Histidine engineered light chain antibodies and genetically modified non-human animals for generating the same

• US 9,334,334 B2
• Filed: 03/15/2013
• Issued: 05/10/2016
• 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
• Assistant Examiner(s): Leonard, Arthur S
• Application Number:
  US13/832,247
• Publication Number:
  US 20130247234A1
• Time in Patent Office: 1,152 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 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• 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: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• 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

A genetically modified non-human animal is provided, wherein the non-human animal expresses an antibody repertoire capable of pH dependent binding to antigens upon immunization. A genetically modified non-human animal is provided that expresses a single light chain variable domain derived from a single rearranged light chain variable region gene in the germline of the non-human animal, wherein the single rearranged light chain variable region gene comprises a substitution of at least one non-histidine encoding codon with a histidine encoding codon. Methods of making non-human animals that express antibodies comprising a histidine-containing universal light chain are provided.

Claims

1. A genetically modified non-human animal comprising in its germline an immunoglobulin light chain locus that comprises a single rearranged human immunoglobulin light chain variable region gene sequence comprising human Vκ

and Jκ

segment sequences,

wherein the Vκ

segment sequence is derived from a human Vκ

1-39 or Vκ

3-20 gene segment, and

wherein the single rearranged human immunoglobulin light chain variable region gene sequence comprises a substitution of at least one non-histidine codon of the Vκ

segment sequence with a histidine codon that is expressed at a position selected from the group consisting of 105, 106, 107, 108, 109, 111 and a combination thereof (according to IMGT numbering).

2. The animal of claim 1, wherein the single rearranged human immunoglobulin variable region sequence is operably linked to an immunoglobulin light chain constant region gene sequence.

3. The animal of claim 2, wherein the immunoglobulin light chain constant region gene sequence is a non-human immunoglobulin light chain constant region gene sequence.

4. The animal of claim 3, wherein the non-human immunoglobulin light chain constant region gene sequence is an endogenous non-human immunoglobulin light chain constant region gene sequence.

5. The animal of claim 1, further comprising in its germline an immunoglobulin heavy chain locus that comprises an unrearranged immunoglobulin heavy chain variable region gene sequence comprising human V_H, D_H, and J_H segments operably linked to an immunoglobulin heavy chain constant region gene sequence.

6. The animal of claim 5, wherein the immunoglobulin heavy chain constant region gene sequence is a non-human immunoglobulin heavy chain constant region gene sequence.

7. The animal of claim 6, wherein the non-human heavy chain constant region gene sequence is an endogenous non-human immunoglobulin constant region gene sequence.

8. The animal of claim 1, wherein the animal lacks a functional unrearranged immunoglobulin κ

light chain variable region.

9. The animal of claim 1, wherein the immunoglobulin light chain locus is at an endogenous non-human immunoglobulin light chain locus.

10. The animal of claim 5, wherein the immunoglobulin heavy chain locus is at an endogenous non-human immunoglobulin heavy chain locus.

11. The animal of claim 1, wherein the substitution of at least one non-histidine codon with a histidine codon is in the nucleotide sequence encoding a complementary determining region (CDR) 3.

12. The animal of claim 11, wherein the substitution is of one, two, three, or four CDR3 codon(s).

13. The animal of claim 1, wherein the single rearranged human immunoglobulin light chain variable region is derived from a rearranged Vκ

1-39/Jκ

5 or Vκ

3-20/Jκ

1 gene sequence.

14. The animal of claim 13, wherein the single rearranged human immunoglobulin light chain variable region is derived from a rearranged Vκ

1-39/Jκ

5 gene sequence, and wherein the Vκ

1-39/Jκ

5 gene sequence comprises a replacement of at least one non-histidine codon with a histidine codon designed to express a histidine at a position selected from the group consisting of 105, 106, 108, 111, and a combination thereof (according to IMGT numbering).

15. The animal of claim 13, wherein the single rearranged human immunoglobulin light chain variable region is derived from a rearranged Vκ

3-20/Jκ

1 gene sequence, and wherein the Vκ

3-20/Jκ

1 gene sequence comprises a replacement of at least one non-histidine codon with a histidine codon designed to express a histidine at position selected from the group consisting of 105, 106, 107, 109, and a combination thereof (according to IMGT numbering).

16. The animal of claim 1, wherein the animal comprises a population of B cells in response to an antigen of interest that is enriched for antibodies that exhibit a decrease in dissociative half-life (t_1/2) at an acidic pH as compared to neutral pH of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, or at least about 30-fold.

17. The animal of claim 16, wherein the decrease in dissociative half-life (t_1/2) at an acidic pH as compared to neutral pH is about 30 fold or more.

18. The animal of claim 1, wherein the animal is a rodent.

19. The rodent of claim 18, wherein the rodent is a rat or a mouse.

20. The rodent of claim 19, wherein the rodent is a mouse.

21. The animal of claim 1, wherein the animal expresses an antibody comprising a human immunoglobulin light chain variable domain with a substitution of at least one non-histidine residue with a histidine at an amino acid position encoded by the at least one codon substituted in the immunoglobulin light chain variable region gene sequence.

22. A genetically modified mouse comprising in its germline an immunoglobulin light chain locus that comprises a single rearranged human immunoglobulin light chain variable region gene sequence comprising human Vκ

and Jκ

segment sequences,

wherein the Vκ

segment sequence is derived from a human Vκ

1-39 or Vκ

3-20 gene segment, and

wherein the single rearranged human immunoglobulin light chain variable region sequence comprises a substitution of at least one non-histidine codon of the Vκ

segment sequence with a histidine codon that is expressed at a position selected from the group consisting of 105, 106, 107, 108, 109, 111 and a combination thereof (according to IMGT numbering), and

wherein the mouse lacks a functional unrearranged immunoglobulin κ

light chain variable region.

