High-affinity T-cell receptor specific for MyD88 L265P mutation for adoptive T-cell therapy of B-cell malignancies

Abstract

BackgroundAdoptive transfer of engineered T cells has shown remarkable success in B-cell malignancies. However, the most common strategy of targeting lineage-specific antigens can lead to undesirable side effects. Also, a substantial fraction of patients have refractory disease. Novel treatment approaches with more precise targeting may be an appealing alternative. Oncogenic somatic mutations represent ideal targets because of tumor specificity. Mutation-derived neoantigens can be recognized by T-cell receptors (TCRs) in the context of MHC–peptide presentation.MethodsHere we have generated T-cell lines from healthy donors by autologous in vitro priming, targeting a missense mutation on the adaptor protein MyD88, changing leucine at position 265 to proline (MyD88 L265P), which is one of the most common driver mutations found in B-cell lymphomas.ResultsGenerated T-cell lines were selectively reactive against the mutant HLA-B*07:02-restricted epitope but not against the corresponding wild-type peptide. Cloned TCRs from these cell lines led to mutation-specific and HLA-restricted reactivity with varying functional avidity. T cells engineered with a mutation-specific TCR (TCR-T cells) recognized and killed B-cell lymphoma cell lines characterized by intrinsic MyD88 L265P mutation. Furthermore, TCR-T cells showed promising therapeutic efficacy in xenograft mouse models. In addition, initial safety screening did not indicate any sign of off-target reactivity.ConclusionTaken together, our data suggest that mutation-specific TCRs can be used to target the MyD88 L265P mutation, and hold promise for precision therapy in a significant subgroup of B-cell malignancies, possibly achieving the goal of absolute tumor specificity, a long sought-after dream of immunotherapy.

Isolation of Neoantigen-Specific Human T Cell Receptors from Different Human and Murine Repertoires

“…Since we have solely investigated the isolation of TCRs against potential HLA-A*02:01-restricted neoepitopes, the possibility remains that specific TCRs against neoepitopes, other than the here predicted ones, could potentially be isolated. Indeed, we have been successful in generating high-affinity TCRs specific to the HLA-B*07:02-restricted MyD88 L265P mutation [ 17 ].…”

“…Recently, we have been able to isolate a specific high-affinity TCR for the recurrent MyD88 L265P mutation. In this case, we screened about 20 donors and were able to isolate 13 TCR sequences, from which seven high-affinity TCRs were identified from five donors [ 17 ]. Here, we tested five different donors for the potential neoepitopes of two patients.…”

“…For bulk sorted CD3+CD8+CD107+ T cells, total RNA was extracted and 5′RACE-ready cDNA was synthesized using the SMARTer RACE kit (Takara Clonetech, Kusatsu, Japan). TCRα and β variable (TRAV/TRBV) chains were amplified as previously described [ 17 ] with TCRA (5′-CGGCCACTTTCAGGAGGAGGATTCGGACC-3′) or TCRB (TCRB: 5′-CCGTAGAACTGGACTTGACAGCGGAAGTGG-3′) gene-specific primers. Amplified PCR products were agarose-gel-purified, and corresponding bands were cloned using the Zero Blunt II TOPO vector (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA).…”

“…This hypothesis is supported by the observation of Strønen and colleagues, who successfully isolated neoantigen-specific T cell clones from PB of healthy donors, which were not detected in the patients’ TIL [ 10 ]. Moreover, we and others have shown that an independent, non-tolerized T cell repertoire of an HLA-compatible healthy donor is a suitable alternative source for the isolation of neoantigen-specific T cells [ 10 , 17 , 18 , 19 ]. Although the risk of cross- and alloreactivity is increased when using donor TCR repertoires compared to the patient’s own repertoire, this strategy has certain advantages: larger amounts of blood can be obtained from healthy donors than from most cancer patients, and the difficulty to isolate and expand cells from a presumably very low precursor frequency can be circumvented.…”

Neo-Splicetopes in Tumor Therapy: A Lost Case?

“…The resulting MyD88 L265P derived neo-epitope however exclusively binds to HLA-B*07:02. Therefore, only patients that carry the HLA-B*07:02 haplotype will potentially benefit from ATT by using a recently published MyD88 L265P specific TCR ( 41 ). So far, problems like these prevented the large-scale use of mutation-specific TCRs using T cells directed against mutational epitopes.…”