CD40 Receptor Agonist Proteins with Peptide Linkers
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Solution Overview
Problem
Current CD40 receptor agonists face challenges such as aggregation, inefficient recombinant manufacturing, and reliance on Fc-gamma-R based crosslinking for biological activity, which limits their stability and effectiveness in therapeutic applications.
Innovation Solution
Development of specific CD40 receptor agonist proteins comprising three soluble CD40L domains and an Fc fragment, with peptide linkers and hinge-linkers to enhance stability and biological activity, reducing aggregation and proteolytic degradation, and prolonging in vivo half-life.
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 trimerization is inefficient
Solution Approach 1:
The patent extracts only the essential trimerization capability from the C1q/collectin multimerization components, using minimal peptide linkers (e.g., (GGGGS)n) to connect three TNF cytokine monomers directly, thereby eliminating the large molecular weight burden while preserving trimer formation
Solution Approach 2:
The patent optimizes the linker length and composition parameters (using short glycine-serine repeats with controlled n values) to achieve efficient trimerization with minimal molecular weight addition, balancing flexibility and compactness
2Stability of the object's composition
If N-terminal stabilization motifs or coiled-coil structures are used, then trimer stability is improved, but the proteins form macromolecular aggregates after pH and ionic strength changes
Solution Approach 1:
The patent uses short, flexible peptide linkers instead of stable coiled-coil structures, accepting that the linkers provide minimal structural constraint while avoiding the aggregation-prone characteristics of rigid helical motifs
Solution Approach 2:
The patent modifies the chemical composition parameters of the linker (using neutral glycine and serine residues) to reduce electrostatic interactions that drive aggregation under varying pH and ionic conditions
3Reliability
If single-chain fusion polypeptides with three TNF family cytokine domains are used, then binding to receptor is achieved, but undesired aggregation occurs
Solution Approach 1:
The patent segments the fusion polypeptide into three distinct TNF cytokine domains connected by separate peptide linkers, allowing each domain to maintain its binding capability while the linkers prevent inter-domain aggregation by providing flexible spacing
4Reliability
If F(ab')2 fragments of anti-CD40 antibodies are used, then CD40 binding is achieved, but agonistic activity requires further crosslinking
Solution Approach 1:
The patent merges three CD40L binding domains into a single trimeric structure, inherently providing the crosslinking capability needed for agonistic activity without requiring additional Fc-gamma-R based crosslinking steps
Data Source
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AI summary
Provided herein are specific CD40 receptor agonist proteins, nucleic acids encoding the same, and methods of treating a subject having a CD40L-associated disease or disorder. The CD40 receptor agonist proteins provided herein comprise three soluble CD40L domains and an Fc fragment. The CD40 receptor agonist proteins are substantially non-aggregating and suitable for therapeutic, diagnostic and/or research applications.