Non-human animals expressing pH-sensitive immunoglobulin sequences

• US 9,648,856 B2
• Filed: 11/20/2013
• Issued: 05/16/2017
• Est. Priority Date: 03/16/2012
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Martin, Joel H., McWhirter, John, Murphy, Andrew J., MacDonald, Lynn
• Primary Examiner(s): Babic, Christopher M
• Assistant Examiner(s): Aron, Kimberly A
• Application Number:
  US14/085,424
• Publication Number:
  US 20140082760A1
• Time in Patent Office: 1,273 Days
• Field of Search: None
• 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 histidine 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 method for making a non-human animal that makes antibody variable domains with a histidine amino acid encoded by a histidine codon substituted or inserted in the germline genome, comprising:

modifying the non-human animal in its germline to comprise either or both

(i) at an endogenous non-human immunoglobulin heavy chain locus an unrearranged immunoglobulin heavy chain variable region gene sequence comprising in a complementary 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, and

(ii) at an endogenous non-human immunoglobulin light chain locus an unrearranged immunoglobulin light chain variable region gene sequence comprising 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;

such that the non-human animal generates 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 heavy chain variable domain and/or a CDR3 of a light chain variable domain.

2. The method of claim 1, wherein the unrearranged immunoglobulin heavy chain variable gene sequence is an unrearranged human immunoglobulin heavy chain variable region nucleotide sequence comprising at least one unrearranged human V_H gene segment, at least one unrearranged human D_H gene segment, and at least one unrearranged human J_H gene segment, and wherein the at least one unrearranged human D_H gene segment comprises the substitution of at least one non-histidine codon with a histidine codon or the insertion of at least one histidine codon.

3. The method of claim 1, wherein the unrearranged immunoglobulin light chain gene sequence is an unrearranged human immunoglobulin light chain variable region nucleotide sequence comprising at least one unrearranged human V_L gene segment and at least one unrearranged human J_L gene segment, and wherein the at least one unrearranged human V_L gene segment comprises in a CDR3 encoding sequence the substitution of at least one non-histidine codon with a histidine codon or the insertion of at least one histidine codon.

4. The method of claim 1, wherein the non-human animal is a rodent.

5. The method of claim 4, wherein the rodent is selected from a mouse, a rat, and a hamster.

9. The method of claim 1, wherein the modifying step comprises

replacing at the endogenous non-human immunoglobulin heavy chain locus an endogenous non-human immunoglobulin heavy chain variable region gene with an unrearranged human immunoglobulin heavy chain variable region gene sequence comprising at least one unrearranged human V_H gene segment, at least one unrearranged human D_H gene segment, and at least one unrearranged human J_H gene segment, and in a CDR3 encoding sequence the substitution of at least one non-histidine codon with a histidine codon or the insertion of at least one histidine codon, and/or

replacing at the endogenous non-human immunoglobulin light chain locus an endogenous non-human immunoglobulin light chain variable region gene sequence with an unrearranged human immunoglobulin light chain variable region gene sequence comprising at least one unrearranged human V_L gene segment and unrearranged human J_L gene segment, and in a CDR3 encoding sequence the substitution of at least one non-histidine codon with a histidine codon or the insertion of at least one histidine codon.

10. The method of claim 1, wherein the modifying step comprises

replacing at the endogenous non-human immunoglobulin heavy chain locus an endogenous non-human immunoglobulin heavy chain variable region gene sequence in a first non-human animal with an unrearranged human immunoglobulin heavy chain variable region gene sequence comprising at least one unrearranged human V_H gene segment, at least one unrearranged human D_H gene segment, and at least one unrearranged human J_H gene segment, and in a CDR3 encoding sequence the substitution of at least one non-histidine codon with a histidine codon or the insertion of at least one histidine codon,

replacing at the endogenous non-human immunoglobulin light chain locus an endogenous non-human immunoglobulin light chain variable region gene sequence in a second non-human animal with an unrearranged human immunoglobulin light chain variable region gene sequence comprising at least one unrearranged human V_L gene segment and at least one unrearranged human J_L gene segment, and in a CDR3 encoding sequence the substitution of at least one non-histidine codon with a histidine codon or the insertion of at least one histidine codon, and

breeding the first and second non-human animals to generate a third non-human animal comprising a replacement of the endogenous non-human immunoglobulin heavy chain variable region gene sequence with the unrearranged human immunoglobulin heavy chain variable region gene sequence and a replacement of the endogenous immunoglobulin light chain variable region gene sequence with the unrearranged human light chain variable region gene sequence,

wherein the first, second and third non-human animals are the same species.

