This last decade, chimeric antigen receptor (CAR) T-cell therapy has become a real treatment option for patients with B-cell malignancies, while multiple efforts are being made to extend this therapy to other malignancies and broader patient populations. However, several limitations remain, including those associated with the time-consuming and highly personalized manufacturing of autologous CAR-Ts. Technologies to establish “off-the-shelf” allogeneic CAR-Ts with low alloreactivity are currently being developed, with a strong focus on gene-editing technologies. Although these technologies have many advantages, they have also strong limitations, including double-strand breaks in the DNA with multiple associated safety risks as well as the lack of modulation. As an alternative, non-gene-editing technologies provide an interesting approach to support the development of allogeneic CAR-Ts in the future, with possibilities of fine-tuning gene expression and easy development. Here, we will review the different ways allogeneic CAR-Ts can be manufactured and discuss which technologies are currently used. The biggest hurdles for successful therapy of allogeneic CAR-Ts will be summarized, and finally, an overview of the current clinical evidence for allogeneic CAR-Ts in comparison to its autologous counterpart will be given.

Chimeric antigen receptor (CAR) T-cell therapy has proven a breakthrough in cancer treatment in the last decade, giving unprecedented results against hematological malignancies. All approved CAR T-cell products, as well as many being assessed in clinical trials, are generated using viral vectors to deploy the exogenous genetic material into T-cells. Viral vectors have a long-standing clinical history in gene delivery, and thus underwent iterations of optimization to improve their efficiency and safety. Nonetheless, their capacity to integrate semi-randomly into the host genome makes them potentially oncogenic via insertional mutagenesis and dysregulation of key cellular genes. Secondary cancers following CAR T-cell administration appear to be a rare adverse event. However several cases documented in the last few years put the spotlight on this issue, which might have been underestimated so far, given the relatively recent deployment of CAR T-cell therapies. Furthermore, the initial successes obtained in hematological malignancies have not yet been replicated in solid tumors. It is now clear that further enhancements are needed to allow CAR T-cells to increase long-term persistence, overcome exhaustion and cope with the immunosuppressive tumor microenvironment. To this aim, a variety of genomic engineering strategies are under evaluation, most relying on CRISPR/Cas9 or other gene editing technologies. These approaches are liable to introduce unintended, irreversible genomic alterations in the product cells. In the first part of this review, we will discuss the viral and non-viral approaches used for the generation of CAR T-cells, whereas in the second part we will focus on gene editing and non-gene editing T-cell engineering, with particular regard to advantages, limitations, and safety. Finally, we will critically analyze the different gene deployment and genomic engineering combinations, delineating strategies with a superior safety profile for the production of next-generation CAR T-cell.

74 Background: CYAD-101 is a first-in-class, non-gene edited allogeneic CAR T-cell product that combines the broad breadth of tumor targeting of the NKG2D-based chimeric antigen receptor (CAR) with a peptide-based approach that controls graft versus host disease (GvHD). NKG2D binds eight ligands commonly over-expressed across many tumors while the co-expressed T-cell receptor (TCR) inhibitory (TIM) peptide interferes with signaling by the endogenous TCR. A bank of CYAD-101 cells was produced from a single donor and evaluated in the AlloSHRINK phase 1 study (NCT03692429) in patients with unresectable metastatic colorectal cancer (mCRC). Methods: Three CYAD-101 infusions, each administered following a FOLFOX standard cycle as preconditioning chemotherapy, were tested in a 3+3 dose-escalation study (dose-levels (DL): 10⁸, 3x10⁸ and 10⁹ T-cells per infusion) in patients with relapsed/refractory mCRC who progressed after previous treatment with oxaliplatin-based chemotherapy, with or without irinotecan-based chemotherapy. Results: Fifteen patients (pts) were enrolled (3 pts at DL-1, 3 pts at DL-2, 9 pts at DL-3). No dose-limiting toxicity (DLT), Grade ≥ 3 related adverse events or GvHD were reported after any of the CYAD-101 infusions, thus confirming the overall good safety profile of CYAD-101 post FOLFOX. Encouraging anti-tumor activity was observed with 2 confirmed partial responses (PR), including one response in a KRAS mutated patient. In addition, 9 pts achieved stable disease (SD), with 7 SD lasting at least 3 months. The median progression-free survival in this heavily pre-treated population was 3.9 months (95% CI). Whilst engraftment of the CYAD-101 cells was observed after each infusion, the relative level of systemic cytokines appeared to be primarily modulated by cell dose with some suggestion that the magnitude of modulation might be associated with clinical response. Interestingly, preliminary analysis of the T-cell repertoire identified some evidence of TCR diversity after therapy in the patient showing the most durable partial response. Conclusions: These clinical results demonstrate the safety and tolerability of a fist-in-human non-gene edited allogeneic CAR T-cell treatment with early promising anti-tumor activity in advanced mCRC pts. Preliminary translational analysis present intriguing observations that the modulation of systemic cytokine levels may be associated with dose which is uncommon in CAR T-cell therapies reported to date while limited T-cell clonal diversification in the best responding patient underscores the likely central role of the adoptively transferred T-cell in driving therapeutic response in this particular patient. Extension cohort evaluating CYAD-101 following other preconditioning chemotherapy is expected to be initiated end 2020. Clinical trial information: NCT03692429.

