The Oncolytic virus VT1092M and an Anti-PD-L1 antibody synergize to induce systemic antitumor immunity in a murine bilateral tumor model

Highlights

• •VT1092 is an oncolytic herpes simplex virus armed with interleukin-12.
• •VT1092M combined with ICIs targeting PD-1, PD-L1, or TIM3 was first explored.
• •VT1092M combined with PD-L1 blockade induces abscopal and tumor-specific effects.
• •CD8⁺T cells play a crucial role in mediating potent systemic antitumor immunity.

Abstract

This study investigated the synergistic potential of an oncolytic herpes simplex virus armed with interleukin 12 (VT1092M) in combination with immune checkpoint inhibitors for enhancing antitumor responses. The potential of this combination treatment to induce systemic antitumor immunity was assessed using bilateral subcutaneous tumor and tumor re-challenge mouse models. The antitumor efficacy of various OV and ICI treatment combinations and the underlying mechanisms were explored through diverse analytical techniques, including flow cytometry and RNA sequencing. Using VT1092M, either alone or in combination with an anti-PD-L1 antibody, significantly reduced the sizes of both the injected and untreated abscopal tumors in a bilateral tumor mouse model. The combination therapy demonstrated superior antitumor efficacy to the other treatment conditions tested, which was accompanied by an increase in T cell numbers and CD8⁺T cell activation. Results from the survival and tumor re-challenge experiments showed that the combination therapy elicited long-term, tumor-specific immune responses, which were associated with tumor clearance and prolonged survival. Immune cell depletion assays identified CD8⁺T cells as the crucial mediators of systemic antitumor immunity during combination therapy. In conclusion, the combination of VT1092M and PD-L1 blockade emerged as a potent inducer of antitumor immune responses, surpassing the efficacy of each monotherapy. This synergistic approach holds promise for achieving robust and sustained antitumor immunity, with potential implications for preventing tumor metastasis in patients with cancer.

Graphical abstract

Introduction

Oncolytic virotherapy holds significant promise as a cancer treatment strategy. It works by leveraging viruses, such as herpes simplex virus (HSV), adenovirus, and vaccinia virus, to selectively target and lyse tumor cells. Large transgene capacity (∼30 kb), a broad host range and the ability to transduce both dividing and nondividing cells make herpes simplex virus type 1 (HSV-1) an attractive vector for discovery and development of oncolytic viruses (OVs) [1,2]. And Talimogene Laherparepvec (T-VEC), a genetically engineered OV based on HSV-1, is the first oncolytic virus therapy to be approved by FDA in 2015 [3]. Since then, oncolytic virotherapy has received more attention in the field of cancer immunotherapy, and these engineered viruses stimulate adaptive immunity, modulate the tumor microenvironment (TME), and enhance immune cell infiltration, to promote tumor cell lysis at the injection site [[4], [5], [6]].

The expression of immune checkpoint molecules (e.g., PD-1, TIM-3, TIGIT, LAG-3 on lymphocytes, and PD-L1 on tumor cells) contributes to an immunosuppressive TME [7,8]. Thus, combining oncolytic viruses with immune checkpoint inhibitors (ICIs) aims to unlock a synergistic antitumor response.

Interleukin (IL)−12 modulates tumorigenicity, inducing antitumor immunity via T and natural killer (NK) cell proliferation and activation, making it a potent candidate for immunotherapy [9,10]. However, systemic IL-12 administration often leads to toxic side effects [11,12].We previously documented the development of VT1092M, an oncolytic HSV-1 armed with IL-12, showing a significant antitumor effects and well-tolerated safety profile [13]. In the present study, we evaluated the antitumor efficacy of VT1092M combined with an anti-PD-L1 antibody in a bilateral CT26 syngeneic tumor mouse model. Our findings revealed that intratumoral injections of VT1092M and the anti-PD-L1 antibody promoted the regression of both primary and untreated abscopal tumors, reduced tumor recurrence, and prolonged survival by inducing systemic antitumor immunity. Moreover, this combination therapy had superior efficacy to either therapy alone.

Materials and methods

Cell lines

The mouse colon cancer cell line CT26 and the breast cancer cell line 4T1 (Cat# CBP61189, Cat# CBP60352)were procured from the National Collection of Authenticated Cell Cultures. The CT26-LUC cell line (Cat# NM-S24A-1) was purchased from Shanghai Model Organisms Center, Inc. All cell lines were cultured in RPMI 1640 medium containing 10 % fetal bovine serum (FBS) at 37 °C, 5 % CO₂.

