Glioblastoma multiforme (GBM) is an aggressive brain cancer with 5-year survival rates below 10% (1). Current treatment includes surgical removal of the tumor followed by chemotherapy and/or radiation therapy; however, successful treatment only increases the median survival to ~15 months (1, 2). This standard-of-care treatment has remained unchanged for decades, creating an urgent need for novel treatments for this disease. Human cytomegalovirus (HCMV) proteins were first shown in GBM tumors in 2002 and were later confirmed by several groups (3–6). Having said this, several studies and major consortia were unable to detect the presence of CMV transcript and/or protein in glioblastoma tissue. In an attempt to address this controversy, HCMV and glioma symposium was convened in 2011, and a consensus was reached that there is sufficient evidence to conclude that HCMV sequences and gene expression exist in most, if not all, malignant gliomas (7). The precise role of HCMV in the GBM pathogenesis remains unclear. From a therapeutic perspective, the specific presence of HCMV antigens in more than 90% of GBM tumors provides an opportunity to develop a targeted therapy for GBM while simultaneously harnessing preexisting antiviral immunity (4).

It has been previously shown using our lysosome-associated membrane protein (LAMP) fusion platform that autologous dendritic cells (DCs) pulsed with LAMP-pp65 mRNA significantly improve survival in GBM patients, providing clinical validation for this strategy (8, 9). Though autologous DC immunotherapy using LAMP-pp65 mRNA demonstrates the effectiveness of HCMV-targeted therapy and has great potential in the clinic, there are some limitations of this treatment. It is highly personalized and requires professional personnel to collect and transfect the DCs under aseptic conditions, thereby increasing the cost of treatment and limiting the availability to specialized hospitals. In addition, certain patients due to their medical condition are not suitable for leukapheresis and hence ineligible for autologous DC immunotherapy.

To overcome these limitations of the autologous DC therapy, we utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) encoding HCMV antigens. The major component of the UNITE platform involves fusion of HCMV antigens with LAMP1. LAMP1 is a glycosylated protein that is transported through the trans-Golgi network into lysosomes and then attached to the internal lysosome membrane through the C-terminus (10, 11). It has been previously shown that the fusion of LAMP1 with antigens induces preferential CD4-mediated T-cell immune responses through the MHC-II–lysosome pathway (12–14). Additionally, the UNITE platform drives CD8 T-cell activation via the MHC-I pathway. Specifically, antigen-LAMP1 fusion construct displayed a significantly higher percentage of CD8 T cells secreting interferon (IFN)γ and tumor necrosis factor (TNF)α leading to better survival compared to construct with antigen alone in a breast cancer mouse model, indicating the advantage of using UNITE platform (14). The ITI-1001 vaccine contains three HCMV antigens: phosphoprotein 65 (pp65; UL83), immediate-early protein 1 (IE-1; UL123), and glycoprotein B (gB; UL55); pp65 and gB are structural proteins, whereas IE-1 is a transcription factor (15–17). The presence of all three antigens has been confirmed in the majority of GBM tumors by Western blotting and immunohistochemistry (4, 5). In addition, all three proteins are highly immunogenic, inducing strong T-cell responses and are considered promising candidates for HCMV vaccine development (18). pp65-specific CD8 and CD4 T cells have been identified in GBM patients, further confirming the immunogenicity of this antigen and preexisting antitumor T-cell repertoire in patients (19). Taking advantage of the presence of HCMV-specific T cells in GBM patients, researchers have isolated and enriched these autologous T cells and succesfully demostrated improved overall survial in primary GBM patients (20–22). Together, these observations provide a strong corroboration for using HCMV antigens to develop a multi-antigen vaccine for GBM.

