Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy

Patent metadata

Field	Value
Publication number	US-11447560-B2
Application number	US-201916297966-A
Country	US
Kind code	B2
Filing date	Mar 11, 2019
Priority date	Oct 22, 2003
Publication date	Sep 20, 2022
Grant date	Sep 20, 2022

Abstract

Methods are provided for the synthesis and secretion of recombinant hetero-multimeric proteins in mating competent yeast. A first expression vector is transformed into a first haploid cell; and a second expression vector is transformed into a second haploid cell. The transformed haploid cells, each individually synthesizing a non-identical polypeptide, are identified and then genetically crossed or fused. The resulting diploid strains are utilized to produce and secrete fully assembled and biologically functional hetero-multimeric protein.

First claim

What is claimed is:

1. A method for the synthesis of a secreted heteromultimeric protein comprising at least two non-identical subunit polypeptide chains in a Pichia pastoris diploid cell, the method comprising:
(a) transforming a first auxotrophic Pichia pastoris haploid cell with a linearized first expression vector, said expression vector comprising a nucleic acid sequence encoding a first subunit of said heteromultimeric protein, operably linked to a first Pichia pastoris promoter whereby the first expression vector comprising a nucleic acid sequence encoding the first subunit of said protein becomes integrated into the gene locus comprising said Pichia pastoris promoter;
(b) transforming a second auxotrophic Pichia pastoris haploid cell with a linearized second expression vector, said expression vector comprising a nucleic acid sequence encoding a second subunit of said heteromultimeric protein, operably linked to a second Pichia pastoris promoter whereby the second expression vector comprising said nucleic acid sequence encoding said second subunit of said heteromultimeric protein becomes integrated into the gene locus comprising said second Pichia pastoris promoter;
(c) generating a putative diploid Pichia pastoris cell by mating or fusion of said first and second Pichia pastoris haploid cells;
(d) determining whether the putative diploid cell is diploid by (i) permitting said putative diploid cell to go through meiosis and form spores; (ii) determining if a significant percentage of the resulting spore products are single or double auxotrophs and (iii) based thereon determining that the putative diploid Pichia pastoris cell is diploid; and
(e) culturing said diploid Pichia pastoris cell in a culture medium under conditions wherein said first and said second subunits are expressed by said diploid Pichia pastoris cell and secreted as said heteromultimeric protein; and
(f) isolating the secreted heteromultimeric protein from the culture medium.

2. The method according to claim 1 , wherein said heteromultimeric protein is an antibody comprising at least a variable region of a heavy and a light chain.

3. The method according to claim 2 , wherein said heteromultimeric protein is an antibody comprising at least a variable and a constant region of a heavy and a light chain.

4. The method according to claim 2 , wherein said first expression vector comprises a nucleic acid sequence encoding a library of light or heavy chain sequences and said second expression vector comprises a nucleic acid sequence encoding a single light or heavy chain sequence.

5. The method according to claim 2 , wherein said first expression vector comprises a nucleic acid sequence encoding a library of light or heavy chain sequences and said second expression vector comprises a nucleic acid sequence encoding a library of light or heavy chain sequences.

6. The method according to claim 1 , wherein said first and said second Pichia pastoris haploid cells are complementary auxotrophs.

7. The method according to claim 6 , wherein said step of generating a diploid cell from said first and second Pichia pastoris haploid cells comprises mating said haploid Pichia pastoris cells.

8. The method according to claim 6 , wherein said step of generating a diploid cell from said first and second Pichia pastoris haploid cells comprises spheroplast fusion of said first and second haploid Pichia pastoris cells.

9. The method according to claim 1 , further comprising the step of calibrating the level of expression of said first or said second subunit prior to generating said diploid cell.

10. The method according to claim 1 , wherein said first Pichia pastoris promoter and said second Pichia pastoris promoter are the same.

11. The method according to claim 1 , wherein said first Pichia pastoris promoter and said second Pichia pastoris promoter are different.

12. The method according to claim 1 , wherein one or both of said Pichia pastoris promoters are constitutive promoters.

13. The method according to claim 1 , wherein one or both of said Pichia pastoris promoters are inducible promoters.

14. The method according to claim 1 , wherein said first and/or second Pichia pastoris promoter is a glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter.

15. The method according to claim 1 , wherein said non-identical subunit polypeptide chains comprises an optimized signal sequence for diploid secretion and expression.

16. The method according to claim 1 , wherein said culturing step is performed in minimal media.

17. The method according to claim 16 , wherein said minimal media lacks selective agents.

18. The method according to claim 1 , wherein said culturing step is performed at a low temperature.

19. The method according to claim 1 , wherein said first and/or second auxotrophic Pichia pastoris haploid cells comprise ade1, ura3, met1 or lys3 auxotrophic strains.

20. The method according to claim 1 , wherein the presence of the genes encoding the heteromultimeric protein in the diploid Pichia pastoris cell is confirmed by a PCR assay and/or by detecting the ability of the diploid cell to synthesize said heteromultimeric protein.

21. The method according to claim 20 , wherein said heteromultimeric protein is an antibody or antigen binding fragment thereof.

22. A culture comprising diploidal Pichia pastoris cells produced according to claim 1 , which express and secrete the heteromultimeric protein.

23. The culture of claim 22 , wherein the heteromultimeric protein is an antibody.

Assignees

• Keck Graduate Inst
• H Lundbeck As

Inventors

• Cregg James M
• Latham John A
• Litton Mark
• Schatzman Randall
• Tolstrorukov Ilya I

Classifications

• C12N15/81

  for yeasts · CPC title

• C07K2317/24

  containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered · CPC title

• C07K16/00 Primary

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C12N15/815

  for yeasts other than Saccharomyces · CPC title

• C07K16/2809

  against the T-cell receptor (TcR)-CD3 complex · CPC title