Anti-PD-1 Binding Protein Framework Mutations for Solubility
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Solution Overview
Problem
Current PD-1 binding proteins, such as scFv's, face challenges with misfolding, instability, and low solubility, limiting their effectiveness in cell-based assays and clinical therapeutics.
Innovation Solution
Engineering PD-1 binding proteins with specific amino acid mutations in framework regions, such as L108G, T110R in VH-FR1 and VH-FR4, and I58R in VL-FR3, to enhance stability and solubility, allowing high-affinity binding to PD-1.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If scFv is generated from PD-1 antibody, then smaller size and greater tissue penetration are achieved, but solubility and stability deteriorate
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations (L108G, T110R in VH-FR1; V21K, L124G, T126R in full length scFv) to modify the physical and chemical properties of the scFv protein. These mutations change the folding kinetics and thermodynamic stability parameters, enabling the small scFv molecule to achieve both compact size and enhanced stability simultaneously.
2Stability of the object's composition
If multiple disulfide bonds are present in scFv, then structural stability is improved, but misfolding and self-aggregation increase
Solution Approach 1:
The patent applies preliminary action by introducing mutations in framework regions before the scFv undergoes folding and disulfide bond formation. The L108G and T110R mutations in VH-FR1 prepare the protein structure by reducing misfolding propensity early in the folding process, preventing aggregation before disulfide bonds are formed, thereby enabling stable folding with multiple disulfide bonds.
3Reliability
If framework region mutations are introduced, then solubility and stability are increased, but protein structure complexity increases
Solution Approach 1:
The patent applies local quality by introducing mutations only in specific framework regions (VH-FR1 and full length scFv) rather than throughout the entire protein structure. The L108G, T110R, V21K, L124G, and T126R mutations are localized to framework regions that do not directly participate in antigen binding, thereby improving solubility and stability while maintaining the simplicity of the overall protein structure and preserving binding function.
Data Source
AI summary
Aspects of the disclosure relate to PD-1 binding proteins comprising immunoglobulin domains which bind specifically to PD-1 and comprise at least three or six specific complementarity determining regions (CDRs) and which comprise a specific feature in a variable domain framework region(s), e.g., heavy variable domain framework 1, heavy domain framework 4, and/or light chain variable domain framework 3 region(s). In some embodiments, the PD-1 protein comprises the substitution(s) L 108G and/or THOR of the heavy chain reference sequence and/or 158R relative to the light chain reference sequence(s). The PD-1 binding proteins are useful, e.g., as immunologic adjuvants, for detecting and quantifying PD-1, monitoring patient responses to therapies, diagnosing PD-1 related conditions, and treating or preventing disorders involving PD-1 expressing cells, such as, e.g., cancers and autoimmune diseases. Also provided herein are antigen binding proteins comprising amino acid substitutions in the heavy chain framework 4 region for improved stability and solubility.