Identification of tumor-reactive T cell receptors through functional single cell interaction analyses for personalized T cell therapy

Cancer immunotherapy approaches such as adoptive transfer of chimeric antigen receptor (CAR) T cells or tumor-infiltrating lymphocytes have yielded promising results in hematological malignancies. Despite inducing deep responses in multiple myeloma, both antigen escape and limited persistence lead to CAR-T cell therapy resistance. Since multiple myeloma remains an incurable malignancy, novel therapeutic approaches are urgently needed. Adoptive transfer of T cells expressing an engineered T cell receptor (TCR-T cells) represent a promising therapeutic alternative. In contrast to CAR-T cells, TCR-T cells can additionally target intracellular antigens, thereby expanding the range of potential immunotherapeutic targets. Furthermore, TCR-T cell therapy may lead to prolonged persistence in vivo and could mediate sustained anti-tumor effects.

In our project, we identified TCRs specifically targeting autologous myeloma cells. Myeloma-reactive T cells were identified applying nanofluidics and opto-electropositioning using the Bruker Cellular Analysis Lightning® platform. This enabled simultaneous functional analysis of up to 1500 individual T cell/target cell interactions on a chip. Myeloma-reactive T cells were discovered based on the secretion of cytokines (IFNγ, IL-2, TNFα) and surface expression of 4-1BB (CD137). Among 15 myeloma patients tested, reactive T cells demonstrating various cytokine secretion patterns and 4-1BB expression were detected (on average 1.2% of T cells screened). Individual myeloma-reactive T cells were isolated and their respective TCR was sequenced. Additionally, single-cell RNA sequencing was conducted for all 15 patients. By mapping TCR sequences of exported T cells to the single-cell RNA sequencing data, a gene expression signature of myeloma-reactive T cells was established. Genomic TCR sequences were in vitro transcribed and electroporated into autologous CD8+ T cells, followed by co-culture with myeloma cells or PBMCs as negative control. Myeloma-reactivity of transgenic T cells was determined for three patients (n = 11 TCRs) by flow cytometric measurement of T cell activation markers CD69 and 4-1BB. Through immuno-precipitation of MHC I molecules and mass spectrometry of the eluted peptides, we identified T cell responses against both shared and private cancer antigens for 12 patients.

To summarize, we established a pipeline for the identification and functional validation of myeloma-reactive T cells. Together with the evidence of expressed shared and private myeloma derived antigens, we envision this approach to facilitate patient-individualized T cell therapy for multiple myeloma patients.