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J Immunol Methods ; 517: 113491, 2023 06.
Article in English | MEDLINE | ID: mdl-37187316

ABSTRACT

Advances in mass spectrometry, genome sequencing techniques, and bioinformatic strategies have accelerated the discovery of cancer-specific neoantigens. Tumors express multiple immunogenic neoantigens, and neoantigen-specific T cell receptors (TCRs) can be identified in peripheral blood's mononuclear cells in cancer patients. Therefore, individualized TCR-based therapies are a promising approach whereby multiple neoantigen-specific TCRs can be selected in each patient, potentially leading to a highly effective treatment for cancer patients. We developed three multiplex analytical assays to determine the quality attributes of the TCR-T cell drug product with a mixture of five engineered TCRs. The identity of each TCR was determined by two NGS-based methods, Illumina MiSeq and PacBio platforms. This approach not only confirms the expected TCR sequences but also differentiates them by their variable regions. The five individual TCR and total TCR knock-in efficiencies were measured by droplet digital PCR using specific reverse primers. A potency assay based on transfection of antigen-encoding-RNA was developed to assess the dose-dependent activation of T cells for each TCR by measuring the surface activation marker CD137 expression and cytokine secretion. This work provides new assays to characterize individualized TCR-T cell products and insights into quality attributes for the control strategy.


Subject(s)
Antigens, Neoplasm , Neoplasms , Humans , Receptors, Antigen, T-Cell , T-Lymphocytes , Neoplasms/therapy , Cell- and Tissue-Based Therapy
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