**3.5 Combination checkpoint inhibition**

Tumor-treating fields (TTFs) work as a non-invasive anti-cancer therapy via alternating electric fields. As stated earlier, TTFs are already FDA-approved for GBM in combination with temozolomide. Voloshin *et al.* expanded upon these findings and found TTFs elicited tumor cell death in murine models of lung and colon cancer. In addition, the authors found TTFs could induce maturation of bone marrow-derived DCs. Furthermore, using an orthotopic model of murine lung cancer, the combination of TTFs and the ICI, anti-PD-1, was found to reduce growth relative to control-treated mice. This anti-tumor effect was found to be mediated by the expansion of macrophages, DCs, and CD8+ T cells within the TME. In addition, when subcutaneous colon cancer-bearing mice were treated with anti-PD-1, TTFs, or the combination, a reduction in tumor growth was observed in combinationtreated mice relative to controls. Combination-treated mice were found to have a decrease in intratumoral DCs and macrophages but increased CD3+ CD4+ and CD3+ CD8+ T cells. These results suggest the combination of an ICI such as anti-PD-1 and TTFs could enhance anti-tumor responses in the context of brain tumors [148].

As stated earlier, ICI as monotherapies has had limited success in patients with CNS-derived malignancies. Therefore, several groups are evaluating combinatorial ICI approaches to enhance anti-tumor effects. Flores *et al.* found the combination of lineage-negative hematopoietic stem and progenitor cells (HSPCs) and the ICI, anti-PD-1 provided significantly prolonged survival relative to HSPC or anti-PD-1 monotherapy. The authors found the enhanced survival is likely due to increased secretion of IFNγ by T cells in the TME. In addition, they found the CCR2+ HSPCs were the population responsible for providing the enhanced anti-tumor efficacy. Interestingly, they observed that utilizing CCR2<sup>+</sup> HSPCs in the context of an adoptive cellular therapy (ACT) platform, which combines tumor RNA-pulsed DCs, tumor-reactive T cells, and radiotherapy, significantly enhanced survival relative to ACT using bulk lineage-negative HSPCs. These results suggest these CCR2+ HSPCs cells may be combined with various types of immunotherapies to enhance antitumor efficacy [149].

Alternatively, Flores-Toro *et al.* identified an expansion of CCR2<sup>+</sup> myeloid cells within the TME using two models of intracranial glioma. The authors used a small molecule inhibitor of CCR2, CCX872, in combination with the ICI, anti-PD-1 to enhance survival using a murine model of high-grade glioma as well as a GSC model. They went on to determine this mechanism of anti-tumor efficacy was likely due to a combination of reduced recruitment of Ly6C+ myeloid cells to the TME,

an increase in intratumoral CD4<sup>+</sup> , CD8+ , CD3+ IFNγ<sup>+</sup> cells, and a reduction in CD8+ TIM3<sup>+</sup> PD-1+ T cells relative to vehicle control-treated mice [150].

Finally, Sabbagh *et al.* used novel combinatorial immunotherapy approach to enhance anti-tumor efficacy. They utilized low-intensity pulsed ultrasound (LIPU) to open the BBB for better penetration of various therapeutics. Although LIPU as monotherapy did not provide a robust anti-tumor response, when combined with anti-PD-1, enhanced median survival was observed relative to IgG control-treated mice. In addition, the authors used EGFRvIII-specific CAR T cells in combination with LIPU and found increased trafficking of administered CAR T cells to the TME as well as enhanced survival relative to CAR T cells alone. These results suggest utilizing combinatorial immunotherapeutic approaches with LIPU may lead to enhanced anti-tumor efficacy [151].

## **4. Conclusions**

Malignant brain tumors pose a unique and difficult set of challenges including high tumor heterogeneity and tumor antigen loss, low mutation burden, an immunosuppressive microenvironment, systemic T cell dysfunction, and relative isolation from systemic circulation due to the blood-brain barrier. These overwhelming obstacles have, thus far, limited immunotherapy efficacy. Despite these hurdles, immunotherapies are making incremental advances to overcome these challenges simultaneously [152, 153]. New developments are occurring in the peptide vaccine platforms by the conjugation with toll-like receptor agonists which can enhance activation of DCs to elicit tuned immune responses [154–156]. Studies are also moving forward to focus on targeting multiple antigens simultaneously to combat tumor antigen loss in CAR T therapy [157]. Other groups are working on addressing the immunosuppressive tumor microenvironment and T cell exhaustion with several studies underway in a variety of cancers that combine vaccines and immune checkpoint inhibitors [158]. In the CAR T therapy arena, groups are overcoming T cell exhaustion by knocking out the checkpoint molecules [159, 160], endowing CAR T cells with the capabilities of secreting anti-PD-L1 antibodies [161], and linking the PD-1 extracellular domain to the CD28 intracellular domain to lead to an activation signal instead of inhibition [162, 163]. Other groups are working on overcoming the blood-brain barrier challenge by using laser interstitial thermal therapy or the aforementioned low-intensity pulsed ultrasound to cause local disruption and permeability which may increase trafficking of therapies to the tumor site [151, 164, 165]. These approaches utilizing various combinations and novel technologies may provide solutions to the aforementioned obstacles.

In summary, the next advances in immunotherapies for CNS malignancies will come from enhanced foundational understanding of immune cells and the tumor microenvironment, better mechanistic understandings of current immunotherapy resistance, increased rational combinations of current immunotherapies with complementary mechanisms of action, and novel immunotherapeutic approaches. Together, the above-mentioned clinical studies and novel preclinical work provide an optimistic future in cancer with much-needed improvement in patient survival.

#### **Acknowledgements**

This work was supported by the University of Florida Clinical and Translational Science Institute, which is supported in part by the NIH National Center for Advancing Translational Sciences under the award TL1TR001428. NIH funding

#### *Immunotherapy against Gliomas DOI: http://dx.doi.org/10.5772/intechopen.101386*

was also received through the National Cancer Institute (F30CA232641 to MS) (T32CA257923 to BDD) and the National Institute of Neurological Disorders and Stroke (R01NS112315 and R01NS111033 to CF). Additionally, this work was supported by Alex's Lemonade Stand Foundation and the University of Florida MD-PhD Training Program.
