mRNA Display Antibody Library with Recombinant CDR Diversification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for generating high-affinity and stable antibodies or binders, such as recombinant phage display libraries and mRNA display, face limitations in diversity, stability, and efficiency, particularly when targeting tumor antigens or neoepitopes, requiring labor-intensive and time-consuming enrichment processes.
Innovation Solution
A high-diversity nucleic acid library is constructed by generating sub-libraries with targeted diversification of V H -CDR1/2, V H -CDR3, and V L domains, using random cassettes with degenerate base positions, allowing for rapid identification of stable, soluble, and functional antibodies or binders through recombination and screening.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If recombinant phage display libraries are used to generate antibody libraries, then library diversity can be achieved, but multiple rounds of enrichment are required which are labor intensive and time consuming
Solution Approach 1:
The antibody variable region is segmented into three separate libraries: VH-CDR1/2, VH-CDR3, and VL sub-libraries. This segmentation allows for independent optimization and recombination of different antibody regions, enabling high diversity generation without requiring multiple enrichment rounds. The segmented approach facilitates rapid assembly of diverse antibody combinations through recombination of the three sub-libraries.
2Reliability
If natural antibody pools are used to identify antibodies, then high-affinity binders can be found, but the diversity is limited depending on exposure frequency to antigens
Solution Approach 1:
The patent performs preliminary action by pre-selecting and constructing three specialized sub-libraries (VH-CDR1/2, VH-CDR3, and VL) with specific randomization patterns before the actual screening process. This preliminary library construction with targeted diversification ensures both high diversity and high affinity potential are built into the library structure beforehand, eliminating the need to rely on limited natural antibody pools and their exposure history.
3Quantity of substance
If library volume is increased to improve diversity, then more binders can be found, but transfection efficiency and practical considerations limit the maximum diversity
Solution Approach 1:
The patent transitions from a single-dimension approach (one large library) to a multi-dimensional approach by creating three separate sub-libraries (VH-CDR1/2, VH-CDR3, and VL) that can be recombined. This dimensional change allows the system to achieve extremely high diversity (10^12 to 10^16 members) without requiring proportionally large physical library volumes or transfection efficiencies, as the diversity is generated through combinatorial recombination of the three sub-libraries rather than through sheer library size.
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
The method enables the isolation of high-affinity binders with K d of equal or less than 100nM in a single or two-pass enrichment, achieving a diversity of at least 10 9< to 10 16< distinct library members, significantly reducing construction time and enhancing the speed of antibody discovery.
Implementation Method 1
mRNA sequences encoding candidate binding molecules (typically scFv) are coupled with a puromycin molecule at their 3'-end, and peptides encoded by the mRNA sequences are generated via in vitro translation to produce a fusion product that coupled the mRNA directly to the protein encoded by the mRNA
Data Source
Figure 1
Figure 2
Figure 3
AI summary
Compositions, methods and uses of high-diversity nucleic acid library that encodes a plurality of antibodies or antibody fragments are presented. The high-diversity nucleic acid library comprises or is derived from (1) a VH-CDR1/2 sub-library, (2) a plurality of VH-CDR3 sub-libraries, and (3) a VL sub-library, each of which comprises a plurality of members. Preferably, each member of the sub-libraries comprises at least one random cassette that has a plurality of degenerate base positions. In an especially preferred embodiment, at least portions of at least two members of the VH-CDR1/2 sub-library, the plurality of VH-CDR3 sub-libraries, and the VL sub-library are recombined to form an expression library member in an expression library, where each member of the expression library encodes a distinct antibody or antibody fragment.