Recombinant Orthopoxvirus Primes Colon Cancer for Checkpoint Inhibitor and Cross-Primes T Cells for Antitumor and Antiviral Immunity

Colon cancer is the third most common cancer in the United States, with approximately 145,000 new cases diagnosed each year, and more than 50,000 deaths in 2019 (1). Although surgery with or without chemotherapy can be curative at early stages, almost 50% of patients will ultimately develop liver metastases (2). Surgery is curative for up to 25% of resected patients with colorectal liver metastases (CRLM) at 10 years (3); however, only a minority of patients are suitable for curative resection and an even smaller proportion of eligible patients get referred for resection (4). Systemic chemotherapies can effectively prolong life for patients with unresectable metastatic disease, but they will ultimately succumb to cancer (5). Thus, there is an urgent need for novel treatment paradigms to eradicate metastatic colorectal cancer and induce durable antitumor immunity for improved patient outcomes.

Oncolytic viruses (OV) selectively infect, replicate in, and kill cancer cells. They are either naturally tumor tropic or are engineered to be tumor tropic, and can be highly immunogenic across multiple OV families and cancer types (6–8). Poxviruses show potent immunogenicity stemming from both standard antiviral responses, as would be seen with any viral infection, and from the immunogenic death of infected cancer cells (8). The current understanding in the field suggests that viral infection results in tumor cell lysis, which in turn releases tumor- and virus-associated antigens to spark antitumor immunity (8). This ultimately results in a cross-primed memory response against tumor antigens and virally infected cancer cells (9).

We and others have shown that OVs can activate T cells against tumor cells (10–16). However, many believe that T cell activation after OV infection is predominantly antiviral and only partly antitumoral (17). Some groups are exploiting this by arming OVs with large swaths of tumor antigens (17–19), whereas others are designing highly replication-competent viruses that can kill cancer cells more efficiently, thereby releasing large quantities of tumor antigens within the tumor microenvironment (TME; ref. 20). We have adopted the latter approach by creating a highly efficient recombinant orthopoxvirus CF33, from the coinfection of nine different orthopoxviruses as previously described (10, 21–24). We have also shown that CF33 is robustly immunogenic and efficacious against colon cancer xenografts (22, 25), and demonstrated its antitumor immune efficacy against triple-negative breast cancer (10). The work herein establishes CF33 derivatives as potent T cell activators in colon cancer models.

Although the field of OV therapy is rapidly gaining momentum, there are two particularly exciting ideas building on viruses as immune activators. First, OVs are robust agents of TME modulation, capable of recruiting and activating immune cells in otherwise immunologically “cold” TMEs devoid of tumor-specific immunity (26). This makes OVs excellent primers for T cell–based immunotherapies like immune-checkpoint inhibitors (ICI; refs. 10, 11, 27). Second, a very recent article by Rosato and colleagues elegantly shows that restimulating known antiviral immunity can trigger antigen presentation and cytotoxic pathways within infected tumors, which in turn can activate dendritic and NK cells to induce adaptive immunity (28). Thus, it is likely that antiviral immunity can both induce and enhance antitumor immunity (9, 29).

In this study, we tested if the antitumor efficacy of CF33-derivatives against colon cancer could be enhanced by immune-checkpoint inhibition (anti–PD-L1). We further characterized the nature of T cell activation following CF33-induced tumor regression. We show that viral infection activates T cells against tumor cells and, in combination with checkpoint inhibition, confers durable antitumor response. Overall, these data suggest that CF33 derivatives in combination with anti–PD-L1 can induce durable antitumor immunity, paving the way for strategies to enhance immune responses to viral therapy.

The chimerization, cloning, competitive selection, and sequence of CF33 backbone virus have been described previously (10, 21–23). Insertion of the hNIS expression cassette or firefly luciferase under the control of the vaccinia H5 synthetic early (SE) promoter at the J2R locus has also been described (22, 25), as has the deletion of the F14.5L gene (10) and insertion of the anti–PD-L1 transgene at the F14.5L locus (30).

