Harnessing the Viral Replication Cycle for Oncolytic Viral and Cancer Immunotherapies Combination Strategies

Abstract

Oncolytic viral therapies have been harnessed to treat tumors. Recent studies have sought to propose to employ combination therapies utilizing oncolytic viral and cancer immunotherapy strategies. The viral replication cycle serves as a “perfect companion” to immunomodulatory drugs such as immune checkpoint inhibitors, bispecific antibodies and adoptive cellular therapies for targeting the tumor microenvironment, and has been described. Oncolytic viruses are known to target multiple steps within the cancer-immunity cycle and are DNA and RNA viruses that are edited without any impairment of viral replication. According to one study, DNA viruses contain efficient DNA polymerases that maintain genomic integrity and replication. This chapter consists of a review of oncolytic viral and cancer immunotherapy combinations for various tumors and compiles the unique replicative and lytic strategies that viruses employ to enhance anti-tumor effects and mitigate immunosuppression.

1. Introduction

The development of viruses as anti-tumor therapies, also known as oncolytic viruses (OVs), has become the subject of recent investigations designed to harness viral replicative abilities to lyse tumor cells. Oncolytic viruses replicate in and destroy tumor cells which lead to systematic anti-tumor immune responses through targeted approaches [1]. OVs operate by lysing cancer cells and leaving non-malignant cells intact by exposing tumor neo-antigens which formerly were not latent to the host’s immune system. This unique combination of tumor oncolysis and activation of immune response and the balance with host self-recognition forms the basis of OVs, since these same OVs could be neutralized by the host [2]. OVs date to the development of other microorganisms that have cancer immunotherapeutic effect such as Coley’s toxin [3].

1.1 Definition of oncolytic viruses

According to Raja et al. [4] “similar to Coley’s toxins, attenuate viruses are able to infect tumor cells and lead to de novo immune response and boost the native immune response” [4]. Oncolytic viruses enhance anti-tumor effects and lead to reduced virulence against the host cells through genetic modification. As described in Figure 1, the OV can either infect a normal cell or a cancer cell with these outcomes in the following manner: (1) an OV replicates in a normal cell or cancer cell. In a normal cell, viral replication is blocked. In an cancer cell, the virus (2) can either replicate, (3) integrate its transgene into the genome, or (4) express its transgenes, (5) in the case where the virus replicates in cancer cells, it is released upon cell lysis, and (6a) the released virus infects more cancer cells, (6b) in the case of a non-replicating virus expressing its transgenes, the virus is released upon cell lysis and the cancer cell with virus is killed, (6c) when the viral transgene is expressed in the newly formed cancer cell, immune activators express epitopes and the cancer cell with virus is killed. Through the generation of a proinflammatory environment, they lead to antigen release and recognition to inhibit tumor evasion by malignant cells. Through their replicative and lytic mechanisms, they harness the tumor’s non-protective mechanisms from the immune system, leading to a cascade of viral transference between the neoplasm and subsequent immune activation [6, 7].

Figure 1.

Mechanisms of oncoviral replication and tumor lysis. (1) An OV replicates in a normal cell or cancer cell. In a normal cell, viral replication is blocked. In an cancer cell, the virus (2) can either replicate, (3) integrate its transgene into the genome, or (4) express its transgenes. (5) in the case where the virus replicates in cancer cells, it released upon cell lysis, (6a) the released virus infects more cancer cells, (6b) in the case of a non-replicating virus expressing its transgenes, the virus is released upon cell lysis and the cancer cell with virus is killed, (6c) when the viral transgene is expressed in the newly formed cancer cell, immune activators express epitopes and the cancer cell with virus is killed (adapted from Sivanandam et al. [5]).

