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. 2021 Jul;9(7):e002644.
doi: 10.1136/jitc-2021-002644.

CD137 and PD-L1 targeting with immunovirotherapy induces a potent and durable antitumor immune response in glioblastoma models

Montserrat Puigdelloses  1   2   3 , Marc Garcia-Moure  1   2   4 , Sara Labiano  1   2   4 , Virginia Laspidea  1   2   4 , Marisol Gonzalez-Huarriz  1   2   4 , Marta Zalacain  1   2   4 , Lucia Marrodan  1   2   4 , Naiara Martinez-Velez  1   2   4 , Daniel De la Nava  1   2   4 , Iker Ausejo  1   2   4 , Sandra Hervás-Stubbs  5 , Guillermo Herrador  1   2   4 , ZhiHong Chen  6 , Dolores Hambardzumyan  6 , Ana Patino Garcia  1   2   4 , Hong Jiang  7 , Candelaria Gomez-Manzano  7 , Juan Fueyo  7   8 , Jaime Gállego Pérez-Larraya  9   2   3 , Marta Alonso  9   2   4
Affiliations

CD137 and PD-L1 targeting with immunovirotherapy induces a potent and durable antitumor immune response in glioblastoma models

Montserrat Puigdelloses et al. J Immunother Cancer. 2021 Jul.

Erratum in

Abstract

Background: Glioblastoma (GBM) is a devastating primary brain tumor with a highly immunosuppressive tumor microenvironment, and treatment with oncolytic viruses (OVs) has emerged as a promising strategy for these tumors. Our group constructed a new OV named Delta-24-ACT, which was based on the Delta-24-RGD platform armed with 4-1BB ligand (4-1BBL). In this study, we evaluated the antitumor effect of Delta-24-ACT alone or in combination with an immune checkpoint inhibitor (ICI) in preclinical models of glioma.

Methods: The in vitro effect of Delta-24-ACT was characterized through analyses of its infectivity, replication and cytotoxicity by flow cytometry, immunofluorescence (IF) and MTS assays, respectively. The antitumor effect and therapeutic mechanism were evaluated in vivo using several immunocompetent murine glioma models. The tumor microenvironment was studied by flow cytometry, immunohistochemistry and IF.

Results: Delta-24-ACT was able to infect and exert a cytotoxic effect on murine and human glioma cell lines. Moreover, Delta-24-ACT expressed functional 4-1BBL that was able to costimulate T lymphocytes in vitro and in vivo. Delta-24-ACT elicited a more potent antitumor effect in GBM murine models than Delta-24-RGD, as demonstrated by significant increases in median survival and the percentage of long-term survivors. Furthermore, Delta-24-ACT modulated the tumor microenvironment, which led to lymphocyte infiltration and alteration of their immune phenotype, as characterized by increases in the expression of Programmed Death 1 (PD-1) on T cells and Programmed Death-ligand 1 (PD-L1) on different myeloid cell populations. Because Delta-24-ACT did not induce an immune memory response in long-term survivors, as indicated by rechallenge experiments, we combined Delta-24-ACT with an anti-PD-L1 antibody. In GL261 tumor-bearing mice, this combination showed superior efficacy compared with either monotherapy. Specifically, this combination not only increased the median survival but also generated immune memory, which allowed long-term survival and thus tumor rejection on rechallenge.

Conclusions: In summary, our data demonstrated the efficacy of Delta-24-ACT combined with a PD-L1 inhibitor in murine glioma models. Moreover, the data underscore the potential to combine local immunovirotherapy with ICIs as an effective therapy for poorly infiltrated tumors.

Keywords: brain neoplasms; oncolytic viruses.

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Conflict of interest statement

Competing interests: CG-M and JF report ownership interest (including patents) in DNATrix. MMA has a research grant from DNAtrix not related to this work.