HCT116 (RRID:CVCL_0291), HT-29 (RRID:CVCL_0320), and African green monkey kidney fibroblasts—CV-1 (RRID:CVCL_0229) cell lines were purchased from the ATCC. All human colorectal cell lines were maintained in McCoy's 5A medium (Gibco), and CV-1 cells were maintained in Dulbecco's modified Eagle's medium (Corning). Pan02-GFP mouse pancreatic cancer cells were purchased from AntiCancer Inc. and cultured in RPMI-1640 medium (Corning). MC38 and MC38-Luc cells were a kind gift from Dr. Laleh Melstrom's laboratory (City of Hope, Duarte, CA). MC38 and MC38-Luc cells were maintained in DMEM. All cells were supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic–antimycotic solution, both purchased from Corning. The cells were maintained in a humidified incubator at 37°C and 5% CO₂. Cell lines were purchased from ATCC and AntiCancer Inc; efforts were made not to perform experiments past 15 passages. All cell lines were tested for Mycoplasma before each experiment initiation. Mice were maintained in a biosafety containment level 2 facility within our vivarium where the environment was temperature and light controlled with 12-hour light and 12-hour dark cycles, and food and water were ingested ad libitum. All animal experiments were performed with approval of the City of Hope Institutional Animal Care and Use Committee. C57Bl/6J mice ages 8 to 12 weeks old were used for most experiments (Jackson Laboratories and Charles River, RRID:IMSR_JAX:000664, RRID:IMSR_CRL:027). Six-week-old Hsd:Athymic Nude-Foxn1nu female mice (Envigo) were purchased and acclimatized for 7 days.

HCT-116, HT-29, and MC38-Luc cells were seeded in 6-well plates (Corning) at 5 × 10⁵ cells/well and incubated. The following day, cells were counted and infected at an MOI of 0.01 plaque forming unit (PFU) per cell with CF33-hNIS or CF33-hNIS-ΔF14.5 in a total volume of 0.5 mL medium containing 2.5% FBS. After 1 hour, the inoculum was aspirated and 2 mL of fresh medium was added to each well, and plates were returned to the incubator. At the indicated times, cell lysates were collected by scraping and virus titers were determined by standard plaque assay as described previously (10, 25).

Cells were seeded in 6-well plates at 5 × 10⁵ cells/well and incubated overnight. The following day, cells were infected at an MOI of 5 with CF33-hNIS-ΔF14.5 and cultured for 24 hours. Total RNA was extracted using the RNeasy Mini kit (Qiagen). Next, cDNA was prepared from the total RNA using the QuantiTect Reverse Transcription Kit (Qiagen). GAPDH was used as an internal control. Levels of PD-L1 mRNA were assessed using the QuantiTect SYBR Green PCR Kit (Qiagen). All primers (Hs_CD274_1_SG QuantiTect Primer, Mm_Pdcd1lg1_1_SG QuantiTect Primer, Hs_GAPDH_1_SG QuantiTect Primer, and Mm_Gapdh_3_SG QuantiTect Primer) were purchased from Qiagen. Real-time PCR was performed on an AB-QuantStudio 3 system (Thermo Fisher Scientific). Relative gene-expression levels were analyzed by the 2^−ΔΔCt method.

Tumors harvested at the time of sacrifice were fixed with 10% formalin. Subsequently, tumors were paraffin-embedded and 5-μm thick sections were obtained. The slides were deparaffinized followed by heat-mediated antigen-retrieval per manufacturer's protocol (IHC World). Tumor sections were then permeabilized with methanol and blocked for 30 minutes with TNB blocking buffer (PerkinElmer). Tumor sections were washed and incubated overnight in a humidified chamber at 4°C with a rabbit anti-vaccinia virus antibody (Abcam, RRID:AB_778768) 1:100 in TNB blocking buffer. The next day, tumor sections were stained with Alexa Fluor-488-conjugated goat anti-rabbit (Abcam, RRID:AB_2630356) for 1 hour at room temperature. Finally, the sections were counterstained with 4′6-diamidino-2-phenylindole (DAPI). IHC for CD8 and granzyme B was performed by the Pathology Core at City of Hope. Images were obtained using the Nanozoomer 2.0HT digital slide scanner (Hamamatsu Photonics) or Ventana iScan HT (Roche).

