A long-term antigen-spreading by novel cancer vaccine with hyaluronic acid nanogel accelerate the anti-neoplastic efficacy of cytotoxicity of infused TCR-T cells and altered neplastic microenvironment Free

Background Recent progress of engineered T cells genetically modified to express tumor-specific T-cell receptors (TCR-T) has demonstrated promising perspectives for treatment of a variety of cancers. Nonetheless, the efficacy of this strategy against solid tumors is still unsatisfactory due to poor expansion, loss of function, and short persistence of the infused cells in vivo. To overcome this, different combination therapies with other modalities, including cancer vaccines and immune-checkpoint inhibitors (ICIs), have attracted attention and are currently being developed to achieve long-term patients' survival. However, the optimal strategy has not been determined yet. Recently, we newly developed a novel cancer vaccine based on hyaluronic acid nanogel (HANG-Vax). HANG can easily embed various substances, such as long peptides, leading to form nanoparticles of about 100 nano meter diameter. HANG-Vax can efficiently deliver vaccine antigens to antigen presenting cells present in lymph nodes after subcutaneous injection. Aims In this study, we investigated the efficacy of the combined treatment of HANG-Vax with TCR-T therapy by using tumor-bearing mouse models.

Methods The efficacy of the combination of TCR-T and HANG-Vax therapy was examined by using each tumor bearing mouse treatment model. Here, we utilized C57BL/6-derived B16F10 melanoma cell line, which is resistant to ICIs and TCR-T therapies. TCR-T cells were prepared from splenic CD8+ T cells of Pmel-1 mice transfected with TCR that specifically recognize gp100. HANG-Vax was prepared by mixing HANG with long peptide encoding gp100. HANG-Vax was subcutaneously injected on day 7, 11 and 15, and CD8+ T cells were intravenously administered on day 8 and 12 after tumor inoculation. Along with monitoring tumor growth, single-cell RNA/TCR seq analysis and spatial gene expression analysis were performed to analyze the dynamics of the infused TCR-T cells in lymph nodes and tumor tissues.

Results Malignant cell transplantation revealed that complete eradication of B16F10 tumors was observed in all mice treated with TCR-T and HANG-Vax therapy. In contrast, the other groups of mice were all dead until 25 days after transplantation of B16F10. Mice that achieved complete remission (CR) survived for long periods and exhibited vitiligo hair. Single-cell RNA/TCR seq analysis of the lymph nodes from each group revealed that HANG-Vax induced the clonal expansion of TCR-T, but much less in TCR-T monotherapy. Projection of reference data identified that HANG-Vax differentiated the TCR-T toward effector memory T cells. In addition, the differential gene expression analysis across the groups showed that HANG-Vax treatment induced the expression of cytotoxic genes such as Granzyme B. KEGG-pathway analysis using the differentially expressed genes highlighted the HANG-Vax accelerated not only “cell-killing”-related gene expression but also “cell metabolism/proliferation”-related genes. These suggested the clonal survival and expansion was induced in HANG-Vax.

To confirm the efficient recruitment of cytotoxic T cells at the tumor site, further analysis utilizing the spatial expression analysis was performed. CD8+T cells were highly co-exisiting with Pmel expressing tumor cells in HANG-Vax treatment group. Pseudo-bulk analysis of these spatial expression data showed significantly higher levels of “cytotoxicity” and “positive activation of immune cell”-related genes. Regarding the non-T population of tumor site, we performed deconvolutional analysis of each spot using Deepcolor method. Interestingly, TCR-T with HANG-Vax induced the higher expression of chemokines, including Ccl4, Ccl5, Cxcl9, and identified that HANG-Vax treatment group possessed higher macrophage invasion with M1 macrophage, suggesting HANG system empowered the macrophage-related anti-neoplastic effect in addition to CD8+ T cell cytotoxicity.

Summary/Conclusion These results indicate that HANG-Vax can potently maximize the efficacy of TCR-T therapy, leading to the cure of immunotherapy-resistant solid tumors and long-term prevention of tumor recurrence. Besides, HANG-Vax can potentially transform the characteristics of resistant-tumors, rendering them more sensitive to immunotherapy. We believe that this study may provide important insights into the clinical application of HANG-Vax combined with adoptive cell therapy for patients with solid tumors.