Fc-Bait Antibody Display System for Pichia Host Cells
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Solution Overview
Problem
Current antibody display systems, such as phage display and yeast surface antibody display, face limitations including the lack of post-translational modifications like glycosylation in bacterial cells and low signal-to-noise ratios, leading to inefficient identification of specific antibodies and high cross-contamination between clones.
Innovation Solution
An antibody display system utilizing a Pichia host cell with a bait comprising a Fc immunoglobulin domain fused to a surface anchor protein, allowing for the display and secretion of antibodies while preserving sequence integrity, enabling screening for parameters like folding and expression, and facilitating the identification of antibodies that specifically bind to antigens.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If phage display is used for antibody screening, then antibody identification can be performed, but post-translational modifications like glycosylation are unavailable in bacterial cells
Solution Approach 1:
The patent uses a yeast cell surface as an intermediary system that bridges the gap between bacterial simplicity and mammalian complexity. The yeast surface display system allows antibodies to be displayed on yeast cells while utilizing yeast's eukaryotic machinery for post-translational modifications, thereby resolving the contradiction between screening capability and modification capability
Solution Approach 2:
The patent changes the biological system parameter from bacterial (prokaryotic) to yeast (eukaryotic) host cells. This parameter change enables post-translational modifications while maintaining the ability to perform antibody screening, thus resolving the contradiction
2Ease of operation
If cold capture method is used for antibody display, then antibodies can be assayed on cell surface, but signal-to-noise ratio is low
Solution Approach 1:
The patent employs preliminary sorting steps before final identification. Yeast cells are first sorted by flow cytometry based on antigen binding, then candidate clones are further validated. This preliminary action enriches for true positives and reduces background noise, improving signal-to-noise ratio
Solution Approach 2:
The patent implements a feedback mechanism where initially selected clones are re-tested and validated through multiple rounds of screening. This feedback loop allows elimination of false positives and confirms true antibody binders, thereby improving measurement precision
3Ease of operation
If affinity matrix system is used to couple antibodies to host cell surface, then antibodies can be assayed for antigen binding, but cross-contamination between antibody clones occurs
Solution Approach 1:
The patent segments the antibody display system by displaying individual antibody molecules on the yeast cell surface rather than coupling full antibodies to the cell surface. Each yeast cell displays a single antibody variant, physically separating different clones and eliminating cross-contamination while maintaining assay capability
4Ease of operation
If full length antibody display systems are used, then antibodies can be tethered on host cell surface, but erroneous binding occurs to host cells that do not express the antibody
Solution Approach 1:
The patent extracts only the essential antigen-binding portion (Fab or scFv) of the antibody for display on yeast cells, rather than tethering the full-length antibody. This extraction eliminates non-specific binding domains while preserving specific antigen recognition, thereby improving measurement precision
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This system effectively identifies and produces antibodies with improved specificity and reduced cross-contamination, enabling the secretion of full antibodies and their display on the cell surface for antigen binding, thereby overcoming the limitations of existing systems.
Implementation Method 1
a bait comprising a Fc immunoglobulin domain or functional fragment thereof fused to a surface anchor polypeptide
Implementation Method 2
identifying antibodies that bind specifically to an antigen
Data Source
AI summary
The present invention provides, in part, an antibody display system that simultaneously uses a secretion and a display mode. A bait complexed with a monovalent antibody fragment can be expressed on the surface of the host cell wherein the fragment may be assayed for antigen binding while full antibody is simultaneously secreted from the host cell. Methods of using the system for identifying antibodies that bind specifically to an antigen of interest are also provided. Polypeptides, polynucleotides and host cells useful for making the antibody display system are also provided along with methods of use thereof.