Non-human animals expressing pH-sensitive immunoglobulin sequences

• US 9,801,362 B2
• Filed: 03/15/2013
• Issued: 10/31/2017
• Est. Priority Date: 03/16/2012
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: McWhirter, John, Macdonald, Lynn, Martin, Joel H., Murphy, Andrew J.
• Primary Examiner(s): Babic, Christopher M
• Assistant Examiner(s): Aron, Kimberly A
• Application Number:
  US13/834,129
• Publication Number:
  US 20130247236A1
• Time in Patent Office: 1,691 Days
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2217/15 Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• 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 2317/10 characterized by their source of isolation or production
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2800/204 of bacterial origin, e.g. BAC

Abstract

Genetically modified non-human animals are provided that express an immunoglobulin variable domain that comprises at least one histidine, wherein the at least one histidine is encoded by a substitution of a non-histidine codon in the germline of the animal with a hisidine codon, or the insertion of a histidine codon in a germline immunoglobulin nucleic acid sequence. Immunoglobulin genes comprising histidines in one or more CDRs, in an N-terminal region, and or in a loop 4 region are also provided. Immunoglobulin variable domains comprising one or more histidines (e.g., histidine clusters) substituted for non-antigen-binding non-histidine residues. Non-human animals that are progeny of animals comprising modified heavy chain variable loci (V, D, J segments), modified light chain variable loci (V, J segments), and rearranged germline light chain genes (VJ sequences) are also provided. Non-human animals that make immunoglobulin domains that bind antigens in a pH-sensitive manner are provided.

Claims

1. A genetically modified non-human animal comprising in its germline an endogenous immunoglobulin locus that comprises an unrearranged immunoglobulin variable gene sequence comprising in a complementarity determining region 3 (CDR3) encoding sequence a substitution of at least one non-histidine codon with a histidine codon or an insertion of at least one histidine codon,

wherein the non-human animal further comprises in vivo a diverse repertoire of antibodies, each of which is specific for an antigen of interest and retains at least one histidine amino acid encoded by the substituted or inserted histidine codon, wherein the at least one histidine amino acid is present in a CDR3 of a variable domain.

2. The non-human animal of claim 1, wherein the non-human animal comprises a first and a second endogenous immunoglobulin variable locus, wherein at least the first or the second immunoglobulin variable locus comprises an unrearranged immunoglobulin variable gene sequence comprising in a CDR3 encoding sequence the insertion of the at least one histidine codon or the substitution of the at least one non-histidine codon with a histidine codon.

3. The non-human animal of claim 2, wherein both the first and the second endogenous immunoglobulin variable locus each comprise an unrearranged immunoglobulin variable gene sequence comprising in a CDR3 encoding sequence the substitution of the at least one histidine codon or the insertion of the at least one non-histidine codon with a histidine codon.

4. The non-human animal of claim 2, wherein the first endogenous immunoglobulin variable locus comprises at least a functional portion of an unrearranged immunoglobulin heavy chain variable gene sequence comprising unrearranged V_H, unrearranged D_H, and unrearranged J_H gene segments.

5. The non-human animal of claim 4, wherein the unrearranged immunoglobulin heavy chain variable gene sequence comprises at least a portion of a human immunoglobulin heavy chain variable gene sequence comprising at least one unrearranged human V_H gene segment, unrearranged human D_H gene segment, and unrearranged human J_H gene segment.

6. The non-human animal of claim 5, wherein the unrearranged immunoglobulin heavy chain variable gene sequence comprising unrearranged human V_H, unrearranged human D_H, and unrearranged human J_H gene segments is operably linked to an endogenous non-human immunoglobulin heavy chain constant region nucleic acid sequence.

7. The non-human animal of claim 6, wherein the unrearranged immunoglobulin heavy chain variable gene sequence comprising unrearranged human V_H, unrearranged human D_H, and unrearranged human J_H gene segments is operably linked to the endogenous non-human immunoglobulin heavy chain constant region nucleic acid sequence at the endogenous immunoglobulin locus.

8. The genetically modified non-human animal of claim 5, wherein the unrearranged immunoglobulin heavy chain gene sequence comprises D gene segments that are inverted with respect to the direction of orientation of the immunoglobulin heavy chain gene sequence.