According to a 2019 published report, three OVs have met with regulatory approval for therapeutic oncologic use, as outlined in Figure 2. Figure 2A outlines the viral mechanistic strategies when infecting normal or cancer cells, similar to the events outline in Figure 1. Figure 2B describes the timeline for the approval of OVs. In 2004, Rigvir, an RNA virus derived from a native picornastrain, was approved for melanoma in Latvia [8]; H101, an engineered adenovirus was approved for melanomas carcinomas in 2005 [9]. OV approvals followed in the United States, when in 2015 the FDA approved Talimogene laherparepvec (T-VEC), an attenuated Herpes simplex virus (HSV-1) which encodes for GM-CSF, for melanomas that had relapse/recurrence [9]. OVs are now considered alternatives to standard treatment with the development of T-VEC. HF10 (Canerpaturev—C-REV) and CVA21 (CAVATAK), are in development and being evaluated in phase II/III trials as monotherapies or with immune checkpoint blockade against melanoma [10].

Figure 2.

A. How an oncolytic virus attacks a tumor cell through replication. B. Timeline of approved oncolytic viruses. In 2004, Rigvir, RNA virus derived from a native picornastrain, was approved for melanoma in Latvia [8]; H101, an engineered adenovirus was approved for nasopharyngeal carcinomas in 2005 [9]. OV approvals followed in the United States, when in 2015 the FDA approved Talimogene laherparepvec (T-VEC), an attenuated Herpes simplex virus (HSV-1) which encode for GM-CSF, for melanomas that had relapse/recurrence [9]. OVs are now considered alternatives to standard treatment with the development of T-VEC. HF10 (Canerpaturev—C-REV) and CVA21 (CAVATAK), are in development and being evaluated in phase II/III trials as monotherapies or with immune checkpoint blockade against melanoma (adapted from Mishra et al. [10]).

Jhawar et al. [11] performed the original research study that reported positive results with T-VEC, also known as talimogene laherparepvec, in a clinical trial against unresectable stage III and IV melanoma. The clinical outcomes showed a 31.5% objective response rate in these tumors [11].

The adenovirus or Ad was discussed by Hemminki et al. [3] as being a potential OV. Adenoviruses due to their capability for entering into cancer cells by binding to tumor receptors and thus leading to viral replication and tumor lysis have been extensively surveyed. The “serotype 5 adenoviruses bind preferentially to the coxsackie- and adenovirus receptor CXADR”; however, this receptor is downregulated in many solid tumors [3]. Adenoviruses are able to retarget the immune system by retargeting CD8+ cytotoxic and CD4+ helper T cells and thereby lift immunosuppression through damage-associated molecular pattern and pathogen-associated molecular pattern receptors [3]. Some adenoviruses have been mutated to replicate in tumor cells. One type of 24-base pair deletion produces a mutated E1A protein that is quiescent in normal tissues but has replicative potential in tumor cells such as retinoblastoma and adenoviral entry, and ensures that the tumor cells remain in the synthesis phase due to ubiquitous defects in the p16/Rb pathway. Similar to HSV, adding a granulocyte macrophage colony stimulating factor cytokine transgene into the adenoviral genome is a commonly used modification. In this approach, virus replication is accompanied by GMCSF production, which results in the recruitment and maturation of dendritic cells (DCs), and subsequent priming of T cells with tumor-associated antigens released by oncolysis [12].

OVs have also demonstrated efficacy in hematologic malignancies with a current slate of clinical trials investigating the efficacy of oncolytic measles virus and vaccinia virus in multiple myeloma, which are beginning to unfold [2]. Yang et al. [12] describe the oncolytic viral strategies utilizing adenovirus and herpes simplex virus as a promising therapeutic strategy for hematologic malignancies, including leukemia and lymphoma. There are over 50 kinds of human infectious serotypes of adenovirus one of which, the Ad5, has a Coxsackie and Ad receptor that targets B lymphoblastic leukemia cells expressing this receptor, and thus leads to anticancer activity [12].

