Anti-TEM1 Antibodies Yeast Display Library Expansion
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Solution Overview
Problem
Existing methods for isolating antigen-specific antibodies, such as yeast-display libraries, are limited by small library sizes, leading to insufficient diversity and potential loss of antigen specificity and affinity when transferring from displayed to secreted forms, due to differences in expression systems and post-translational modifications.
Innovation Solution
The use of electroporation combined with buffer modifications and a single expression system (Saccharomyces cerevisiae) for both display and secretion of scFv, with N-terminus modifications to minimize conformational changes, resulting in a 1×10^9-member yeast scFv display library and the development of 'biobodies' for high-throughput screening and validation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If yeast-display libraries are used for antibody isolation, then screening efficiency is improved, but library size is limited resulting in insufficient diversity
Solution Approach 1:
The patent transforms the yeast display library from a limited chemical transformation system to an electroporation-based system with modified buffer conditions, achieving a 10,000-fold increase in transformant numbers and enabling libraries exceeding 1×10^9 members while maintaining high-throughput screening capability
Solution Approach 2:
The patent introduces an intermediary expression system where scFv is first expressed as a surface-displayed form on yeast, then transferred to a secreted form through controlled expression system switching, preserving antigen-binding activity while enabling library expansion
2Productivity
If scFv is transferred from displayed to secreted forms, then antibody production is improved, but antigen specificity and affinity are lost due to expression system differences
Solution Approach 1:
The patent performs preliminary screening and selection of high-affinity scFv clones in the displayed form before transferring them to secreted form expression, ensuring that only clones with proven antigen-specific binding are produced in secreted form, thus preserving reliability
Solution Approach 2:
The patent creates a copy of the selected scFv sequence from the displayed form and expresses it in the secreted form using the same yeast expression system, maintaining identical amino acid sequence and thus preserving antigen-binding properties while changing the expression output form
3Adaptability or versatility
If different expression systems are used for displayed and secreted forms, then production flexibility is improved, but conformational changes occur resulting in loss of scFv function
Solution Approach 1:
The patent uses a single yeast expression system that can perform both surface display and secreted form production functions, eliminating the need to switch between different expression systems and thereby preventing conformational changes that would compromise scFv function
Data Source
AI summary
The invention relates to Anti-TEM 1 antibodies or antigen-binding fragments thereof, yeast libraries comprising the same, and prophylactic, diagnostic, and therapeutic methods using the same.