437 Personalized tumor-trained lymphocytes – a neoantigen targeted T cell therapy product for treatment of colorectal cancer

<h3>Background</h3> Adoptive T cell therapy utilizing high-precision neoantigen targeting is a promising concept for the treatment of solid tumors. Personalized tumor trained lymphocytes (pTTL) is an autologous T cell product originating from regional lymph nodes (RLN) and targeting patient-specific neoantigens. A phase I/II First in Human (FIH) clinical trial of pTTL in Stage IV colorectal cancer (CRC) patients is ongoing. Methods pTTL is produced through <i>in vitro</i> expansion of T cells derived from tumor-adjacent RLNs. The T cells are stimulated with a patient-specific array of neoantigens utilizing the proprietary EpiTCer® technology. Tumor specific mutations are identified from next generation sequencing data obtained from tumor and normal tissue samples, and personal neoantigen epitopes are selected and ranked using the bioinformatic software PIOR®. Neoantigen epitope containing proteins are produced and linked to EpiTCer® beads (paramagnetic micro-particles), used for tumor-selective T cell expansion in pTTL manufacturing. pTTL is characterized by FACS phenotyping of T cell differentiation and activation markers. Neoantigen specificity is assessed by FACS and/or Fluorospot analysis of IFNγ, TNFα, degranulation marker CD107a and co-expression of cell activation induced markers (CD25, OX40, 4-1BB, and CD40-L) after restimulation of pTTL with EpiTCer® beads. TCR-sequencing of RLN cells and corresponding pTTL measures clonality of starting material and T cell product. <h3>Results and Conclusions</h3> Characterization of pTTL reveals that the pTTL manufacturing process generates a T cell product with individually varying CD4/CD8 ratios and minor proportions of other cells, mainly NK-cells. It contains a significant proportion of memory T cells with limited percentages of terminally differentiated T cells, indicating functionality. Functionality is also supported by upregulation of IFN-γ and TNF-α production and increased surface expression of CD107a, i.e. markers of anti-tumor killing potential, in response to specific EpiTCer® beads restimulation. TCR sequencing shows that pTTL is oligoclonal, indicating antigen-specific expansion during manufacturing from polyclonal RLNs. Moreover, neoantigen-specificity is corroborated by the upregulation of T cell activation-induced marker coexpression of varying magnitude after stimulation with separate neoantigens on a subgroup of CD4+ and CD8+ T cells within pTTL. In the ongoing FIH trial, up to 16 patients with Stage IV CRC will be treated with a single-dose pTTL after chemotherapy-based preconditioning with cyclophosphamide and fludarabine. A dose-escalation design is applied. The primary endpoint is safety. Secondary outcomes include objective response, overall survival, and progression-free survival. Biomarkers for pTTL persistence, pTTL characteristics, and response will be evaluated. <h3>Trial Registration</h3> Trial Registration EUDRA CT #2022-000394-96. Clinicaltrials.gov Identifier #NCT05908643. <h3>Ethics Approval</h3> The study was approved by Swedish Ethical Review Authority, approval number 2022-01842-01.