In the present study, using ITI-1001 vaccination of naive mice, we demonstrated strong antigen-specific humoral response and T-cell activation in both CD8 and CD4 T-cell compartments as evaluated by their ability to secrete high levels of IFNγ and TNFα. Therapeutic vaccination with ITI-1001 conferred more than 50% survival in an orthotopic syngeneic GBM mouse model expressing HCMV antigens. Furthermore, based on our tumor microenvironment (TME) studies, ITI-1001-treated tumors showed significant CD4 T-cell infiltration and activation and enhanced activation of CD8 T cytotoxic killer cells, along with an increase in Natural Killer T (NKT) cells thereby providing possible clues into the mechanism(s) of action of ITI-1001. Interrogating the bimodal separation of activated CD4 T cells and their negative correlation with tumor burden identified responders and non-responders of the ITI-1001 treatment group. Further characterization of responders showed higher TME infiltration of both CD4 and CD8 T cells along with increased CD8 T-cell activation. Taken together, our data show that ITI-1001 generates strong cellular and humoral immune responses, triggering significant antitumor activity that leads to enhanced survival in this preclinical evaluation of the vaccine.

GBM, a grade IV astrocytoma, is considered a cold tumor due to the low number of immune cell infiltration within the TME and has low tumor mutational burden, resulting in fewer targetable neoantigens (25). These factors make GBM refractive to most existing checkpoint inhibitor immunotherapies and present a formidable challenge for developing novel treatments (26). The recent failure of checkpoint inhibitor monotherapies was in part attributable to the scarce presence of tumor-specific T cells in the TME. In order to tackle this obstacle, we aimed to boost the immune system with a GBM tumor-targeted approach. We developed ITI-1001, a therapeutic DNA vaccine that contains three immunogenic HCMV antigens (pp65, IE-1, and gB). GBM-specific expression of these HCMV antigens provided a targeted approach, and utilization of our LAMP1-based UNITE platform ensured a strong CD4 and CD8 T cell-driven adaptive response with long-term immunologic memory. As HCMV infects most of the human population, we believe that in an immunologically cold GBM tumor, reeducating the immune system via HCMV antigens would result in an effective antitumor immune response leading to increased infiltration and further activation of immune cells in the TME. The use of the three antigens in ITI-1001 minimizes the possibility of antigen escape. Another advantage of using multiple antigens would be to maintain the vaccine’s efficacy even with the known heterogeneous expression of HCMV antigens in patient tumors (7). An alternative to DNA vaccine could be to use immunogenic CD4 and CD8 peptides to induce an immune response, but LAMP1 platform provides such peptides in a physiological manner. And based on our internal work with oncology targets (data not shown), we found that whole or truncated protein fused to LAMP1 generates a much better immune response than that of peptides alone.

ITI-1001 contains HCMV antigens fused to LAMP1 in two plasmid vectors. After injection and subsequent internalization of ITI-1001 pDNA, the glycosylated LAMP1-antigen fusion proteins are expressed in APCs. These fusion proteins are trafficked through the lysosome membrane by LAMP1 and are linked to the internal lysosomal membrane (14). In addition to the MHC-II–lysosome pathway that activates antigen-specific CD4 T cells via a Th1 response, LAMP1 also drives the antigens through the MHC-I pathway, leading to cytotoxic CD8 T-cell activation (14, 27, 28). Of note, the plasmid backbone containing CpG islands and the double-stranded plasmid DNA itself further augments the immune response via foreign DNA-sensing pathways in the APCs (29).

Results from both in vitro and in vivo immunogenicity studies of ITI-1001 showed that gB gave the strongest cellular and humoral responses followed by pp65 and then IE-1. Flow cytometry data showed strong CD4 and CD8 T-cell activation, indicating that LAMP1 elicits an immune response via both the MHC-II and MHC-I pathways.

