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. 2024 Apr 6;32(2):200799.
doi: 10.1016/j.omton.2024.200799. eCollection 2024 Jun 20.

Enhanced therapeutic efficacy for glioblastoma immunotherapy with an oncolytic herpes simplex virus armed with anti-PD-1 antibody and IL-12

Lei Wang  1   2 , Xusha Zhou  3 , Xiaoqing Chen  3 , Yuanyuan Liu  3 , Yue Huang  3 , Yuan Cheng  4 , Peigen Ren  1 , Jing Zhao  2 , Grace Guoying Zhou  2
Affiliations

Enhanced therapeutic efficacy for glioblastoma immunotherapy with an oncolytic herpes simplex virus armed with anti-PD-1 antibody and IL-12

Lei Wang et al. Mol Ther Oncol. .

Abstract

Glioblastoma is the most common and aggressive malignant brain tumor and has limited treatment options. Hence, innovative approaches are urgently needed. Oncolytic virus therapy is emerging as a promising modality for cancer treatment due to its tumor-specific targeting and immune-stimulatory properties. In this study, we developed a new generation of oncolytic herpes simplex virus C5252 by deletion of a 15-kb internal repeat region and both copies of γ34.5 genes. Additionally, C5252 was armed with anti-programmed cell death protein 1 antibody and interleukin-12 to enhance its therapeutic efficacy for glioblastoma immune-virotherapy. In vitro and in vivo experiments demonstrate that C5252 has a remarkable safety profile and potent anti-tumor activity against glioblastoma. Mechanistic studies demonstrated that C5252 specifically induces cell apoptosis by caspase-3/7 activation via downregulating ciliary neurotrophic factor receptor α. Furthermore, the enhanced anti-tumor therapeutic efficacy of C5252 in a subcutaneous glioblastoma model and an orthotopic glioblastoma model was confirmed. Moreover, syngeneic mouse models showed that the murine surrogate of C5252 has superior anti-tumor activity compared to the unarmed backbone virus, with enhanced immune activation. Taken together, our findings support C5252 as a promising therapeutic option for glioblastoma treatment, positioning it as a highly promising candidate for clinical translation.

