Rabbit Antigen-Binding Protein Libraries with C80 Mutation

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Solution Overview

Problem

Existing methods for isolating rabbit antibodies with high affinity for intracellular peptide-MHC antigens are limited by the free thiol group at position C80, reducing the selectable diversity of rabbit antibody libraries and lowering the chances of finding antibodies with desired specificity and affinity.

Innovation Solution

Mutating the C80 position in rabbit variable kappa light chains using polymerase chain reaction (PCR) to introduce amino acid substitutions, expanding the diversity of rabbit antigen binding protein nucleic acid libraries and enhancing the chances of isolating antibodies with high specificity for target antigens.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If the free thiol group at position C80 in rabbit variable kappa light chains is present, then the natural antibody structure is maintained, but the selectable diversity of rabbit antibody libraries is reduced and the chances of finding antibodies with desired specificity and affinity are lowered

Engineering Contradiction:

Improveselectable diversity of antibody librariesVSAvoidprobability of finding antibodies with desired specificity and affinity

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent removes the harmful free thiol group at position C80 from the variable kappa light chain by mutating it to alanine. This extraction of the problematic residue enables the antibody library to achieve full diversity without the restrictive effects of the free thiol group, while maintaining the essential antibody structure and function through the conserved framework regions.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent changes the amino acid parameter at position C80 from cysteine (with free thiol) to alanine (without free thiol). This parameter change fundamentally alters the chemical properties at the critical position, eliminating the steric and chemical constraints imposed by the free thiol group while preserving the overall antibody fold and binding capability through the conserved framework.

Inventive Principle:

Principle #35Parameter changes

2Productivity

If conventional hybridoma generation and antigen-specific B cell sorting strategies are used, then rabbit antibodies can be isolated, but the survival of B cells is low and the time window to screen or immortalize B cells is short, resulting in loss of antibody diversity

Engineering Contradiction:

Improveefficiency of antibody isolationVSAvoidantibody diversity

Core Design Contradiction:

ProductivityVSQuantity of substance

Solution Approach 1:

The patent performs preliminary action by creating a phage display library that captures and preserves the complete antibody repertoire diversity before the B cells can be lost. The library is constructed by amplifying and cloning variable region genes from immunized rabbit B cells, thereby preserving diversity in advance and eliminating the need for time-sensitive B cell isolation and immortalization steps.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent creates a copy of the antibody repertoire in the form of a phage display library. Instead of working with the original, fragile B cells that have low survival rates, the patent copies the antibody genetic information into a stable, easily manipulated phage display system, thereby preserving diversity and enabling unlimited screening time.

Inventive Principle:

Principle #26Copying

3Ease of operation

If the free thiol group at position C80 is present in rabbit light chains, then the natural immune response is maintained, but the display and selection of antibody scFv fragments is restricted

Engineering Contradiction:

Improvedisplay and selection of antibody scFv fragmentsVSAvoid selectable diversity of rabbit libraries

Core Design Contradiction:

Ease of operationVSAdaptability or versatility

Solution Approach 1:

The patent extracts the problematic free thiol group at position C80 from the variable kappa light chain sequence. By mutating cysteine to alanine at this critical position, the patent removes the chemical constraint that prevented proper display and selection of scFv fragments in phage display systems, while maintaining the essential antibody structure through the conserved framework regions.

Inventive Principle:

Principle #2Taking out (Extraction)

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 significantly expands the diversity of rabbit antibody libraries, increasing the probability of identifying antibodies with high affinity for target antigens, such as peptide-MHC complexes, thereby improving the efficiency of targeting cancer cells or virally-infected cells.

Implementation Method 1

Mutating the C80 position in rabbit variable kappa light chains using polymerase chain reaction (PCR) to introduce amino acid substitutions

Methodology Applied

Scientific EffectPolymerase chain reaction (PCR):

Data Source

PatentUS12435444B2Rabbit-derived antigen binding protein nucleic acid libraries and methods of making the same

Publication Date: 2025.10.07 CDR LIFE AG

AI summary

Rabbit antigen binding protein nucleic acid libraries are provided (e.g., nucleic acid libraries encoding antigen binding proteins that specifically recognize a target peptide-MHC (pMHC)). Methods of producing the rabbit antigen binding protein nucleic acid libraries are also provided.