9. The non-human animal of claim 5, wherein the at least one unrearranged human D_H gene segment comprises the at least one inserted or substituted histidine codon.

10. The non-human animal of claim 4, wherein the unrearranged immunoglobulin heavy chain variable gene sequence is an unrearranged human immunoglobulin heavy chain gene sequence comprising unrearranged human V_H gene segments, a synthetic D_H segment that comprises a linker, and an unrearranged human J_H gene segment.

11. The non-human animal of claim 10, wherein the synthetic D_H segment comprises the at least one inserted or substituted histidine codon.

12. The non-human animal of claim 11, wherein the endogenous heavy chain locus comprises a replacement of 90-100% of all endogenous non-human V_H gene segments and all endogenous non-human D_H and J_H gene segments with the at least one unrearranged human V_H gene segment, the synthetic D segment, and the at least one unrearranged human J_H gene segment, respectively.

13. The non-human animal of claim 12, wherein the synthetic D segment comprises a sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3 and SEQ ID NO;

4.

14. The non-human animal of claim 13, wherein the non-human animal is a rodent.

15. The non-human animal of claim 14, wherein the rodent is a mouse or a rat.

16. The non-human animal of claim 2, wherein the second endogenous immunoglobulin variable locus comprises at least a functional portion of an unrearranged immunoglobulin light chain variable gene sequence comprising unrearranged V_L and unrearranged J_L gene segments.

17. The non-human animal of claim 16, wherein the immunoglobulin light chain variable gene sequence comprising unrearranged V_L and J_L gene segments is operably linked to an endogenous non-human immunoglobulin light chain constant region sequence at the endogenous non-human immunoglobulin locus.

18. The non-human animal of claim 17, wherein the unrearranged immunoglobulin light chain variable region gene sequence comprises at least a portion of a human immunoglobulin light chain variable sequence.

19. The non-human animal of claim 18, wherein the unrearranged immunoglobulin light chain variable region gene sequence comprises no more than two human V_L gene segments.

20. The non-human animal of claim 19, wherein each of the no more than two human V_L gene segments comprises in a CDR3 encoding sequence a substitution of at least one non-histidine codon with a histidine codon or an insertion of at least one histidine codon.

21. The non-human animal of claim 20, wherein the endogenous light chain locus comprises a replacement of an endogenous light chain variable region gene sequence with the no more than two human V_L gene segments.

22. The non-human animal of claim 21, wherein the non-human animal is a rodent.

23. The non-human animal of claim 22, wherein the rodent is a mouse or a rat.

24. The non-human animal of claim 16, wherein the unrearranged light chain gene sequence is an unrearranged human light chain gene sequence comprising unrearranged human V_L and unrearranged human J_L gene segments.

25. The non-human animal of claim 2, wherein the second endogenous immunoglobulin variable locus comprises a rearranged immunoglobulin light chain variable gene sequence comprising a V_L gene segment rearranged with a J_L gene segment.

26. The non-human animal of claim 25, wherein the rearranged immunoglobulin light chain variable gene sequence is a rearranged human immunoglobulin light chain variable gene sequence comprising a V_L gene segment rearranged with a J_L gene segment.

27. The non-human animal of claim 2, wherein at least one of the first endogenous immunoglobulin variable locus or the second endogenous immunoglobulin variable locus is operably linked to an endogenous non-human immunoglobulin constant region nucleic acid sequence at the endogenous immunoglobulin locus.

28. The non-human animal of claim 1, further comprising at least a second inserted or substituted histidine codon in a sequence that encodes a variable domain selected from an N-terminal region of an immunoglobulin chain, a loop 4 region of an immunoglobulin chain, a complementarity determining region 1 (CDR1) of an immunoglobulin heavy chain, a complementarity determining region 2 (CDR2) of an immunoglobulin heavy chain, a CDR3 of an immunoglobulin heavy chain, a CDR1 of an immunoglobulin light chain, a CDR2 of an immunoglobulin light chain, a CDR3 of an immunoglobulin light chain, and a combination thereof.

29. The genetically modified non-human animal of claim 1, wherein an immunoglobulin variable region CDR3 encoding sequence in the immunoglobulin variable locus comprises 2, 3, 4, or 5 histidines that are substitutions of a histidine codon for a non-histidine codon and/or insertions of histidine codons.

30. The non-human animal of claim 1, wherein the histidine amino acid is not encoded by a corresponding germline gene segment in a wild-type non-human animal.