Animal ethics and housing

All animal experimental protocols were approved by the Ethics Committee of Yantai University (IACUC No. 2021-DA-12) and were conducted according to the Care and Use of Laboratory Animals of the National Institutes of Health. Female BALB/c mice (5–6 weeks old) were purchased from Jinan Pengyue Laboratory Animal Breeding Co. Ltd (Animal License Number: SCXK (lu) 2022–0006). All BALB/c mice were housed in specific pathogen-free conditions and were sacrificed after being anesthetized at the end of the experiments.

Virus engineering and amplification

Two viruses were employed in this study. VT09X was engineered by deleting of both copies of γ34.5 genes and the α47 gene from a wild HSV-1 strain isolated in China, HL-1, via homologous recombination. The second virus, VT1092M was generated by fusing mouse IL-12 to the VT09X backbone. Both viruses were amplified in Vero cells, purified according to established protocols, and stored at −80 °C [14].

Bilateral subcutaneous tumor models and therapeutic protocols

Bilateral subcutaneous tumor models were utilized to evaluate systemic antitumor effects. To establish the model, approximately 3 × 10⁶ and 1 × 10⁶ CT26 cells were subcutaneously inoculated into the right (injective tumor) and left (abscopal tumor) flanks, respectively. Upon the tumors reaching 100–130 mm³, the mice were grouped by stratified randomization to receive intratumoral OV injections (administered to the injective tumors in a 50-μL volume), intraperitoneal immune checkpoint antibodies, or both. Tumor dimensions and body weights were monitored every 3 days, whereby Day 1 was defined as the first day of OV injection. Mice were euthanized when a tumor grew to a volume of 2000 mm³. For the combination therapy experiments, 108 mice were divided into the following nine treatment groups: vehicle, single therapy (VT09X, VT1092M, anti-PD-L1, anti-PD1, or anti-TIM3 antibodies), or combination therapy (Anti-PD-L1+VT1092M, Anti-PD1+VT1092M, or Anti-TIM3+VT1092M). Survival was monitored over 34 days.

Efficacy evaluation and immunodepletion assay

The vehicle, VT09X, VT1092M, anti-PD-L1, and Anti-PD-L1+VT1092M groups were subjected to additional evaluation. On Day 5, four mice/group were sacrificed and their interferon (IFN)-γ and IL-12 levels were analyzed by enzyme-linked immunosorbent assay (ELISA). On Day 13, six mice/group were sacrificed for immune cell subset analysis (spleens), immune cell-mediated cytotoxicity assay (peripheral blood), and RNA sequencing (RNA-seq) (tumors). The survival of the remaining six mice/group was monitored until Day 70. An additional bilateral subcutaneous tumor model was established for use in the immunodepletion assay. To this end, the mice received intraperitoneal anti-CD8α (Clone YTS 169.4, Cat# BP0117, BioXCell) or anti-CD20 (Clone 2H7, Cat# BE0276, BioXCell) antibodies to deplete their CD8⁺ T cells or B cells, respectively; control mice were treated with anti-IgG (Clone MPC-11, Cat #BE0276, BioXCell). Successful in vivo depletion was confirmed by flow cytometry. The Anti-PD-L1+VT1092M regimen was subsequently administered to the immunodepleted mice to evaluate its effect on the TME.

Re-Challenge experiment

The established unilateral subcutaneous tumor models were also used in the re-challenge experiments. Mice were euthanized when a tumor reached a volume of 2000 mm³. Day 1 was defined as the first day of OV injection. On Day 35, the surviving tumor-free mice were re-challenged with CT26 and 4T1 cells; their tumor volumes were then monitored for another 30 days. On Day 65, one mouse from the Anti-PD-L1+VT1092M group died of injurious home-cage fighting and one mouse from the VT1092M group was euthanized because its 4T1 tumor volume reached 2000 mm³. On Day 66, the surviving mice were intraperitoneally re-challenged with CT26-LUC cells and monitored every 3 days using an IVIS® Lumina III In Vivo Imaging System (PerkinElmer) following the intraperitoneal injection of 150 mg/kg d-luciferin (Cat# 122,799, PerkinElmer). Mice were anesthetized by inhalation of isoflurane (4 % in oxygen) during Image acquisition.

