Express Delivery of Next-Generation CAR T Cells with Preserved Naive and Stemness Phenotypes for the Treatment of Aggressive Lymphomas

CD19-directed chimeric antigen receptor (CAR) T-cell therapy has shown impressive efficacy in patients with different relapsed/refractory (R/R) B-cell lymphomas for which tisagenlecleucel, axicabtagene ciloleucel, and/or lisocabtagene maraleucel are approved. However, more than 50% of patients eventually relapse. Current challenges in CAR T-cell therapy include antigen escape, immunosuppressors, and the metabolic barrier in the tumor microenvironment; CAR T-cell exhaustion or lack of persistence; on-target, off-tumor effects; and CAR T cell–associated toxicities. Among them, CAR T-cell fitness and persistence are known to be associated with T-cell properties and could be improved through modification of the CAR T-cell manufacturing process.

In 2008, Berger and colleagues found that antigen-specific CD8⁺ T-cell clones derived from central memory T cells (T_CM) persisted for a long time in vivo and reacquired phenotypic and functional properties of memory T cells (T_MEM) (1), suggesting that a less differentiated state confers T-cell persistence in vivo. This finding was further supported by a report from Gattinoni and colleagues in 2011 that less differentiated stem cell memory T cells (T_SCM) and T_CM maintained superior properties, especially the stem cell–like self-renewal ability and capacity of T_SCM to differentiate into all subsets of memory and effector T cells, which facilitates their engraftment and persistence in the host (2).

In 2014, a clinical trial conducted by Xu and colleagues (3) further demonstrated that CD19-positive relapse after CD19 CAR T-cell therapy was associated with T-cell exhaustion and loss of persistence, whereas patients with durable responses to therapy had relatively elevated levels of naive and memory T cells (T_N/MEM). The expansion of CD19 CAR T cells in patients correlated with the frequency of CD8⁺CD45RA⁺CCR7⁺ cells within the infused product that phenocopied the T_SCM population (3). The idea that naive or stem memory T cells confer superior T-cell persistence and immune responses in vivo became increasingly appreciated with time. Since 2016, numerous reports from both preclinical research and clinical trials further confirmed that the extended longevity, persistence, and robust potential for immune reconstitution of T_SCM are associated with improved antitumor efficacy in adoptive T-cell therapy (4). Despite their low frequency, T_SCM clones contribute substantially to the circulating CAR T-cell pools during early expansion and long-term persistence (5).

Traditional CAR T-cell manufacturing requires a substantial and undesirably long expansion in vitro (9–14 days), which leads to depletion of T_N and T_SCM cells in the final product. In in vitro or ex vivo culture, T cells tend to undergo differentiation and aging accompanied by progressive cell proliferation. Therefore, it has become a consensus view that it is necessary to dramatically shorten the ex vivo culture time to preserve naive and stem cell populations. High-speed manufacturing of CAR T cells in a significantly shorter time without T-cell activation or extensive culture in vitro has been demonstrated to significantly preserve the naive state and stemness of the T cells.

Inspired by the above findings, Gracell Biotechnologies in 2017 introduced their FasTCAR platform that shortened the manufacturing time of CAR T cells from 2 weeks to 1 day (https://ir.gracellbio.com/news-releases/news-release-details/gracell-biotechnologies-presents-longer-term-results-fastcar-t). Since then, published results from clinical trials have demonstrated the potent efficacy of CD19 FasTCAR T cells in the treatment of patients with CD19⁺ relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL; ref. 6). Two years later, Ghassemi and colleagues presented their pioneering work showing that shortened ex vivo culture improves the antileukemic effect of CD19-targeted CAR T cells in a murine xenograft model of ALL. In that study, they generated functional CAR T cells within 24 hours from T cells that were isolated from peripheral blood without activation or ex vivo expansion (7).

In 2021, Novartis announced the development of T-Charge, a next-generation CAR-T platform with promising first-in-human clinical data presented at the American Society of Hematology Annual Meeting. According to the company's claim, the platform is capable of preserving T-cell stemness and enhancing CAR T-cell efficacy with a manufacturing time under 48 hours (8).

Joining these studies, Ghassemi and colleagues in early 2022 presented a fast-manufacturing protocol for generating functional CAR T cells within 24 hours from T cells isolated from peripheral blood without activation or ex vivo expansion. At nearly the same time, Agarwalla and colleagues introduced the Multifunctional Alginate Scaffold for T Cell Engineering and Release (MASTER) technology that facilitated production of faster CAR T-cell therapy within the body using a “sponge-like” bioengineered implant, thereby streamlining in vivo CAR T-cell manufacturing and reducing processing time to a single day (9).

