Whereas adoptive T cell therapy has been extensively studied for cancer treatment, the response is still limited primarily due to immune dysfunction related to poor cell engraftment, tumor infiltration and engagement, and lack of a target. In addition, the modification of therapeutic T cells often suffers from being complex and expensive. Here, we present a strategy to load T cells with SHP099, an allosteric SHP2 inhibitor, to enhance the therapeutic efficacy of the T cells. Remote-loading of SHP099 into lipid nanoparticles decorated with triarginine motifs resulted in nanocrystal formation of SHP099 inside the lipid vesicles and allowed high loading efficiency and prolonged retention of SHP099 nanocrystals within T cells. Cell-loaded SHP099 enabled sustained inhibition of the PD-1/PD-L1 signaling and increased cytolytic activity of the T cells. We show in a mouse model that tumor-homing T cells can circulate with the cargos, improving their tumor accumulation compared to systemically administered lipid nanoparticles. On an established solid tumor model, adoptively transferred SHP099 loaded T cells induced complete tumor eradication and durable immune memory against tumor rechallenging on all treated mice by effectively inhibiting the PD-1/PD-L1 checkpoint signal. We demonstrate that the combination of T cell therapy with SHP2 inhibition is a promising therapeutic strategy, and the lipid nanocrystal platform could be generalized as a promising approach for T cell loading of immunomodulatory drugs.

We used novel quantitative methods to study the interactions between reconstituted high-density lipoproteins (rHDL) and human leukocytes – showing that rHDL cargo are preferentially taken up by monocytes.

TLR 7/8 agonists are highly potent immunostimulators, though their clinical translation has been met with mixed success, due to their high toxicity as a result of an unregulated systemic immune activation. There is enormous potential to augment cancer immunotherapies with synthetic TLR 7/8 agonists, though a thorough control of pharmacokinetics and localization is needed for the general use of TLR 7/8 agonists in cancer immunotherapy. Herein, we control localization of TLR 7/8 agonists, by exploiting the extensive tissue retention of poly(acrylic acid-co-styrene). In a murine CT26 model, we find that covalently attaching TLR 7/8 agonists to the copolymer allows for retaining the drug in the tumor microenvironment for at least 15 weeks, after intratumoral injection, and results in a curative monotherapy. The copolymer itself is a new avenue for attaining prolonged tissue rentention for covalently attached drugs.

BackgroundAdoptive T cell therapy (ACT) is often accompanied by supporting immunomodulatory drugs to protect T cells from the suppressive tumor microenvironment (TME) [1]. However, systemic administration of these immunomodulators can cause serious side effects and fail to distribute optimally to exert sufficient lymphocyte stimulation within the tumor and lymphoid compartments. Loading T cells with adjuvant drugs or cytokines prior to cell transfer provides a solution to this issue, showing the potential to use T cells as vehicles to carry immunomodulatory molecules to target sites [2]. SHP2 is an important hub connecting several intracellular oncogenic signaling pathways including PD-1/PD-L1, representing a notable target for cancer immunotherapy. SHP2 inhibition has been shown to elicited tumor regression by improving CD8+ T cells activity [3]. Herein we present a lipid nanoparticle system encapsulating an SHP2 inhibitor (SHP2i) that allows high T cell loading capacity and enhances their therapeutic activity.MethodsRemote-loading gradients were used to achieve high encapsulation efficiency of SHP2i into the lipid nanoparticle platform. Mouse cytotoxic T cells were loaded with SHP2i, and loading efficiency and release rates from the T cells were evaluated in vitro. Flow cytometry was used to assess T cell viability, proliferation, and phenotype. In vivo biodistribution of loaded T cells was evaluated by labeling lipid nanoparticles with gadolinium and T cells with Cell-trace-marker, which were measured with ICP-MS and Flow respectively. The therapeutic anti-tumor efficacy of the loaded T cells was demonstrated on EG.7-OVA tumor-bearing mice.ResultsThe developed formulation allowed high T cell loading efficiency of SHPi and extended-release over 5 days. Loading T cells with lipid formulated SHP2i did not compromise cell viability and proliferation and resulted in T cells retaining a central memory phenotype than unloaded counterparts. Adoptively transferred T cells loaded with lipid nanoparticles showed the same distribution and proliferation behavior as the unloaded T cells in vivo, accumulating into tumor tissue three days post cell infusion. Loaded OT.I T cells significantly improved tumor growth inhibition and overall survival than OT.I T cells alone, with 5 out of 6 mice completely tumor-free, resulting in durable long-term responders.ConclusionsLoading T cells with liposomal SHP2i before ACT allowed specific and controlled delivery of immunomodulatory drugs by T cells. The loaded T cells showed improved anti-tumor efficacy. The developed lipid formulation allows the loading of a variety of immunomodulatory drugs to T cells, which serve both as a drug delivery vehicle and enhance the tumor efficacy of the transferred cells.ReferencesWaldman AD, Fritz JM, Lenardo MJ. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat. Rev. Immunol. 2020. p. 651–68.Combes F, Meyer E, Sanders NN. Immune cells as tumor drug delivery vehicles. J Control Release. Elsevier; 2020;327:70–87.Yuan X, Bu H, Zhou J, Yang CY, Zhang H. Recent Advances of SHP2 Inhibitors in Cancer Therapy: Current Development and Clinical Application. J Med Chem. 2020;63:11368–96.Ethics ApprovalThe study has been approved by the Danish Animal Experiments Inspectorate with the permit number 2020-15-0201-00482. The participants gave informed consent before taking part.