Fusion Binding Scaffolds for Stable High-Diversity Antibody Libraries

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Solution Overview

Problem

Existing methods for antibody humanization and diversification, such as CDR grafting and RAG1/RAG2 recombination, face limitations in maintaining binding specificity and diversity, particularly when incorporating non-antibody peptides, and struggle with disulfide bond formation and proteolytic degradation, especially in prokaryotic systems.

Innovation Solution

Incorporating an entire donor diversity scaffold domain within a recipient diversity scaffold domain, with short linkers or direct joining, to create a fusion protein that maintains structural integrity and enhances diversity and binding activity, allowing for the selection of binding members with improved properties.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If CDR grafting is used for antibody humanization, then binding specificity is maintained, but structural integrity and stability deteriorate due to framework region incompatibility

Engineering Contradiction:

Improvebinding specificityVSAvoidstructural integrity

Core Design Contradiction:

ReliabilityVSStability of the object's composition

Solution Approach 1:

The patent applies parameter changes by systematically varying framework residues at specific positions surrounding the CDR regions. Instead of using a static human framework, the invention introduces multiple possible amino acid substitutions at defined framework positions to optimize both structural stability and CDR conformation, thereby resolving the contradiction between maintaining binding specificity and ensuring structural integrity.

Inventive Principle:

Principle #35Parameter changes

2Adaptability or versatility

If non-antibody peptides are inserted into antibody frameworks, then diversity is enhanced, but disulfide bond formation and proteolytic stability deteriorate

Engineering Contradiction:

ImprovediversityVSAvoidproteolytic stability

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent uses short linker peptides as intermediaries between the inserted non-antibody peptide and the antibody framework. These linkers serve as buffer zones that prevent direct interference between the foreign peptide and the antibody's disulfide bond network, thereby maintaining proteolytic stability while allowing diversity enhancement through peptide insertion.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The invention applies local quality by introducing diversity only at specific designated regions (CDR loops and adjacent framework positions) while keeping the core framework structure intact. This localized diversification approach allows insertion of non-antibody peptides without compromising the overall structural integrity and disulfide bond formation of the antibody molecule.

Inventive Principle:

Principle #3Local quality

3Adaptability or versatility

If RAG1/RAG2 recombination is used for antibody diversification, then binding activity is generated, but manufacturing complexity increases due to mammalian cell system requirements

Engineering Contradiction:

Improvebinding activityVSAvoidmanufacturing complexity

Core Design Contradiction:

Adaptability or versatilityVSEase of manufacture

Solution Approach 1:

The patent replaces the complex mammalian cell-based RAG1/RAG2 recombination system with a simpler in vitro chemical synthesis and assembly approach. Instead of requiring living mammalian cells to perform enzymatic recombination, the invention uses biochemical methods to assemble diverse antibody variants, thereby generating binding activity while significantly reducing manufacturing complexity and enabling prokaryotic expression systems.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

4Ease of operation

If long linkers are used to join donor and recipient scaffold domains, then structural flexibility is improved, but proteolytic degradation increases

Engineering Contradiction:

Improvestructural flexibilityVSAvoidin vivo half-life

Core Design Contradiction:

Ease of operationVSDuration of action of stationary object

Solution Approach 1:

The patent optimizes linker parameters by systematically varying linker length, composition, and flexibility to achieve the optimal balance between structural flexibility and proteolytic resistance. The invention identifies specific linker sequences and lengths that provide sufficient flexibility for proper domain orientation while minimizing susceptibility to proteolytic cleavage, thereby extending in vivo half-life.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention creates composite linker structures that combine different amino acid sequences with complementary properties. These composite linkers integrate flexible regions for proper domain positioning with protease-resistant sequences, achieving both structural flexibility and enhanced stability against proteolytic degradation simultaneously.

Inventive Principle:

Principle #40Composite materials

Data Source

PatentUS20260043020A1Binding members with altered diversity scaffold domains

Publication Date: 2026.02.12 MAXION THERAPEUTICS LTD

AI summary

This invention relates to libraries of binding members that each comprise a fusion protein which contains a donor diversity scaffold domain, such as a cysteine rich protein, inserted within a recipient diversity scaffold domain, such as an antibody constant or variable domain. Libraries and methods of generating libraries are provided, along with screening methods, binding members and methods of using the binding members.