Mice that produce antigen-binding proteins with pH-dependent binding characteristics

• US 9,301,510 B2
• Filed: 03/15/2013
• Issued: 04/05/2016
• Est. Priority Date: 03/16/2012
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: McWhirter, John, MacDonald, Lynn, Murphy, Andrew J.
• Primary Examiner(s): Long, Scott
• Application Number:
  US13/832,309
• Publication Number:
  US 20130247235A1
• Time in Patent Office: 1,117 Days
• US Class Current: 1/1
• CPC Class Codes: A01K 2207/15 Humanized animals
• 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 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• 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
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins

Abstract

Genetically modified non-human animals are provided that comprise an immunoglobulin heavy chain locus comprising an unrearranged human heavy chain variable region nucleotide sequence comprising an addition of at least one histidine codon or a substitution of at least one endogenous non-histidine codon with a histidine codon. Compositions and methods for making the genetically modified non-human animals as described herein are provided. Non-human animals capable of expressing an antigen-binding protein characterized by pH-dependent antigen binding, enhanced recyclability and/or enhanced serum half-life are also provided.

Claims

1. A non-human animal comprising

(i) in its germline genome a genetically modified immunoglobulin heavy chain locus comprising an unrearranged human immunoglobulin heavy chain variable region nucleotide sequence, wherein the unrearranged heavy chain variable region nucleotide sequence comprises an addition of at least one histidine codon or a substitution of at least one non-histidine codon with a histidine codon, wherein the histidine codon is not encoded by a corresponding human germline heavy chain variable region gene segment;

and

wherein the added or substituted histidine codon is present in a complementary determining region 3 (CDR3) encoding sequence.

2. The non-human animal of claim 1, wherein the non-human animal is a mammal.

3. The non-human animal of claim 2, wherein the mammal is a rodent selected from the group consisting of a mouse, a rat, and a hamster.

4. The non-human animal of claim 1, wherein the CDR3 encoding sequence is selected from a human V_H gene segment sequence, a human D gene segment sequence, a human J_H gene segment sequence, and a combination thereof.

5. The non-human animal of claim 1, wherein the CDR3 encoding sequence is selected from a human germline V_H gene segment sequence, a human germline D gene segment sequence, a human germline J_H gene segment sequence, and a combination thereof.

6. The non-human animal of claim 5, further comprising at least a second additional or substituted histidine codon in at least one reading frame of the human immunoglobulin heavy chain gene segment that encodes a heavy chain variable domain selected from an N-terminal region, a loop 4 region, a complementary determining region 1 (CDR1), a complementary determining region 2 (CDR2), the complementary determining region 3 (CDR3), and a combination thereof.

7. The non-human animal of claim 1, wherein the endogenous non-histidine codon that is substituted with the histidine codon encodes the amino acid selected from the group consisting of Y, N, D, Q, S, W, and R.

8. The non-human animal of claim 1, wherein the added or substituted histidine codon is present in at least one reading frame of a human D gene segment.

9. The non-human animal of claim 8, wherein the reading frame is a hydrophilic frame of the human D gene segment, and the hydrophilic frame comprises a nucleotide sequence that encodes the amino acid sequence selected from the group consisting of SEQ ID NO:

46, SEQ ID NO;

48, SEQ ID NO;

50, SEQ ID NO;

52, SEQ ID NO;

54, SEQ ID NO;

56, SEQ ID NO;

58, SEQ ID NO;

60, SEQ ID NO;

62, SEQ ID NO;

64, SEQ ID NO;

66, SEQ ID NO;

68, SEQ ID NO;

70, SEQ ID NO;

72, SEQ ID NO;

74, SEQ ID NO;

76, SEQ ID NO;

78, SEQ ID NO;

80, SEQ ID NO;

82, SEQ ID NO;

84, SEQ ID NO;

86, and a combination thereof.

10. The non-human animal of claim 1, wherein the unrearranged human immunoglobulin heavy chain variable region nucleotide sequence is operably linked to a human or non-human heavy chain constant region nucleotide sequence.

11. The non-human animal of claim 10, wherein the unrearranged human immunoglobulin heavy chain variable region nucleotide sequence is operably linked to an endogenous non-human heavy chain constant region nucleotide sequence selected from the group consisting of a C_H1, a hinge, a C_H2, a C_H3, and a combination thereof.

12. The non-human animal of claim 10, wherein the human heavy chain constant region nucleotide sequence comprises a modification that increases an affinity of a C_H2-C_H3 region of an IgG heavy chain constant region amino acid sequence to neonatal Fc receptor (FcRn) at a pH ranging from about 5.5 to about 6.0, wherein the modification is a mutation in the IgG heavy chain constant region amino acid sequence selected from the group consisting of M428L, N434S, V259I, V308F, N434A, M252Y, S254T, T256E, T250Q, H433K, N434Y, and a combination thereof.

13. The non-human animal of claim 1, wherein the non-human animal is homozygous for the genetically modified immunoglobulin heavy chain locus in the germline.

14. The non-human animal of claim 1, wherein the non-human animal further comprises an unrearranged human immunoglobulin light chain V gene segment and an unrearranged human immunoglobulin light chain J gene segment.

15. The non-human animal of claim 1, wherein the non-human animal comprises a B cell population that is capable of producing a diverse population of antigen-binding proteins that exhibit pH-dependent binding, each comprising a heavy chain variable domain having at least one histidine residue derived from the added or substituted histidine codon.

