GITR Agonist Proteins with Engineered Trimerization Domains
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Solution Overview
Problem
There is a need for novel GITR receptor agonists that exhibit high biological activity independent of Fc-gamma-R based crosslinking in vivo, with high stability and efficient recombinant manufacturing, as existing technologies face challenges in achieving these criteria.
Innovation Solution
The development of specific GITR receptor agonist proteins comprising three soluble GITRL domains connected by peptide linkers and an antibody Fc fragment, which are designed to mimic the GITR:GITRL interaction, exhibit low proteolytic degradation, and are engineered for stability and efficient production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If fusion proteins comprising TNF cytokine and multimerization component (C1q or collectin) are used, then trimerization is achieved, but the trimerization domain has large molecular weight and/or trimerization is inefficient
Solution Approach 1:
The patent changes the molecular weight parameter of the trimerization domain by replacing large C1q or collectin domains with smaller, engineered trimerization domains derived from TNF superfamily cytokines, achieving efficient trimerization with reduced molecular weight
Solution Approach 2:
The patent creates composite fusion proteins combining TNF cytokine domains with engineered trimerization domains from the same TNF superfamily, producing a composite structure that achieves both trimerization and cytokine function with optimized properties
2Stability of the object's composition
If coiled-coil structures are used for stabilization, then trimerization is achieved, but macromolecular aggregates form after alteration of pH and/or ionic strength
Solution Approach 1:
The patent modifies the structural parameters of the trimerization domain by using TNF superfamily-derived domains with specific beta-sheet structures instead of coiled-coil structures, changing the stabilization mechanism to be less sensitive to pH and ionic strength variations
Solution Approach 2:
The patent uses simplified, engineered TNF-derived trimerization domains that are more stable and less prone to aggregation under varying conditions compared to complex coiled-coil structures
3Object-affected harmful factors
If single-chain fusion polypeptides comprising three TNF family cytokine domains are used, then non-aggregating properties are achieved, but Fc-gamma-R based crosslinking is required for in vivo activity
Solution Approach 1:
The patent extracts and removes the Fc fragment from the fusion protein, eliminating the dependence on Fc-gamma-R crosslinking mechanisms while retaining the essential trimerization and cytokine activity functions
Solution Approach 2:
The patent designs the TNF cytokine domain fusion protein to be self-sufficient, where the three cytokine domains themselves provide both trimerization and receptor activation functions without requiring additional Fc-mediated mechanisms
Data Source
AI summary
Provided herein are specific GITR receptor agonist proteins, nucleic acids encoding the same, and methods of treating a subject having a GITRL-associated disease or disorder. The GITR receptor agonist proteins provided herein comprise three soluble GITRL domains and an Fc fragment. The GITR receptor agonist proteins are substantially non-aggregating and suitable for therapeutic, diagnostic and/or research applications.