Naïve Antibody Phage Display Library Construction
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Solution Overview
Problem
Current methods for producing antibody phage display libraries, particularly in the Fab format, face challenges in achieving large sizes and high diversity, leading to low yields of antigen-specific binders and high chances of generating false positives or non-binders, due to limitations in recombinant antibody assembly and phage propagation issues.
Innovation Solution
A process is developed to create a large naïve antibody phage display library with a size ranging from 8.86×10^10 to 9.13×10^11 cfu, involving RNA isolation, cDNA synthesis, amplification of VL and VH domains, overlap PCR to form Fabs, and a method for manufacturable antibody retrieval through target-specific panning, periplasmic qELISA, kinetic ranking, and bioassay, resulting in a phenotype-to-genotype correlation of >90%.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional recombinant antibody assembly methods are used to create phage display libraries, then the library can be constructed, but the library size and diversity are limited, resulting in low yields of antigen-specific binders
Solution Approach 1:
The patent performs preliminary enrichment of high-quality IgG producing B-cells from peripheral blood mononuclear cells (PBMCs) before library construction. This preliminary selection ensures that only B-cells capable of producing functional, high-affinity antibodies are included in the library, thereby increasing the proportion of antigen-specific binders and resolving the contradiction between library size and binder yield
Solution Approach 2:
The patent creates multiple copies of the enriched B-cell population through clonal expansion and generates large numbers of phage particles displaying antibody fragments. This copying approach maintains the high quality of the enriched cell population while achieving the large library sizes needed for comprehensive antigen-specific binder discovery
2Quantity of substance
If conventional phage display library methods are used, then libraries can be generated, but there is a high chance of generating false positives or non-binders
Solution Approach 1:
The patent performs preliminary enrichment of IgG producing B-cells using fluorescence-activated cell sorting (FACS) based on surface IgG expression and intracellular IgG staining. This preliminary enrichment step ensures that only B-cells producing functional, high-affinity antibodies are included in the library, thereby increasing the proportion of antigen-specific binders and reducing false positives
Solution Approach 2:
The patent implements multiple rounds of biopanning with stringent washing conditions and includes negative selection steps where phages binding to control proteins (e.g., BSA, IgG) are removed. This feedback mechanism continuously enriches for specific binders while eliminating false positives throughout the screening process
3Ease of manufacture
If scFv format is used for phage display, then the library can be constructed, but the secreted scFvs show low binding affinity to cognate antigens
Solution Approach 1:
The patent uses Fab fragments displayed on phage surfaces as copies of the original antibody structure, preserving the native VH-VL interface and CDR regions. This copying approach maintains the high binding affinity of the original antibody while enabling phage display, resolving the contradiction between ease of manufacture and binding affinity
Solution Approach 2:
The patent changes the antibody format from scFv to Fab fragment, which fundamentally alters the structural parameters of the antigen-binding unit. The Fab format with separate heavy and light chains connected by disulfide bonds provides better structural stability and preserves native antigen-binding interfaces, thereby improving binding affinity while maintaining phage display compatibility
Data Source
AI summary
The present invention discloses a naïve antibody phage display library (APDL), a process for producing the same and a method of obtaining manufacturable antibodies as soluble Fabs from the antibody phage display library.