Isolation and analysis of T cell receptors against a drug-selected secondary mutation in BCR-ABL for adoptive T cell therapy

Abstract

Adoptive T cell therapy with T cell receptor (TCR) engineered T cells is a promising approach for cancer treatment. Target antigens are categorized into tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). TAAs are overexpressed on cancer cells and often shared between different cancers, however, targeting TAAs has the risk of inducing autoimmune responses. In contrast to TAAs, TSAs are expressed exclusively on cancer cells, but only some of them are shared between patients. Since drug-selected secondary mutations in chronic myeloid leukemia (CML) are both cancer-specific and shared between patients, they become attractive targets. CML is a myeloproliferative disease. Over 95% of CML cases are associated with Philadelphia (Ph) chromosome t(9;22). Such abnormal chromosome results in the BCR-ABL hybrid gene that encodes a constantly active tyrosine kinase. Currently, tyrosine kinase inhibitors (TKIs) are used as the first-line treatment, as they effectively avoid uncontrollable cell division by blocking the ATP binding site of the kinase. Nevertheless, drug resistance is often observed due to drug-selected secondary mutations that prevent the TKIs from binding to the BCR-ABL protein. As relapsed CML patients with compound mutations are resistant to most or all TKIs, TCR gene therapy could benefit these patients.

In this project, we aimed to discover neoepitopes from drug-selected secondary mutations in CML and to generate neoepitope-specific TCRs for clinical use. ABabDII mice expressing a human TCR repertoire and HLA-A2 molecules were immunized with two selected mutant peptides and analyzed. Although the peptide comprising mutation T315I did not elicit immune responses, two specific TCRs against the peptide comprising mutation E255V (ABL-E255V) were isolated from ABabDII mice. The T9141 TCR exhibiting a superior avidity was further analyzed for off-target toxicity. As no allo- and cross-reactivities were detected, T9141 was constructed to express minimal murinized human TCR constant region (T9141-mmc) as in the clinic. The expression and the functionality of T9141-mmc were comparable to T9141. Furthermore, ABL-E255V was demonstrated to be endogenously processed and presented by the ABL-minigene-E255V-transduced cancer cell lines. Taking a step forward, we introduced the point mutation E255V into Ph+ CML cell lines to simulate the antigen expression of blast cells in CML patients. Although no T cell responses against the genome-edited BV173 cells were found, detectable T cell responses against the genome-edited K562-A2 cells were observed, suggesting that the induced T cell responses varied with the BCR-ABL expression level in CML cell lines. Accordingly, the potency of T9141 could differ among CML patients. Therefore, it is recommended to analyze the BCR-ABL expression level in corresponding CML patients, which should allow us to evaluate the efficacy of TCR gene therapy with T9141. Taken together, T9141 is optimal for TCR gene therapy; ABL-E255V could be a potential target.