Protein Nanocage-Based Immunomodulatory Strategies to Enhance Immunotherapy for Glioblastoma

Introduction: Glioblastoma (GBM) carries a bleak prognosis with a median survival < 15 months despite the aggressive therapeutic regimen given to the patients comprising surgery, chemotherapy, and radiation. Immunotherapy has shown success as a promising treatment method in various cancers. However, in contrast to positive results in clinical trials for other non-brain tumors, patients with GBM have demonstrated minimal clinical benefits. This may be attributed to the high immune-suppressive microenvironment in GBM tissues and their surroundings. Among the key contributors to the immune-suppressive environment, Transforming Growth Factor-Beta (TGFβ) plays a crucial role in promoting the immune-suppressive tumor microenvironment in GBM through various mechanisms, including the inhibition of CD8+ effector T cells from migrating to the tumor. A recent noteworthy discovery is that TGFβ inhibition has been found to enhance the accumulation of T cells within the tumor microenvironment, thereby mitigating the therapeutic efficacy of antibodies blocking Programmed Death-1 (PD1) and Programmed Death-Ligand 1. We have developed a nanocage-based TGFβ trap to inhibit the TGFβ signaling pathway, aiming to revive the therapeutic efficacy of immunotherapy and alleviate the immune-suppressive tumor environment in GBM. An important aspect is that the nano-cage can traverse the blood-brain barrier (BBB) through transferrin receptors, enhancing the delivery of treatment to the brain after systemic administration.

Materials and

Methods: A custom-made plasmid DNA was transformed into BL21 cells to express a fusion protein of the TGFβ receptor type II ectodomain and heavy chain ferritin (TβR2-HFt, Fig. A and B). The purified TβR2-HFt was evaluated using SDS-PAGE and DLS. Binding assays were performed using a standard ELISA method. To establish a syngeneic orthotopic mouse model of brain tumors, GL261-Luc2 cells (1 x 10^5 cells) were intracranially implanted into the right striatum of 6-8-week-old female C57BL/6J mice. TβR2-HFt and PD1 antibodies were administered via tail vein injection every 5 days, and survival was monitored. In parallel, tumor tissues were harvested, sectioned, and immunostained for key intracellular mediators of the TGFβ signaling pathway (phosphorylated SMAD2 or pSMAD2), CD3, and CD8, or stained with Masson's Trichrome.

Results, Conclusions, and Discussions: TβR2-HFt binds to both TGFβ1 (Fig. C) and human type 1 transferrin receptor (Fig. D), enabling BBB penetration. Following tail vein injection into mice, TβR2-HFt did not induce any body weight loss over a broad dose range. In contrast to the PD1 antibody or TβR2-HFt monotherapy the combination therapy of PD1 antibody and TβR2-HFt significantly prolonged the survival of the GBM mouse model (Fig. E). In the tumor tissue, we observed enhanced infiltration of CD3+ and CD8+ T cells (Fig. F and G). As a result of staining brain sections harvested from GBM model mice, TβR2-HFt treatment reduced pSMAD2 in tumor tissue (Fig. H and I). Additionally, we found that collagen density was significantly reduced in tumor tissue after TβR2-HFt treatment (Fig. J, K). These results suggest that modulation of the TGFβ pathway can regulate collagen density in the tumor tissue, thereby allowing immune cell infiltration. Overall, TβR2-HFt mitigated the tumor stromal barrier to promote intratumoral T cell infiltration, thereby enhancing therapeutic efficacy of immune checkpoint blockade.

Acknowledgements (Optional): This work was supported by NIH (R01NS119609)