Intentional heterogeneity in autologous cell-based gene therapies: strategic considerations for first-in-human trials

Abstract

Cell-based gene therapies, including chimeric antigen receptor-T, T-cell receptor-T, and tumor-infiltrating lymphocyte therapies, have transformed the treatment landscape for certain cancers, yet their efficacy in solid tumors remains limited. Next-generation therapies aim to overcome biological barriers, enhance potency and safety, and streamline development timelines through innovative approaches. Recent advances in genome editing technologies have identified hundreds of gene edits that improve T-cell functionality in preclinical models. However, the limited direct translatability of these findings and the impracticality of testing each of the individual edits in a traditional clinical trial highlight the need for more efficient strategies.This article provides an overview of genome-wide screens that identify gene knockouts and knock-ins to enhance T-cell function and the limitations with translating these results to human trials. Next, we propose a novel clinical trial design for testing multiple gene modifications simultaneously within a single T-cell infusion product. This approach would enable head-to-head evaluation of edits in an internally controlled setting, accelerating the identification of promising candidate edits. Key considerations for Chemistry, Manufacturing, and Controls, non-clinical evaluation, and clinical protocols are discussed, with an emphasis on patient safety and ethical transparency.This framework is informed by insights shared at the "Unlocking Complex Cell-based Gene Therapies" workshop, held on May 6, 2024. Co-hosted by Friends of Cancer Research and the Parker Institute for Cancer Immunotherapy, the event brought together participants from academia, the US Food and Drug Administration, and patient advocacy groups. By fostering collaboration among these stakeholders, this innovative approach aims to accelerate the development of effective cell-based therapies for complex diseases.

Keywords: Adoptive cell therapy - ACT; Chimeric antigen receptor - CAR; Gene therapy; Solid tumor; T cell Receptor - TCR.

Figure 1. Testing a heterogeneous pool of genetically modified T cells. On the left, the infusion product is depicted, consisting of T cells uniformly modified to target one specific tumor-associated antigen. Distinct subpopulations within this infusion product would carry unique secondary gene edits (denoted as Edits A through D), each introduced to potentially enhance antitumor efficacy. Following infusion, analyses of tumor biopsies and blood samples would examine the persistence, expansion, function, and trafficking patterns of each genetically distinct subpopulation, as illustrated on the right. This would enable the assessment of how individual gene edits may influence the cells’ in vivo behavior, such as Edits A and C appearing in both tumor and blood, Edit B localized exclusively in blood, and Edit D not prevailing in either. Patient safety and efficacy would be continuously monitored throughout the study. Created in BioRender. Yang (2025) https://BioRender.com/j69j443.