Directed evolution of a stable scaffold for T-cell receptor engineering

Area of Science:

• Immunology
• Protein Engineering
• Biotechnology

Background:

• Single-chain T-cell receptors (scTCRs) are crucial for adaptive immunity.
• Existing scTCRs often lack the stability and expression efficiency required for therapeutic applications.
• Improving scTCR stability and solubility is key for developing novel immunotherapies.

Purpose of the Study:

• To engineer a highly stable and soluble single-chain T-cell receptor (scTCR) scaffold.
• To investigate the correlation between thermal stability and intracellular processing efficiency.
• To create a versatile scTCR scaffold for therapeutic applications and advanced selection strategies.

Main Methods:

• Directed evolution and yeast surface display were employed to enhance scTCR properties.
• scTCRs were evolved for thermal denaturation resistance and improved intracellular processing.
• Mutations were recombined to generate a superior scTCR scaffold.

Main Results:

• An engineered scTCR scaffold demonstrated high stability (over 1 hour at 65°C) and solubility (>4 mg/mL).
• Expression levels reached 7.5 mg/L in shake flasks, comparable to single-chain antibodies.
• The scaffold retained specific ligand binding to peptide-major histocompatibility complexes (pMHCs) and bacterial superantigen.
• Yeast displaying the scTCR formed conjugates with antigen-presenting cells (APCs).

Conclusions:

• Thermal stability is a reliable predictor of scTCR kinetic processing efficiency.
• The developed scTCR scaffold offers significant improvements over existing constructs.
• This scaffold enables engineering of high-affinity soluble scTCRs for antagonizing cell-mediated immunity.
• Novel in situ cell-to-cell selection strategies for scTCR evolution are feasible using APC conjugation.