Antibody Library Generation via CDR Segmentation
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Solution Overview
Problem
Current methods for generating antibody libraries face challenges such as low expression and folding issues due to randomly diversified sequences, particularly with CDR3, leading to a significant proportion of non-functional antibodies and reduced library quality, with frame shift mutations further complicating the process.
Innovation Solution
The method involves combining stable antibody frameworks with naturally encoded CDRs or amino acid sequences that can fulfill the role of CDRs, using Type IIs restriction enzymes to introduce diversity outside the immunoglobulin coding region, allowing for the capture and insertion of correctly folded CDRs from various sources into human antibody frameworks, thereby minimizing frame shifts and increasing the frequency of functional antibodies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If randomly diversified sequences are used to generate CDR regions, then library diversity is increased, but the proportion of functional antibodies decreases due to folding issues and frame shift mutations
Solution Approach 1:
The antibody variable domain is segmented into framework regions (stable) and CDR regions (diverse). The patent separates the CDR regions from the framework, allowing independent optimization of each part. This segmentation enables the framework to provide structural stability while CDRs provide diversity, resolving the contradiction between library diversity and functional proportion.
Solution Approach 2:
Different regions of the antibody are assigned different qualities: framework regions are designed for stability and proper folding, while CDR regions are designed for diversity and antigen binding. This local quality differentiation allows the molecule to simultaneously achieve high diversity in binding regions while maintaining overall structural integrity and functionality.
2Productivity
If synthetic diversity is introduced into CDR regions, then the number of antibody variants increases, but expression levels and folding efficiency decrease
Solution Approach 1:
The framework regions are pre-designed and optimized for proper folding and high expression levels before CDR diversification. This preliminary action ensures that the structural foundation is stable, allowing subsequent introduction of diverse CDR sequences without compromising overall expression and folding efficiency.
Solution Approach 2:
The patent optimizes specific parameters of the framework regions, such as amino acid composition and structural stability, to compensate for the destabilizing effect of diverse CDR sequences. By adjusting framework parameters, the system maintains high expression levels and folding efficiency even with extensive CDR diversification.
3Adaptability or versatility
If natural repertoires are captured for library generation, then all framework and diversity variants are included, but antibodies with low intrinsic stability are present, limiting shelf life and expression
Solution Approach 1:
The patent extracts only the CDR regions from natural antibody repertoires, separating them from their original framework regions. This extraction allows the diverse CDR sequences to be transferred onto stable, pre-selected framework regions, thereby preserving repertoire diversity while eliminating frameworks with low intrinsic stability.
Solution Approach 2:
The patent creates composite antibody molecules combining synthetic stable framework regions with naturally derived diverse CDR regions. This composite approach merges the advantages of both natural and synthetic antibodies: the stability and proper folding of synthetic frameworks with the diverse antigen-binding capabilities of natural CDRs.
Data Source
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AI summary
The invention provides compositions and methods for generating libraries of DNA sequences encoding homologous polypeptides, and uses of the libraries to identify naturally diversified polypeptide variants. The invention also provides compositions and methods for generating collections of synthetic antibody fragments in which one or several complementary determining regions (CDR) are replaced by a collection of the corresponding CDR captured from a natural source. The invention further provides compositions and methods for diversifying a portion of a polypeptide by inserting a diversified sequence of synthetic or natural origin without the need for modification of the original polypeptide coding sequence.