Programming Antitumor Gamma Delta T Cells via a Phosphoantigen Nanoparticle Delivery Vehicle

Introduction: Gamma delta (γδ) T cells are a set of ‘unconventional’ T cells that can recognize and eradicate cancer tumors upon activation (Olofsson, Front. Immunol. 2021). Adoptive cell therapies utilizing ex vivo activated Vγ9Vδ2 T cells, the most cytotoxic subset of γδ T cells, are being explored clinically as treatments for cancer (Saura-Esteller, Front. Immunol. 2022). However, autologous γδ T cell therapies require expensive on-site cell isolation and activation protocols with burdensome quality control and sterility standards (Zhang, J. Hematol. Oncol. 2023). Activating γδ T cells in vivo would overcome the issues associated with adoptive therapy, but delivery challenges of activation agents have stymied the clinical development of such approaches.
Vγ9Vδ2 T cell activation and subsequent tumor cell killing are mediated by internalization of phosphoantigens (pAgs) by malignant cells (Rigau, Sci. 2020). While bolus injections of pAg can modestly activate T cells in the tumor microenvironment, systemically toxic doses of pAg are required to activate cytotoxic Vγ9Vδ2 T cell responses. Although nanoparticulate vehicles have been used to target small molecules to tumors, pAgs are small, water-soluble and therefore difficult to encapsulate efficiently in traditional hydrophobic polymer delivery vehicles.
We present a self-assembling nanoparticle (NP) composed of a hydrophilic, tumor-targeting block and a hydrophobic polymer block containing phosphoantigen monomers (pAgMAs) as pAg prodrugs (Scheme 1a). We hypothesize that these high-dose pAg nanoparticles will program Vγ9Vδ2 T cells in situ to an anti-tumor, interferon gamma (IFNγ)-expressing phenotype, resulting in tumor cell cytotoxicity and recruitment of other anti-tumor immune cells (Scheme 1b).

Materials and

Methods: Particle synthesis: An azide-reactive dibenzocyclooctyne (DBCO)-terminated hydrophobic polymer was synthesized via RAFT polymerization from a biologically inert co-monomer and a phosphoantigen-decorated monomer (pAgMA) that degrades at physiological pH into Vγ9Vδ2 T cell-activating pAg. This material was coupled to an azide-terminated, hydrophilic, tumor-targeting block comprised of polyethylene glycol (PEG) conjugated to an internalizing RGD peptide. The resultant block copolymers were self-assembled into RGD-pAg-nanoparticles (RGD-pAg-NPs) in PBS at pH = 7.4.
Uptake assay: Fluorescein-loaded RGD-pAg-NPs (Fl-RGD-pAg-NPs) and un-targeted pAg-NP lacking internalizing RGD (Fl-PEG-pAg-NPs) were synthesized as above. Tumor cells were incubated with dye-loaded Fl-RGD-pAg-NPs or Fl-PEG-pAg-NPs for 6 hours at 4°C and 37°C, and the mean fluorescence intensity was determined by flow cytometry.
Co-culture assay: Tumor cells were pre-incubated with various concentrations of RGD-pAg-NPs (10, 50, 100 µM of pAg), pAgMA, or zoledronate—a powerful commercial Vγ9Vδ2 T cell activator. The treated tumor cells were then co-cultured with human PBMC-derived γδ T cells in the presence of interleukin-2 (IL-2). After three days of coculture, tumor cell viability was assessed (CellTiter-Glo® 2.0 Assay) and normalized to equivalent monocultured wells. Concentration of IFNγ in the supernatant media was assessed via enzyme-linked immunosorbent assay (ELISA).
Statistical analyses were performed using one-way analysis of variance (ANOVA) with Dunnett’s test comparing conditions to untreated controls (** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).

Results, Conclusions, and Discussions: Results and

Discussion: Fl-RGD-pAg-NPs displayed superior uptake compared to un-targeted Fl-PEG-pAg-NPs at physiological temperature (Fig. 1a). However, at 4C, there was no statistical difference between the targeted or untargeted particles. Taken together, these results demonstrate that RGD-pAg-NPs are taken up via an energy-dependent process and that RGD is essential to the enhanced uptake of pAg-NPs. In addition, RGD-pAg-NPs showed significant cytotoxic effect in the presence of γδ T cells (Fig. 1b). Finally, tumor cell/γδ T cell co-cultures treated with 100 µM of RGD-pAg-NPs produced 100-fold more IFNγ than untreated co-cultures (Fig. 1c) - a particularly exciting result given that IFNγ expression defines the cytotoxic Vγ9Vδ2 T cell phenotype and the essential role played by IFNγ in Vγ9Vδ2 T cell-mediated tumor clearance.

Conclusions: The demonstration of active uptake and cytotoxicity due to γδ T cell anti-tumor activation in vitro indicates promise for this therapy in vivo. Based on our tumor cell uptake, cytotoxicity, and cytokine results, we anticipate that upon entry into the tumor microenviroment, RGD-pAg-NPs will localize and continually release their large payloads to pAgs into tumor cells, minimizing off-target effects and activating Vγ9Vδ2 T cell tumor eradication.
Future directions: We are currently translating this therapy into a mouse tumor model. Immune compromised mice bearing human tumors will be treated with RGD-pAg-NPs or appropriate controls via i.v. or intratumoral injection. The mice will then receive an infusion of PBMC-derived γδ T cells. Tumor size, animal weight, serum cytokine levels, and γδ T cell activation will be measured to determine efficacy and safety.

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