*3.3. H-1PV + PD-1 Immune Checkpoint Inhibitors (Nivolumab)*

Checkpoint blockade, a strategy which aims at overcoming immune system tolerance towards the tumor through the release from negative regulators of immune activation (immune checkpoints), is presently at the leading edge of cancer immunotherapy. Although efficient in controlling various other solid tumors, immune checkpoint inhibitors (ICIs) frequently fail to achieve a significant response in glioblastoma patients [63]. Several preclinical studies and clinical trials have therefore been initiated, in order to determine the optimal ICI-based combinations and redefine the future standards of care for this deadly disease [64].

*Cancers* **2021**, *13*, x 9 of 15

**Figure 3.** Rationale for combining H-1PV administration with bevacizumab treatment in patients with recurrent glioblastoma multiforme (GBM). Bevacizumab antibody and H-1PV infection share the capacity for inhibiting vascular endothelial growth factor (VEGF) (production) (upper row) and triggering distinct immuno-modulations (lower rows), raising hopes to improve antitumor immunity by combining both treatments. In support of this strategy, bevacizumab and H-1PV were found to jointly achieve significant clinical improvement in GBM patients at second or third recurrence, leading to remission of the recurrent tumor. The precise mechanisms of this therapeutic potentiation remain to be determined. However, the establishment by H-1PV of an immunologically "improved" proinflammatory background, which facilitates bevacizumab-mediated immuno-stimulating effects, is a likely scenario. For details and references, see the main text. **Figure 3.** Rationale for combining H-1PV administration with bevacizumab treatment in patients with recurrent glioblastoma multiforme (GBM). Bevacizumab antibody and H-1PV infection share the capacity for inhibiting vascular endothelial growth factor (VEGF) (production) (upper row) and triggering distinct immuno-modulations (lower rows), raising hopes to improve antitumor immunity by combining both treatments. In support of this strategy, bevacizumab and H-1PV were found to jointly achieve significant clinical improvement in GBM patients at second or third recurrence, leading to remission of the recurrent tumor. The precise mechanisms of this therapeutic potentiation remain to be determined. However, the establishment by H-1PV of an immunologically "improved" proinflammatory background, which facilitates bevacizumab-mediated immuno-stimulating effects, is a likely scenario. For details and references, see the main text.

*3.3. H-1PV + PD-1 Immune Checkpoint Inhibitors (Nivolumab)*  Checkpoint blockade, a strategy which aims at overcoming immune system tolerance towards the tumor through the release from negative regulators of immune activation (immune checkpoints), is presently at the leading edge of cancer immunotherapy. Although efficient in controlling various other solid tumors, immune checkpoint inhibitors (ICIs) frequently fail to achieve a significant response in glioblastoma patients [63]. Several preclinical studies and clinical trials have therefore been initiated, in order to determine the optimal ICI-based combinations and redefine the future standards of care for this deadly disease [64]. First clinical hints of improved antitumor effects of H-1PV virotherapy upon combination with checkpoint blockade were obtained through compassionate virus uses. A series of three patients with rapidly progressing recurrent glioblastoma were treated with First clinical hints of improved antitumor effects of H-1PV virotherapy upon combination with checkpoint blockade were obtained through compassionate virus uses. A series of three patients with rapidly progressing recurrent glioblastoma were treated with H-1PV (two were irradiated prior to virus administration), followed by bevacizumab and the programmed cell death protein 1 (PD-1) inhibitor nivolumab. In addition, all patients received the HDACi VPA. This innovative PV-based multimodal strategy led to radiologically confirmed tumor regression accompanied by clinical improvement in all subjects 4 to 8 weeks after virus injection [65]. An objective tumor response was also seen in another group of primary or recurrent glioblastoma patients, who received H-1PV in combination with bevacizumab and checkpoint blockade. Complete to partial tumor remission was documented in 78% of the cases, which is a significantly higher response rate than the one reported in the literature for bevacizumab- and ICI-based monotherapies [66].

H-1PV (two were irradiated prior to virus administration), followed by bevacizumab and the programmed cell death protein 1 (PD-1) inhibitor nivolumab. In addition, all patients received the HDACi VPA. This innovative PV-based multimodal strategy led to radiolog-Altogether, the above data provide a strong impetus for further clinical development of H-1PV combinations with radiation and/or immunomodulators (in particular bevacizumab and ICIs) in the fight against glioblastoma.

### ically confirmed tumor regression accompanied by clinical improvement in all subjects 4 to 8 weeks after virus injection [65]. An objective tumor response was also seen in another **4. Parvovirus-Based Combinatorial Immunotherapy against Colorectal Cancer**

group of primary or recurrent glioblastoma patients, who received H-1PV in combination with bevacizumab and checkpoint blockade. Complete to partial tumor remission was documented in 78% of the cases, which is a significantly higher response rate than the one reported in the literature for bevacizumab- and ICI-based monotherapies [66]. CRC is another major cause of cancer-related deaths worldwide. Although the implementation of early-detection screening programs has substantially improved the 5-year overall survival, prognosis for CRC patients with stage 4 metastatic disease remains poor [67]. Immunotherapy, in particular checkpoint blockade, has proved efficient against

heavily mutated colorectal tumors. However, it fails to elicit sufficiently strong therapeutic responses in carcinomas, which are mismatch-repair-proficient (pMMR) and possess low levels of microsatellite instability (MSI-L). Low mutational burden, together with the lack of immune cell infiltration, contribute to pMMR-MSI-L immune resistance [68]. Novel approaches are therefore needed for the treatment of patients with advanced metastatic or low mutational burden CRC. One such approach, combinatorial immunotherapy, holds much potential for extending the scope of checkpoint blockade so as to bring benefit also to CRC patients with unfavorable prognosis.