Single cells were generated from tumors of mice using mouse Tumor Dissociation Kit utilizing gentleMACS dissociator (Miltenyi Biotec). Cells were washed with PBS and stained for different extracellular and intracellular targets. Before staining for targets, cells were stained with LIVE/DEAD Fixable dye (Invitrogen) in PBS for 30 minutes at 4°C in dark. Next, Fc receptors on the cells were blocked using an anti-CD16/32 antibody (BD Biosciences, RRID: AB_394657 in FACS buffer (PBS containing 2% FBS) for 10 minutes and then stained for extracellular targets for 30 minutes at 4°C in the dark using the following antibodies: mouse CD3- fluorescein isothiocyanate (FITC; eBioscience, RRID:AB_2572431), mouse CD11b- allophycocyanin (APC; BioLegend, RRID:AB_389327), mouse PD-L1–phycoerythrin (PE; BioLegend, RRID:AB_2073557), mouse CD45–phycoerythrin (PE; BD Biosciences, RRID:AB_394611) or peridinin chlorophyll protein complex (PerCP; BioLegend, RRID:AB_893340), mouse CD8- VioGreen (Miltenyi Biotec, RRID:AB_2659495), mouse CD4-APC (BioLegend, RRID:AB_389325), and mouse F4/80-PerCP(BioLegend, RRID:AB_893496). For intracellular (IFNγ) staining, cells were stained with extracellular markers and then permeabilized using the BD Cytofix/Cytoperm Solution Kit according to the manufacturer's protocol (BD Biosciences). Cells were then incubated with mouse IFNγ-APC (BioLegend, RRID:AB_315404) antibody and incubated for 30 minutes at 4°C in dark. Finally, cells were washed twice, resuspended in FACS buffer, and data were acquired using the MACSQuant Analyzer 10 (Miltenyi Biotec). Data were analyzed using the FlowJo software (v10, TreeStar).

For the athymic nude mouse models, flank tumors were generated by injecting 2 × 10⁶ to 3 × 10⁶ HCT116 or HT-29 cells in a total of 100 μL PBS containing 50% matrigel for each tumor. When the average tumor size approached 150 mm³, mice were divided into experimental groups and treated with 10⁴ PFU of CF33-hNIS-ΔF14.5 in 50 μL PBS by intratumoral injection.

Flank tumors of MC38-Luc were established using 1 × 10⁵ to 5 × 10⁵ cells in matrigel. Tumor measurements and mouse weight were monitored twice weekly using calipers to calculate tumor volume, V (mm³) = (1/2) × A² × B, where A is the shortest and B is the longest measurement. Treatment typically occurred when tumors reached 50 to 100 mm³ (approximately 10 days after cell injection), following which mice were randomized into treatment groups (n = 4–10) such that average tumor volume in each group is similar. In virus with and without anti–PD-L1 experiments, tumors were treated on the same day with intratumoral (IT) virus and intraperitoneal (IP) anti–PD-L1 injection. Medication dosages were 50 μL PBS IT, 1 × 10⁸ pfu of CF33-hNIS-ΔF14.5 in 50 μL IT, 150 to 200 μg mouse anti–PD-L1 (Bio X Cell, RRID:AB_10949073) in 100 μL of PBS IT, or 100 μL of PBS IP. Groups included: (i) PBS IT/PBS IP, (ii) PBS IT/mouse anti–PD-L1 IP (m-aPDL1), (iii) CF33-hNIS-ΔF14.5 IT/PBS IP, and (iv) CF33-hNIS-ΔF14.5 IT/m-aPDL1 IP.

Regression and rechallenge experiments for the double-deleted virus were performed using MC38-Luc cells to create unilateral flank tumors on the right flank of each animal. All tumors measuring greater than 130 mm³ at treatment initiation were excluded. Iterations of this experiment were performed with similar results. Representative tumor measurements and survival curves are shown in Fig. 3.

Mouse splenocytes were obtained from naïve C57BL/6 mice or MC38-Luc tumor-bearing C57BL/6 mice that received indicated treatments. T cells were isolated using the EasySep Mouse T Cell Isolation Kit (STEMCELL Technologies) according to the manufacturer's instructions. Freshly isolated mouse T cells were cultured overnight in RPMI medium containing 10% FBS, 10 ng/mL recombinant murine IL7 (PeproTech), 50 U/mL recombinant human IL2 (Novartis Oncology), and 50 μmol/L 2-Mercaptoethanol (Gibco). Next day, T cells were centrifuged at 300 × g for 10 minutes and resuspended in RPMI medium containing 10% FBS. T cells were counted and used for coculture with MC38-Luc cells.