23. The mouse of claim 22, wherein the single rearranged immunoglobulin light chain variable region gene sequence is operably linked to an immunoglobulin light chain constant region gene sequence.

24. The mouse of claim 23, wherein the immunoglobulin light chain constant region gene sequence is selected from a rat or a mouse immunoglobulin light chain constant region gene sequence.

25. The mouse of claim 22, further comprising in its germline an immunoglobulin heavy chain locus that comprises an unrearranged immunoglobulin heavy chain variable region sequence comprising human V_H, D_H, and J_H segments operably linked to an immunoglobulin heavy chain constant region gene sequence.

26. The mouse of claim 25, wherein the immunoglobulin heavy chain constant region gene sequence is a rat or a mouse heavy chain constant region gene sequence.

27. The mouse of claim 22, wherein the immunoglobulin light chain locus is at an endogenous mouse immunoglobulin light chain locus.

28. The mouse of claim 25, wherein the immunoglobulin heavy chain locus is at an endogenous mouse immunoglobulin heavy chain locus.

29. The mouse of claim 22, wherein the substitution of at least one non-histidine codon with a histidine codon is in the nucleotide sequence encoding a complementary determining region (CDR) 3.

30. The mouse of claim 29, wherein the substitution is of one, two, three, or four CDR3 codon(s).

31. The mouse of claim 22, wherein the single rearranged human immunoglobulin light chain variable region is derived from a rearranged Vκ

1-39/Jκ

5 or Vκ

3-20/Jκ

1 gene sequence.

32. The mouse of claim 31, wherein the single rearranged human immunoglobulin light chain variable region is derived from a rearranged Vκ

1-39/Jκ

5 gene sequence, and wherein the Vκ

1-39/Jκ

5 gene sequence comprises a replacement of at least one non-histidine codon with a histidine codon designed to express a histidine at a position selected from the group consisting of 105, 106, 108, 111, and a combination thereof (according to IMGT numbering).

33. The mouse of claim 32, wherein the Vκ

1-39/Jκ

5 gene sequence comprises a replacement of non-histidine codons designed to express histidines at positions 105, 106, 108, and 111 (according to IMGT numbering).

34. The mouse of claim 32, wherein the Vκ

1-39/Jκ

5 gene sequence comprises a replacement of non-histidine codons designed to express histidines at positions 106, 108 and 111 (according to IMGT numbering).

35. The mouse of claim 31, wherein the single rearranged human immunoglobulin light chain variable region is derived from a rearranged Vκ

3-20/Jκ

1 gene sequence, and wherein the Vκ

3-20/Jκ

1 gene sequence comprises replacement of at least one non-histidine codon with a histidine codon designed to express a histidine at a position selected from the group consisting of 105, 106, 107, 109, and a combination thereof (according to IMGT numbering).

36. The mouse of claim 35, wherein the Vκ

3-20/Jκ

1 gene sequence comprises a replacement of non-histidine codons designed to express histidines at positions 105, 106, 107, and 109 (according to IMGT numbering).

37. The mouse of claim 35, wherein the Vκ

3-20/Jκ

1 gene sequence comprises a replacement of non-histidine codons designed to express histidines at IGMT positions 105, 106 and 109 (according to IMGT numbering).

38. The mouse of claim 22, wherein the mouse expresses a population of antigen-specific antibodies in response to an antigen of interest wherein all antibodies comprise:

immunoglobulin light chain variable domains derived from the same single rearranged human light chain variable region gene sequence which comprises a substitution of at least one non-histidine codon with a histidine codon, and

immunoglobulin heavy chains comprising human heavy chain variable domains derived from a repertoire of human heavy chain V, D, and J segments.

39. A method of generating an antibody that exhibits pH-dependent binding to an antigen of interest comprising:

generating a mouse of claim 22;

immunizing the mouse with an antigen of interest; and

selecting an antibody that binds to the antigen of interest with a desired affinity at a neutral pH while displaying reduced binding to the antigen of interest at an acidic pH.

40. The method of claim 39, wherein the antibody exhibits a dissociative half-life (t_1/2) at acidic pH and 37°

C. of about 2 minutes or less.

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

modifying a genome of a non-human animal to delete or render non-functional endogenous immunoglobulin light chain Vκ and

Jκ

segments in an immunoglobulin light chain locus, and

placing in the genome a single rearranged human light chain variable region gene sequence comprising human Vκ and

Jκ

segment sequences,

wherein the Vκ

segment sequence is derived from a human Vκ

1-39 or Vκ

3-20 gene segment, and

wherein the single rearranged human immunoglobulin light chain variable region gene sequence comprises a substitution of at least one non-histidine codon of the Vκ

segment sequence with a histidine codon that is expressed at a position selected from the group consisting of 105, 106, 107, 108, 109, 111 and a combination thereof (according to IMGT numbering).

42. The method of claim 41, wherein the method results in a genetically modified non-human animal that comprises a population of B cells enriched for antibodies exhibiting pH-dependent binding to an antigen of interest.

43. The method of claim 41, wherein the animal is a rodent.

44. The method of claim 43, wherein the animal is a mouse or a rat.