Flow cytometry for immune cell subset analysis

Mouse spleens were gently homogenized on a cell strainer using a syringe plunger to obtain a single-cell suspension. Subsequently, any erythrocytes were lysed with RBC Lysis Buffer (Cat# 00–4300, Thermo Fisher Scientific). Following centrifugation, the pellet was resuspended in phosphate-buffered saline (PBS) for flow cytometric analysis. The cells were stained using two panels. Panel 1 contained the following reagents: anti-mCD16/32 antibody (Cat# 156,604, BioLegend), Fixable Viability Dye (Cat# 65–0866–18, eBioscience), BV605-anti-mCD45 antibody (Cat# 103,140, BioLegend), PerCP/Cy5.5-anti-mCD3 antibody (Cat# 100,328, BioLegend), APC/Cy7-anti-mCD4 antibody (Cat# 100,414, BioLegend), FITC-anti-mCD8 antibody (Cat# 100,706, BioLegend), BV421-anti-mCD44 (Cat# 103,040, BioLegend), and PE-anti-mCD62L (Cat# 104,407, BioLegend). Panel 2 consisted of an anti-mCD16/32 antibody (Cat# 156,604, BioLegend), Fixable Viability Dye (Cat# 65–0866–18, eBioscience), BV605-anti-mCD45 antibody (Cat# 103,140, BioLegend), PerCP/Cy5.5-anti-mCD3 antibody (Cat# 100,328, BioLegend), FITC-anti-mCD45R/B220 antibody (Cat# 103,205, BioLegend), and APC-anti-mCD335 (NKp46) antibody (Cat# 137,608, BioLegend). After a 30-minute incubation in the dark, cells were washed and analyzed using a BD FACSCelesta flow cytometer (BD Biosciences) equipped with the 405-nm, 488-nm, and 640-nm excitation lasers.

Tumor homogenization and cytokine analysis

Isolated tumors were cut into small pieces and homogenized in M tubes (Cat# 130–094–392, Miltenyi Biotec) containing 3 mL PBS using a gentleMACS Octo Dissociator with Heaters (Cat# 130–096–427, Miltenyi Biotec). The samples were centrifuged at 3000 rpm for 10 min at 4 °C. The resulting supernatant was transferred to an Amicon® Ultra 10 K device-10,000 MWCO (Cat# UFC901024, Millipore) and spun at 12,000 × g for 20 min using a fixed-angle rotor. The final supernatant was analyzed by ELISA using ELISA kits specific for IL-12 (Cat# SEKM-0013, Solarbio) and IFN-γ (Cat# SEKM-0031, Solarbio).

Isolation of CD45⁺ cells from mouse blood and the cytotoxicity assay

This assay was performed as previously described [15]. Briefly, mouse blood was collected following eyeball removal. Red blood cells were then lysed with RBC Lysis Buffer. Dead cells were further removed using the Dead Cell Removal Kit (Cat# 130–090–101, Miltenyi Biotec). After passing through a 70-μm nylon filter, a single-cell suspension was obtained. The CD45⁺ cells were positively selected by magnetically labelling the collected cells with CD45 MicroBeads (Cat# 30–052–301, Miltenyi Biotec). The labeled cell suspension was then loaded onto an LS Column (Cat# 130–042–401, Miltenyi Biotec) and placed in the magnetic field of a QuadroMACS™ Separator (Cat# 130–090–976, Miltenyi Biotec). The magnetically labeled CD45⁺cells were eluted from the column using an appropriate amount of buffer. In the cytotoxicity assay, the CT26 and 4T1 cell lines were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) dye (Cat# C34554, Thermo Fisher Scientific) for 20 min. The labeled CT26 or 4T1 cells were then incubated with the isolated CD45⁺ cells at effector-to-target (E:T) ratios of 20:1 and 40:1 for 4 h in an incubator. Cells were stained with propidium iodide (PI) dye before being analyzed on a flow cytometer. The cytotoxicity assay allowed for the assessment of the cytotoxic activity of the isolated CD45⁺ cells against CT26 and 4T1 target cells, providing valuable insights into the immune response dynamics.

RNA-Seq analysis of tumor samples

Three mice were randomly selected for RNA-seq. Both the injective and abscopal tumors were collected and frozen in liquid nitrogen, before being sent to Anzenda Biotechnology Co., LTD for RNA-seq. Because insufficient amounts of tumor tissue were collected from some of the mice, all of the injective and abscopal tumors from combined therapy group were mixed together to ensure sufficient material for analysis. In addition, one tiny injective tumor from VT1092M group was removed from the test samples. The mRNA libraries, each with different indices, were multiplexed and loaded onto an Illumina HiSeq/Illumina Novaseq/MGI2000 instrument for sequencing. The sequencing data underwent filtering with Cutadapt, and the clean data were aligned to the reference genome using the Hisat2 software (v2.0.1). Differential expression analysis was performed using DESeq2, with criteria set at a fold change > 2 and a Q value ≤ 0.05. Gene Ontology (GO) term enrichment was identified using GOSeq (v1.34.1). Moreover, in-house scripts were employed to determine which differentially expressed genes were significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The RNA-seq analysis aimed to elucidate the transcriptional landscape and identify key molecular pathways associated with the observed treatment effects, providing valuable insights into the underlying mechanisms of the antitumor immune responses induced by the different therapies.