16. The non-human animal of claim 15, wherein at least one B cell of the B cell population comprises a rearranged human immunoglobulin heavy chain variable region sequence that is derived from the modified immunoglobulin heavy chain locus and that comprises at least one somatic hypermutation (SHM).

17. The non-human animal of claim 15, wherein the antigen-binding protein produced by the B cell population exhibits a decreased antigen-binding affinity at a pH ranging from about 5.5 to about 6.0 as compared with at a neutral pH ranging from about 7.0 to about 7.4.

18. The non-human animal of claim 1, wherein the non-human animal is heterozygous for the genetically modified immunoglobulin heavy chain locus in the germline.

19. The non-human animal of claim 1, wherein the non-human animal comprises an Adam6a gene, an Adam6b gene, or both.

20. A method of making a non-human animal that comprises a genetically modified immunoglobulin heavy chain locus in its germline genome, the method comprising:

(a) modifying a genome of a non-human animal to delete or render non-functional endogenous immunoglobulin heavy chain V, D, and I gene segments in an immunoglobulin heavy chain locus; and

(b) placing in the genome an unrearranged human heavy chain variable region nucleotide sequence comprising an addition of at least one histidine codon or a substitution of at least one endogenous non-histidine codon with a histidine codon, wherein the histidine codon is not encoded by a corresponding human germline heavy chain variable region gene segment; and

wherein the added or substituted histidine codon is present in a complementary determining region 3 (CDR3) encoding sequence.

21. The method of claim 20, wherein the CDR3 encoding sequence is selected from a human V_H gene segment, a human D gene segment, a human J_H gene segment, and a combination thereof.

22. The method of claim 20, wherein the CDR3 encoding sequence is selected from a human germline V_H gene segment sequence, a human germline D gene segment sequence, a human germline J_H gene segment sequence, and a combination thereof.

23. The method of claim 20, further comprising at least a second additional or substituted histidine codon in at least one reading frame encoding a heavy chain variable domain selected from an N-terminal region, a loop 4 region, a complementary determining region 1 (CDR1), a complementary determining region 2 (CDR2), the complementary determining region 3 (CDR3), and a combination thereof.

24. The method of claim 20, wherein the endogenous non-histidine codon that is replaced with the histidine codon encodes the amino acid selected from the group consisting of Y, N, D, Q, S, W, and R.

25. The method of claim 20, wherein the added or substituted histidine codon is present in one or more reading frame of a human D gene segment.

26. The method of claim 25, wherein the reading frame is a hydrophilic frame of the human D gene segment, and the hydrophilic frame comprises a nucleotide sequence that encodes the amino acid sequence selected from the group consisting of SEQ ID NO:

46, SEQ ID NO;

48, SEQ ID NO;

50, SEQ ID NO;

52, SEQ ID NO;

54, SEQ ID NO;

56, SEQ ID NO;

58, SEQ ID NO;

60, SEQ ID NO;

62, SEQ ID NO;

64, SEQ ID NO;

66, SEQ ID NO;

68, SEQ ID NO;

70, SEQ ID NO;

72, SEQ ID NO;

74, SEQ ID NO;

76, SEQ ID NO;

78, SEQ ID NO;

80, SEQ ID NO;

82, SEQ ID NO;

84, SEQ ID NO;

86, and a combination thereof.

27. The method of claim 20, wherein the unrearranged immunoglobulin human heavy chain variable region nucleotide sequence is operably linked to a human or non-human heavy chain constant region nucleotide sequence selected from the group consisting of a C_H1, a hinge, a C_H2, a C_H3, and a combination thereof.

28. The method of claim 27, wherein the unrearranged immunoglobulin human heavy chain variable region nucleotide sequence is operably linked to an endogenous non-human heavy chain constant region nucleotide sequence selected from the group consisting of a C_H1, a hinge, a C_H2, a C_H3, and a combination thereof.

29. The method of claim 27, wherein the human heavy chain constant region nucleotide sequence comprises a modification that increases an affinity of a C_H2-C_H3 region of an IgG heavy chain constant region amino acid sequence to neonatal Fc receptor (FcRn) at a pH ranging from about 5.5 to about 6.0, wherein the modification is a mutation in the IgG heavy chain constant region amino acid sequence selected from the group consisting of M428L, N4345, V2591, V308F, N434A, M252Y, 5254T, T256E T250Q, H433K, N434Y, and a combination thereof.

30. The method of claim 20, wherein the non-human animal is homozygous for the genetically modified immunoglobulin heavy chain locus in the germline genome.

31. The method of claim 20, wherein the non-human animal comprising the genetically modified immunoglobulin heavy chain locus comprises a B cell population that is capable of producing an diverse population of antigen-binding proteins that exhibit pH-dependent binding, each comprising a heavy chain variable domain having at least one histidine residue derived from the added or substituted histidine codon.

32. The method of claim 31, wherein at least one B cell of the B cell population comprises a rearranged human immunoglobulin heavy chain variable region sequence that is derived from the modified immunoglobulin heavy chain locus and that comprises at least one somatic hypermutation (SHM).

33. The method of claim 31, wherein the antigen-binding protein produced by the B cell population exhibits a decreased antigen-binding affinity at a pH ranging from about 5.5 to about 6.0 as compared with at a neutral pH ranging from about 7.0 to about 7.4.

34. The method of claim 20, wherein the non-human animal comprises an Adam6a gene, an Adam6b gene, or both.

35. The method of claim 20, wherein the method results in a genetically modified animal that comprises a population of B cells for antibodies exhibiting enhanced pH-dependent binding to an antigen of interest.