2 × 10⁴ to 3 × 10⁴ MC38-Luc cells/well were plated in round-bottom 96-well tissue culture plates (Corning). After 2 hours, isolated T cells were added to the wells at a ratio of 1:1 (MC38-Luc cells: T cell) with the addition of varying MOIs of the virus. Plates were incubated for 18 or 48 hours. After culturing, the expression of T cells activation markers was evaluated by flow cytometry using mouse CD45-PerC (BioLegend, RRID:AB_893340), mouse CD8-VioGreen (Miltenyi Biotec, RRID:AB_2659495), mouse CD137-PE (BioLegend, RRID:AB_2287565), and mouse CD25-PeCy7 (BioLegend, RRID:AB_2616762) as described above.

Supernatants were collected from T cells and MC38-Luc cells coculture as described above. For IFNγ ELISA with MHC blockade, 10 μg/mL of anti-mouse MHC class I (H-2; Bio X Cell, RRID:AB_1125537) or anti-mouse MHC Class II (I-A/I-E; Bio X Cell, RRID:AB_10949298) were used to block MHC. IFNγ was quantified using mouse IFNγ ELISA Ready-SET-GO! (eBioscience) following the manufacturer's protocol. Plates were read at 450 nm using a spectrophotometer (Tecan Spark 10M).

Statistical analysis was performed using GraphPad Prism (Version 7.01). Student t test or one-way ANOVA was used to evaluate for statistical significance. P < 0.05 was considered significant. Where present in figures, error bars indicate SD or SEM as defined in legends.

We have constructed a recombinant orthopoxvirus (CF33) and modified it as previously described to carry either a single deletion [J2R gene replaced with human sodium iodide symporter (hNIS) transgene] (21, 25) or a double deletion (deletion of J2R and F14.5L genes) to allow for future transgene insertion at F14.5L locus (Fig. 1A; refs. 10, 30). Other configurations of double deletion have included the addition of an anti–PD-L1 transgene as described previously at the F14.5L locus (30). We hypothesized that there would be minimal to no alteration in viral replication between single- and double-deleted CF33 derivatives in human and murine colon cancer cells. Indeed, we observed no attenuation in viral replication in vitro (Fig. 1B) with a single or double deletion. Similarly, we did not observe significant differences in tumor volumes and the survival of nude mouse models with established human xenografts of colon cancer infected with either the single- or double-deleted virus (Fig. 1C–F). These results indicate that single or double-deleted CF33 derivatives have similar efficacy against colon cancer. In previous publications, we have also shown that double-deleted derivatives bearing deleted F14.5L or anti–PD-L1 expression at F14.5L have similar efficacy (30).

We have equipped the CF33 virus with transgenes and have previously demonstrated immunogenic cell death in colorectal cancer cells in response to CF33-derivative infection (22). To further understand the ability of our virus to enhance the induction of antitumor immunity, we examined PD-L1 expression following viral infection and found that CF33-derivative indeed upregulates PD-L1 mRNA in vitro (Fig. 2A) in human and mouse colon cancer cells.

Building on previous work, wherein we showed increased immune cell infiltration and activation following CF33-derivative infection in syngeneic models of murine breast and lung cancer (10, 21), we hypothesized that similar effects would be observed in the current MC38-Luc model. To investigate this, we injected MC38-Luc tumors in immunocompetent C57Bl/6 mice with a single dose of 1 × 10⁸ PFU of CF33-hNIS-ΔF14.5. Flow-cytometric analysis of single cells from the tumors revealed a trend of higher levels of PD-L1 in virus-treated tumors compared with PBS-treated tumors at day 2 after treatment; however, this difference did not reach statistical significance. The observed increase in PD-L1 levels in virus-treated tumors was abated by day 10 after infection (Fig. 2B and C). Nevertheless, our data show an increased influx of tumor-infiltrating lymphocytes on day 2 and to a lesser extent on day 10 following virus treatment. We observed a concomitant increase in macrophage and T cell infiltration on day 2, and later an increase in CD8⁺ T cells (Fig. 2B–D). On day 2, no difference in IFNγ-positive T cells was seen between the treatment groups (Fig. 2B), but by day 10, significantly more activated CD8⁺ T cells were seen in the TME (Fig. 2C).