Figures

Figure 1
Figure 1
Characterization of Delta-24-ACT in glioma cell lines. (A) Schematic representation of engineered Delta-24-ACT. (B) Infectivity of Delta-24-ACT in GL261-5 (left panel) and CT-2A (right panel) cells measured by flow cytometry as the percentage of GFP+ cells at 48 hours after infection with Delta-24-RGD-GFP at MOIs ranging from 0.1 to 100 PFUs/cell. The values represent the mean percentages of GFP+ cells ±SDs (n=3). (C) Assessment of viral protein expression (Fiber and E1A) in GL261-5 and CT-2A cells by western blot analysis. Cells were infected with Delta-24-ACT at the indicated MOIs, and 48 hours later, whole-cell lysates were collected. Samples were also collected at 16 hours postinfection with the highest dose (MOI of 300) as a control. Grb2 was used as a protein-loading control. (D) Replication and quantification of Delta-24-ACT in murine CT-2A and GL261-5 cells and human U251-MG and U87-MG cells that were infected at MOIs of 300 and 10, respectively. Delta-24-ACT replication was determined 16 and 72 hours postinfection. The dashed lines indicate the total initial viral input, and the results are expressed as the mean viral titers±SDs (n=3, one-tailed Mann-Whitney test). Oncolytic effect of Delta-24-ACT on murine and human glioma cells (E) and NHAs (F). To quantify the oncolytic effect of Delta-24-ACT on murine CT-2A and GL261-5 cells and human U251-MG, U87-MG and NHA cells, the cells were infected at the indicated MOIs, and 5 days later, their viability was evaluated by MTS assays. The values indicate the percentages of viable cells in infected cultures compared with those in noninfected cultures (means±SDs, n=3). (G) Oncolytic effect of Delta-24-ACT on U87-MG cells and NHAs. U87-MG cells and NHAs were infected at the indicated MOIs, and 3 days later, their viability was measured by crystal violet staining. MOI, multiplicities of infection; NHA, normal human astrocyte.
Figure 2
Figure 2
Delta-24-ACT can be expressed in glioma cells and induces T cell activation. (A) 4-1BBL protein expression in CT-2A, GL261, GL261-5 and U87-MG cells infected with Delta-24-ACT and Delta-24-RGD at the indicated MOIs analyzed by flow cytometry (one-way ANOVA). (B) 4-1BBL protein expression in GL261-5 and CT-2A cells infected with Delta-24-ACT at the indicated MOIs analyzed by western blot analysis. A representative image is shown. (C) In vivo evaluation of 4-1BBL mRNA expression. The mRNA expression of 4-1BBL in mice bearing CT-2A tumors 3 days after treatment with Delta-24-ACT was analyzed by real-time PCR. The contralateral hemisphere was used as the negative control (Student’s t-test). (D) In vivo assessment of 4-1BBL mRNA expression in mice bearing CT-2A and U87-MG subcutaneously and treated with either Delta-24-RGD or Delta-24-ACT for 48 hours. Mice treated with PBS were used as controls. (E) IFN-gamma production by lymphocytes after Delta-24-ACT infection. CD8+ T cells from PMEL mice were cocultured with CT-2A cells infected with Delta-24-RGD or Delta-4-ACT at an MOI of 100. The secretion of IFN-gamma in the collected supernatants was quantified by ELISA. MOI, multiplicities of infection.
Figure 3
Figure 3
Comparison of the in vivo antitumor effects exerted by Delta-24-RGD and Delta-24-ACT in murine glioma models. Schedule of survival experiments with CT-2A (A) and GL261 (B) tumor models. Survival of mice bearing CT-2A tumors (C) or GL261 tumors (D) treated with Delta-24-ACT or Delta-24-RGD (1×108 PFUs/mouse, 3 µL, n=10). A control group of mice treated with PBS was included in every survival experiment. (E) Biochemical studies performed with the serum of mice bearing CT-2A tumors (n=5) treated with mock infection or Delta-24-ACT analyzed by a Cobas 311 analyzer. (F) Rechallenge experiment involving the inoculation of naïve mice (n=5) or long-term survivors with CT-2A cells. All survival experiment results are shown as Kaplan-Meier survival curves (log-rank test). The shaded area represents a 10-day interval from the time of cell implantation.
Figure 4
Figure 4
Modulation of the tumor immune microenvironment by Delta-24-ACT treatment. IFN-gamma secretion by splenocytes isolated from the spleen of mice treated with PBS or Delta-24-ACT and cocultured with mock- or Delta-24-ACT-infected CT-2A cells; the secretion was measured by ELISA (A). Assessment of mean counted spots (left panel, B) and mean spot size (right panel, B) obtained by ELISPOT of the coculture of splenocytes isolated from the spleen of mice treated with PBS or Delta-24-ACT with mock-infected CT-2A cells. (C) Analysis of tumor-infiltrating lymphocyte populations in the brains of mice bearing CT-2A tumors treated with Delta-24-ACT or PBS. The studied populations were CD45+ cells, CD4+ cells, CD8+ cells, CD4+ T regulatory (FOXP3+) cells, microglia, macrophages (F4/80+ cells), M1 cells and DCs. All markers were analyzed by flow cytometry (one-way ANOVA).
Figure 5
Figure 5
The combination of Delta-24-ACT and PD-L1 blockade in GL261 tumors increases survival and generates long-term protection. (A) GL261 cells were infected with Delta-24-ACT for 48 hours. The PD-L1 marker was analyzed by flow cytometry. (B) Schedule of the survival experiment with the GL261 tumor model. (C) Survival of mice bearing GL261 tumors. Mice were treated with an anti-PD-L1 antibody or Delta-24-ACT as a monotherapy or with the combination of Delta-24-ACT and the anti-PD-L1 antibody. (D) Rechallenge experiment performed with mice bearing GL261 tumors. GL261 cells were implanted in naïve mice or long-term survivors. All survival results are shown as Kaplan-Meier survival curves (log-rank test).
Figure 6
Figure 6
Modulation of the immune compartment in GL261 tumor-bearing mice after treatment with Delta-24-ACT in combination with PD-L1 antibody. Analysis of tumor-infiltrating lymphocyte populations in the brains of mice bearing GL261 tumors treated with PBS (control), αPD-L1, Delta-24-ACT or Delta-24-ACT/αPD-L1. The studied populations were CD45+ cells (A), myeloid cell populations, including macrophages, DCs and microglia (B), and lymphoid cell populations, including CD8+, CD4+, Tregs and NK cells (C). The analyses of the functional status of the myeloid cells described above included analyses of PD-L1 (D), CD86+MHCI+I (E), CD137L (F) markers and the percentage of M1 macrophages (G). Percentage of DCs present in the lymph nodes. (H) DC functional status. The CD40, CD86 and PD-L1 markers were analyzed (I). Phenotype of CD8+ and CD4+ T cells (J) based on studies of the CD137, GITR, PD-1 and GrzB markers. (K) Quantification of splenocyte IFN-gamma production by ELISPOT after the indicated treatment. Top panel: mean counted spots. Lower panel: mean spot size (one-way ANOVA).
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