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Glycoengineered yeast Pichia pastoris Expression System

Yeast expression system, especially Pichia pastoris (P. pastoris), has many benefits over other expression systems for the production of recombinant protein and antibody. It is ease to manipulate genetically, has low cost for high protein production, and can perform higher eukaryotic protein modifications, such as glycosylation, disulfide bond formation and proteolytic processing. Unlike mammalian cells, yeast P. pastoris does not need serum in culture medium, and can be grown to very high cell densities using minimal media. The fermentation processes can be easily scaled up to meet higher demands, and the integrated vectors have genetic stability in continuous and large-scale fermentation. However, yeast P. pastoris also has a major drawback for expressing glycoprotein. Protein N-glycosylation in yeast P. pastoris has glycans of the high mannose type with up to 100 or more mannose residues (hypermannosylation), which differ greatly from those of mammalian cells, and affect protein structure and function.
Genekine has genetically re-engineered N-glycosylation modification in yeast P. pastoris to mimic N-glycosylation modification in mammalian cells. By deleting the intrinsic N-glycosylation genes and integrating mammalian N-glycosylation genes in the genome of P. pastoris, we have created glycoengineered yeast P. pastoris strains, which produce human-like N-glycans on protein and antibody. Since the hypermannosylation of N-glycosylation is eliminated, our glycoengineered P. pastoris offer significant advantages over P. pastoris and other expression systems to produce high level of protein and antibody for both basic research and industrial manufacture.

Yeast Surface Display of Full-length IgG

Surface display technologies are powerful tools for high-throughput screening in antibody discovery and optimization. Phage display and yeast display are commonly used for displaying and screening the library of antibody fragment such as single-chain variable fragment (ScFv). However, for pharmaceutical development, scFv needs format conversion into full-length IgG (two heavy chain and two light chain) for further characterization, namely IgG reformatting. The converted full-length IgG may lose activity and often need further affinity maturation. The selected scFv fragment may be hard to re-engineer and generate full-length IgG，which has good expression and biophysical properties required for pharmaceutical development. Mammalian cells are idea system to display full-length IgG in native form and allow selection of antibody for functional properties. However, mammalian cell display system has some drawbacks, such as small library size, difficulty in genetic manipulation, slow generation time, and high cost. Mammalian cell display system is not an efficient system for antibody maturation.
Like mammalian cell expression system, glycoengineered yeast P. pastoris cells have eukaryotic internal quality control apparatus for antibody correct folding and post-translational modification (such as human-like N-glycosylation). We have used glycoengineered yeast P. pastoris to develop a novel approach to display full-length IgG on yeast surface. The approach uses genetic engineering tools to efficiently direct site-specific integration of antibody library genes into predefined chromosome sites. This enables construction of libraries of many millions of monoclonal stable cell lines displaying full-length IgG antibodies on their surface. The libraries can be screened directly for high affinity and improved developability profiles by using fluorescence-activated cell sorting (FACS). This novel approach combines the high throughput of yeast display with mammalian-cell quality control in one platform. Full-length IgG antibodies are displayed as native forms in molecular structure, biophysical property, and biological function. This display approach is well applicable in real-time selection of full-length IgG antibody libraries, which may not be possible by other display systems, such as ribosome display, phage display, traditional yeast display, and mammalian cell display. It can be used for antibody discovery, humanization, and optimization in native IgG format. The selected antibody can be used directly for animal study and clinical trial. Based on our proprietary technology of full-length IgG yeast surface display, Genekine can provide comprehensive solutions for antibody humanization, affinity maturation, and developability improvement. Our clients can increase their chances of finding the best therapeutic or diagnostic antibody leads for the most challenging targets.

Schematic illustration of IgG yeast surface display

The yeast surface display of IgG antibody libraries can be generated by transforming yeast P. pastoris with plasmids encoding for anchor protein, heavy chains and light chains. The use of genetic engineering tools ensures plasmid integration into predefined chromosome sites and thus, full-length IgG antibody libraries are expressed and displayed on the surface of yeast cells. IgG-displaying yeast cells are incubated with the fluorophore-labeled antigen and fluorophore-labeled detection antibody (i.e. fluorophore-labeled anti-kappa-antibody) for sorting by FACS. FACS sorting of IgG display libraries is based on both antibody affinity and display level. It can eliminate the bias caused by expression levels and identify clones with small affinity differences.