Statistical analysis

Data were presented as the mean ± standard deviation (SD) and evaluated using analysis of variance (ANOVA), followed by Dunnett's test for multiple comparisons. Kaplan-Meier analysis was employed to assess the significance of survival rate differences between groups. Statistical analysis was conducted using SPSS 22.0 software, and a P-value < 0.05 was considered as a measure of statistical significance.

Results

Evaluation of combination therapies on bilateral subcutaneous tumors

We assessed the antitumor activity of VT1092M combined with anti-PD-L1, anti-PD1, or anti-TIM3 antibodies in immunocompetent BALB/c mice with bilateral subcutaneous tumors (Fig. 1A). Animals with bilateral tumors received OV injections in the injective tumors only; tumor progression on both flanks was then monitored (Fig. 1C, D).

Fig. 1.

The antitumor efficacy of VT1092M was assessed in conjunction with antibodies targeting anti-PD-L1, anti-PD1, or anti-TIM3 in bilateral colon tumor mouse models. (A) Mice bearing CT26 subcutaneous tumors were treated with diverse therapeutic agents as outlined in the treatment scheme. (B) The Kaplan-Meier survival curves of tumor-bearing mice were examined to evaluate the impact of different therapies on overall survival. (C) Assessment of the mean tumor volumes (of both injective and abscopal tumors) and body weights of mice receiving different forms of treatment. (D) Individual tumor growth curves were generated, depicting tumor diameters and body weight measurements taken every 3 days. Oncolytic viruses (OVs) were injected into the injective tumors in a volume of 50 μL. Mice were euthanized upon the tumor reaching a volume of 2000 mm³. Day 1 was defined as the day of OV injection. Error bars denote standard deviation; ns, no significant differences; *p < 0.05; **p < 0.01; ***p < 0.001.

The Anti-PD-L1+VT1092M regimen demonstrated superior antitumor effects to the Anti-PD-L1 and VT09X monotherapies in reducing the size of the injective tumors; however, the combination treatment was not significantly more effective than the VT1092M monotherapy. The Anti-PD1+VT1092M regimen exhibited superior antitumor efficacy to the Anti-PD1 group, but not the VT09X or VT1092M groups. Similarly, the Anti-TIM3+VT1092M group had a superior antitumor response to the Anti-TIM3 group, but not the VT09X and VT1092M groups.

We next evaluated the abscopal tumors and found that the Anti-PD-L1+VT1092M regimen displayed superior antitumor effects to the Anti-PD-L1, VT09X, and VT1092 monotherapies. Neither the Anti-PD1+VT1092M group nor the Anti-TIM3+VT1092M group exhibited superior antitumor effects to their respective monotherapy groups. Notably, the Anti-PD-L1+VT1092M combination significantly prolonged the survival of mice relative to the use of anti-PD1 antibody alone; however, it was not significantly superior to the antitumor efficacy of VT1092M monotherapy (Fig. 1B). None of the other treatment combinations significantly improved the survival of mice relative to their respective monotherapies (Fig. 1B). Throughout the experiment, no changes in body weight were observed among the groups (Fig. 1C). These results suggest that the combination of the anti-PD-L1 antibody and VT1092M was more effective at reducing both injected and untreated tumor burden than the other two combination therapies.

Enhanced systemic antitumor immunity following Anti-PD-L1+VT1092M treatment of injective tumors

To further elucidate the impact of VT1092M in conjunction with the anti-PD-L1 antibody on both the injective and abscopal tumors, a bilateral subcutaneous tumor model was established (Fig. 2A). Consistent with previous findings, the administration of Anti-PD-L1+VT1092M significantly reduced the volumes of both injective and untreated abscopal tumors when compared with the use of Anti-PD-L1, VT09X, or vehicle alone; however, this reduction in tumor volume was not significantly greater than that of the VT1092M group (Fig. 2B, D, Fig. S1).

Fig. 2.

Superior antitumor efficacy was observed in the model mice receiving a combination of anti-PD-L1 antibody and VT1092M. (A) Treatment scheme detailing how the combination therapy was administered to mice bearing CT26 subcutaneous tumors. (B) Endpoint tumor images from the injective and abscopal sites. (C) Kaplan-Meier survival curves for the tumor-bearing mice. (D) Individual growth curves for injective and abscopal tumors, with measurements taken every 3 days. Oncolytic viruses (OVs) were administered to injective tumors in a 50 μL volume. Mice were euthanized upon the tumor reaching a volume of 2000 mm³. The initiation of OV injection was considered as Day 1. “Tumor-free” denotes complete tumor remission. Error bars indicate standard deviation; ns, no significant differences; *p < 0.05; **p < 0.01; ***p < 0.001.