To assess vaccine efficacy, we developed an orthotopic syngeneic GBM mouse model expressing HCMV antigens. The CT2A mouse GBM cell line was utilized due to its low tumor burden and refractive nature to immune checkpoint inhibitor monotherapies similar to that of human GBM (25). Interestingly, the ITI-1001 vaccine effectively eliminated GBM in more than 50% of treated mice, suggesting that ITI-1001 generated a strong antigen-specific immune response that could eliminate GBM in these mice and maintain their tumor-free survival for 106 days (study duration) post intracranial injection. This long-term efficacy is even more meaningful considering both the immunologically cold nature of GBM and the aggressive nature of the mouse CT2A GBM cell line.

Tumor cells have a complex relationship with several stromal, vascular, and immune cells that form the TME. The TME plays a crucial role in tumor initiation and growth and in antitumor activities (30, 31). To investigate how ITI-1001 reshapes the TME and to gain insights into the mechanism of action of the vaccine, we evaluated the TME after vaccination. Total T cells (CD3+) showed higher infiltration in the treated tumors compared to control, whose levels were indistinguishable in the two groups. Though CD8 T-cell infiltration did not increase in the treatment group, we observed a significant increase in CD8 T-cell activation, determined by increases in the expression of IFNγ, TNFα, and granzyme B in treated tumors. This strong activation of cytotoxic killer CD8 T cells explains the robust antitumor activity and the corresponding efficacy.

Several recent studies in immunotherapy have shown that in order to have a strong and sustained antitumor immune response, activation of CD8 T cells alone is not sufficient, and additional activation of CD4 T cells is required (32, 33). As a result of our LAMP1 fusion platform, we observed a significant infiltration of CD4 T cells and a concurrent increase in activated CD4 T cells expressing the Th1 pro-inflammatory cytokines IFNγ and TNFα in the treatment group. The preferential activation of CD4 T cells using antigen-LAMP1 fusion proteins has been shown previously in other cancers (10–12, 34). Based on our recent observations using breast cancer model and CD4 depletion studies, CD4 T-cell activation (which results from LAMP1-antigen fusion) was found to be essential for the initial stages of CD8 T-cell activation (14). We hypothesized that ITI-1001 vaccination in our mouse GBM model would result in the generation of activated CD4 Th1 cells that in turn would result in the enhanced activation of CD8 T cytotoxic killer cells by DC licensing and cytokine release (27, 33). CD4 T cells are also known to show direct tumor cytotoxicity and are indirectly involved in the generation of a humoral response (32, 33).

In addition to T-cell infiltration and activation, we also observed significant infiltration of NKT cells in tumors after ITI-1001 vaccination. NKT cells are activated by glycosylated lipids, and the reason behind their increased infiltration in the vaccinated TME is unclear in the absence of any glycosylated lipids in the ITI-1001 vaccine. It remains to be determined whether the heavily glycosylated LAMP1-antigen fusion proteins could be responsible for this infiltration of NKT cells. Interestingly, Duane Mitchell’s group, using single-cell RNA sequencing on human GBM tumors treated with pp65-LAMP1-transfected autologous DCs (35), also found a similar increase in NKT cells in addition to T-cell activation. NKT cells are known to show direct antitumor activity and also help activate CD8 killer T cell via DC licensing along with CD4 T helper cells (36, 37). It is noteworthy that although we are unclear on the reason behind the increase in NKT cell infiltration, the similarity in the data with human GBM patients treated with pp65-LAMP1-transfected DCs corroborates the relevance of our preclinical TME data and further supports the therapeutic efficacy of our vaccine in a mouse model.

Upon TME evaluation of immunosuppressive response post vaccination, we observed Tregs to be significantly higher in treated compared to those in the untreated group. Other probable candidates including PD-1, MDSCs, and M1/M2 macrophage populations remained unaltered.

