Antibody Libraries with Variable CDR3 Lengths
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Solution Overview
Problem
Existing antibody libraries that focus on amino acid sequence diversity fail to replicate the natural diversity of antibodies, including length variation, leading to inefficient construction, increased immunogenicity, and potential degradation issues, as they do not accurately mimic the natural diversity of antibodies, which is crucial for proper folding and antigen binding.
Innovation Solution
Development of libraries that encode human antibodies with heavy chain CDR3s of varying lengths (3 to 35 amino acids) enriched with Tyr and Ser residues, incorporating diversified D regions and extended JH regions, along with methods for constructing and screening these libraries to ensure diversity in both sequence and length, thereby mimicking natural antibody diversity and reducing immunogenicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If antibody libraries focus only on amino acid sequence diversity, then library construction is simplified, but the libraries fail to replicate natural antibody diversity including length variation, leading to reduced functionality and increased immunogenicity
Solution Approach 1:
The patent extends the diversity dimension from single-dimensional amino acid sequence variation to multi-dimensional diversity by incorporating CDR length variation as an additional dimension. This is achieved through controlled insertion of amino acids at specific positions (N1, N2, N3) in the CDR regions, particularly CDR3, thereby replicating the natural diversity of antibodies more accurately and improving functional antibody isolation efficiency
Solution Approach 2:
The patent systematically varies the length parameter of CDR regions by controlling the number of amino acid insertions at defined positions. By adjusting the length of CDR3 (and to a lesser extent CDR1 and CDR2) while maintaining sequence diversity, the library more closely mimics natural antibody variability, thereby improving the reliability of isolating functional antibodies with reduced immunogenicity
2Quantity of substance
If antibody libraries include only sequence diversity with fixed lengths, then the library size is manageable, but they do not reflect natural antibody diversity where length varies, compromising proper folding and antigen binding
Solution Approach 1:
The patent divides the CDR3 region into distinct segments with specific insertion points (N1 between residues 96-97, N2 between residues 100-101, N3 between residues 104-105). This segmentation allows controlled length variation in manageable increments while maintaining the overall structure and functionality of the antibody, thereby reflecting natural diversity without exponentially increasing library complexity
Solution Approach 2:
The patent pre-defines specific positions and numbers of amino acid insertions in the CDR regions before library construction. By establishing a structured framework for length variation in advance (with predetermined insertion sites and limited length ranges), the library can capture natural diversity patterns without requiring exhaustive sampling of all possible length combinations, thus managing library size effectively
3Reliability
If libraries use diverse CDR3 lengths and sequences, then natural antibody diversity is better captured, but library construction complexity increases
Solution Approach 1:
The patent applies diversity selectively at specific local positions within the CDR regions rather than uniformly across the entire antibody sequence. By concentrating length variation at defined insertion points (N1, N2, N3) in CDR3 and limited positions in CDR1 and CDR2, the library captures essential natural diversity while minimizing construction complexity. The framework regions and non-CDR portions remain relatively conserved
Solution Approach 2:
The patent implements a controlled, partial approach to diversity by limiting CDR3 length variation to specific ranges (e.g., 5-15 amino acids) and using a finite set of predetermined insertion positions, rather than allowing unlimited length and sequence variation. This partial action approach captures the most critical aspects of natural antibody diversity (CDR3 length and sequence variation) without the excessive complexity of complete randomization, thereby improving functional antibody isolation efficiency while managing construction complexity
Data Source
AI summary
Provided are compositions and methods for preparing and identifying antibodies having CDR3s that vary in sequence and in length from very short to very long which in certain embodiments may bind to a carbohydrate moiety or the active site of an enzyme. Libraries coding for antibodies with the CDR3s are also provided. The libraries can be provided by modifying a pre-existing nucleic acid library.