Next, the overall survival (OS) of six mice per group was assessed in parallel with the main experiment over a period of 70 days. We found that all the mice in the Anti-PD-L1+VT1092M group were free of injective tumors and achieved complete remission (CR), while 4/6 and 2/6 of mice in the VT1092M and Anti-PD-L1 groups achieved CR, respectively (Fig. 2C, D). All the mice in the combination group survived until the end of the experiment; 5/6 of the mice were free of their untreated abscopal tumors while one mouse experiencing tumor recurrence. Two mice achieved CR in both the VT1092M and Anti-PD-L1 groups. Notably, the four deceased mice treated with VT1092M had longer survival times than the mice from the Anti-PD-L1 group. Meanwhile, all the mice from the vehicle and VT09X groups perished before Day 28. These results suggest that the combination treatment regimen significantly improved the antitumor efficacy of each monotherapy.

Additionally, a comprehensive analysis of systemic immune cell subsets was conducted to evaluate the underlying systemic antitumor immunity. Immune cell subset analysis of splenocytes harvested from the different mouse groups revealed that the Anti-PD-L1+VT1092M combination significantly increased the proportions of CD3⁺, CD4⁺, and CD8⁺ T cells compared with either monotherapy alone, although the regimen had no significant effect on the numbers of natural killer (NK) cells (Fig. 3A–C). Subsequent analysis of CD8⁺ T cell subsets demonstrated a significant increase in both central memory (CD62L⁺CD44⁺) and effector memory (CD62L⁻CD44⁺) subpopulations. Additionally, ELISA evaluation of injective tumors revealed that the Anti-PD-L1+VT1092M combination significantly increased the secretion of IL-12 and IFN-γ, compared with the vehicle, VT09X, or anti-PD-L1 antibody; however, no significant differences in these cytokine levels were noted relative to the VT1092M group (Fig. 3D). Unfortunately, the abscopal tumors were too small to be subjected to ELISA evaluation.

Fig. 3.

Evaluation of immune cell subsets and cytokines modulated by the various therapeutic agents. (A) Representative flow cytometry dot plots showing the gating strategy for identifying different immune cell subsets in mouse spleens. (B) Bar graphs showing the proportions of various lymphocyte subsets isolated from the spleens of mice treated with different agents. (C) Representative flow cytometry dot plots (left) and bar graph (right) showing how the different therapeutic agents affected NK cell numbers in the mouse spleens. (D) ELISA evaluation of the levels of two key cytokines in the injective tumors. Error bars represent standard deviation; ns, no significant differences; *p < 0.05; **p < 0.01; ***p < 0.001.

In summary, these results suggest that tumor regression was associated with an increase in the numbers of cytotoxic T lymphocytes and the levels of IFN-γ and IL-12. The Anti-PD-L1+VT1092M regimen demonstrated greater efficacy in mediating the regression of distal untreated tumors than VT1092M or Anti-PD-L1 alone, indicating that the combination therapy induced potent circulating antitumor immunity.

Combination treatment establishes long-term tumor-specific immunological memory and systemic antitumor immunity

Next, we used a cytotoxicity assay and tumor re-challenge experiments to assess the ability of the different therapeutic agents to induce antitumor immunity. CFSE, known for its spontaneous and irreversible binding to intracellular proteins, was used to track cells and evaluate their proliferation. To determine tumor specificity for the colon cancer cell line CT26, a breast cancer cell line, 4T1, was included in the cytotoxicity assays. Both the CT26 and 4T1 cell lines were initially stained with CFSE and then co-incubated with CD45⁺ cells isolated from the blood of tumor-bearing mice at E:T ratios of 20:1 or 40:1 for 4 h (Fig. 4A). PI was used for dead cell staining. We first confirmed that CD45⁺ cells from the different treatment groups exhibited minimal killing of 4T1 cells, and the extent of cytotoxicity did not correlate with increasing E:T ratios. Consistent with the earlier CD8⁺ T cell subset analysis, CD45⁺ cells from mice treated with VT1092M, either alone or in combination, displayed a higher degree of CT26 target cell killing (CFSE⁺PI⁺) than those isolated from mice treated with the other agents. In accordance, the extent of target cell killing increased as the E:T ratio rose from 10:1 to 40:1. Overall, we found that VT1092M treatment, either alone or in combination, established tumor-specific immunological memory.

Fig. 4.

