Background Approximately 30–40% of AML patients relapse following allogeneic hematopoietic stem cell transplant therapy, leaving them with very few treatment options.1 2 Rare patients that naturally develop an HA-1-specific graft-versus-leukemia T cell response, however, show substantially lower relapse rates.3 4 HA-1 (VLHDDLLEA, genotype RS_1801284 A/G or A/A) is an HLA-A*02:01-and hematopoietically restricted minor histocompatibility antigen, making it an ideal candidate for TCR immunotherapy for liquid tumors.5
Methods We developed a high-throughput TCR discovery platform that enables rapid cloning of antigen-specific TCRs from healthy donors. We then used this platform to screen 178.3 million naïve CD8+ T cells from six unique HA-1- (VLRDDLLEA, genotype RS_1801284 G/G) donors, identifying 329 HA-1-specific TCRs. We tested each TCR for expression and the ability to kill HA-1+ target cells, using a previously published, clinical-stage HA-1-specific TCR as a benchmark for these studies.6 In parallel, we tested TCR constant region modifications to promote expression and proper pairing of exogenous TCR alpha and beta chains and designed a lentiviral vector to co-deliver CD8 coreceptors as well as a CD34 enrichment tag to enable purification of engineered T cells. The top 11 candidates were cloned into our optimized backbone and evaluated for cytotoxicity, cytokine production, and T cell proliferation using a panel of HLA-A*02:01+ HA-1+ cell lines. Finally, the top two TCRs were evaluated for allo-reactivity and off-target cross-reactivity using our proprietary genome-wide T-Scan platform.
Results The TCR discovery and evaluation platform described here identified 329 HA-1-specific TCRs from a total of 178.3 million naïve T cells, and TSC-100 as the most active TCR. Defined mutations in the constant region of TSC-100 enhanced its surface expression while decreasing expression of endogenous TCRs, and co-introduction of CD8 enabled efficient engagement and function of engineered CD4 cells. Overall, TSC-100 exhibited comparable activity to a clinical-stage benchmark TCR when challenged with cell lines expressing moderate to high levels of HA-1, and superior activity when incubated with cell lines expressing low levels of both HA-1 and MHC-I.6 In addition, TSC-100 exhibited no detectable allo-reactivity to 108 different HLA types tested, and minimal off-target effects when challenged with a genome-wide library expressing peptides derived from human proteins.
Conclusions TSC-100 exhibits comparable or superior activity to a clinical-stage therapeutic TCR, with minimal allo-reactivity or off-target effects. Based on these results, TSC-100 has been advanced to IND-enabling activities to prepare for first-in-human testing in 2021.
Ethics Approval All clinical samples used in the study were collected by STEMCELL Technologies, StemExpress and HemaCare using their IRB approved protocols.
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