Insertable Variable Fragments for Antibody Loop Integration

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Solution Overview

Problem

Current methods for producing polypeptides with specific antigen-binding properties, such as scFv structures, face challenges in inserting variable fragments into protein loops without disrupting the protein structure or stability, limiting their utility in targeting antigens effectively.

Innovation Solution

Development of insertable variable fragment (iFv) peptides with two linker regions and a split variable domain, allowing for insertion into protein loops without destabilizing the protein fold, and enabling specific antigen binding and targeting.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If variable fragments are inserted into protein loops using conventional scFv structures, then antigen-binding properties are achieved, but the protein structure and stability are disrupted

Engineering Contradiction:

Improveantigen-binding capabilityVSAvoidprotein structure stability

Core Design Contradiction:

Adaptability or versatilityVSStability of the object's composition

Solution Approach 1:

The variable domain is divided into two separate fragments (VH and VL) that are connected by flexible linker regions. This segmentation allows the variable fragment to be inserted into protein loops without disrupting the overall protein fold, as each fragment can independently bind to its corresponding antigen while the linker provides flexibility to accommodate structural constraints

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Flexible linker regions serve as intermediaries between the VH and VL domains, allowing them to connect without rigidly constraining the protein structure. The linkers act as mediators that provide the necessary flexibility and distance for antigen binding while maintaining compatibility with the host protein's structural framework

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If insertable variable fragment peptides with two linker regions and split variable domain are used, then thermal stability and antigen-binding capabilities are enhanced, but device complexity increases

Engineering Contradiction:

Improvethermal stability and antigen-binding capabilityVSAvoidpeptide structure complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The iFv peptide structure with two linker regions and split variable domain serves multiple functions simultaneously: it provides antigen-binding capability through the variable domains, maintains thermal stability through the stable framework structure, and enables flexible insertion into various protein loops. This multi-functional design justifies the increased structural complexity by delivering multiple benefits in a single construct

Inventive Principle:

Principle #6Universality (Multi-functionality)

Data Source

PatentUS11117969B2Insertable variable fragments of antibodies and modified α1-α2 domains of NKG2D ligands

Publication Date: 2021.09.14 XYPHOS BIOSCIENCES INC

AI summary

This application relates generally to the production of polypeptides having specific antigen-binding properties of Fv domains, for example, insertable variable fragments of antibodies, and modified α1-α2 domains of NKG2D ligands. This application further relates to modified α1-α2 domains of NKG2D ligands attached to polypeptides, in some embodiments antibodies or fragments of antibodies. This application further relates to antigen-binding peptides derived from light and heavy chain antibody variable domains, which contain two linker regions and a split variable domain.