Table 3.

Combinations of immunotherapies including ICB in preclinical GBM models

Combination treatment	Protocol	Cell line and model	Outcome	Reference
anti-PD-1 anti-LAG-3	• Tumor implantation: 1.3 × 105 cells, striatum • anti-PD-1: 200 μg, IP  - on d7, d10, d12, and d14 (early schedule)  - on d10, d12, and d14 (late schedule) • anti-LAG-3: 200 μg, IP  - on d7 and d10 (early schedule)  - on d10 and d12 (late schedule)	GL261-Luc orthotopic syngeneic	• Early treatment with anti-LAG-3 and/or anti-PD-1 significantly improved survival • Late treatment with anti-LAG-3 did not significantly improve survival, but the combination did • The global immunological profiles were not different between the different treatment arms • Immune memory was established in long-term survivors	Harris-Bookman S., et al. (2018) [137]
anti-PD-1 anti-TIGIT	• Tumor implantation: 1.3 × 105 cells, striatum • anti-PD-1: 200 μg, on d10, d12, and d14, IP • anti-TIGIT: 200 μg, IP, every other day for a total of 5 doses starting on d8, d10, d12 or d14 (4 ≠ schedules: A, B, C, D)	GL261-Luc orthotopic syngeneic	• Combination therapy improved long-term survival following each schedule • Combination therapy increased immune cell tumor infiltration and cytokine production • Tumor-infiltrating DCs were reduced following anti-TIGIT and anti-PD-1 combination treatment • Immune memory was established in long-term survivors	Hung AL., et al. (2018) [138]
anti-CTLA-4 anti-PD-L1 1-MT	• Tumor implantation: 4 × 105 cells • anti-CTLA-4: 100 μg loading dose followed by 3 × 50 μg maintenance doses every 3 days, IP • anti-PD-L1: 500 μg loading dose followed by 3 × 200 μg maintenance doses every 3 days, IP • 1-MT: in the drinking water over 30 days, starting on d7 for early blockade or on d14 for late blockade	GL261 orthotopic syngeneic	• Early blockade with the triple combination cured 100% of mice, reduced Treg infiltration and increased IFN-γ-secreting CD8 T cell infiltration • Late blockade prolonged survival (78% long-term survival rate) and reduced Treg infiltration but also reduced brain-infiltrating T cells	Wainwright DA., et al. (2014) [139]
anti-GITR agonist SRS	• Tumor implantation: 1.3 × 105 cells, striatum • SRS: 10 Gy radiation (1.9 Gy/min), d10, focal • anti-GITR: 10 mg/kg, 3x, on d10, d13, and d16, IP	GL261-Luc orthotopic syngeneic	• Combination therapy improved survival • The combination increased the CD8 effector T cell/Treg ratio • Immune memory was established in long-term survivors	Patel MA., et al. (2016) [140]
anti-PD-1 anti-OX40 agonist GVAX (whole tumor cell vaccine)	• Tumor implantation: 7.5 × 104 cells, right striatum • anti-PD-1: 200 μg, on d3, d6, and d9, IP • anti-OX40: 250 μg, on d3, d6, and d9, IP • GVAX: 1 × 106 irradiated (35 Gy) GL261-GMCSF cells, on d3, d6, and d9, SC	GL261 orthotopic syngeneic	• The anti-PD-1 + anti-OX40 dual combination improved survival and the CD8/Treg ratio • The anti-PD-1 + GVAX dual combination improved survival and the CD8/Treg ratio and increased brain-infiltrating CD8 T cells • The triple combination led to 100% long-term survival with an increase in IFN-γ- and IL-2-secreting splenocytes and the CD4/CD8 ratio • Immune memory is established in long-term survivors	Jahan N., et al. (2019) [141]
anti-PD-L1 Neoantigen peptide vaccine	• Tumor implantation: 5 × 104 cells • anti-PD-L1: on d7, d9, and d11, IP • Vaccine: 50 μg of each peptide + 100 μg polyIC adjuvant, on d3, d6, and d9, SC	CT2A orthotopic syngeneic	The combination therapy significantly improved mouse survival (60% long-term survivors)	Liu CJ., et al. (2020) [142]
anti-PD-1 anti-TIM3 SRS	• Tumor implantation: 1.3 × 105 cells, left striatum • SRS: 10 Gy radiation (1.9 Gy/min), d10, focal, using the Small Animal Radiation Research Platform (SARRP) • anti-PD-1: 200 μg, on d10, d12, and d14 • anti-TIM-3: 250 μg, on d7, d11, and d-15	GL261-Luc orthotopic syngeneic	• The anti-PD-1 and dual therapies improved survival and led to long-term survival • The triple combination led to 100% remission • The triple combination improved the immune profile of the TME and the cytokine profile of both CD4 and CD8 T cells (increased the CD8/Treg ratio, decreased the frequency of FoxP3+ Tregs, and increased the production of the inflammatory cytokines IFN-γ, TNF-α, and IL-17a) • Immune memory was established in long-term survivors	Kim JE., et al. (2017) [143]