For further confirmation of our TME flow cytometry data showing CD4 and CD8 T-cell activation, we found an increase in Th1 pro-inflammatory cytokine (IFNγ, TNFα, IL-2, and KC/GRO) levels in the tumor lysate. Interestingly, the anti-inflammatory cytokine IL-10 and the Th2 cytokine IL-4 were also upregulated in treated tumors. Elevated IL-10 levels could be explained by the increased number of Treg cells found in the TME in treated tumors and might represent some of the immunosuppressive mechanisms utilized by tumor cells to combat vaccination. It is a known observation that immunosuppressive mechanisms such as expression of cytokine IL-10 is induced to counter the inflammatory reaction induced by the vaccine. In order to increase the efficacy of the vaccine, some researchers have utilized mechanisms that block IL-10 by siRNA, antibody, or by other means (38).

A closer evaluation of the CD4+IFNγ+ population demonstrated two distinct populations based on the number of IFNγ−expressing cells. We separated the treated tumors into two groups based on their CD4 IFNγ expression levels and compared their tumor weights. We found a significant difference between the two groups where the group showing a higher CD4 T-cell IFNγ expression had a lower tumor burden (responders) and the group with a lower CD4 T-cell IFNγ expression had a higher tumor burden (non-responders). To further corroborate the relationship between CD4 T-cell activation and tumor burden, we performed an unbiased correlation analysis of all the treated tumor samples for tumor burden and CD4 T IFNγ+ cell frequency and found a strong negative correlation between these two parameters. This confirmed the significance of CD4 T-cell activation in our vaccine efficacy and also reinforced the responder/non-responder segregation. These two groups were defined further by evaluating other T-cell populations and where we found that CD4 T-cell infiltration and activation including CD4 T-cell expression of TNFα were significantly higher in our responder group. Since CD4 T-cell activation is a critical step in the cellular immune response for LAMP1-fused antigens, this difference is in line with our proposed mechanism of action for the ITI-1001 vaccine. Furthermore, an increased number of total infiltrating CD3+ T cells were observed in the responder group. Also, in correlation with the smaller tumors observed in the responders, we found higher numbers of activated CD8 T cells (IFNγ+ and granzyme B+) in this population, in part explaining the strong antitumor activity of the vaccine. Our objective of identifying and characterizing the responder and non-responder populations was to probe into the lack of CD4 T-cell activation in the non-responders to potentially find ways to further improve the overall efficacy of ITI-1001. In this regard, Tregs were the only cells that were significantly different between the responders and non-responders, but unexpectedly, responders showed higher numbers of Tregs as compared with those in non-responders ( Supplementary Figure S4 ). Recent studies have shown antitumor properties of Tregs through their conversion into effector T cells (32, 33). Further studies are needed in our model treated with ITI-1001 to verify such Treg to effector T cell (Teff) conversion. Based on the data from this study, it appears that the non-responding property of tumors to ITI-1001 is independent of M1/M2 macrophages, MDSCs, PD-1 expression and needs further scrutiny.

In summary, based on our preclinical proof-of-concept investigation, ITI-1001 is a potential candidate for human GBM treatment given the robust cellular and humoral response in naive mice, as well as 56% survival efficacy in a GBM mouse model. TME analysis showed strong dependence of CD4 activation to reduce tumor burden and that the vaccine responders showed a high frequency of CD8 T-cell activation. Based on these properties, an Investigational New Drug Application is being submitted with a plan to perform a Phase I clinical trial evaluating the efficacy of ITI-1001 vaccine in the treatment of newly diagnosed GBM patients. Based on the preclinical study outcomes, ITI-1001 can be a new approach for treating GBM patients to reduce tumor burden and prolong progression-free survival.

All data relevant to the study are included in the article or uploaded as online Supplementary Information . Any clarification on the data, protocols, or statistical methods used will be available on request.

All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee.

AA designed, performed, analyzed the studies and wrote the article. JM, YJ, and MC performed the studies. TC and TH supervised the studies and reviewed the article. All authors contributed to the article and approved the submitted version.

This study received funding from Immunomic Therapeutics. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

AA, JM, YJ, MC, TC, and TH are employees of Immunomic Therapeutics, and TH is also a shareholder.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.