Single-Chain Fusion Polypeptides for TNF Cytokine Stability
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Solution Overview
Problem
Existing recombinant fusion proteins comprising TNF superfamily cytokines face challenges in efficient recombinant manufacturing and stability against aggregation, particularly trimerization domains with high molecular weight and inefficient trimerization, as well as issues with coiled-coil structures forming aggregates under pH and ionic strength changes.
Innovation Solution
A single-chain fusion polypeptide comprising three soluble TNF-family cytokine domains connected by two peptide linkers, designed to be substantially non-aggregating, with optimized linker sequences and domain structures to maintain stability and functionality, allowing for efficient recombinant production and therapeutic or diagnostic applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If trimerization domains with large molecular weight are used to stabilize TNF cytokine trimers, then trimer stability is improved, but manufacturing efficiency and solubility deteriorate
Solution Approach 1:
The patent removes the problematic large molecular weight trimerization domain (C1q or collectin) from the fusion protein construct and instead relies on the natural trimerization capability of the TNF cytokine receptor binding domain itself, which is stabilized by N-terminal amino acid domains. This extraction of the unnecessary component resolves the contradiction by maintaining trimer stability without the manufacturing inefficiencies of large trimerization domains.
Solution Approach 2:
The patent introduces small peptide linkers (5-20 amino acids) as intermediaries to connect the N-terminal stabilizing domains to the receptor binding domains. These short linkers serve as mediators that maintain the spatial arrangement necessary for trimer stability while avoiding the aggregation and solubility problems associated with large trimerization domains, thus improving both stability and manufacturability.
2Stability of the object's composition
If coiled-coil structures are used for trimerization, then trimer formation is achieved, but aggregation occurs under pH and ionic strength changes
Solution Approach 1:
The patent changes the structural parameters of the trimerization mechanism by using N-terminal amino acid domains with specific sequences (such as those found in CD95L) that are naturally resistant to aggregation under varying pH and ionic strength conditions. This parameter change from coiled-coil structures to N-terminal domain-mediated trimerization resolves the reliability issue while maintaining trimer formation capability.
3Adaptability or versatility
If single-chain fusion polypeptides with multiple TNF ligand domains are constructed, then receptor binding capability is enhanced, but aggregation tendency increases
Solution Approach 1:
The patent segments the fusion polypeptide into distinct functional modules: N-terminal stabilizing domains, peptide linkers, and receptor binding domains. This segmentation allows each component to perform its specific function independently, with the N-terminal domains preventing aggregation of the multiple TNF ligand domains while the linkers maintain appropriate spacing, thus resolving the contradiction between enhanced binding capability and reduced aggregation.
Data Source
AI summary
The present invention refers to single-chain fusion proteins comprising three soluble TNF superfamily (TNFSF) cytokine domains and nucleic acid molecules encoding these fusion proteins. The fusion proteins are substantially non-aggregating and suitable for therapeutic, diagnostic and/or research applications.