Figure 1.
A schematic representation of the Fc-Sed1p antibody display system.
The DNA sequence of the hinge region along with the CH2 and CH3 domains comprising the IgG Fc fragment are fused through a flexible linker to a cell wall anchoring partner, in this case the S. cerevisiae GPI anchor Sed1p is used. When co-expressed in the same host with a secretable full length IgG molecule, the Fc portion of the anchored fusion (the bait) heterodimerizes in the ER with the Fc region of the IgG molecule, forming two disulfide bridges. Since the CH1 domain of the heavy chain can still pair with the CL domain of the secreted light chain, this tripartite complex results in surface display of the monovalent (H+L) IgG molecule. Meanwhile the assembly of soluble full length IgG occurs with equal probability resulting in secretion of the bivalent (H2+L2) in the culture medium.
Figure 2.
Efficiency of the surface anchored Fc bait (Fc-Sed1p) in capturing monovalent IgG molecules on the cell wall of Glyco-engineered Pichia pastoris.
A) (I) Strain expressing soluble anti-PCSK9 antibody (solid histogram) and anti-PCSK9 strain co-expressing Fc-Sed1p fusion (dotted) were incubated with DyeLight 488 anti-Fc antibody and florescence intensities were assayed by flow cytometry (II). Same strains as in (I) were incubated with APC-conjugated anti-Kappa antibodies to detect light chain on cell surface (III). Flow cytometry was used to compare strain co-expressing anti-PCSK9 and Fc-Sed1p anchor (solid) against strain co-expressing anti-Her2 and Fc-Sed1p (dotted) for binding to DyeLight 488 anti-Fc or (IV) APC-anti Kappa. B) Detection of co-secreted full length IgG in antibody producing strains with and without Fc-Sed1p. Culture medium was passed through a protein A column, IgGs were eluted and protein was resolved in a native or reduced form through Protein Chip analysis C) Bar graph of percentage human N-glycosylation abundances of protein A purified mAbs isolated in B. D) Cells co-expressing anti-PCSK9 and Fc-Sed1p fusion were induced and incubated for 10 minutes at room temperature in 1XPBS buffer (solid); 1XPBS buffer containing 6 M Urea (dotted); 1XPBS buffer containing 0.5 M DTT (dashed); or 1XPBS containing 6 M Urea and 0.5 M DTT (complex). Cells were washed and labeled with anti-human Fc (I) (DyeLight 488) and anti-human Kappa Lc (II) (APC). Fluorescence intensities were analyzed by flow cytometry.
Figure 3.
Antigen binding capacity of monovalent IgGs displayed by Fc-Sed1p measured by flow cytometry.
The cells were dually labeled with goat anti-human Fc DyeLight 488, biotinylated PCSK9, and APC 635 labeled Streptavidin A) FACS analysis of labeled Pichia pastoris strains displaying Fc-Sed1p complexed with monovalent anti-Her2 antibody fragment (H+L) or B) an monovalent anti-PCSK9 (H+L) antibody fragment.
Figure 4.
The construction of a proof of principle mating library and the isolation of high affinity, and high expression antigen binders using Fc-Sed1p.
A) Haploid Pichia pastoris strains containing Fc-Sed1p expression cassette were transformed with a library of Anti-PCSK9 Hc or a library of Anti-PCSK9 Lc. Following mating and selection, diploids were cultured to express full length IgG library. Cultures were labeled using 20nM biotinylated human PCSK9 and APC 635 labeled antihuman Kappa. DyeLight 488 labeled Streptavidin was used to detect biotinylated PCSK9. B) Analysis and enrichment of high affinity anti-PCSK9 binders using three rounds of sequential sorting (S1 > S2 > S3). Known anti-PCSK9 strain co-expressing Fc-Sed1p was labeled as above as a control. C) Sensograms showing binding and dissociation kinetics of rhPCSK9 to immobilized anti-PCSK9 antibodies. Each anti-PCSK9 antibody lead was captured on the chip to ~ 500 RU followed by analyte injections of wild-type human PCSK9 at concentrations ranging from 0.156–2.5nM. Kinetics for the highest PCSK9 concentration (2.5 nM) is depicted for these antibodies.
Figure 5.
An antibody discovery and optimization scheme that utilizes Fc-Sed11p display-secretion technology in humanized Pichia pastoris.
This paradigm enables progression from lead discovery to preclinical validation and development using a single host.