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

• US 9,332,742 B2
• Filed: 05/06/2015
• 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:
  US14/705,916
• Publication Number:
  US 20150250151A1
• Time in Patent Office: 370 Days
• US Class Current: 1/1
• 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 2317/21 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 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• 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 2317/92 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

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 human immunoglobulin light chain variable domains derived from a limited repertoire of human immunoglobulin light chain variable gene segments that comprise histidine modifications in their germline sequence. Methods of making non-human animals that express antibodies comprising histidine residues encoded by histidine codons introduced into immunoglobulin light chain nucleotide sequences are provided.

Claims

1. A genetically modified non-human animal comprising in its germline an immunoglobulin light chain locus comprising two unrearranged human Vκ

gene segments and one or more unrearranged human Jκ

gene segment(s) operably linked to an immunoglobulin light chain constant region sequence,

wherein the two unrearranged human Vκ

gene segments are human Vκ

1-39 and Vκ

3-20 gene segments each comprising one or more substitutions of a non-histidine codon with a histidine codon, and

wherein the human Vκ and

Jκ

gene segments are capable of rearranging and the human Vκ and

Jκ

gene segments encode a human light chain variable domain comprising one or more histidines at a position selected from the group consisting of 105, 106, 107, 108, 109, 111 (according to IGMT numbering), and a combination thereof, wherein the one or more histidines are derived from the one or more substitutions.

2. The animal of claim 1, wherein the animal does not comprise an endogenous κ

light chain variable region gene segment that is capable of rearranging to form an immunoglobulin light chain.

3. The animal of claim 1, wherein the immunoglobulin light chain constant region sequence is a non-human immunoglobulin light chain constant region sequence.

4. The animal of claim 3, wherein the non-human immunoglobulin light chain constant region sequence is a mouse or a rat sequence.

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

6. The animal of claim 1, 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 gene segments operably linked to an immunoglobulin heavy chain constant region sequence.

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

8. The animal of claim 7, wherein the non-human immunoglobulin heavy chain constant region sequence is a mouse or a rat sequence.

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

10. The animal of claim 1, wherein the two unrearranged human Vκ

gene segments and the one or more unrearranged human Jκ

gene segment(s) are present at the endogenous immunoglobulin light chain locus.

11. The animal of claim 1, wherein the immunoglobulin light chain constant region is a Cκ

region.

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

13. The rodent of claim 12, wherein the rodent is a rat or a mouse.

14. The rodent of claim 13, wherein the rodent is a mouse.

15. 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.

16. The animal of claim 15, wherein the enrichment in antibodies that exhibit a decrease in t_1/2 is at least about 2 fold.

17. The animal of claim 1, wherein the animal comprises five human Jκ

segments, and the five human Jκ

segments are human Jκ

1, Jκ

2, Jκ

3, Jκ

4, and Jκ

5 segments.

18. The animal of claim 1, wherein the immunoglobulin light chain locus comprises no more than the human Vκ

1-39 and Vκ

3-20 gene segments and the one or more unrearranged human Jκ

gene segment operably linked to an immunoglobulin light chain constant region sequence.

19. The animal of claim 18, wherein either or both of the unrearranged human Vκ

1-39 and Vκ

3-20 gene segments comprise a substitution of three or four non-histidine codons with the histidine codons.

20. The animal of claim 19, wherein the substitution is of three non-histidine codons of the human Vκ

1-39 gene segment, and the substitution is designed to express histidines at positions 106, 108, and 111 of the human Vκ

1-39 gene segment.

21. The animal of claim 19, wherein the substitution is of four non-histidine codons of the human Vκ

1-39 gene segment, and the substitution is designed to express histidines at positions 105, 106, 108, and 111 of the human Vκ

1-39 gene segment.

22. The animal of claim 19, wherein the substitution is of three non-histidine codons of the human Vκ

3-20 gene segment, and the substitution is designed to express histidines at positions 105, 106, and 109 of the human Vκ

3-20 gene segment.

23. The animal of claim 19, wherein the substitution is of four non-histidine codons of the human Vκ

3-20 gene segment, and the substitution is designed to express histidines at positions 105, 106, 107, and 109 of the human Vκ

3-20 gene segment.

24. The animal of claim 1, wherein the animal expresses a population of antigen-specific antibodies in response to an antigen wherein all antibodies in the population comprise:

immunoglobulin light chain variable domains derived from a rearrangement of the unrearranged human Vκ

1-39 and Vκ

3-20 gene segments and the one or more unrearranged human Jκ

gene segment(s), and

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

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

immunizing the animal of claim 1 with an antigen of interest.

30. The method of claim 29, further comprising 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.

25. 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 germline genome of the non-human animal to delete or render non-functional endogenous immunoglobulin light chain Vκ and

Jκ

gene segments in an immunoglobulin light chain locus, and

placing in the germline genome of the non-human animal an immunoglobulin light chain variable region comprising two unrearranged human Vκ

gene segments and at least one unrearranged human Jκ

gene segment, such that the immunoglobulin light chain variable region sequence is operably linked to an immunoglobulin constant region sequence,

wherein the two unrearranged human Vκ

gene segments are human Vκ

1-39 and Vκ

3-20 gene segments each comprising one or more substitutions of a non-histidine codon with a histidine codon, and

wherein the unrearranged human Vκ and

Jκ

gene segments are capable of rearranging and the unrearranged human Vκ and

Jκ

gene segments encode a human light chain variable domain comprising one or more histidines at a position selected from the group consisting of 105, 106, 107, 108, 109, 111 (according to IGMT numbering) and a combination thereof, wherein the one or more histidines are derived from the one or more substitutions.

26. The method of claim 25, wherein the immunoglobulin light chain variable region is at the endogenous non-human immunoglobulin light chain locus.

27. The method of claim 25, wherein the animal is a rodent.

28. The method of claim 27, wherein the rodent is a mouse or a rat.