11. The method of claim 1, wherein the unrearranged immunoglobulin heavy chain variable region gene sequence and/or the unrearranged immunoglobulin light chain variable region gene sequence further comprises an additional substituted or inserted histidine codon in a complementary determining region 1 (CDR1) encoding sequence, a complementary determining region 2 (CDR2) encoding sequence an N-terminal encoding sequence or a loop encoding sequence.

12. The method of claim 2, wherein the unrearranged immunoglobulin heavy chain variable gene sequence is operably linked to an endogenous non-human immunoglobulin heavy chain constant region gene sequence and/or the unrearranged immunoglobulin light chain variable gene sequence is operably linked to an endogenous non-human immunoglobulin light chain constant region gene sequence.

19. The method of claim 3, wherein the unrearranged human immunoglobulin light chain variable region light chain gene sequence comprises no more than two human V_L gene segments.

20. The method 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.

6. A method for making a non-human animal that makes antibody variable domains with a histidine amino acid encoded by a histidine codon substituted or inserted in the germline genome, comprising:

modifying the non-human animal in its germline to comprise

(i) at an endogenous non-human immunoglobulin heavy chain locus an unrearranged immunoglobulin heavy chain variable region gene sequence comprising 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, and

(ii) at an endogenous non-human immunoglobulin light chain locus a rearranged immunoglobulin light chain variable region gene sequence comprising 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;

such that the non-human animal generates 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 light chain variable domain.

7. The method of claim 6, wherein the non-human animal is a rodent.

8. The method of claim 7, wherein the rodent is selected from a mouse, a rat, and a hamster.

13. The method of claim 6, wherein the modifying step comprises

replacing at the endogenous non-human immunoglobulin heavy chain locus an endogenous non-human immunoglobulin heavy chain variable region gene with an unrearranged human immunoglobulin heavy chain variable region gene sequence comprising at least one unrearranged human V_H gene segment, at least one unrearranged human D_H gene segment, and at least one unrearranged human J_H gene segment, and in a CDR3 encoding sequence the substitution of at least one non-histidine codon with a histidine codon or the insertion of at least one histidine codon;

and

replacing at the endogenous non-human immunoglobulin light chain locus an endogenous non-human immunoglobulin light chain variable region gene sequence with a rearranged human immunoglobulin light chain variable region gene sequence comprising in a CDR3 encoding sequence the substitution of at least one non-histidine codon with a histidine codon or the insertion of at least one histidine codon.

14. The method of claim 6, wherein the modifying step comprises

replacing at the endogenous non-human immunoglobulin heavy chain locus an endogenous non-human immunoglobulin heavy chain variable region gene sequence in a first animal with an unrearranged human immunoglobulin heavy chain variable region gene sequence comprising unrearranged human V_H, unrearranged human D_H, and unrearranged human J_H gene segments, and in a CDR3 encoding sequence the substitution of at least one non-histidine codon with a histidine codon or an insertion of at least one histidine codon,

replacing at the endogenous non-human immunoglobulin light chain locus an endogenous non-human immunoglobulin light chain variable region gene sequence in a second animal with a rearranged human immunoglobulin light chain variable region gene sequence comprising in a CDR3 encoding sequence the substitution of at least one non-histidine codon with a histidine codon or an insertion of at least one histidine codon, and

breeding the first and second non-human animals to generate a third non-human animal comprising a replacement of the endogenous non-human immunoglobulin heavy chain variable region gene sequence with the unrearranged human immunoglobulin heavy chain variable region gene sequence and a replacement of the endogenous immunoglobulin light chain variable region gene sequence with the rearranged human light chain variable region gene sequence,

wherein the first, second and third non-human animals are the same species.

15. The method of claim 6, wherein the unrearranged immunoglobulin heavy chain variable region gene sequence and/or the rearranged immunoglobulin light chain variable region gene sequence further comprises an additional substituted or inserted histidine codon in a CDR1 encoding sequence, a CDR2 encoding sequence, an N-terminal encoding sequence or a loop encoding sequence.

16. The method of claim 6, wherein the rearranged immunoglobulin light chain variable region gene sequence is derived from a human Vκ

1-39 or Vκ

3-20 gene segment.

17. The method of claim of claim 16, wherein the rearranged immunoglobulin light chain variable region is derived from a rearranged human Vκ

1-39/Jκ

5 or Vκ

3-20/Jκ

1 gene sequence.

18. The method of claim 6, wherein the unrearranged immunoglobulin heavy chain variable gene sequence is operably linked to an endogenous non-human immunoglobulin heavy chain constant region gene sequence.