Antibody Humanization via Multi-Stage CDR Library Selection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Traditional methods for humanizing and optimizing antibodies face challenges such as limited affinity improvement, immunogenicity issues due to unwanted mutations, and the inability to capture clones with better binding in combinations of complementarity determining regions (CDRs), leading to suboptimal therapeutic antibodies.
Innovation Solution
A multi-stage approach involving the construction of targeted CDR libraries, phage display, and overlap PCR mutagenesis to select and combine libraries with increasing stringency, ensuring cooperative mutations across multiple CDRs, and incorporating heat treatment to enhance stability and developability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-generated harmful factors
If CDR grafting into human frameworks is performed, then humanization is achieved, but affinity and specificity may be lost
Solution Approach 1:
The patent applies local quality by making different parts of the antibody have different properties: CDR regions are engineered for high affinity binding to the target antigen, while framework regions are humanized to reduce immunogenicity. This spatial differentiation allows simultaneous optimization of binding function and reduced immune recognition.
Solution Approach 2:
The patent performs preliminary action by pre-selecting human framework sequences that are most compatible with the CDR regions before assembly. The method also pre-identifies potential immunogenic epitopes and addresses them in advance through framework humanization, preventing immunogenicity issues before they manifest.
2Manufacturing precision
If saturation mutagenesis is used to investigate all possible single mutations, then comprehensive coverage is achieved, but combinations of mutations cannot be obtained
Solution Approach 1:
The patent merges multiple CDR libraries containing different mutations into a single combined library. This allows simultaneous evaluation of multiple mutation combinations that would be impossible to assess individually through saturation mutagenesis alone, capturing cooperative effects between different CDR mutations.
Solution Approach 2:
The patent employs dynamic library construction where the composition of the library evolves through sequential steps: individual CDR libraries are created, then combined in various configurations, and finally selected based on binding performance. This dynamic approach allows the system to explore a vast combinatorial space that static saturation mutagenesis cannot access.
3Manufacturing precision
If CDR walking with sequential optimization is performed, then individual CDR optimization is achieved, but cooperative effects among CDRs are missed
Solution Approach 1:
The patent merges multiple CDR libraries into a combined library that allows simultaneous evaluation of multiple CDR mutations together. This captures cooperative binding effects that arise when multiple CDRs work in concert, which would be missed by sequential optimization where each CDR is optimized in isolation and then fixed.
4Adaptability or versatility
If error prone PCR is used for mutagenesis, then diversity is generated, but unwanted mutations in framework regions occur causing immunogenicity
Solution Approach 1:
The patent segments the antibody into distinct functional regions: CDRs that are engineered for diversity and affinity, and framework regions that are humanized to minimize immunogenicity. By applying mutagenesis strategies selectively to CDR regions while maintaining human frameworks, the patent generates necessary sequence diversity without introducing immunogenic mutations in the framework.
Solution Approach 2:
The patent applies local quality by treating different regions of the antibody differently: high diversity is introduced in CDR regions through targeted mutagenesis, while framework regions maintain human sequences to reduce immunogenicity. This spatial differentiation allows diversity generation without compromising on immunogenicity reduction.
5Adaptability or versatility
If parallel CDR optimization is performed, then sequence diversity is limited to selected CDRs, but no further selection is possible
Solution Approach 1:
The patent employs a dynamic, multi-stage selection process where libraries are constructed, selected, and then recombined for further selection rounds. This dynamic approach allows continuous improvement of affinity by iteratively selecting the best clones and using them as templates for the next round of library construction and selection, rather than relying on a single static selection event.
Data Source
AI summary
The present disclosure discloses a method for identifying an optimized protein. The method involves constructing and targeting targeted libraries against a target antigen using a first set of selection conditions to select a pool of binders in each library, combining the selected libraries into one or more libraries; and selecting the combined library against the target antigen using a second set of selection conditions to identify at least one protein having an optimized functional profile. An exemplar protein that can be identified with this method is an antibody or a fragment thereof.