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

Various formats, such as single-chain variable fragment (scFv) and antigen-binding fragment (Fab) are available for engineering of IgG monoclonal antibody by yeast surface display, but they do not all lead to efficient expression of functional molecules. Some antibodies do not seem to be active in scFv format when they are expressed at the surface of yeast cells. For pharmaceutical development, scFv needs format conversion into full-length IgG for further characterization. Some affinity enhanced molecules engineered in scFv format lose affinity after conversion into IgG. Fab is a common format to express functional antibody by yeast surface display. However, it has the issue of low expression. A selected antibody was respectively displayed as full-length IgG, Fab and scFv formats on the surface of yeast cells. Yeast cells were labeled with biotinylated antigen and Streptavidin-APC for flow-cytometry (FC) analysis. Cells expressing full-length IgG showed a high level of fluorescence (Fig. A), indicating quantitative binding of the antigen; whereas cells expressing Fab had weak signal (Fig. B), indicating weak antigen binding. Cells expressing scFv had very weak signal (Data not shown). In antibody engineering or affinity maturation, a strong signal would favor an easy discrimination of high-affinity clones in libraries containing very high numbers of mutants. Yeast surface display of full-length IgG is superior for high-throughput screening. Most of therapeutic antibodies on the market are antibodies in the IgG format. Thus, it is ideal to screen antibody in IgG format by using yeast surface display of full-length IgG antibody.
Fig. A Cells expressing full-length IgG showed a high level of fluorescence.
Fig. B Cells expressing Fab had weak signal of fluorescence.

Genotype-phenotype Coupling for Library Screening

To confirm that a genotype-phenotype linkage exists for our yeast surface display of full-length IgG antibody, a demo experiment was performed, in which TNF-binding adalimumab-displaying cells were mixed with trastuzumab-displaying cells at a 1 to 1,000,000 ratio, mimicking the immune library. Cells were double labeled with biotinylated TNF, streptavidin-APC, and FITC-labeled anti-kappa-antibody, then examined simultaneously for its antigen binding and IgG display level by two-dimensional FACS. In bi-variate dot plot, each dot represented two fluorescent signals of a separate yeast cell in the display library. The x-axis was a measure of the amount of the APC fluorescent TNF that is bound, whereas the y-axis gave an indication of the IgG display level by FITC fluorescence. Yeast cell subpopulation in Q4 of Fig. C did not express and display IgGs on the cell surface (Unstained control). Subpopulation in Q1 of Fig. D expressed no TNF-affinity trastuzumab IgGs on the cell surface. Subpopulation in gate Q2 of Fig. D expressed high TNF-affinity adalimumab IgGs on the cell surface. After several rounds of sorting, the yeast cells with high fluorescence in Q2 of Fig. D were plated on a selective medium and the individual clones were sequenced. All sequenced clones were confirmed to be adalimumab, demonstrating the preservation of genotype-phenotype linkage for library screening in yeast surface display of full-length IgG.
Fig. C Cell subpopulation in Q4 did not express and display IgGs on the cell surface (Unstained control).
Fig. D Cell subpopulation in Q1 expressed no TNF-affinity trastuzumab IgGs on the cell surface. Cell subpopulation Q2 expressed TNF-affinity adalimumab IgGs on the cell surface.

Yeast Surface Display for Affinity Maturation

The affinity maturation technology mimics the in vivo antibody maturation occurring in B cells during the immune response, but can achieve higher affinities than those obtained in vivo. It is based on mutation of the antibody binding sites and the subsequent selection of the antibody variants showing the highest affinity for the target antigen, using a variety of display methods, such as ribosome display, phage display, traditional yeast display, and mammalian cell display. Yeast surface display of full-length IgG antibody will become the most widely used affinity maturation platform because it combines many advantages compared with the other approaches; in particular, the eukaryotic quality control machinery ensures the correct folding and post-translational modifications of the displayed antibodies; more importantly, above all, clones with improved affinity can be selected by fluorescence-activated cell sorting (FACS). This allows a real-time quantification of both the antibody display level and the antigen-binding strength directly during the screening process, distinguishing even small differences in the binding properties of the antibody variants.
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).
Fig. E FACS of unenriched antibody mutant library.
Fig. F FACS of enriched antibody mutant library.
Table 1 The binding affinities of parental antibody and mutants measured by ForteBio Octet.

Yeast Surface Display of Common Light Chain Bispecific Antibody

A full-length IgG contains two identical heavy chains and light chains. Each heavy chain associates with a light chain through a disulfide bond and non-covalent interactions to form a heterodimer, and both heterodimers associate to forming a complex Y-shaped antibody. Bispecific antibodies (bsAbs) combine specificities of two antibodies and simultaneously bind to two different antigens or epitopes. Bispecific antibodies are emerging as the next wave of antibody-based therapies. Advances in genetic engineering technology has resulted in a range of recombinant bispecific antibody formats. Generally, bispecific antibody can be divided into two major categories, those with an Fc region and those without an Fc region. However, most of these formats have been limited by some of their liabilities, such as instability, short half-life, poor manufacturability, and immunogenicity.
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.

Schematic illustration of yeast surface display of common light chain bispecific antibody

The yeast surface display of common light chain bispecific antibody libraries can be generated by transforming yeast P. pastoris with plasmids encoding for anchor protein, two heavy chains and light chain libraries. The use of genetic engineering tools ensures plasmid integration into predefined chromosome sites and thus, common light chain bispecific antibody libraries are expressed and displayed on the surface of yeast cells. Yeast cells are incubated with two fluorophore-labeled antigens for sorting by FACS. The bispecific antibody libraries can be simultaneously selected by FACS against two antigens to isolate common light chains that, in combination with two heavy chains, keep their binding affinity for two antigens.
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