Enhancing adoptive cell therapy: future strategies for immune cell radioprotection in neuro-oncology

Abstract

Adoptive cell therapy (ACT), particularly chimeric antigen receptor T cell (CAR T) therapy, has emerged as a promising approach in cancer treatment, demonstrating efficacy in hematological malignancies but facing challenges in brain tumors. The combination of ACT with radiation therapy (RT) offers a potential strategy to enhance therapeutic outcomes, as RT can stimulate immune responses by promoting antigen presentation and T cell recruitment. However, a major hurdle is the radiosensitivity of immune cells, leading to their rapid depletion within the radiation field, which undermines the benefits of this combination. This review explores strategies to increase the radioresistance of immune cells, highlighting the need for innovative radioprotective approaches. We discuss the potential of extremophile-derived molecules, such as the Damage Suppressor protein from tardigrades, as novel radioprotectants that could be integrated into ACT protocols. Furthermore, we address key considerations for clinical trial design, including the sequencing of RT and ACT, dosing parameters, and safety considerations. By bridging insights from extremophile biology and immuno-oncology, this work aims to optimize the efficacy of ACT in the challenging context of brain tumors, paving the way for enhanced treatment strategies in neuro-oncology.

Fig. 1. Using an ultra-radioresistant extremophile gene to radioprotect T cells and potentiate ACT.

a Poor immunogenicity is a key barrier to efficacy for CAR T cells in brain tumors. b RT added to CAR T therapy has immunostimulatory effects that may potentiate CAR T therapy. But RT also kills T cells that enter the radiation field which has a counterproductive effect. c Radioprotectants such as a gene from the ultra-radioresistant extremophile Tardigrade are expressed in CAR T cells to protect them from RT, allowing immune stimulation from RT to synergize with CAR T.

Fig. 2. Baseline radiosensitivity of cell types used for ACT.

This figure summarizes the inherent radiosensitivity and other effects of irradiation on various immune-cell types commonly used in adoptive cell therapy (ACT). While radiosensitivity is likely highly context-dependent, in general B cells are highly sensitive to radiation and undergo apoptosis at even low doses. T cells are sensitive, with sublethal doses (<2 Gy) causing activation, while higher doses (>2 Gy) induce apoptosis. γδ T cells are relatively resistant to radiation and maintain their cytotoxic functions. Invariant natural killer T cells (iNKT cells) exhibit moderate resistance to radiation. Natural killer (NK) cells and macrophages are relatively radioresistant.

Fig. 3. Anti-tumor effects of radiation on CAR immune cells.

This figure summarizes the anti-tumor impacts of radiation on CAR T, NK, γδ, iNKT, and macrophage cells. Radiation may illicit an immune response via a variety of mechanisms, including upregulation of tumor antigen presentation and increased cytokine release. TAA: tumor associated antigen; CAR: chimeric antigen receptor; GzmB: granzyme B; PFN: perforin; PPA: phosphoantigen; LRP1: low-density lipoprotein receptor 1; MHC I: major histone compatibility complex I; IFNγ: interferon gamma; TNFα: tumor necrosis factor alpha; Chmk: chemokines; CRT: calreticulin; CTL: cytotoxicity T lymphocyte; CD: cluster of differentiation; MICA/B: MHC class I chain-related protein A and B; TCR: T cell receptor; iTCR: invariant T cell receptor.

Fig. 4. Mechanisms of radioprotection.

Radioprotective agents mitigate radiation-induced damage through multiple mechanisms. These include protection of DNA from direct damage, reduction of indirect DNA damage by scavenging reactive oxygen species (ROS), and promotion of DNA damage repair. Additionally, radioprotective strategies may involve decreasing apoptosis and cell death. While reducing ROS is a broadly applicable and potentially safer strategy, inhibiting apoptosis could carry risks, such as increasing the potential for carcinogenesis. Mechanisms related to ROS scavenging are likely to be more transferable between species, while apoptosis modulation may depend on species-specific pathways, raising concerns about their safety and efficacy across different models.

Fig. 5. Radioresistant extremophile organisms.

Select radioresistant extremophile organisms are shown, compared to humans. Radiation dose in Gray (Gy) for D10 (dose required to kill 90% of organism sample) when available or LD50 (dose required to kill 50% of organism sample) is shown. Note log scale indicating >1000X radioresistance compared to humans for many organisms.

Fig. 6. Approaches to screen for immune-cell specific radioprotectors.

Various strategies can be employed to identify radioprotectors that specifically target immune cells. High-throughput screening of chemical libraries, genetic screens, and functional assays offer ways to discover agents that selectively shield immune cells from radiation damage. Depending on the type of ACT being studied, screens can be conducted in a range of immune-cell types, from easily manipulated cell lines to more clinically relevant, human-derived cells. Screening can be performed in vitro, which allows for more practical, controlled experiments, or in vivo, where complex biological interactions are better represented. Radiation is applied as needed, using tools like cabinet irradiators for in vitro cultures or small animal irradiators for in vivo studies. Readouts can vary depending on the specific goals of the screen, ranging from simple measurements of cell survival or function post-irradiation to more sophisticated next-generation sequencing (NGS) approaches that assess the selection of different perturbations after exposure. Analytical methods can involve straightforward ranking of top perturbations that improve immune-cell survival or function, as well as deeper molecular pathway analyses to gain mechanistic insight. Radioprotectors are also assessed for their ability to maintain immune-cell functionality, prevent apoptosis, or preserve immune-cell subsets during or after radiation. Validation of hits is critical to confirm radioprotective efficacy and ensure that identified candidates not only protect immune cells but also preserve their therapeutic potential. These methods hold promise for identifying radioprotectors that maintain immune competence in ACT, while ensuring therapeutic safety and efficacy.

Fig. 7. Formulation of radioresistant CAR T cells.

CAR T cells are developed from peripheral blood mononuclear cells (PBMCs) which are harvested, expanded, sorted into T cells, and then tranduced with lentiviral vectors to deliver the CAR gene playload. Our proposal will identify the most potent radioprotector genes to deliver in a similar fashion during the CAR T manufacturing process, producing radioresistant CAR T cells.

Fig. 8. Considerations for RT delivery in combination with ACT.

Key factors in optimizing the combination of radiation therapy (RT) with adoptive cell therapy (ACT) are presented. Fractionation options include conventionally-fractionated RT (e.g., 60 Gy in 30 fractions) and hypofractionated RT (e.g., 20 Gy in 1 fraction or 9 Gy in 3 fractions). Dose-rate considerations compare the use of conventional dose rates with FLASH-RT, where doses greater than 40 Gy per second are delivered. Anatomic targets are categorized into conformal vs. wide-field approaches, focusing on elective lymph node fields. Sequencing and timing of treatment strategies include concurrent, neoadjuvant ACT followed by RT, RT followed by adjuvant ACT, and using ACT with salvage RT for restimulation of immune responses.

Fig. 9. Concept for clinical trial of radioprotected CAR T and RT.

Patients with suspected glioma are recruited to the trial. Patients undergo standard-of-care surgical resection followed by radiation therapy. PBMCs are collected and manufactured into CAR T cells including the radioprotector(s) identified here. CAR T cells are re-infused by day 56, providing time for CAR manufacturing but delivering CAR T early in the RT course when immunogenicity may be highest. Concurrent standard-of-care temozolomide may be given or omitted depending on rapid tumor molecular analysis.