PD-1 Binding Protein Framework Mutations for Stable scFv Folding
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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 therapeutic applications.
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
1Volume of moving object
If single-chain antibody fragments (scFv's) are used instead of full antibodies, then tissue penetration and manufacturing cost are improved, but folding stability and solubility deteriorate
Solution Approach 1:
The patent applies local quality by making specific amino acid substitutions at particular positions within the scFv framework regions (e.g., positions 108, 110, 58 in the variable domains). These localized changes modify disulfide bond formation and framework structure specifically where needed to improve folding and stability, without altering the entire molecule or sacrificing the size advantages of scFv fragments.
2Strength
If multiple disulfide bonds are present in the scFv structure, then binding affinity is improved, but folding complexity and misfolding tendency increase
Solution Approach 1:
The patent applies preliminary action by pre-engineering the disulfide bond framework before antigen binding occurs. Specific cysteine residues are positioned in the framework regions to form predetermined disulfide bonds that stabilize the folded structure during production and storage, ensuring correct folding precedes and enables high-affinity binding without the complexity of forming multiple bonds de novo.
3Productivity
If scFv production yield is increased, then therapeutic efficacy is improved, but aggregation and insolubility worsen
Solution Approach 1:
The patent applies parameter changes by modifying amino acid residues in the framework regions (such as substitutions at positions 108, 110, and 58) to alter the physicochemical properties of the scFv. These changes improve solubility and reduce aggregation propensity, allowing high-yield production without the harmful effects of self-aggregation that typically limit productivity.
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) L108G and/or T110R of the heavy chain reference sequence and/or I58R 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.