The above findings suggest that CF33 derivatives can recruit and activate T cells and may thus serve as an effective primer for immune-checkpoint inhibitors. Based on the optimal timing of anti–PD-L1 administration suggested by the experience of other groups using vaccinia-based vectors (11, 12), we planned a single dose of virus concurrently with systemic anti–PD-L1 (Fig. 3A). We examined mice in four treatment groups, including local CF33-hNIS-ΔF14.5 or systemic anti–PD-L1 alone or in combination, and found that the combination results in optimal and consistent tumor regression (Fig. 3B and D) and long-term survival (Fig. 3C). Mice that experienced complete tumor regression were rechallenged with MC38-Luc cells, which did not show regrowth of tumors except in one mouse (Fig. 4A and B), suggesting that these mice developed antitumor immunity. To further decipher the specificity of the antitumor immunity, we performed a second round of rechallenge studies in these same mice (Fig. 4C). This time, mice that had been cured by the combination treatment were challenged with MC38-Luc and concurrently with different murine cancer cells (Pan02-GFP) in the opposite flank. All but one mouse developed Pan02-GFP tumors and none regrew MC38 tumors (Fig. 4D), indicating that the “cured” mice developed tumor-specific immunity.

C57BL/6 mice that experienced complete tumor regression following treatment with anti–PD-L1 alone, CF33-derivative alone, and CF33 derivative in combination with anti–PD-L1 were euthanized after 45 days following rechallenge along with age-matched non–tumor-bearing mice that never received any treatment. Their spleens were collected and T cells were isolated and cocultured with MC38-Luc under different treatment conditions as shown in Fig. 5. Treatment conditions of cocultured MC38-Luc and isolated T cells included: no treatment, anti–PD-L1 alone, CF33-derivative alone, and combination. Flow-cytometric analyses were performed at 18 hours (Supplementary Fig. S1) and at 48 hours (Fig. 5A) after infection to examine T cell activation signatures. T cell activation signatures (CD25 and CD137 positivity) from spleens of mice cured by any means were uniformly higher compared with T cells from naïve mice under all coculture treatment conditions. Furthermore, activation signatures were consistently higher in mice cured by virus treatment either as a single agent or as combination therapy, compared with those cured with anti–PD-L1 treatment alone in all treatment conditions. In the case of CD25⁺ activation signatures, T cells from animals cured with the virus were significantly more activated in the presence of the virus (Fig. 5A). In the case of CD137⁺ activation signatures, T cells isolated from virus-treated mice were more highly expressed under all treatment conditions (Fig. 5A). In addition to flow-cytometric analyses, we also performed ELISA to quantify IFNγ in the supernatant of T cells cocultured with the target cells (MC38-Luc) at 18 hours (Supplementary Fig. S1) and 48 hours (Fig. 5B) after coculture. Intriguingly, despite the same levels of T cell surface markers of activation among varied coculture treatment conditions, IFNγ levels were markedly higher in the supernatants of T cells that had been previously exposed to the virus in vivo and were again treated with the virus in vitro. This suggests that a robust systemic antiviral response may be necessary to maximally prime a TME to induce systemic antitumor immunity.

To further characterize virus-induced T cell activation, we sought to understand the relevance of MHC in viral antigen presentation to T cell activation in coculture in the treatment groups described above. Again, mice having experienced tumor regression following treatment with virus combined with anti–PD-L1 were rechallenged with MC38-Luc tumors. “Cured” mice demonstrating no tumor regrowth were euthanized after 45 days following rechallenge. T cells isolated from the spleens of these mice were cocultured with MC38-Luc cells as described above, this time with blockade of either MHC-I or MHC-II. ELISA of coculture supernatant showed that T cells in the presence of the MHC-I–blocked MC38-Luc cells demonstrated substantially less IFNγ production than their un-blocked counterparts in response to viral infection (Fig. 5C). MHC-II blockade did not appear to affect IFNγ production. Additional support for the idea that T cell activation from “cured” mice is MHC-I dependent was found after IHC analysis at 10 days after infection with a single-deleted CF33 derivative. We observed that CD8⁺ T cells colocalize with replicating virus (Fig. 5E). Areas of active viral replication and CD8⁺ T cell predominance were also observed to stain positive for granzyme B (Fig. 5E). Given that granzyme B is produced by cytotoxic T cells responding to foreign antigens presented through MHC-I molecules, these findings further support the idea that T cells found in spleens of “cured” mice are activated in an antigen-specific manner against tumor cells and are possibly more strongly activated against viral antigens. Taken together, these data demonstrate that CF33-hNIS and CF33-hNIS-ΔF14.5 potently activate tumor-specific immunity in the MC38 colon cancer model. Additionally, the strong antiviral immunity seems to further enhance the tumor immunity, which was demonstrated by higher levels of CD8⁺ T cells activation against virus-infected MC38 cells compared with mock-infected cells.