The combination therapy established long-term tumor-specific immunological memory in the tumor re-challenged model mice. (A) Representative flow cytometry dot plots (left) and corresponding bar graphs (right) showing the cytotoxic activity of CD45⁺cells isolated from the peripheral blood of mice treated with the different agents. CD45⁺ cells were isolated, stained with CSFE, and incubated with CT26 or 4T1 cells at E:T ratios of 20:1 and 40:1 for 4 h; the extent of tumor cell killing was then evaluated by flow cytometry. (B) Re-challenge experimental scheme. (C) Tumor volumes of previously cured mice which were subcutaneously re-challenged with CT26 or 4T1 cells. (D) Representative bioluminescent images of CT26-LUC ascites model mice subjected to the indicated treatments. (E) Quantification of CT26-LUC bioluminescence in mice 5 min after the intraperitoneal injection of d-luciferin. Error bars represent standard deviation; ns, no significant differences; *p < 0.05; **p < 0.01; ***p < 0.001.

To further assess the ability to establish tumor-specific immunological memory in vivo, 5/6 and 4/6 tumor-free mice from the Anti-PD-L1+VT1092M and VT1092M groups, respectively, were subcutaneously re-challenged with CT26 and 4T1 cells, which were injected simultaneously but separately into each flank (Fig. 4B, C). All the age-matched treatment-naïve mice exhibited CT26 and 4T1 tumor progression. At the end of the first re-challenge experiment (Day 64), one mouse in the VT1092M group exhibited CT26 tumor recurrence, while all the mice in the Anti-PD-L1+VT1092M group were CT26-tumor-free; all the mice exhibited 4T1 tumor progression.

On Day 66, the mice that survived the first re-challenge experiment (4/5 from the Anti-PD-L1+VT1092M group and 3/4 from the VT1092M group) were intraperitoneally re-challenged twice with CT26-LUC cells. At the study endpoint, all age-matched treatment-naïve mice developed cancerous ascites in the peritoneum, while all previously CT26-tumor-free mice did not develop ascites (Fig. 4D, E). These data indicate that the combination of Anti-PD-L1+VT1092M was a more effective inducer of long-term tumor-specific immunological memory and systemic antitumor immunity than either monotherapy.

RNA-Seq analysis of differentially expressed genes in the various treatment groups

Tumors were next subjected to RNA-seq to delve deeper into the mechanisms underlying the induction of systemic immunity and the regression of the untreated abscopal tumors in the mice. The differential gene expression analysis revealed that a number of genes were upregulated or downregulated in the injective and abscopal tumors within each treatment group (Fig. 5A, B).

Fig. 5.

RNA sequencing analysis was conducted to examine gene expression in both injective and abscopal tumors under different treatment conditions. (A) Heatmap showing the expression of genes implicated in immune-related pathways, including cytokine-cytokine receptor interaction, hematopoietic cell lineage, antigen processing and presentation, cell apoptosis, and cell adhesion molecules. (B) Identification of differentially expressed genes between the injective and abscopal tumors of each treatment group. (C) The expression levels of specific representative genes in the different treatment groups. Error bars represent standard deviation. A, abscopal tumor; I, injective tumor.

We found that numerous genes were upregulated in the injective tumors of the Anti-PD-L1 and VT09X groups relative to those of the combination group, while only 303 distinct genes were differentially upregulated between the injective tumors of theVT1092M and combination groups. Further analysis of the differentially expressed genes highlighted the significance of VT1092M-mediated IL-12 secretion in the injective tumors. Conversely, thousands of differentially expressed genes were identified in the abscopal tumors of the VT1092M, VT09X, and Anti-PD-L1 groups relative those of the combination group, indicating the effective and synergistic induction of systemic antitumor immunity by VT1092M and the anti-PD-L1 antibody.

Moreover, the limited number of differentially expressed genes between the injective and abscopal tumors in the groups treated with Vehicle, VT09X, or anti-PD-L1, which all had a weak therapeutic effect on the injective tumors, resulted in poor inhibition of abscopal tumor growth, aligning with previous studies. By contrast, the potent antitumor efficacy observed in injective tumors in both the VT1092M and Anti-PD-L1+VT1092M groups induced an effective immune response against abscopal tumors.

KEGG pathway enrichment analysis highlighted cytokine-cytokine receptor interaction, hematopoietic cell lineage, antigen processing and presentation, cell apoptosis, and cell adhesion molecules as the most significant pathways associated with the differentially expressed genes induced by Anti-PD-L1+VT1092M administration. The combination treatment induced the robust secretion of cytokines, as evidenced by the upregulation of genes belonging to the IL superfamily (e.g., Ifng, Il10rb, Il10ra, Il15ra, Il2rb), chemokines (e.g., Cxcl9, Ccl8, Cxcl16), and the tumor necrosis factor (TNF) superfamily (e.g., Tnfsf10, Tnfrsf1b) [10,16,17]. Hematopoietic cell lineage-associated genes such as Cd4, Cd3e, Cd8a, and Cd86 were significantly upregulated, indicating effective immune cell infiltration into the tumors. Additionally, the expression of genes with crucial roles in major histocompatibility complex class II (MHCII)-mediated antigen processing and presentation (e.g., H2-DMb2, H2-DMa, H2-Eb1, H2-Ab1) was increased following Anti-PD-L1+VT1092M treatment [18,19]. Fig. 5C shows the differentially expressed genes enriched in the various KEGG pathways associated with each treatment type.