However caveats remain since many of these cells lack the receptor, and prevents Ad5 from widely used. However, the Ad26 and Ad48 lead to strong tumor cell targeting and lymphoblastic cell growth in vivo [12]. According to Yang, human acute myeloid leukemia cells were apoptotic upon infection of a “tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-coated oncolytic Ad virus malignancies” [12]. Now, there are over 50 kinds of human infectious serotypes, which can be divided into seven subtypes (A-G). Additionally, it was shown that third-generation herpes simplex virus-1, termed T-01 had anti-tumor effects in vivo in hematopoietic cells [12].

The tumor microenvironment, or TME, contributes to a great deal to the efficacy of OVs. The TME is a vast assortment of innate and adaptive immune cells and neoplastic cells that can be modulated by oncolytic viruses and enable the immune system to become more capable for inducing an anti-tumor response. These include cancer stem cells, mesenchymal stem cells, and endothelial cells as well as macrophages and natural killer cells, B cells, dendritic cells and neutrophils, include cytokines such as IL-10 and TGF-beta and chemokines. A number of mechanisms mediate an immunosuppressive environment since cytokines play an immunosuppressive role in dampening cytotoxic T cells and natural killer cells and promote regulatory T cells [13].

These oncolytic viruses have been employed in combination with immunomodulatory agents such as adoptive cellular therapies, immune checkpoint inhibitors and bispecific antibodies. Earlier studies used approaches with chemotherapy and radiation therapy [11]. Cancer immunotherapies such as immune checkpoint inhibitors, CAR-T cell therapy and bispecific T cell engagers in combination with OVs have been the subject of ongoing investigations and reported in the literature [1].

This article consists of a literature review on OVs and their oncolytic strategies such as replication and lysis in combination with standard-of-care cancer immunotherapies, with an emphasis on solid tumors, although recent data is emerging for hematologic malignancies. The emphasis in this chapter are genetically modified oncoviruses, such as adenovirus, an enveloped viruses with large dsDNA genome, replicate in the nucleus consisting of large transgene insertion capacity; and herpes simplex virus, a non-enveloped viruses with intermediate-sized dsDNA genome that replicates in the nucleus with medium transgene insertion capacity [13].

2. Results

2.1 Immunomodulatory and oncolytic virus combinations

2.1.1 Oncolytic viruses and immune checkpoint inhibitors

The mechanism of OV and immunomodulatory strategies is described in Figure 3. Since tumor cells are able to selectively induce a systemic immune response, OVs can act as immune adjuvants to enhance the effectivity of cancer immunotherapies such as CAR T cell therapy and Immune checkpoint blockade. The mechanism in tumor cell lysis mediated by OVs is through release of tumor-associated antigens, cytokines, pathogen-associated molecular patterns and damage associated molecular patterns, turning a non-immunogenic tumor to an immunogenic one [14].

Figure 3.

Employing OVs as genetic vectors with immunogenic properties to effect tumor lysis. Since tumor cells are able to selectively induce a systemic immune response, OVs can act as immune adjuvants to enhance the effectivity of cancer immunotherapies such as CAR T cell therapy and immune checkpoint blockade. The mechanism in tumor cell lysis mediated by OVs is through release of tumor antigens, cytokines, PAMPs and DAMPs, turning a “cold” tumor to an “hot” one. TAAs, tumor-associated antigens; DAMPs, damage associated molecular patterns; HMGB1, high mobility group box 1; HSP, heat shock protein; PAMPs, pathogen-associated molecular patterns; dsDNA, double-stranded DNA; ssRNA, single-stranded RNA; ROS, reactive oxygen species; TLR, Toll-like receptor (adapted from Guo [14]).

The clinical combination of OVs with ICIs led synergistic effects on anti-tumor activity as was seen in a phase Ib study using T-VEC in combination with ipilimumab in patients with advanced melanoma. In the T-VEC + ipilimumab combination therapy, the objective response rate was 50%, and 44% of patients had a durable response lasting for 6 months or longer. Importantly, the combination had a tolerable safety profile, and appeared to have greater efficacy than either T-VEC or ipilimumab monotherapy [13].