This study confirmed our hypothesis that CF33-derivative infection activates T cells against cancer cells, resulting in synergy with inhibition of the immune-checkpoint protein, PD-L1. We found that CF33-hNIS-ΔF14.5 induced durable antitumor immunity when combined with anti–PD-L1 treatment against a syngeneic colon cancer model. Durable cure, defined as complete tumor regression and resistance to regrow tumors upon rechallenge with the same cells, was most often seen when virus treatment was combined with anti–PD-L1 administration. Failure of the “cured” mice to develop MC38 is suggestive of the presence of MC38-specific immunity in those mice. However, the ability of the mice to develop Pan02 tumors but not MC38 tumors when rechallenged concomitantly with MC38 and Pan02 cells provides definitive evidence that the virus alone or in combination with anti–PD-L1 generates tumor-specific immunity in mice that undergo complete tumor regression. Although this confirmed a tumor-specific T cell response, our findings showed that memory-based T cell activation is far more robust against virus-infected cancer cells than noninfected cancer cells. We further confirmed that this recognition appears to be MHC-I dependent. Thus, viral infection with CF33 derivatives appears to cross-prime T cells against both tumor and viral antigens.

Several studies have demonstrated the ability of OVs to sensitize, otherwise resistant, tumors to immunotherapies. Although others have demonstrated that antitumor immunity induced after combining vaccinia with immune-checkpoint inhibition is dependent upon CD8⁺ T cells, there is a lack of clear evidence implicating viral-specific T cells in the success of oncolytic vaccinia virus. Our study is novel in its quantification of T cell memory-based activation against virus-infected versus uninfected tumor cells. Interestingly, we have shown that when T cells from spleens of cured mice are cocultured with cancer cells, they have activation signatures that are more robust if the mice were cured by viral treatment (either as a single agent or as part of combination therapy). Still more intriguing, we found that T cells from mice previously cured with the virus (alone or in combination with anti–PD-L1) when cocultured with MC38-Luc cells and retreated with the virus in vitro, showed the same activation signatures as T cells that are only in coculture with MC38-Luc cells, without any virus treatment. However, the activated T cells in the virus treatment groups produce markedly higher levels of IFNγ than their untreated counterparts. This suggests a role for serial virus dosing and further confirms that a robust antiviral response plays a major role in reprogramming a TME to prime for immunotherapy. This builds on the findings of Rosato and colleagues who have shown that reactivation of antiviral T cells can abrogate the growth of poorly immunogenic tumors in mice and render the tumors more susceptible to PD-L1 blockade (28). It also hints at a possible mechanism outside of variable PD-L1 expression as to how viral infection could prime a microenvironment for checkpoint inhibition.

One ongoing debate in the field of oncolytic viroimmunotherapy is whether we should be optimizing tumor cell destruction and then optimizing a host immune response to the resultant exposed tumor antigens, or arming viruses with as many tumor antigens as possible such that a host immune response to virus infection is conveniently also an antitumor response (17). Many OV investigators believe that tumor antigen exposure and antitumor immunity create only a fraction of the immune response generated by a viral infection (17, 18). In other words, antiviral immunity is far stronger than antitumor immunity. This theory has resulted in the creation of viruses armed with large swaths of tumor antigens (18). Yet, in a heterogeneous tumor, it is unclear if this would be enough to ensure a durable cure. We believe that sufficiently potent viruses that can directly infect tumor cells and thus expose tumor antigens in an immunogenic fashion will be just as effective as antigen-loaded viruses, with the added benefit of creating a tumor-specific cancer vaccine in real time. This avoids the limitations of more inefficient vectors loaded with some but not all the antigens associated with a given tumor. In this study, we demonstrate that tumor-specific T cell activation induced by CF33 derivatives is, in fact, more strongly activated against viral infection. Further studies are being considered to determine whether a CF33 derivative encoding tumor antigens or other immune stimulants would offer a different pattern of T cell activation.