Chimeric Antigen Receptor (CAR) T cells expressing the fusion of the NKG2D protein with CD3ζ (NKG2D-CAR T Cells) acquire a specificity for stress-induced ligands expressed on hematological and solid cancers. However, these stress ligands are also transiently expressed by activated T cells implying that NKG2D-based T cells may undergo self-killing (fratricide) during cell manufacturing or during the freeze thaw cycle prior to infusion in patients. To avoid target-driven fratricide and enable the production of NKG2D-CAR T cells for clinical application, two distinct approaches were investigated. The first focused upon the inclusion of a Phosphoinositol-3-Kinase inhibitor (LY294002) into the production process. A second strategy involved the inclusion of antibody blockade of NKG2D itself. Both processes impacted T cell fratricide, albeit at different levels with the antibody process being the most effective in terms of cell yield. While both approaches generated comparable NKG2D-CAR T cells, there were subtle differences, for example in differentiation status, that were fine-tuned through the phasing of the inhibitor and antibody during culture in order to generate a highly potent NKG2D-CAR T cell product. By means of targeted inhibition of NKG2D expression or generic inhibition of enzyme function, target-driven CAR T fratricide can be overcome. These strategies have been incorporated into on-going clinical trials to enable a highly efficient and reproducible manufacturing process for NKG2D-CAR T cells.

Allogeneic chimeric antigen receptor (CAR) T holds the promise of taking this therapeutic approach to broader patient populations while avoiding the intensive manufacturing demands of autologous cell products. One limitation to delivering an allogeneic CAR T is T-cell receptor (TCR) driven toxicity. In this work, the expression of a peptide to interfere with TCR signaling was assessed for the generation of allogeneic CAR T cells. The expression of a truncated CD3ζ peptide was shown to incorporate into the TCR complex and to result in blunted TCR responses. When coexpressed with a natural killer group 2D (NKG2D) CAR, the allogeneic T cells (called CYAD-101) failed to induce graft-versus-host disease in mouse models while maintaining antitumor activity driven by the CAR in vitro and in vivo. Two clinical grade discrete batches of CYAD-101 cells were produced of single donor apheresis resulting in 48 billion CAR T cells sufficient for the entire dose-escalation phase of the proposed clinical trial. The 2 batches showed high consistency producing a predominantly CD4⁺ T-cell population that displayed an effector/central memory phenotype with no evidence of exhaustion markers expression. These clinical grade CYAD-101 cells secreted cytokines and chemokines in response to ligands expressing target cells in vitro, demonstrating effector function through the CAR. Moreover, CYAD-101 cells failed to respond to TCR stimulation, indicating a lack of allogeneic potential. This bank of clinical grade, non–gene-edited, allogeneic CYAD-101 cells are used in the alloSHRINK clinical trial (NCT03692429).