Table 2.

Combinations of immunotherapy and chemotherapy in preclinical GBM models

Combination treatment	Protocol	Cell line and model	Outcome	Reference
anti-PD-1 TMZ	• Tumor implantation: 2 × 105 cells • TMZ: 30 mg/kg, 5 consecutive days starting at d8, IP • anti-PD-1: 10 mg/kg 2x on d13 and d15, IV	GL261-Luc orthotopic syngeneic	• Combined therapy showed better antitumor efficacy than monotherapies with 100% tumor regression • TMZ abrogated the favorable immunological effects of anti-PD-1 (increased TIL numbers, decreased Treg and exhausted T cell frequencies, increased immunological memory)	Park J., et al. (2018) [123]
anti-PD-1 TMZ (standard or metronomic dose)	• Tumor implantation: / • TMZ:  - Standard dose (SD): 50 mg/kg for 5 consecutive days, IP  - Metronomic dose (MD): 25 mg/kg for 10 consecutive days, IP • anti-PD-1: 10 mg/kg 4x every 5 days, IP	GL261 orthotopic syngeneic	• SD TMZ increased exhaustion markers on T cells, while MD TMZ did not lead to T cell exhaustion • anti-PD-1 reversed the exhaustion induced by SD TMZ in peripheral T cells but not in TILs • The survival benefit of anti-PD-1 therapy was abrogated by SD TMZ but not by MD TMZ	Karachi A., et al. (2019) [124]
anti-PD-1 TMZ (low dose)	• Tumor implantation: 5 × 104 cells, right cerebral cortex • TMZ: 50 μg/kg, 5 consecutive days, IP • anti-PD-1: 200 μg 3x, IP	GL261 orthotopic syngeneic	• Combined therapy synergistically inhibited GBM tumor growth with a higher median survival time, a reduced tumor volume and 40% long-term survivors • Combined therapy increased CD4 and CD8 T cell infiltration in tumor lesions	Dai B., et al. (2018) [126]
anti-PD-1 TMZ or carmustine (BCNU) (systemic or local administration)	• Tumor implantation: 1.3 × 105 cells, left striatum • Systemic chemotherapy (SC):  - TMZ: 66 mg/kg, daily from d10 to d14, IP  - BCNU: 5, 15 and 30 mg/kg, 3x/week for 2 weeks starting at d14, IP • Local chemotherapy (LC):  - TMZ: implanted at d10  - BCNU: implanted at d14  Polymer impregnated with chemotherapy (wafer), allowing constant release in the TME for at least 2 weeks, placed directly on top of the tumor • anti-PD-1: 200 μg 3x, on d0, d12, and d14, IP	GL261-Luc orthotopic syngeneic	• Combination of LC and anti-PD-1 induced a robust immune response and survival benefit, with higher numbers of TILs and IFN-γ-secreting CD8 T cells in the brain, a higher Teff/Treg ratio and a higher tumor-infiltrating DC % • LC preserved the memory response upon rechallenge; SC abrogated it • SC abrogated the immunological benefits of anti-PD-1, did not provide survival benefit and resulted in severe lymphodepletion and severe depletion of TILs • SC alone or in combination with anti-PD-1 delayed tumor progression, but tumors recurred	Mathios D., et al. (2016) [125]
TMZ (systemic) ICD-based DC vaccine	• Tumor implantation: 5 × 105 cells • ICD-based DC vaccine: 1 × 106 DCs, IP  - On d2, d9 and d15 – vaccine alone  - On d21, d28 and d35 – combination  Production: cancer cells were incubated with hypericin followed by light irradiation, and then, Hyp-PDT-treated cells were mixed with DCs • TMZ: 40 mg/kg, 6x on d5, d7, d9, d12, d14, and d16	GL261 orthotopic syngeneic	• Combined therapy provided a strong survival benefit with improved median survival and 50% long-term survivors • The ICD-based vaccine partially overcame the immune-ablating effects of chemotherapy. TMZ decreased the levels of brain-infiltrating CD8 T cells, but the combination decreased the levels of Tregs in the brain	Garg AD., et al. (2016) [127]
TMZ (systemic or local) GL-GM (whole tumor cell vaccine)	• Tumor implantation: 5 × 103 cells, right frontal lobe • GL-GM: 2 × 106 irradiated (40 Gy) GL261-GMCSF cells, on d5, d19, and d33, IP • TMZ:  - Systemic (SC): 50 mg/kg, at d7, d8 and d9, IP  - Local (LC): 4.2 mg/kg/day, from d7 to d9, intratumoral	GL261 orthotopic syngeneic	• Local administration of TMZ induced a higher survival rate than systemic administration, and the effect was T cell-dependent • SC but not LC TMZ depleted blood leukocytes • Combination of TMZ IC and GL-GM increased survival and induced immune benefits with increased CD4 and CD8 TILs • Immune memory was established in long-term survivors (SC TMZ + GL-GM)	Fritzell S., et al. (2013) [128]
TMZ (local) Whole cell vaccine	• Tumor implantation: 5 × 103 cells, right frontal lobe • Whole cell immunization: 2 × 106 irradiated (40 Gy) cells (GL261 or KR158) on d5, d19, and d33, SC • TMZ: 180 μg administered over 3 days, starting on d7, convection-enhanced delivery (CED), intratumoral	GL261 or KR158-Luc orthotopic syngeneic	• CED-TMZ and the whole cell vaccine synergized in the GL261 model resulting in 93% long-term survivors • The whole cell vaccine cured some mice of the KR158 model, and CED-TMZ prolonged median survival; however, there was no synergy between chemotherapy and immunotherapy • CED-TMZ plus the vaccine significantly decreased tumor volume and increased the intratumoral influx of T cells in both models	Enriquez Pérez JE., et al. (2020) [129]
TMZ anti-CD47 anti-PD-1	• Tumor implantation: 1 × 105 cells, caudate putamen • TMZ:  - Concurrent: 80 mg/kg, at d11, d13 and d15, IP  - Sequential: metronomic dose (20 mg/kg) at d7–9 + 80 mg/kg at d11, d13 and d15, IP • anti-CD47 (MIAP-140): 100 μg, at d11, d13 and d15, IP • anti-PD-1: 100 μg, on d16, d18 and d20, IP	GL261 or CT2-A orthotopic syngeneic	• Sequential TMZ treatment combined with anti-CD47 improved tumor growth inhibition and mice survival; monotherapies and concurrent treatment did not • Combination of sequential TMZ and anti-CD47 activated immune response in vivo, with significant increase of CD4 and CD8 T cell, IFN-γ-secreting cell and activated TAM numbers • Triple combination of TMZ, anti-CD47 and anti-PD-1 further improved the survival	von Roemeling CA., et al. (2020) [130]