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

Patent metadata

Field	Value
Publication number	US-10259883-B2
Application number	US-201514853292-A
Country	US
Kind code	B2
Filing date	Sep 14, 2015
Priority date	Oct 22, 2003
Publication date	Apr 16, 2019
Grant date	Apr 16, 2019

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 diploidal Pichia yeast culture which secretes a desired heteromultimeric protein comprising at least two non-identical subunit polypeptide chains, produced by a method comprising the following steps:
(i) obtaining a first haploid Pichia yeast strain which comprises a nucleic acid fragment encoding at least one subunit of a heteropolymeric polypeptide which is not endogenously expressed by said Pichia strain, which coding sequence is operably linked to a promoter and a signal peptide that directs the secretion of said polypeptide by a Pichia yeast;
(ii) obtaining a second haploid Pichia yeast which comprises a nucleic acid encoding at least one subunit of the same heteropolymeric polypeptide, wherein the subunits encoded by the first and second yeast strains comprise all of the subunits of the desired heteropolymeric polypeptide, which heteropolymeric polypeptide is not endogenously expressed by said Pichia strain, and which coding sequence is operably linked to a promoter and a signal peptide that directs the secretion of said polypeptide by a Pichia yeast; and
(iii) fusing said first and second haploid Pichia yeast strains by spheroplast fusion or by mating to produce a diploid Pichia yeast strain which is capable of expressing and secreting the desired heteropolymeric polypeptide, wherein said first and second haploid Pichia yeast strains are of the same species.

2. The culture of claim 1 , wherein the diploid is produced by mating.

3. The culture of claim 1 , wherein the diploid is produced by spheroplast fusion.

4. The culture according to claim 1 , wherein both said first and second haploid Pichia yeast strains are Pichia pastoris, Pichia methanolica , or Pichia angusta.

5. The culture according to claim 1 , wherein said heteropolymeric protein is a mammalian protein.

6. The culture according to claim 1 , wherein said heteromultimeric protein is an intact antibody comprising heavy and light variable regions and human constant regions.

7. The culture according to claim 1 , wherein both of said haploids are auxotrophic.

8. The culture according to claim 1 , wherein in steps (i) and (ii) the nucleic acid fragments encoding the polypeptide subunits are respectively operably linked to a first promoter and a second promoter which first and second promoters are the same or different.

9. The culture according to claim 8 , wherein at least one of said first and second promoters is a GAP promoter.

10. The culture of claim 1 , wherein the haploids are Pichia pastoris strains.

11. The culture of claim 1 , which comprises a minimal media.

12. The culture of claim 11 wherein said minimal media lacks selective agents.

13. The culture of claim 1 , wherein said heteromultimeric protein is a full-length antibody and is secreted by said diploid cells at a concentration of at least about 100 mg/liter culture.

14. The culture of claim 1 , wherein the yeast cell density is at least about 100 g/L.

15. The culture of claim 14 , wherein said cell density is at least about 300 g/L.

16. The culture of claim 14 , wherein said cell density is at least about 400 g/L.

17. The culture of claim 14 , wherein said cell density is at least about 500 g/L.

Assignees

• Keck Graduate Inst
• Alderbio Holdings Llc

Inventors

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

Classifications

• C07K2317/24

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

• C12N15/81

  for yeasts · CPC title

• C07K16/2809

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

• C07K16/00 Primary

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

• C07K2317/14

  Specific host cells or culture conditions, e.g. components, pH or temperature · CPC title