Overall, the express CAR-T platforms not only dramatically shorten the time for CAR T-cell production but also maintain the naive state and stemness of the T cells. The FasTCAR platform requires isolation and activation of resting T cells from peripheral blood mononuclear cells (PBMC) from leukapheresis of either healthy donors or patients before they are transduced using XLenti lentiviral particles with high quality and efficient transduction. The transduced cells are then administered into patients without the need for ex vivo cell expansion (6). With enhanced stemness and proliferation, FasTCAR T cells may lead to lower dosage requirements and improved therapeutic outcomes. Thus far, not all details of the protocol have been sufficiently disclosed.

Using the T-Charge platform, T cells enriched from leukapheresis of healthy donors and patients are activated and transduced with CAR-encoding lentiviral particles. After a short culture period, CAR T cells are harvested, washed, and formulated within 2 days. With the T-Charge platform, the expansion of produced CAR T cells occurs primarily within the patient's body, avoiding the extended culture time outside of the body. Here, too, many details of the platform remain to be sufficiently disclosed.

The MASTER platform integrates PBMCs, CAR-encoding retroviral particles, and the cytokine IL2 in an implantable MASTER scaffold, which, after implantation in the body, allows the generation of CAR T cells in vivo without activation or ex vivo expansion. Compared with CAR T cells generated using conventional protocols, MASTER-generated CAR T cells contain increased percentages of CCR7⁺CD45RA⁺ T_CM, CCR7⁺CD45RA⁺ stem cell–like T cells, and CCR7⁺CD62L⁺ T cells with lymphoid homing capacity, which may contribute to the improved anticancer potency in a mouse lymphoma model (9).

In this issue of Cancer Discovery, Dickinson and colleagues (10) report their data on the treatment of diffuse large B-cell lymphoma (DLBCL), in which they successfully generated next-generation YTB323 CAR T cells using the T-Charge platform to preserve the naive state and stemness of the T cells and enhance their proliferation, expansion, and antitumor efficacy. YTB323, a novel manufactured autologous CD19-directed CAR T-cell therapy, is generated in under 48 hours. YTB323 retained a higher percentage of naive/T_SCM cells (CD45RO^–/CCR7⁺) from the leukapheresis product, compared with a much higher percentage of T_CM cells (CD45RO⁺/CCR7⁺) generated with the traditional manufacturing process.

Likely due to the preserved stemness of their therapeutic T cells and the proliferative potential in vivo, YTB323 dramatically decreases the cell dosage to as much as 25-fold lower than previous CD19-targeted CAR T-cell products such as tisagenlecleucel, with both good overall safety and high overall response rates (ORR). However, the transduction efficiency was not revealed in this report, so it is unclear whether the actual effector T cells are the CAR T cells or just the enriched untransduced naive stem T cells, which is a theoretical possibility.

In preclinical mouse models, YTB323 exhibited enhanced in vivo expansion and antitumor activity at lower doses than traditionally manufactured CAR T cells. Specifically, YTB323 controlled NALM6 B-ALL tumor growth at a low dose of only 0.1 × 10⁶ CAR⁺ viable T cells compared with that achieved with five times as many CAR⁺ viable CTL*019 (lab-grade tisagenlecleucel) T cells. The tumor-killing potency corresponds to the preservation of T-cell stemness and higher expansion capacity in YTB323 T cells generated with the new manufacturing process.

To further extend the preclinical findings, this group initiated a first-in-human study of YTB323 in patients with R/R DLBCL, chronic lymphocytic leukemia, or adult ALL. At doses of 2.5 × 10⁶ and 12.5 × 10⁶ CAR⁺ viable YTB323 T cells, up to 25-fold lower than tisagenlecleucel, treatment with YTB323 achieved impressive 75% and 80% ORRs and 25% and 73% complete responses, respectively, with a manageable overall safety profile. Biologically, YTB323 exhibited enhanced proliferative potential, preservation of the T-cell subpopulation distribution, and stemness phenotype as compared with tisagenlecleucel. A shortcoming of the study is that acquisition of some of the proposed clinical trial data has not been completed because the optimized clinical achievable doses were not identified before their work was published. Moreover, the stemness phenotype could have been further elucidated by the data for persistence or longevity of YTB323 T cells and their differences among patients, which are not reported.

In the clinic, preservation of T-cell stemness, with high proliferation and tumor-killing efficacy of the novel CAR-T product, is expected to overcome the drawbacks of CAR T cells made with conventional procedures. The study has the potential to expand our perspective for current CAR T-cell therapy and promote the development of fast, potent CAR T-cell therapy for the treatment of relapsed/refractory B-cell lymphoma.