Glycoengineered yeast Pichia pastoris Expression System
Yeast Surface Display of Full-length IgG
Schematic illustration of IgG yeast surface display
Benefits of Full-length IgG Yeast Surface Display
- In glycoengineered yeast P. pastoris, full-length IgG antibody is displayed as native form in molecular structure, biophysical property, and biological function. ScFv in phage or yeast display needs format conversion into full-length IgG, which may lose activity.
- Comparing to the mammalian cell display, full-length IgG yeast surface display has many benefits, such as large library size, easy genetic manipulation, fast generation time, and low cost screening.
- FACS sorting of full-length IgG in yeast surface display system is based on both IgG antibody affinity and display level. It can eliminate the bias caused by expression levels and identify clones with small affinity difference. However, phage display screen is usually influenced not only by affinity, but also by the expression level.
- FACS sorting technology can directly select high, medium, and low affinity clones at once.
IgG is the Most Efficient Format in Yeast Display
Genotype-phenotype Coupling for Library Screening
Yeast Surface Display for Affinity Maturation
For affinity maturation of a parental antibody, its heavy chain complementarity determining region 3 (CDR3-H) was randomly mutated using error-prone mutagenesis. The mutated antibody library was displayed on the cell surface of yeast P. pastoris to generate a library with a diversity over a million. Yeast cells were double labeled with biotinylated antigen, streptavidin-APC, and FITC-labeled anti-kappa-antibody, then examined simultaneously for its antigen binding and IgG display level by two-dimensional FACS. After several rounds of sorting, the yeast cells with high fluorescence in Q2 of Fig. F were plated on a selective medium and the individual clones were sequenced. In this way, a couple of antibody mutants were isolated, which showed an affinity improvement for target compared to the parental antibody (Table 1).
Yeast Surface Display of Common Light Chain Bispecific Antibody
Heterodimeric IgG-like bispecific antibody, which is based on the heterodimerization of two different IgG molecules, is a promising format because it maintains the overall size and natural structure of human IgG with good stability, half-life, and pharmacokinetics profile. To generate heterodimeric IgG-like bispecific antibody, two challenges have to overcome. One is to facilitate heterodimerization of two distinct heavy chains and prevent homodimerization of two identical heavy chains. The second is to have correct pairing of cognate heavy and light chain. An efficient strategy to overcome these challenges is to use knobs-into-holes (KIH) technology to prioritize heterodimerization of two different heavy chains and combine with a common light chain, based on generally accepted fact that the affinity and specificity of an antibody is predominantly defined by the heavy chain, and it can be maintained when an antibody has a non-cognate light chain. Genekine apply a versatile approach to directly generate common light chain bispecific antibodies by using yeast surface display of heterodimeric IgG-like bispecific antibody. It can provide service to efficiently isolate common light chains for various combinations of two existing heavy chains. In this approach, two heavy chains from existing antibodies to two different antigens are introduced into human naïve and synthesized light chain libraries. The resulting bispecific antibody libraries are displayed on yeast cell surface in heterodimeric IgG-like bispecific antibody format. The libraries can be simultaneously selected by fluorescence-activated cell sorting (FACS) against two target antigens to isolate common light chains that, in combination with two heavy chains, keep their binding affinity for two antigens. This straightforward approach can efficiently isolate common light chains for various combinations of two existing heavy chains. It generates functional and developable IgG-like bispecific antibodies, which exhibit high affinities in the nanomolar range and have the similar biochemical and biophysical properties to parental antibodies.