31. A genetically modified non-human animal, comprising

at an endogenous immunoglobulin heavy chain locus at least a portion of an unrearranged human immunoglobulin heavy chain variable region nucleic acid sequence comprising unrearranged human V_H, unrearranged human D_H, and unrearranged human J_H gene segments operably linked to an immunoglobulin heavy chain constant region gene sequence, wherein one or more of the unrearranged human V_H, D_H, and J_H gene segments comprise in a CDR3 encoding sequence at least one substitution of a non-histidine codon with a histidine codon, or at least one histidine codon insertion;

and

at an endogenous immunoglobulin light chain locus at least a portion of an unrearranged human immunoglobulin light chain variable region nucleic acid sequence comprising unrearranged human V_L and unrearranged human J_L gene segments operably linked to an immunoglobulin light chain constant region gene sequence, wherein one or more of the unrearranged human V_L and J_L gene segments comprise in a CDR3 encoding sequence at least one substitution of a non-histidine codon with a histidine codon, or at least one histidine codon insertion;

wherein the non-human animal comprises a diverse repertoire of antibodies, each of which is specific for an antigen of interest and retains at least one histidine amino acid encoded by the substituted or inserted histidine codon, wherein the at least one histidine amino acid is present in a CDR3 of an immunoglobulin heavy chain variable domain and/or in a CDR3 of an immunoglobulin light chain variable domain.

32. The genetically modified non-human animal of claim 31, wherein the non-human animal is a mammal.

33. The genetically modified mammal of claim 32, wherein the mammal is a rodent.

34. The rodent of claim 33, wherein the rodent is selected from the group consisting of a mouse, a rat, and a hamster.

35. The genetically modified non-human animal of claim 31, wherein the immunoglobulin heavy chain constant region gene sequence is an endogenous non-human immunoglobulin heavy chain constant region sequence at the endogenous immunoglobulin heavy chain locus.

36. The genetically modified non-human animal of claim 31, wherein the immunoglobulin light chain constant region gene sequence is an endogenous non-human immunoglobulin light chain constant region nucleic acid sequence at the endogenous light chain locus.

37. A genetically modified non-human animal, comprising

at an endogenous heavy chain locus at least a portion of a human unrearranged immunoglobulin heavy chain variable region nucleic acid sequence comprising unrearranged human V_H, unrearranged human D_H, and unrearranged human J_H gene segments operably linked to an immunoglobulin heavy chain constant region gene sequence, wherein one or more of the unrearranged V_H, D_H, and J_H gene segments comprise in a CDR3 encoding sequence at least one substitution of a non-histidine codon with a histidine codon, or at least one histidine codon insertion;

and

at an endogenous light chain locus a rearranged human immunoglobulin light chain variable region nucleic acid sequence comprising a human V_L gene segment rearranged with a human J_L gene segment operably linked to an immunoglobulin light chain constant region gene sequence, wherein the rearranged human immunoglobulin light chain variable region nucleic acid sequence comprises in a CDR3 encoding sequence at least one substitution of a non-histidine codon with a histidine codon, or at least one histidine codon insertion;

wherein the non-human animal comprises in vivo a diverse repertoire of antibodies, each of which are specific for an antigen of interest and retains at least one histidine amino acid encoded by the substituted or inserted histidine codon, wherein the at least one histidine amino acid is present in a CDR3 of an immunoglobulin heavy chain variable domain and/or in a CDR3 of an immunoglobulin light chain variable domain.

38. The genetically modified non-human animal of claim 37, which is a mammal.

39. The genetically modified mammal of claim 38, which is a rodent.

40. The rodent of claim 39, selected from the group consisting of a mouse, a rat, and a hamster.

41. The genetically modified non-human animal of claim 37, wherein the immunoglobulin heavy chain constant region gene sequence is an endogenous non-human immunoglobulin heavy chain constant region sequence at the endogenous heavy chain locus.

42. The genetically modified non-human animal of claim 37, wherein the immunoglobulin light chain constant region gene sequence is an endogenous non-human immunoglobulin light chain constant region sequence at the endogenous immunoglobulin light chain locus.

43. The non-human animal of claim 37, wherein the histidine amino acid is not encoded by a corresponding human germline gene segment in a wild-type non-human animal.