In vitro proteasome processing of neo-splicetopes does not predict their presentation in vivo

Area of Science:

• Immunology
• Oncology
• Proteasome Biology

Background:

• Proteasome-catalyzed peptide splicing (PCPS) can create neoepitopes from cancer antigens for T cell receptor (TCR)-based therapies.
• Predictive algorithms aim to identify these neo-splicetopes for therapeutic targeting.

Purpose of the Study:

• To evaluate the in vivo generation and presentation of KRASG12V- and RAC2P29L-derived neo-splicetopes.
• To assess the efficacy of TCRs generated against predicted neo-splicetopes.
• To question the reliability of predictive algorithms and in vitro PCPS for simulating in vivo processes.

Main Methods:

• Development of TCRs against putative KRASG12V- and RAC2P29L-neo-splicetopes using a prediction algorithm.
• Testing TCR efficacy against target peptides in mice with a diverse human TCR repertoire.
• Assessing in vivo neo-splicetope generation and presentation.

Main Results:

• TCRs recognized target peptides with high efficacy in vitro.
• No neo-splicetope-specific T cell response was detected in vivo.
• Experimental evidence for natural processing and presentation of KRASG12V- and RAC2P29L-neo-splicetopes was lacking.
• Only RAC2P29L-neo-splicetopes were generated by in vitro PCPS.

Conclusions:

• Available algorithms and in vitro PCPS may not reliably simulate in vivo splicing and presentation.
• The study challenges the general applicability of algorithm-driven 'reverse immunology' for identifying cancer-specific neo-splicetopes.
• Further research is needed to validate in vivo neo-splicetope generation and presentation for cancer immunotherapy.