In summary, the aforementioned alterations in the injective and abscopal tumors suggest that the synergy between VT1092M and anti-PD-L1 remodels the TME and triggers systemic antitumor activity, ultimately mediating the regression of the untreated abscopal tumors.

CD8⁺T cells as mediators of antitumor immunity induced by Anti-PD-L1+VT1092M therapy

To elucidate the contribution of CD8⁺ T cells and B cells to tumor clearance following VT1092M and anti-PD-L1 antibody administration, both lymphocyte types were selectively depleted through the intraperitoneal injection of anti-CD8α or anti-CD20 antibodies, respectively (Fig. 6A). In these experiments, an anti-IgG antibody served as an isotype control. Depleting CD8⁺ T cells significantly reduced their numbers in the spleen, resulting in higher levels of tumor growth than observed in anti-IgG-treated mice (Fig. 6B–E). When these CD8⁺-T-cell-depleted mice were treated with a combination of anti-PD-L1 antibody and VT1092M, however, their immune cell counts were slightly restored, resulting in a moderate inhibition of injected and abscopal tumor growth relative to the Anti-PD-L1+VT1092M group. Notably, CD8⁺ depletion did not significantly affect the NK and B cell subsets.

Fig. 6.

The antitumor efficacy of the VT1092M and anti-PD-L1 antibody combination therapy was evaluated in CD8⁺T cell or B cell depletion mouse models. (A) Treatment scheme illustrating how the combination therapy was administered to mice bearing subcutaneous (s.c.) CT26 tumors following CD8⁺T cell or B cell depletion. (B) Mean volumes of the injective and abscopal tumors and the mouse corresponding body weights. (C) Images of the injective and abscopal tumors at the experiment's endpoint. (D) Flow cytometric confirmation of CD8⁺T cell and B cell depletion in the mouse spleens. (E) Flow cytometric quantification of the proportions of CD3⁺T, CD8⁺T, NK, and B cells in the mouse spleens. The tumor diameters and body weights were measured every 3 days. Oncolytic viruses (OVs) were administered to the injective tumors in a volume of 50 μL, with the first OV injection defined as Day 1. Error bars represent standard deviation; ns, no significant differences; *p < 0.05; **p < 0.01; ***p < 0.001.

Anti-CD20 antibody treatment markedly reduced B cell numbers relative to the anti-IgG antibody treatment (Fig. 6D). When the Anti-PD-L1+VT1092M combination was administered to these B-cell-depleted mice, their B cell numbers were partially restored relative to the Anti-PD-L1+VT1092M group which were not treated with the anti-CD20 antibody. Surprisingly, mice in the Anti-CD20+ Anti-PD-L1+VT1092M group were able to effectively clear both tumors, just like the Anti-PD-L1+VT1092M group. Additionally, B cell depletion slightly reduced the frequencies of total, and specifically, CD8⁺ T cells, possibly due to diminished antigen presentation by B cells [17,18]. B cell depletion did not have an obvious impact on NK cell numbers.

In summary, the depletion of CD8⁺ T cells partially attenuated the antitumor efficacy of the Anti-PD-L1+VT1092M combination therapy, while the depletion of B cells had no discernible impact. This suggests that CD8⁺ T cells, rather than B cells, were the key mediators of tumor regression during Anti-PD-L1+VT1092M combination therapy.

Discussion

This study investigated the potential of OVs, specifically VT1092M, to increase the antitumor efficacy of immune checkpoint blockade. Recent drug discovery efforts have highlighted the promising therapeutic potential of OV and ICI combinations [20,21]. The feasibility of using T-VEC, an OV, in combination with ICIs such as ipilimumab (an anti-CTLA-4 antibody) and pembrolizumab (an anti-PD-1 antibody) has been evaluated in clinical trials; for instance, the combination of ipilimumab and T-VEC proved more effective than ipilimumab alone in melanoma patients [22,23]. However, in a Phase Ib/III and pivotal Phase III study, the combination of T-VEC and pembrolizumab did not significantly improve PFS or OS compared with the placebo and pembrolizumab combination in patients with advanced melanoma [24]. Thus, new combination strategies are needed to improve the efficacy of currently available therapies without exacerbating their toxicity.