These positive results were later confirmed in a follow-up phase II study showing a significant increase in confirmed objective response rate with T-VEC+ ipilimumab compared with ipilimumab alone (39%versus18%, respectively; p = 0.002) [15]. Recently, Ribas et al. showed, in a phase Ib study using T-VEC combined with pembrolizumab, exceptionally high overall and complete response rates of 62% and 33%, respectively, in patients with advanced melanoma [16].

Shi reiterated the T-VEC data reported in Jhawar et al., that the combinations of immune checkpoint antibodies with unmodified OVs or OVs mobilized with assisted cytokines and chemokines, such as TNFa, IL-2, IL12, IL-15, GM-CSF, and IFN-b, synergistically act as therapies against metastatic or locally unresectable tumors [1]. They also describe an open-label phase II study evaluating T-VEC in combination with anti-CTLA-4 immune checkpoint inhibitor ipilimumab in resected melanoma, and found improved ORRs alone (39% vs. 18%, P = 0.002) [1, 16]. PD-1 blockade combination with adenovirus and TNF-a and IL-2 resulted in complete tumor regression in a preclinical model [1, 17]. Similarly, pembrolizumab was shown to be efficacious with oncolytic virotherapy in a melanoma mouse model [1, 15]. Shi et al. [1] conclude that OVs are at high clinical efficacy when administered pre-operatively and in combination with ICIs post-operatively for these solid tumors.

A study utilizing human papillomavirus as an OV directed against cervical cancer in combination with immune checkpoint blockade, transforming a “cold” tumor into “hot” tumor [18]. After demonstrating the efficacy of the oncolytic HSV T-01 in human papillomavirus in mouse models, Kagabu et al. [18] investigated whether T-01 leads to an enhancement of antitumor effect of ICIs in tumor models. Six-week-old female C57BL/6 mice harboring bilateral subcutaneous TC-1 tumors co-injected with T-01 and anti-PD-L1 inhibitor demonstrated a marked increase in tumor-infiltrating cytotoxic T cells 13 days after inoculation, further showing that HPV-related OVs have the potential for tumor regression in cervical cancer [18].

2.1.2 Oncolytic viruses and cellular therapies

Chimeric antigen T cell therapy also known as CAR T cell therapy, is considered an adoptive cellular therapy where T cells are engineered ex vivo with the CD19 epitope to allow for cytotoxic T cells to kill tumor cells and then reintroduced to effect this tumor lysis. Park et al. [19], “engineered an oncolytic virus to express a non-signaling, truncated CD19 (CD19t) protein for tumor-selective delivery, enabling targeting by CD19-CAR T cells.” The vaccinia virus that ultimately would infect tumor cells coded for CD19t (OV19t) producing cell surface de novo CD19 followed by viral mediated tumor lysis. According to the study, the CAR-T cells that resulted secreted inflammatory cytokines that had “potent cytolytic activity against infected tumors.” They used several mouse tumor models to control the delivery of OV19t and promote tumor control following CD19-CAR T cell administration. They also state that “[i]mportantly, OV19t induced local immunity characterized by tumor infiltration of endogenous and adoptively-transferred T cells. CAR T cell-mediated tumor killing also induced release of virus from dying tumor cells, which propagated tumor expression of CD19t” [19].

The study demonstrated tumor lytic activity due to OV19t through phase contrast microscopic analysis over a 72-hour time period, which was quantified using flow cytometry, showing that at 24 h greater killing of tumor cells occurred, and at 48 and 72 h the lowest infectivity and tumor lysis were observed with virus alone. However, in combination with OV19t and CD19-CAR T cells, 60–70% killing was observed.

