Antibodies and methods for generating genetically altered antibodies with enhanced effector function

• US 20050054048A1
• Filed: 07/29/2004
• Published: 03/10/2005
• Est. Priority Date: 07/29/2003
• Status: Abandoned Application

• Current Assignee: Eisai Incorporated (Eisai Medical Research Incorporated)
• Original Assignee: Eisai Incorporated (Eisai Medical Research Incorporated)
• Inventors: Nicolaides, Nicholas C., Grasso, Luigi, Sass, Philip M., Sands, Howard
• Application Number:
  US10/901,650
• Publication Number:
  US 20050054048A1
• Time in Patent Office: Days
• US Class Current: 435/69.100
• 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 16/4291 against IgE
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C12N 15/1024 In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair

Abstract

Dominant negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. By introducing these genes into cells and transgenic animals, new cell lines and animal varieties with novel and useful properties can be prepared more efficiently than by relying on the natural rate of mutation. These methods are useful for generating genetic diversity within immunoglobulin genes directed against an antigen of interest to produce altered antibodies with enhanced biochemical activity. Moreover, these methods are useful for generating antibody-producing cells with increased level of antibody production. The invention also provides methods for increasing the effector function of monoclonal antibodies and monoclonal antibodies with increased effector function.

Claims

1. A method of producing an antibody with enhanced effector function comprising introducing into an antibody-producing cell a dominant negative allele of a mismatch repair gene, whereby said antibody-producing cell becomes hypermutable;

screening said hypermutable cells for cells that produce antibodies with increased effector function, thereby producing an antibody with enhanced effector function.

2. The method of claim 1 wherein said dominant negative allele is a PMS2 allele.

3. The method of claim 2 wherein said PMS2 allele comprises a truncation mutation at codon 134.

4. The method of claim 3 wherein said truncation mutation is a thymidine at nucleotide 424 of wild-type PMS2.

5. The method of claim 3 wherein said PMS2 allele encodes the first 133 amino acids of wild-type PMS2.

6. The method of claim 2 wherein the PMS2 allele is human PMS2.

7. The method of claim 1 wherein said effector function is antibody-dependent cytotoxicity (ADCC) activity.

8. The method of claim 1 further comprising restoring genetic stability to said antibody-producing cell.

9. The method of claim 1 further comprising exposing said antibody-producing cell to a chemical mutagen.

10. The method of claim 1 wherein said antibody comprises an amino acid substitution with proline or hydroxyproline.

11. The method of claim 1 wherein said antibody comprises an amino acid substitution in a heavy chain variable region.

12. The method of claim 1 wherein said antibody comprises an amino acid substitution in a light chain variable region.

13. The method of claim 1 wherein said antibody comprises an amino acid substitution in a first framework region of a heavy chain.

14. The method of claim 13 wherein said substitution occurs at position 6 of a first framework region comprising the amino acid sequence of SEQ ID NO:

18.

15. The method of claim 1 wherein said antibody comprises an amino acid substitution in a second framework region of a light chain.

16. The method of claim 14 wherein said amino acid substitution comprises proline or hydroxyproline.

17. The method of claim 15 wherein said substitution occurs at position 22 of a second framework region comprising the amino acid sequence of SEQ ID NO:

21.

18. The method of claim 1 wherein said antibody comprises an amino acid substitution in a first framework region of a heavy chain and an amino acid substitution in a second framework region of a light chain.

19. The method of claim 17 wherein said amino acid substitution comprises proline or hydroxyproline.

20. An antibody having enhanced effector function produced according to the method of claim 1.