VT1092M was designed to deliver IL-12 locally to tumors, thus, avoiding systemic toxicity [[25], [26], [27]]. The objective of the study was to evaluate the antitumor response elicited by VT1092M, particularly in combination with various ICIs targeting PD-1, PD-L1, and TIM3, and delineate the underlying mechanisms.

We used the bilateral subcutaneous tumor model to show that the combination of anti-PD-L1 antibody and VT1092M demonstrated superior efficacy to other combination therapies at reducing both the injected and untreated tumor burden. As a result, the Anti-PD-L1+VT1092M combination was subsequently selected for further investigation.

The development of antibodies targeting PD-1 or PD-L1 has significantly advanced cancer immunotherapy. Both the anti-PD-1 and anti-PD-L1 antibodies have demonstrated efficacy in treating various cancers. However, a systematic review and meta-analysis of the literature suggested that anti-PD-1 antibodies may be superior to anti-PD-L1 antibodies in prolonging the OS and progression-free survival (PFS) of cancer patients [28,29]. However, the present study reported that combining VT1092M with an anti-PD-L1 antibody rather than an anti-PD-1 antibody led to better outcomes. A potential reason for this difference may be that VT1092M infection increases PD-L1 expression on tumor cells [[30], [31], [32]]. Additionally, differences in the half maximal effective concentration (EC50) values of the anti-PD-1 and anti-PD-L1 antibodies may impact their antitumor efficacy. Given that the mechanisms underlying these findings are unclear, we acknowledge that further investigation is required. These mechanistic insights will enable the optimization of combination therapies involving OVs and ICIs for more effective cancer treatment.

Our data revealed that VT1092M alone or in combination with an anti-PD-L1 antibody induced the regression of both the injected and untreated distant tumors. However, the combination therapy was associated with a higher rate of tumor regression, a lower rate of tumor recurrence, reduced tumor progression, prolonged survival, and a more potent antitumor immune response than VT1092M alone. Mechanistically, the superior antitumor response of the combination therapy may be attributed to the induction of circulating tumor-antigen-specific cytotoxic CD8⁺ T cells or the increased transfer of cytokines from the injection site to distal tumors [21,33,34].

Our flow cytometric analysis showed a significant increase in the frequencies of various T cell subsets (e.g., CD3⁺, CD4⁺, central memory and effector memory CD8⁺ T cells) in the combination group relative to the monotherapy groups. Moreover, IL-12 and IFN-γ levels were increased in the injective tumors following combination therapy administration; it is possible that these elevated cytokine concentrations contributed to the clearance of distal tumors.

Meanwhile, our RNA-seq results revealed that the combination treatment induced profound changes in the expression of genes associated with cytokine-mediated systemic antitumor immunity. The KEGG analysis further demonstrated that these genes were enriched in pathways related to cytokine-cytokine receptor interaction, hematopoietic cell lineage, and antigen processing and presentation.

Moreover, we demonstrated that the combination of anti-PD-L1 antibody and VT1092M induced potent antitumor immunity, as evidenced by specific target cell killing in vitro and a reduction in the rate of tumor progression in vivo upon re-challenge. Depletion of CD8⁺ T cells markedly reduced the therapeutic efficacy of the anti-PD-L1 antibody and VT1092M combination, highlighting their essential role in the antitumor response during OV and ICI therapy. We also found that B cells, traditionally considered as the central mediators of humoral immunity [[35], [36], [37]], did not significantly contribute to the antitumor activity of the combination therapy.

Conclusions

In conclusion, our study revealed that VT1092M synergizes with PD-L1 blockade, leading to long-term and potent antitumor immunity. These finding will have potential implications for advancing cancer treatment and preventing metastatic spread.

Data availability

Data will be made available on request.

CRediT authorship contribution statement

Wei Zhu: Writing – review & editing, Writing – original draft, Visualization, Supervision, Project administration, Methodology, Investigation, Formal analysis, Conceptualization. Mingxia Shao: Visualization, Methodology, Investigation. Chao Tian: Methodology, Investigation. Jianshuai Yang: Methodology, Investigation. Hua Zhou: Methodology, Investigation. Jiajia Liu: Methodology, Investigation. Chunyang Sun: Methodology, Investigation. Min Liu: Methodology, Investigation. Jinyu Wang: Methodology, Investigation. Lijun Wei: Methodology, Investigation. Shuzhen Li: Methodology, Investigation. Xiaopeng Li: Supervision, Project administration, Funding acquisition, Conceptualization. Jingfeng Li: Writing – review & editing, Supervision, Project administration, Methodology, Investigation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.