Keywords: GBM; IL-12; checkpoint inhibitor; glioblastoma; immunotherapy; oHSV; oncolytic herpes simplex virus.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Virus construction and identification (A) Schematic representation of the virus genome. Wild-type HSV-1(F) is the prototype strain used in this laboratory and R3616 has a deletion of two copies of γ34.5. C5252 is a genetically modified replication-competent HSV-1 in which the internal repeat (IR) sequence (IR region) of 15 kb and the remaining copy of γ34.5 genes in the terminal repeat (TR) region were deleted, resulting in the removal of both copies of the γ34.5 genes as well as one copy each of diploid genes α0, α4, and LAT. The IR region is replaced with an expression cassette of heterodimer of IL-12. Another expression cassette of antigen-binding fragment (Fab) of an anti-PD-1 Ab is inserted into the genome between UL3 and UL4. (B) Growth curves. Vero cells were exposed to HSV-1(F), R3616, or C5252 (MOI = 0.1) over 48 h. Virus progeny were collected at 3, 6, 12, 24, and 48 h and titered via plaque assay using Vero cells (∗p < 0.05). (C) Accumulation of viral protein. Vero cells were infected with HSV-1(F), R3616, or C5252 (MOI = 1) for 6, 12, and 24 hpi. Cell samples were collected at indicated time points. Proteins were separated on 10% denaturing gels and analyzed via immunoblotting with antibodies targeting ICP0, ICP4, ICP27, ICP8, US11, γ34.5, and GAPDH. (D) The expression of human IL-12 p70 and anti-PD-1 Ab in HSV-1(F)-, R3616-, and C5252-infected Vero cells. Vero cells were mock infected (Mock) or infected with of HSV-1(F), R3616, or C5252. After 48 h, cell culture media were collected. Human IL-12 and anti-PD-1 Ab levels were quantified using ELISA, following a standard curve constructed with purified IL-12 protein and anti-PD-1 Ab as described in the materials and methods.
Figure 2
Figure 2
Virus replication and cytotoxicity in glioblastoma cells (A) Glioblastoma cells (A172, D54, U138, U87, and D458) were exposed to 0.1 PFU of HSV-1(F), R3616, or C5252 per cell. Virus progeny were collected at 48 hpi and titrated using Vero cells. (B) Similar to (A) but cells were exposed to 1.0 PFU per cell. (C) Glioblastoma cells were infected with HSV-1(F), R3616, or C5252 at 0.1 PFU per cell, and cytotoxicity was assessed via CCK8 assay at 48 hpi. (D) Similar to (C) but cells exposed to 1.0 PFU per cell. Data represent mean ± SD; N.S. p > 0.05, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Student’s t test (two-tailed) was used for statistical analysis.
Figure 3
Figure 3
Caspase-3/7 activity and CNTFRα expression during C5252 infection in glioblastoma cells (A) Caspase-3/7 activity in glioblastoma cells. Caspase-3/7 activity was assessed at 8 h post-infection (hpi) with HSV-1(F), R3616, or C5252 in glioblastoma cells. Luminescence-based measurements are expressed as relative light units (RLUs). Data are from three independent trials and presented as mean ± SD. Statistically significant differences between C5252 and R3616 indicated (N.S. p > 0.05, ∗p < 0.05, and ∗∗p < 0.01). (B) CNTFRα mRNA levels in glioblastoma cells. Glioblastoma cells were infected with 0.1 PFU of HSV-1(F), R3616, or C5252 per cell. At 48 hpi, mRNA levels of CNTFRα were measured and normalized to HSV-1(F)-infected cells. Data are from three independent trials and presented as mean ± SD. Statistically significant differences between C5252 and R3616 are indicated (∗p < 0.05). (C) CNTFRα overexpression in glioblastoma cells. CNTFRα overexpression was achieved using the Pc-CNTFRα plasmid. Real-time PCR assessed the fold change in CNTFRα mRNA levels compared to control (Pc-DNA3.1)-transfected cells. Statistically significant differences between Pc-CNTFRα and Pc-DNA3.1 are indicated (∗p < 0.05 and ∗∗p < 0.01). (D) Caspase-3/7 activity in overexpressed CNTFRα glioblastoma cells. After plasmid transfection, cells were infected with 1.0 PFU of HSV-1(F), R3616, or C5252 per cell. Caspase-3/7 activity was assessed 24 h post-infection. Data are from three independent trials and presented as mean ± SD. Statistically significant differences between C5252 and R3616 are indicated (∗p < 0.05 and ∗∗p < 0.01). (E) CNTFRα knockdown in glioblastoma cells. Glioblastoma cells were transfected with siRNA targeting CNTFRα (si-CNTFRα) or non-target siRNA (si-NT). CNTFRα downregulation efficiency was assessed by real-time PCR. Statistically significant differences between si-CNTFRα and si-NT are indicated (∗p < 0.05 and ∗∗p < 0.01). (F) Caspase-3/7 activity in CNTFRα knockdown glioblastoma cells. Caspase-3/7 activity was measured using the Caspase-Glo 3/7 kit in CNTFRα knockdown glioblastoma cells. Results are presented as RLUs. Data are from three independent trials and presented as mean ± SD. Statistically significant differences between C5252 and R3616 are indicated (∗p < 0.05 and ∗∗p < 0.01).
