	US 12,084,651 B2
	Surface display of whole antibodies in eukaryotes
Bianka Prinz, Lebanon, NH (US); Natarajan Sethuraman, Hanover, NH (US); Dongxing Zha, Houston, TX (US); Stefan Wildt, Somerville, MA (US); and Piotr Bobrowicz, Hanover, NH (US)
Assigned to Merck Sharp & Dohme LLC, Rahway, NJ (US)
Filed by Merck Sharp & Dohme LLC, Rahway, NJ (US)
Filed on Jun. 10, 2021, as Appl. No. 17/343,905.
Application 15/007,622 is a division of application No. 13/251,410, filed on Oct. 3, 2011, granted, now 9,260,712, issued on Feb. 16, 2016.
Application 13/251,410 is a division of application No. 12/489,900, filed on Jun. 23, 2009, granted, now 8,067,339, issued on Nov. 29, 2011.
Application 17/343,905 is a continuation of application No. 16/150,674, filed on Oct. 3, 2018, granted, now 11,046,951.
Application 16/150,674 is a continuation of application No. 15/007,622, filed on Jan. 27, 2016, abandoned.
Claims priority of provisional application 61/208,583, filed on Feb. 25, 2009.
Claims priority of provisional application 61/134,331, filed on Jul. 9, 2008.
Prior Publication US 2021/0317441 A1, Oct. 14, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/10 (2006.01); C07K 16/00 (2006.01); C07K 16/28 (2006.01); C07K 16/32 (2006.01); G01N 33/68 (2006.01)

CPC C12N 15/1037 (2013.01) [C07K 16/00 (2013.01); C07K 16/2887 (2013.01); C07K 16/32 (2013.01); C12N 15/1034 (2013.01); G01N 33/6854 (2013.01); C07K 2319/30 (2013.01)]

11 Claims

1. A method for producing yeast host cells that express an immunoglobulin of interest, comprising:

a. providing yeast host cells that include a first nucleic acid molecule operably linked to a Pichia pastoris GUT1 promoter and which encodes a capture moiety comprising a Sed1p protein fused to protein A or ZZ-domain from Protein A integrated into the genome of the yeast host cells;

b. transfecting the yeast host cells with a plurality of nucleic acid molecules encoding immunoglobulin heavy and light chains, wherein the nucleic acid molecules encoding the heavy chain and light chains are operably linked to a Pichia pastoris GAPDH promoter;

c. (i) growing the yeast host cells under conditions that selectively induce expression of the Pichia pastoris GUT1 promoter to produce the capture moiety on the surface of the yeast host cells and (ii) growing the yeast host cells under conditions that selectively induce expression of the Pichia pastoris GAPDH promoter to produce yeast host cells in which the immunoglobulins expressed in the yeast host cells are secreted and captured by the capture moiety on the surface of the yeast host cells;

d. contacting the yeast host cells with a detection means that specifically binds to the immunoglobulin of interest; and

e. isolating yeast host cells in which the detection means is bound to the immunoglobulin of interest to produce the yeast host cells that express the immunoglobulin of interest.