Antibody CDR Library Construction via Controlled Degenerate Codons
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Solution Overview
Problem
Existing methods for constructing synthetic antibody libraries face challenges in accurately controlling the amino acid distribution at specific positions within complementarity determining regions (CDRs), leading to low precision in achieving desired amino acid ratios, which is crucial for mimicking natural antibody repertoires.
Innovation Solution
A method involving the determination of all allowable CDR amino acid sequences based on predetermined amino acid residues and specific distribution ratios, followed by random selection and addition to the library, ensuring that the cumulative number of amino acids at each position does not exceed calculated limits, thereby generating a library with precise amino acid distribution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If random combinations of mononucleotide or trinucleotide units are used to generate sequence differences in CDR regions, then the library construction is simple and cost-effective, but the control accuracy of amino acid distribution is very low
Solution Approach 1:
The patent changes the parameters of sequence generation by transitioning from random mononucleotide/trinucleotide combinations to a systematic approach using degenerate codons with defined amino acid preferences. Each position in the CDR region is assigned a degenerate codon that encodes a specific set of amino acids with controlled frequencies, thereby precisely controlling the amino acid distribution while maintaining synthetic library construction methods
Solution Approach 2:
The patent introduces dynamic control over amino acid distribution by allowing different degenerate codons to be selected at different positions based on desired amino acid frequencies. The library design dynamically adjusts the composition of degenerate codons at each position to achieve the target amino acid distribution pattern, rather than using a static random approach throughout
2Manufacturing precision
If degenerate codons are used to encode specific amino acid distributions, then amino acid distribution control is improved, but the design complexity increases
Solution Approach 1:
The patent segments the CDR region into individual positions, and for each position, selects an appropriate degenerate codon from a predefined set. This segmentation allows the complex task of controlling amino acid distribution across the entire CDR to be broken down into manageable position-by-position decisions, where each position can be independently optimized with a specific degenerate codon choice
Solution Approach 2:
The patent performs preliminary selection of degenerate codons for each position before actual library synthesis. By pre-calculating and assigning the optimal degenerate codon at each position based on desired amino acid distributions, the complex design work is completed in advance, simplifying the subsequent synthesis and library construction processes
3Manufacturing precision
If trinucleotide-directed mutagenesis is used to freely insert any desired codon combination, then amino acid distribution precision is improved, but the cost and synthesis complexity increase significantly
Solution Approach 1:
The patent employs degenerate codons that can be synthesized using standard, cost-effective oligonucleotide synthesis methods rather than requiring expensive and complex trinucleotide-directed mutagenesis. The degenerate codon approach uses readily available chemical synthesis techniques to introduce controlled variability, achieving precise amino acid distribution at a fraction of the cost and complexity of TRIM methods
Data Source
AI summary
Disclosed are a method and a device for constructing an antibody complementarity determining region (CDR) library. Also disclosed are a method, a device and a computer program product for determining the occurrence frequency of member sequences of an antibody CDR library, by means of which an antibody CDR library with a specific amino acid distribution at one or more positions can be obtained.