ANTIGEN-BINDING MOLECULE HAVING REGULATED CONJUGATION BETWEEN HEAVY-CHAIN AND LIGHT-CHAIN

• US 20140370020A1
• Filed: 10/31/2012
• Published: 12/18/2014
• Est. Priority Date: 10/31/2011
• Status: Active Grant

• Current Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Original Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Inventors: Kuramochi, Taichi, Kawazoe, Meiri, Hironiwa, Naoka, Igawa, Tomoyuki
• Granted Patent: US 11,851,476 B2
• Time in Patent Office: Days
• US Class Current: 424/136.1
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 16/303 Liver or Pancreas
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/522 CH1 domain

Abstract

It was found that association between CH1 and CL can be suppressed by substituting amino acids that exist on the interface between CH 1 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.

Claims

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).

16. The antigen-binding molecule of any one of claims 1 to 15, 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.

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).

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).

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.

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).

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).

46. The method of any one of claims 34 to 45, wherein the antigen-binding molecule is a bispecific antibody.

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).