SUCCESSFUL REDIRECTION OF TCR SPECIFICITY AGAINST CRYPTIC LEUKEMIA ANTIGENS USING NON-VIRAL TCR ENGINEERING

Abstract

Background & Aim

Acute myeloid leukemia (AML) remains highly lethal despite significant treatment advances, with a high relapse rate and an urgent need for novel therapies. The discovery and characterization of new antigenic targets capable of eliciting therapeutic responses remain critical to treating more patients. Leukemia cells can express tumor-specific antigens (TSA) absent in healthy tissues. Proteogenomic analysis have identified several MHC Class I-associated peptides eluted from AML cells that are derived from extra-exomic regions of the genome and selectively expressed in AML. These cryptic antigens present as promising targets for immunotherapy, prompting us to characterize T-cell responses against them and develop a T-cell receptor (TCR) transgenic strategy for clinical application.

Methodology

Using synthesized peptides, we generated cryptic TSA-specific CD8⁺ T cell lines by co-culturing healthy donor T cells with autologous dendritic cells presenting the peptide of interest. Antigen-specific T cells were identified via cytokine secretion assays and fluorescent MHC-peptide multimers. Sorted multimer-positive T cells underwent RNA 5’RACE PCR to amplify their TCR alpha and beta chains. These were cloned into a nanoplasmid and inserted in situ into the TRAC locus of the endogenous TCR via single-step TCR replacement using CRISPR-Cas9, ensuring deletion of native TCR chains. Antigenic redirection was confirmed through cytokine secretion after exposure to cryptic TSA peptide and cytotoxicity assay.

Results

After establishing that cryptic TSAs were recognized by T cells in healthy donors' peripheral blood, we demonstrated that at least 60% (6/10) of tested peptides were antigenic. T-cell responses were highly oligoclonal, and as few clonotypes were present among antigen-specific cells, alpha and beta chain pairing following 5’RACE PCR was facilitated. Assessment of CD3 and TCR expression using flow cytometry confirmed successful TCR replacement. Functional assays, including ELISpot, cytokine secretion, and cytotoxicity assays, confirmed that these engineered TCRs could redirect T cells' antigenic specificity towards the recognition of cryptic leukemia antigens.

Conclusion

Ex vivo expansion of cryptic antigen-specific T cells enables TCR identification that can be used to devise new T-cell immunotherapies through TCR engineering. This strategy allows the generation of large numbers of AML-specific, reactive T lymphocytes towards cryptic TSA, offering promising new therapeutic avenues.