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

• US 9,422,370 B2
• Filed: 07/23/2014
• Issued: 08/23/2016
• Est. Priority Date: 03/16/2012
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Murphy, Andrew J., Macdonald, Lynn, McWhirter, John
• Primary Examiner(s): Long, Scott
• Assistant Examiner(s): Leonard, Arthur S
• Application Number:
  US14/339,376
• Publication Number:
  US 20140329711A1
• Time in Patent Office: 762 Days
• Field of Search: None
• 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 method of generating an antibody that exhibits pH-dependent binding to an antigen of interest comprising:

(a) co-expressing in vitro in a host cell;

(i) a first immunoglobulin heavy chain nucleotide sequence comprising a heavy chain variable region sequence encoding a heavy chain variable domain of a first antibody, wherein the first antibody binds to the antigen of interest with a desired affinity at a neutral pH, wherein the heavy chain variable region sequence is generated in a non-human animal comprising in its germline genome a nucleic acid sequence comprising a single rearranged human immunoglobulin light chain variable region sequence that comprises a human germline Vκ

1-39 or Vκ

3-20 gene segment rearranged with a human germline Jκ

gene segment and that is expressed in the antibody repertoire of the non-human animal, and

(ii) an immunoglobulin light chain nucleotide sequence that is derived from the single rearranged human immunoglobulin light chain variable region sequence and is modified to comprise a substitution of at least one non-histidine codon with a histidine codon designed to express a histidine at a position selected from 105, 106, 107, 108, 109, 111 and a combination thereof (according to IMGT numbering), and

(b) selecting a second antibody expressed in the host cell that is encoded by the first immunoglobulin heavy chain nucleotide sequence and the immunoglobulin light chain nucleotide sequence, wherein the second antibody retains the desired affinity for the antigen of interest at neutral pH and displays reduced binding to the antigen of interest at an acidic pH.

2. The method of claim 1, wherein the non-human animal further comprises an immunoglobulin heavy chain sequence derived from a repertoire of human VH, DH, and JH segments present in the non-human animal germline genome.

3. The method of claim 1, wherein the single rearranged human immunoglobulin light chain variable region sequence is Vκ

1-39/Jκ

5, and the substitution of at least one non-histidine codon with a histidine codon in the immunoglobulin light chain nucleotide sequence is designed to express a histidine at a position selected from 105, 106, 108, 111, and a combination thereof.

4. The method of claim 1 wherein the single rearranged human immunoglobulin light chain variable region sequence is Vκ

3-20/Jκ

1, and the substitution of at least one non-histidine codon with a histidine codon in the immunoglobulin light chain nucleotide sequence is designed to express a histidine at a position selected from 105, 106, 107, 109, and a combination thereof.

5. The method of claim 1, wherein the second antibody displays 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.

6. The method of claim 1, wherein the second antibody exhibits a dissociative half-life (t_1/2) at acidic pH and 37°

C. of about 2 minutes or less.

7. The method of claim 1, wherein the non-human animal is a mouse.

8. The method of claim 2, wherein the non-human animal is a mouse.

9. The method of claim 1, wherein the second antibody comprises a fully human heavy chain and a fully human light chain.

10. The method of claim 1, wherein the immunoglobulin light chain nucleotide sequence of the first antibody further comprises at least one codon that encodes for a somatic mutation not encoded by the human germline Vκ

1-39 or Vκ

3-20 gene segment rearranged with a human germline Jκ

gene segment.

11. The method of claim 10, wherein the second antibody comprises a fully human first immunoglobulin heavy chain and a fully human immunoglobulin light chain.

12. A method of generating a bi-specific antibody that exhibits pH-dependent binding to a first antigen of interest and a second antigen of interest comprising:

(a) co-expressing in vitro in a host cell;

(i) a first immunoglobulin heavy chain nucleotide sequence comprising a first heavy chain variable region sequence encoding a first heavy chain variable domain of a first antibody, wherein the first antibody binds the first antigen of interest with a desired affinity at a neutral pH,

(ii) a second immunoglobulin heavy chain nucleotide sequence comprising a second heavy chain variable region sequence encoding a second heavy chain variable domain of a second antibody, wherein the second antibody binds the second antigen of interest with a desired affinity at a neutral pH,

wherein the first and second heavy chain variable region sequences are generated in a non-human animal comprising in its germline genome a nucleic acid sequence comprising a single rearranged human immunoglobulin light chain variable region sequence that comprises a human germline Vκ

1-39 or Vκ

3-20 gene segment rearranged with a human germline Jκ

gene segment and that is expressed in the antibody repertoire of the non-human animal and (2) comprise a same light chain component encoded by the human germline Vκ

1-39 or Vκ

3-20 gene segment rearranged with a human germline Jκ

gene segment; and

(iii) an immunoglobulin light chain nucleotide sequence that is (1) derived from the single rearranged human immunoglobulin light chain variable region sequence and (2) modified to comprise a substitution of at least one non-histidine codon with a histidine codon designed to express a histidine at a position selected from 105, 106, 107, 108, 109, 111 and a combination thereof (according to IMGT numbering), and

(b) selecting a third antibody expressed in the host cell that is encoded by the first and second immunoglobulin heavy chain nucleotide sequences and the immunoglobulin light chain nucleotide sequence, wherein the third antibody retains a desired affinity for the first and second antigens of interest at a neutral pH, and displays reduced binding to the first and second antigens of interest at an acidic pH.

13. The method of claim 12, wherein first and second antibodies are generated in a non-human animal further comprising an immunoglobulin heavy chain sequence derived from a repertoire of human VH, DH, and JH segments present in a non-human animal germline genome.

14. The method of claim 12, wherein the single rearranged human immunoglobulin light chain variable region sequence is Vκ

1-39/Jκ

5, and the substitution of at least one non-histidine codon with a histidine codon in the immunoglobulin light chain nucleotide sequence is designed to express a histidine at a position selected 105, 106, 108, 111, and a combination thereof.

15. The method of claim 12 wherein the single rearranged human immunoglobulin light chain variable region sequence is Vκ

3-20/Jκ

1, and the substitution of at least one non-histidine codon with a histidine codon in the immunoglobulin light chain nucleotide sequence is designed to express a histidine at a position selected from 105, 106, 107, 109, and a combination thereof.

16. The method of claim 12, wherein the non-human animal is a mouse.

17. The method of claim 13, wherein the non-human animal is a mouse.

18. The method of claim 12, wherein the third antibody comprises a fully human first immunoglobulin heavy chain, a fully human second immunoglobulin heavy chain, and a fully human immunoglobulin light chain.

19. The method of claim 12, wherein the third antibody displays 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.

20. The method of claim 12, wherein the third antibody exhibits a dissociative half-life (t_1/2) at acidic pH and 37°

C. of about 2 minutes or less.