There are limitations with using murine models, and the durable cure observed in murine models in the current study is not perfectly reflective of human immune responses. Moreover, when examining antitumor immunity, it must be noted that MHC-related differences along with species-specific responses to vaccinia virus make it difficult to predict the extent to which these findings will predict virus function in humans. Furthermore, use of luciferase-tagged cells may stimulate an immune response in and of themselves. Nevertheless, we have clearly and consistently shown durable antigen-specific antitumor immunity against a syngeneic mouse model of colon cancer that was most robust when using a combination of CF33-derivative infection and anti–PD-L1 treatment. We have also shown that the antiviral T cell response is even more robust than the antitumor immune response, which warrants further study.

Although OVs may have a role as first-line single-agent treatments for some already immunogenic malignancies, it seems that the most promising role for OVs in clinical practice will be to recruit immune cells and “heat” immunologically cold TMEs bereft of activated immune cells (26). Viruses, and particularly orthopoxviruses, can both destroy tumor cells and modulate the surrounding microenvironment to recruit and activate immune cells while exposing tumor antigens to a newly inflamed TME and tumor-infiltrating lymphocytes (18). This study elucidated the strength of CD8⁺ T cell activation against CF33 infection and tumor cells. Viruses like CF33 derivatives that are both immunogenic agents for tumor destruction and primers for other immunotherapies – like immune checkpoint inhibition and CAR-T cell therapy – are likely to find a strong foothold in the climb toward clinical relevance (31).

CF33 derivatives induce durable antitumor immunity when combined with anti–PD-L1 immune-checkpoint inhibition. T cell activation occurring in association with viral infection appears to be far more robust against infected tumor cells than against untreated cells. These findings provide support for serial viral dosing strategies and consideration of incorporation of tumor antigens into viral design to enhance antitumor T cell activity. Further study of cross-priming between antiviral and antitumor immune responses following viroimmunotherapy will enhance understanding of existing and future clinical treatment paradigms, especially those involving checkpoint inhibition.

S.-I. Kim, S. Kang, J. Lu, A. Yang, and Z. Zhang report CF33 derivatives are licensed by City of Hope to Imugene, Inc. Y. Fong reports personal fees from Imugene (scientific consultant), Sangamo (data monitoring board), Merck (royalties), Medtronics (scientific consultant), Johnson and Johnson (scientific consultant), and Eureka (scientific consultant) outside the submitted work; in addition, Dr Fong has a patent for patent pending and with royalties paid from Imugene. S.G. Warner reports personal fees from Coloplast (spouse (urologist) is a consultant), Olympus (spouse (urologist) is a consultant), and Boston Scientific (spouse (urologist) is a consultant) and grants from American Cancer Society (MRSG-16-047-01-MPC) outside the submitted work; and CF33 derivatives are licensed by City of Hope to Imugene, Inc. CF33 derivatives are licensed by City of Hope to Imugene, Inc. No disclosures were reported by the other authors.

S.-I. Kim: Conceptualization, data curation, formal analysis, investigation, methodology, writing–original draft, project administration, writing–review and editing. A.K. Park: Conceptualization, data curation, formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing. S. Chaurasiya: Conceptualization, investigation, methodology, project administration, writing–review and editing. S. Kang: Data curation, investigation, writing–review and editing. J. Lu: Resources, software, supervision, investigation, visualization, project administration, writing–review and editing. A. Yang: Data curation, investigation, visualization. V. Sivanandam: Validation, investigation, visualization, writing–review and editing. Z. Zhang: Data curation, software, formal analysis, investigation, visualization, methodology, writing–review and editing. Y. Woo: Resources, data curation, supervision, funding acquisition, writing–review and editing. S.J. Priceman: Conceptualization, resources, formal analysis, project administration, writing–review and editing. Y. Fong: Resources, formal analysis, supervision, funding acquisition, investigation, methodology, project administration, writing–review and editing. S.G. Warner: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, visualization, methodology, writing–original draft, project administration, writing–review and editing.

This work is supported by the American Cancer Society Mentored Research Scholar Grant: MRSG-16-047-01-MPC (S.G. Warner). S. Chaurasiya and S.G. Warner are supported through the generosity of Natalie and David Roberts. The authors wish to thank them for their philanthropy. This project used the Beckman Research Institute–shared facilities that are supported in part by the NCI of the NIH, grant award P30CA033572. The authors thank Lorna Rodriguez, MD, PhD, for her extensive scientific guidance, and would further like to thank Supriya Deshpande, PhD, for her expert editorial assistance.

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