anti-CTLA-4 anti-4-1BB agonist SRS	• Tumor implantation: 1.3 × 105 cells, left striatum • SRS: 10 Gy radiation (1.9 Gy/min), d10, focal, using the SARRP • anti-4-1BB: 200 μg 3x, on d11, d14, and d17, IP • anti-CTLA-4: 800 μg 3x, on d11, d17, and d23, IP	GL261-Luc orthotopic syngeneic	• The SRS + anti-CTLA-4 dual therapy prolonged survival, but only the triple combination led to long-term survival • The triple therapy and double immunotherapy led to higher TILs (CD4 and CD8 T cells) • Immune memory was established in long-term survivors and was glioma-specific	Belcaid Z., et al. (2014) [144]
anti-PD-1 anti-CXCR4	• Tumor implantation: 1.3 × 105 cells, left striatum • anti-PD-1: 200 μg, on d10, d12, and d14, IP • anti-CXCR4: 200 μg, on d10, d12, and d14, IP	GL261-Luc orthotopic syngeneic	• The combination improved survival • Combination therapy decreased populations of suppressive myeloid cells in the brain • Combination therapy decreased the CD4/CD8 and Treg/CD8 ratios in the brain to a higher extent than did the monotherapies • The combination and monotherapies increased the levels of circulating inflammatory antitumor cytokines • Immune memory was established in long-term survivors	Wu A., et al. (2019) [145]
anti-PD-1 Antiangiogenic therapy (anti-VEGF + anti-Ang-2)	• Tumor implantation: 1 × 105 cells, striatum • anti-PD-1: 10 mg/kg, 2x/week starting on d10 for a total of 8 doses, IP • Antiangiogenic therapy: 25 mg/kg (anti-VEGF) and 5.6 mg/kg (anti-Ang-2), 2x/week starting on d5 until symptom apparition, SC	GL261 orthotopic syngeneic	• The triple therapy significantly improved survival compared to antiangiogenic therapy alone • The triple therapy increased CD8 TIL numbers and decreased MDSCs and Tregs in the brain The antiangiogenic therapy efficacy was improved by the addition of anti-PD-1 therapy	Di Tacchio M., et al. (2019) [146]
anti-PD-1 anti-CTLA-4 G47Δ-mIL12 (= Oncolytic herpes simplex virus expressing IL-12)	• Tumor implantation: 2 × 104 cells (005 GSCs) or 1 × 104 cells (CT-2A), striatum • anti-PD-1: 10 mg/kg, on d8, 11 and d14 (005 GSCs) or on d10, d13 and d16 (CT-2A), IP • anti-CTLA4: 5 mg/kg, on d8, d11 and d14 (005 GSCs) or on d10, d13 and d16 (CT-2A), IP • G47Δ-mIL12: 5 × 105 PFU, on d8 (005 GSCs) or on d10 (CT-2A), intratumoral	mouse 005 GSCs or CT-2A orthotopic syngeneic	• Individual ICB only minimally extended survival in 005 GBM; combination of G47Δ-mIL12 with individual ICB modestly improved efficacy • G47Δ-mIL12 decreased the % of 005 cells and Tregs and induced M1-like polarization in TAMs; these effects are further increased with the triple combination • Dual ICBs significantly increased CD8 T cells in the brain, triple combination increased CD8 T cells and decreased Tregs • Triple combination resulted in 89% or 50% long term survival (005 GSCs or CT-2A, respectively) Immune memory was established in long-term survivors	Saha D., et al. (2017) [147]
anti-PD-1 anti-CTLA-4 anti-IL-6 anti-CD40 agonist	• Tumor implantation: 3 × 105 genetically engineered mouse tumor cells or 2 × 105 GL261 cells • anti-PD-1: 200 μg on d9, d12, d15 and d18, IP • anti-CTLA-4: 200 μg on d9, d12, d15 and d18, IP • anti-IL-6: 200 μg on d9, d12, d15 and d18, IP • anti-CD40: 100 μg on d12, IP	RCAS-genetically engineered model or GL261 orthotopic syngeneic	• Dual targeting of IL-6 and CD40 sensitized GBM to ICBs; survival was significantly improved following triple combination • Dual targeting of IL-6 and CD40 reduced tumor growth, triple combination blocked it • All treatments induced a decrease in immunosuppressive TAM activity • All treatments, except anti-CD40 alone, decreased the expression of immunosuppressive cytokines (IL-10, TGFβ) • Only triple combination induced an increase in TILs and in IFN-γ-secreting CD8 T cells	Yang F. et al. (2021) [148]