Another study recently reported “an oncolytic adenovirus expressing TNF-a and IL-2 (Ad-mTNFa-mIL2) was combined with mesothelin-redirected CAR-T cell (meso-CAR-T) therapy to treat human-PDA (pancreatic ductal adenocarcinoma)-xenograft immunodeficient mice”. Researchers found that Ad-mTNFa-mIL2 increased both CAR-T cell and host T cell infiltration into immunosuppressive PDA tumors and altered immune status in the TME, causing M1 polarization of macrophages and increased dendritic cell (DC) maturation [20]. “Another construct theVV.CXCL11 demonstrated the ability to recruit total and antigen-specific T cells into the TME after CART cell injection and significantly enhanced anti-tumor efficacy compared with direct delivery of CXCL11 by CAR-T cells [1, 21]”.

2.2 Oncolytic viruses and bispecific antibodies

Bispecific T cell engagers also known as BiTEs, consist of two antibodies combined with two variable fragments of single chain antibodies target CD3, on the surface of T cells and CD19, a tumor antigen thus creating contacts between the two cell surface receptors that lead to the killing of tumor cells through cytokine production. Figure 4 describes the mechanism of OV-BiTEs. 1. After infecting the tumor cell, the OV-BiTE replicates, leading to the expressed BiTE being secreted on the exterior of the tumor cell. 2. Lysis of the tumor cell takes place, mediated by the OV-BiTE, as the secreted BiTE targets tumor cells. This is termed a new multimodal anti-tumor model, since cancer-associated fibroblasts (CAFs) are targeted and killed, which reduces TME-mediated immunosuppression, transforming the immunogenicity of a tumor [10].

Figure 4.

OV-BiTE structure and mechanism. One type of OV-BiTE, EnAd. 1. After infecting the tumor cell, the OV-BiTE replicates, leading to the expressed BiTE being secreted on the exterior of the tumor cell. 2. Lysis of the tumor cell takes place, mediated by the OV-BiTE, as the secreted BiTE targets tumor cells. This is termed a new multimodal anti-tumor model, since cancer-associated fibroblasts (CAFs) are targeted and killed, which reduces TME-mediated immunosuppression, transforming the immunogenicity of a tumor from a “cold” one to a “hot” one (adapted from Huang et al. [22]).

In 2017, investigators engineered an oncolytic adenovirus ICOVIR-15K to express EGFR/CD3-targeting antibodies (ICOVIR-15K-cBiTE) in human lung and colorectal carcinoma mouse models. According to Ylosmaki, “the results suggested that tumor-infiltrating T cells could be more effectively activated and redirected by ICOVIR-15K-cBiTE, compared with treatment by ICOVIR-15K alone” [13].

In 2017, another preclinical study by Freedman et al. reported that an adenovirus expressing EpCAM/CD3–specific BiTEs enhanced T-cell-mediated destruction of tumor cells by activated CD4+ and CD8+ T-cells in clinical tissue biopsy samples that contained the EpCAM-positive tumor cells [1]. The OV that expressed a BiTE molecule bound to EpCAM and led to overexpression on target cancer cells (EnAd-SA-EpCAM) [21].

Also described are promising preclinical studies and clinical trials of OV-BiTE with adenovirus expressing p53 and HSV expressing GM-CSF. These studies describe how bispecific T cell engagers have a synergistic effect with OVs, such as adenoviru-BiTE and measles virus BiTE, however no clinical data have been reported [22] (Table 1).