Figure 4
Figure 4
The safety evaluation of C5252 in vivo (A) The results of LD50 after intracranial injection. Neurovirulence was observed after intracranial administration of HSV-1(F), R3616, and C5252. LD50 values were determined for each group. LD50 for C5252 exceeded the maximum administered dose (1.5 × 105 PFUs/animal), while HSV-1(F) had an LD50 of 1.88 × 102 PFUs/animal. (B) Quantification of viral DNA expression in mice TG during latency. BALB/c mice were infected with HSV-1(F) or C5252 via the corneal route. TGs were extracted at various time points, and viral genome DNA copy numbers were quantified and normalized to cellular DNA. (C) TGs excised 30 days after inoculation of HSV-1(F) were processed immediately or after 24 h of incubation with anti-NGF antibody. Fold changes in mRNA levels encoding ICP27, TK, UL41, VP16, and LAT compared to 0 h are shown (∗p < 0.05 and ∗∗p < 0.01). (D) TGs excised 30 days after inoculation of C5252 were processed immediately or after 24 h of incubation with anti-NGF antibody. Fold changes in mRNA levels encoding ICP27, TK, UL41, VP16, and LAT compared to 0 h are shown. Data are presented as mean ± SD.
Figure 5
Figure 5
The anti-tumor therapeutic efficacy of C5252 in a subcutaneous glioblastoma model and an orthotopic glioblastoma model (A and B) Subcutaneous glioblastoma model. Subcutaneous U87 (A) and D54 (B) tumors were established in BALB/c-derived nude mice. Tumor-bearing mice were treated with intratumoral injections of control, R3616 (5 × 106 PFUs/animal), or C5252 (5 × 106 PFUs/animal) on specified days. Tumor volumes are represented as mean ± SD for 6 animals per group. Statistically significant differences between C5252 and R3616 group are indicated (∗p < 0.05). (C) C5252 in orthotopic tumor model. Female BALB/c nude mice received intracerebral inoculation with U-87MG-Luc tumor cells. Mice were treated with intratumoral injections of C5252 (3 × 105 PFUs/animal in 5 μL) at specific intervals. Tumor volume (ROI) is shown as mean ± SD. Statistically significant differences between C5252 and control group are indicated (∗p < 0.05). The Luc images are presented in Figure S5A. (D) Survival curves. Survival curves demonstrating the significant survival benefit of C5252 treatment are shown. Statistically significant differences between C5252 and control group are indicated (∗∗p < 0.01) using Kaplan-Meier analysis. (E) C8282 in orthotopic tumor model. Female C57BL/6 mice received intracerebral inoculation with CT-2A-GFP-Luc tumor cells (2 × 103 cells/animal). Mice were treated with intratumoral injections of C8282 (1 × 105 PFUs/animal in 5 μL) at specific intervals. Tumor volume (ROI) is shown as mean ± SD. The images are presented in Figure S5B. (F) Survival curves. Survival curves demonstrating the significant survival benefit of C8282 treatment are shown. Statistically significant differences between C8282 and control group are indicated (∗∗p < 0.01) using Kaplan-Meier analysis.
Figure 6
Figure 6
In vivo anti-tumor efficacy with syngeneic mouse model (A) C57BL/6 mice were used as the syngeneic host for the CT-2A cell line. In tumor treatment studies, C57BL/6 mice (n = 6 per group) were treated with C8282 or C1212 virus (1 × 107 PFUs/mouse) via intratumoral injection on days 1, 4, 7, 10, 13, and 16 six times in total. The sizes of tumors were measured on days 1, 4, 7, 10, 13, 16, and 19 before every administration using a caliper, and the volume was calculated as (length × width2) × 0.5. The results are shown as mean tumor volume (mm3) ± SD (n = 6). Statistically significant differences between C8282 and C1212 group were analyzed by Student’s t test (two-tailed) using GraphPad Prism software (∗p < 0.05). On the final day of the experiment, the tumors in the mice were dissected, and the images are shown in the Figure S6. Murine IFN-γ (B) and murine TNF-α (C) production in CT-2A syngeneic mice infected with control (DPBS+10% glycerin), C1212, or C8282 (1 × 107 PFUs/mouse) on days 0. CT-2A tumor samples from each mouse were collected on days 1, 3, 6, and 14 following infection (n = 3 mice per time point) for the determination of murine IFN-γ and murine TNF-α levels by ELISA. The results are reported as pg/g of tumor.
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