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Published on: September 10, 2017
Assessing TCR identity, knock-in efficiency, and potency for individualized TCR-T cell therapy
Julien Camperi1, Srinidhi Devarajan1, Andrew McKay2
1Cell Therapy Engineering and Development, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
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.
Insights
New analytical assays were developed to ensure the quality of T cell therapies targeting cancer neoantigens. These methods characterize individual T cell receptors (TCRs) for effective cancer treatment.
Area of Science:
- Oncology
- Immunology
- Biotechnology
Background:
- Cancer neoantigen discovery is accelerated by advances in sequencing and bioinformatics.
- Tumor neoantigens elicit T cell responses, making T cell receptors (TCRs) targets for cancer therapy.
- Individualized TCR-based therapies offer a promising strategy for effective cancer treatment by selecting patient-specific TCRs.
Purpose of the Study:
- To develop and validate multiplex analytical assays for characterizing TCR-T cell drug products.
- To establish quality control strategies for individualized TCR-T cell therapies.
- To assess the identity, efficiency, and potency of engineered TCRs in a multi-TCR product.
Main Methods:
- Next-generation sequencing (NGS) using Illumina MiSeq and PacBio platforms to confirm TCR identity and variable regions.
- Droplet digital PCR (ddPCR) with specific primers to quantify individual and total TCR knock-in efficiencies.
- A potency assay involving RNA transfection to measure dose-dependent T cell activation via CD137 expression and cytokine secretion.
Main Results:
- Successful development of three multiplex assays for TCR-T cell product characterization.
- Confirmation of TCR identity and differentiation by variable regions using two NGS platforms.
- Accurate measurement of TCR knock-in efficiencies and dose-dependent T cell activation, indicating product potency.
Conclusions:
- The developed assays provide robust methods for characterizing individualized TCR-T cell products.
- These assays offer critical insights into quality attributes essential for a comprehensive control strategy.
- This work supports the advancement of TCR-based immunotherapies for cancer treatment.