Tumor type/model	OV	Immunotherapy	Trial/study	Outcomes	Safety profile
Advanced melanoma	T-VEC	Ipilimumab	Phase Ib study	ORR: 50% vs. 44% (combination vs. ipilimumab alone)	Tolerable
Advanced melanoma	T-VEC	Ipilimumab	Follow-up Phase II study	ORR: 39% vs. 18% (combination vs. ipilimumab alone)	N/A
Advanced melanoma	T-VEC	Pembrolizumab	Phase Ib	ORR: 62% versus 33%	N/A
HPV-related Cervical Cancer:	T-01	Anti-PD-1 inhibitor	Pre-clinical mouse models	Marked increase in cytotoxic T cells leading to tumor regression	N/A
Pancreatic ductal adenocarcinoma	Oncolytic adenovirus expressing TNF-a and IL-2 (Ad-mTNFa-mIL2)	Mesothelin-redirected CAR-T cell	Pre-clinical Xenograft immunodeficient mice x	Increase in both CAR-T cell and host T cell infiltration into immunosuppressive environment of these PDA tumors and also modified immune status in the TME, leading to M1 polarization of macrophages and increased dendritic cell (DC) maturation
human lung and colorectal carcinoma	Adenovirus ICOVIR-15K	(ICOVIR-15K-cBiTE	Mouse models	Activation of tumor-infiltrating T cells
CLINICAL tissue biopsy samples containing EpCAM-positive tumor cells	Adenovirus	EpCAM/CD3–specific BiTEs	Preclinical study	Penetration and activation of both CD4C and CD8C T cells, thus enhancing T cell-mediated tumor killing

Table 1.

Summary of immunomodulatory and oncolytic viral combinations.

2.3 Limitations and potential drawbacks

While several studies and review articles propose the promising strategy of OVs, there are limitations and drawbacks to their clinical use suggested by the literature. One major limitation is ensuring effective delivery and administration for systematic use of OVs through either intravenous or intratumoral injection. Even though the clinical data suggest tolerability and good safety profiles, the effectivity as determined by adequate viral titers from IV delivery has not been clearly shown, and quantitative PCR assay is not considered completely reliable in confirming live virus. Also while, as detailed earlier, OV infection into normal cells leads to abortive effects, these normal cells may include potential immune cells and hamper viral delivery. As one study suggested, rapid viral clearance could take place by inducing antibody titers and viral binding to serum proteins, which may over time become pronounced with multiple infusions [5]. This issue may become more pronounced over time in patients treated with multiple viral infusions. The most common adverse events reported have been influenza-like symptoms and local reactions at the injection site even though local administrated is well-tolerated.

3. Discussion

Current research encompassing other potential oncoviruses in combination with other cancer immunotherapies such as antibody drug conjugates combinations particularly against hematologic malignancies are ongoing. The vaccinia virus, measles virus, respiratory syncytial virus, and reovirus were also considered as viable OVs, particularly for multiple myeloma, a hematologic malignancy with poor prognosis, as reported by Meyers et al. [2]. Lymphoma and leukemia regressions were observed after measles virus infection. Intravenous injection is the typical route of administration for OVs and subcutaneous injections are being explored as an alternative route of delivery. Vaccinia virus has been shown to target tumor cells through various genomic insertions and deletions. Ways of improving oncolytic efficiency are being explored through increasing injection concentration and co-administration with cytokines [2]. Future investigations could focus on combining these approaches to harness the viruses’ natural abilities for replication and tumor cell lysis while evading the host cells and leading to immunostimulation and mitigating immunosuppression.

4. Summary and future directions

Oncoviruses with their viral replicative strategies are able to serve as cancer therapies alone or in concert with developed cancer immunotherapies. At least three are approved for clinical use, and studies are ongoing for their use in combination with bispecific antibodies, immune checkpoint blockade and CAR T cell therapy. Adenovirus and herpes simplex virus are frequently employed as genetic constructs for their high transgene capability and effective tumor lysis effects. As described earlier, other viruses have the potential for using these approaches in oncology.

Finally, strategies for immunomodulatory-OV combination strategies will benefit greatly from an improved understanding of how viruses replicate and their subsequent localization, and kinetics in humans [23]. As one review articulated, “prior to embarking on large combination clinical trials, it may be prudent to understand the biology of OV delivery in more detail to better optimize dosing, schedules and routes of administration”. Designing future clinical trials that compare IV and IT delivery would also be paramount for understanding the nuances and potential of each approach [24].

Conflict of interest

No conflict of interests to disclose.