (1), modifying nucleic acids so that two or more amino acid residues forming the interface between the heavy chain variable region and light chain variable region are amino acid residues that do not mutually repel electrically. 
     
     
         30 . The method for producing an antigen-binding molecule of  claim 29 , wherein the amino acid residues that do not mutually repel electrically are amino acid residues of any one set selected from the group consisting of the sets of amino acid residues shown in (a) or (b) below:
 (a) the amino acid residue comprised in the heavy chain variable region at position 39 as indicated by Kabat numbering, and the amino acid residue comprised in the light chain variable region at position 38 as indicated by Kabat numbering; or   (b) the amino acid residue comprised in the heavy chain variable region at position 45 as indicated by Kabat numbering, and the amino acid residue comprised in the light chain variable region at position 44 as indicated by Kabat numbering.   
     
     
         31 . The method for producing an antigen-binding molecule of  claim 29  or  30 , wherein the amino acid residues that do not mutually repel electrically are amino acid residues selected from each of two sets selected from the group consisting of (X) to (Z) below, and wherein the two sets are selected from among the combinations of (X) and (Y), (X) and (Z), (Y) and (Z), and (Z) and (Z):
 (X) glutamic acid (E) or aspartic acid (D); 
 (Y) lysine (K), arginine (R) or histidine (H); and 
 (Z) alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), or valine (V). 
 
     
     
         32 . An antigen-binding molecule produced according to the method for producing an antigen-binding molecule of any one of  claims 17  to  31 . 
     
     
         33 . The antigen-binding molecule of  claim 32 , wherein the antigen-binding molecule is a bispecific antibody. 
     
     
         34 . A method for regulating association of the heavy chain and light chain of an antigen-binding molecule, comprising:
 modifying nucleic acids so that one set or two or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) to (c) below are amino acid residues that mutually repel electrically:   
       (a) the amino acid residue comprised in CH1 at position 147 as indicated by EU numbering, and the amino acid residue comprised in CL at position 180 as indicated by EU numbering; 
       (b) the amino acid residue comprised in CH1 at position 147 as indicated by EU numbering, and the amino acid residue comprised in CL at position 131 as indicated by EU numbering; and 
       (c) the amino acid residue comprised in CH1 at position 175 as indicated by EU numbering, and the amino acid residue comprised in CL at position 160 as indicated by EU numbering. 
     
     
         35 . The method of  claim 34 , further comprising modifying nucleic acids so that the amino acid residues in the set of amino acid residues shown in (d) below are amino acid residues that mutually repel electrically: 
       (d) the amino acid residue comprised in CH1 at position 213 as indicated by EU numbering, and the amino acid residue comprised in CL at position 123 as indicated by EU numbering. 
     
     
         36 . The method of  claim 34  or  35 , wherein the amino acid residues that mutually repel electrically are selected from amino acid residues comprised in either set of (X) and (Y) below:
 (X) glutamic acid (E) or aspartic acid (D); and 
 (Y) lysine (K), arginine (R), or histidine (H). 
 
     
     
         37 . The method of any one of  claims 34  to  36 , wherein two or more amino acid residues forming the interface between the heavy chain variable region and light chain variable region are amino acid residues that mutually repel electrically. 
     
     
         38 . The method of  claim 37 , wherein the amino acid residues that mutually repel electrically are amino acid residues of any one set selected from the group consisting of the sets of amino acid residues shown in (a) or (b) below:
 (a) the amino acid residue comprised in the heavy chain variable region at position 39 as indicated by Kabat numbering, and the amino acid residue comprised in the light chain variable region at position 38 as indicated by Kabat numbering; or   (b) the amino acid residue comprised in the heavy chain variable region at position 45 as indicated by Kabat numbering, and the amino acid residue comprised in the light chain variable region at position 44 as indicated by Kabat numbering.   
     
     
         39 . The method of  claim 37  or  38 , wherein the amino acid residues that mutually repel electrically are selected from amino acid residues comprised in either set of (X) and (Y) below:
 (X) glutamic acid (E) or aspartic acid (D); and 
 (Y) lysine (K), arginine (R), or histidine (H). 
 
     
     
         40 . A method for regulating association of the heavy chain and light chain of an antigen-binding molecule, comprising:
 modifying nucleic acids so that one set or two or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) to (c) below in the associating heavy chain and light chain in the antigen-binding molecule are amino acid residues that do not mutually repel electrically:   
       (a) the amino acid residue comprised in CH1 at position 147 as indicated by EU numbering, and the amino acid residue comprised in CL at position 180 as indicated by EU numbering; 
       (b) the amino acid residue comprised in CH1 at position 147 as indicated by EU numbering, and the amino acid residue comprised in CL at position 131 as indicated by EU numbering; and, 
       (c) the amino acid residue comprised in CH1 at position 175 as indicated by EU numbering, and the amino acid residue comprised in CL at position 160 as indicated by EU numbering. 
     
     
         41 . The method of  claim 40 , further comprising modifying nucleic acids so that the amino acid residues in the set of amino acid residues shown in (d) below are amino acid residues that do not mutually repel electrically: 
       (d) the amino acid residue comprised in CH1 at position 213 as indicated by EU numbering, and the amino acid residue comprised in CL at position 123 as indicated by EU numbering. 
     
     
         42 . The method of  claim 40  or  41 , wherein the amino acid residues that do not mutually repel electrically are amino acid residues selected from each of two sets selected from the group consisting of (X) to (Z) below, and wherein the two sets are selected from among the combinations of (X) and (Y), (X) and (Z), (Y) and (Z), and (Z) and (Z):
 (X) glutamic acid (E) or aspartic acid (D); 
 (Y) lysine (K), arginine (R), or histidine (H); and 
 (Z) alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), or valine (V). 
 
     
     
         43 . The method of any one of  claims 40  to  42 , wherein the amino acid residues that do not mutually repel electrically are the amino acid residue comprised in CH1 at position 175 as indicated by EU numbering which is lysine (K), and the amino acid residues comprised in CL at position 180, position 131, and position 160 as indicated by EU numbering which are all glutamic acid (E). 
     
     
         44 . The method of any one of  claims 40  to  42 , wherein the amino acid residues that do not mutually repel electrically are the amino acid residues comprised in CH1 at position 147 and position 175 as indicated by EU numbering which are glutamic acid (E), and the amino acid residues comprised in CL at position 180, position 131, and position 160 as indicated by EU numbering which are all lysine (K). 
     
     
         45 . The method of  claim 44 , wherein the amino acid residue comprised in CH1 at position 213 as indicated by EU numbering is glutamic acid (E), and the amino acid residue contained in CL at position 123 as indicated by EU numbering is lysine (K). 
     
     
         46 . The method of any one of  claims 34  to  45 , wherein the antigen-binding molecule is a bispecific antibody. 
     
     
         47 . A composition containing the antigen-binding molecule of any one of  claims 1  to  16 ,  32 , and  33 , and a pharmaceutically acceptable carrier. 
     
     
         48 . A nucleic acid encoding the antigen-binding molecule of any one of  claims 1  to  16 ,  32 , and  33 . 
     
     
         49 . A host cell having the nucleic acid of  claim 48 .