Production of high-complexity frameshift neoantigen peptide microarrays

Area of Science:

• Biotechnology
• Immunology
• Proteomics

Background:

• Peptide microarray technologies facilitate the parallel measurement of numerous antigen-antibody interactions.
• Profiling patient-specific antibodies against neoantigens is crucial for understanding immune responses.

Purpose of the Study:

• To develop and demonstrate a novel peptide microarray system for synthesizing and analyzing a large number of frameshift neoantigens.
• To profile patient-specific antibodies reactive to frameshift neoantigens in a single assay.

Main Methods:

• Utilized mask-based photolithographic peptide synthesis for *in situ* synthesis of over 400,000 frameshift neoantigens.
• Developed a system producing 208 replicate microarrays per wafer, enabling simultaneous synthesis of over 300 million peptides.
• Demonstrated detection of peripheral-blood antibody binding to frameshift neoantigens across multiple synthetic lots.

Main Results:

• Successfully synthesized >400,000 unique frameshift neoantigens on a single peptide microarray.
• The system demonstrated feasibility in detecting peripheral-blood antibody binding to these neoantigens.
• High-throughput synthesis capability of over 300 million peptides simultaneously was achieved.

Conclusions:

• The developed peptide microarray system is a feasible and powerful tool for profiling patient-specific antibodies against a vast array of frameshift neoantigens.
• This technology offers a high-throughput solution for immunological studies and potential diagnostic applications.
• The system's scalability supports routine synthesis for large-scale neoantigen antibody profiling.