Harnessing live vectors for cancer vaccines: Advancing therapeutic immunotherapy.

Introduction

Cancer vaccines represent a promising therapeutic branch within immuno-oncology. 1 , 2 Unlike traditional prophylactic vaccines designed to prevent diseases, therapeutic cancer vaccines treat existing cancers by stimulating the immune system to recognize and kill tumor cells. 1 , 3 These vaccines are generally precision therapies that target tumor-specific antigens (TSAs). The integration of cancer vaccines with other immunotherapies underscores their potential to complement existing treatments (i.e., immune checkpoint inhibitors (ICI), adoptive cell therapies, and monoclonal antibodies) to enhance efficacy and improve patient outcomes. 3

Key in the development of therapeutic cancer vaccines is the selection of appropriate vaccine target and vaccine platform (delivery system). Ideal vaccine targets include tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). TAAs are proteins overexpressed in tumor cells but also in normal cells at lower levels, 4 while TSAs are unique proteins expressed exclusively by tumor cells. Neoantigens, a subset of TSAs, are newly formed antigens specifically generated from tumor-specific mutations, providing highly distinct and personalized targets for vaccine-induced immune responses. 5 Along with TAAs and TSAs, human endogenous retroviral elements (HERVs) have been used to develop cancer vaccines and chimeric antigen receptor (CAR)-expressing T cells. 5 , 6 Activation of the HERVs can lead to a state of viral mimicry, inducing an innate immune response and production of cytokines. 6 , 7 By mimicking viral infections, HERVs function as an intrinsic adjuvant sensitizing cancer cells for immune recognition. 5

The platforms used to deliver cancer vaccines are diverse and have evolved to enhance the immune response. Cancer vaccines can be characterized by these platforms and include vector-based vaccines, recombinant vectors, peptide-based vaccines, deoxyribonucleic acid (DNA)-based vaccines, messenger ribonucleic acid (mRNA) vaccines, whole-cell vaccines, gangliosides, genes and dendritic cell (DC) vaccines. 1 , 8 Vector-based vaccines use viral, yeast or bacterial vectors to deliver genetic material encoding tumor antigens, leveraging the vectors’ natural ability to infect cells and induce immunity. Whole-cell vaccines employ whole tumor cells, often attenuated via genetic modification or irradiation, to stimulate a broad immune response against multiple antigens. Tumor antigen-loaded DC vaccines utilize DCs loaded with tumor antigens to activate T cells, the primary effectors of the immune response. 1 , 8

Live cancer vaccines leverage various biological entities, including live or recombinant vectors to stimulate an immune response. Each of these biological entities has unique properties that can be harnessed to target and destroy cancer cells while eliciting a robust immune response. 1 , 9 The delivery methods for live cancer vaccines are crucial as they influence the vaccine’s ability to reach and stimulate the immune system. 10 , 11 The strategy of using the immune system to treat cancer dates back to the 1800s, when physicians observed significant tumor shrinkage in patients following acute infections. Dr. William Coley pioneered an early vaccine product (known as Coley’s Toxins) developed from inactivated Streptococcus and Serratia species. 12 In the early 1900s, patients with tuberculosis were noted to develop cancers less frequently than patients without tuberculosis. 13 This observation spurred interest in exploring Bacillus Calmette-Guérin (BCG) as a potential cancer treatment, with initial experiments beginning in the 1930s. 14 By the 1970s, BCG vaccine had been established as the standard of care for non-muscle invasive bladder cancer (NMIBC) and later received FDA approval in the 1990s. 15 During this time, vaccine development expanded bacterial vectors to include other pathogens (i.e., viruses, yeast, and fungi), which were also found to exhibit anti-tumor. This broadened the scope of interest in pathogen-based immunotherapy, laying the groundwork for further advancements in cancer vaccines.

This review provides an in-depth exploration of various live cancer vaccines, examining the biological entities used as vectors involved with a focus on the most extensively researched bacteria, yeast, and viruses, including oncolytic viruses. This comprehensive analysis offers valuable insights into the current landscape of live cancer vaccines and potential future directions in the field. To ensure a focused scope, this review will concentrate on the most extensively studied vector platforms and highlight ongoing clinical trials as of September 15, 2024.

Bacterial vectors

Listeria monocytogenes

Listeria monocytogenes (Lm) as a vector for cancer vaccines marks a pivotal development in immunotherapy. Leveraging the unique properties of this intracellular bacterial pathogen, researchers have enhanced the delivery and presentation of TAAs, thereby eliciting robust immune responses. 16–18 Lm possesses an inherent ability to infect both phagosomal and cytosolic compartments of Antigen Presenting Cells (APC). 19 This dual access facilitates the presentation of antigens via both major histocompatibility complex (MHC) class I and class II pathways, thereby stimulating both CD8+ cytotoxic T lymphocytes and CD4+ helper T cells. 18–20 The innate response is initially mediated by the activation of toll-like receptors (TLR), which results in downstream signaling and the production of pro-inflammatory cytokines. 21 Once phagocytized, Lm may be processed in the phagolysosomal compartment and peptides presented on MHC Class II to activate Lm-specific CD4+ T cell responses. Alternatively, Lm can escape the phagosome and enter the cytosol, leading to CD8+ T cell activation. 19 , 22 This combination of responses is crucial for a sustained anti-tumor immune response and makes Lm a powerful vaccine vector. 18–20

Aduro Biotech, now Chinook Therapeutics, developed two Lm platforms: LADD (Live Attenuated Double Deleted) and pLADD (personalized LADD). LADD utilizes genetically modified bacteria to express TAAs. The pLADD platform builds upon LADD by formulating vaccines incorporating predicted neoantigen epitopes for a more targeted immune response. One such vaccine, CRS-207, expresses mesothelin, a TAA overexpressed in pancreatic, ovarian, and lung cancers. 19 , 23 Initial clinical trials demonstrated its safety and ability to induce mesothelin-specific T-cell responses. 24 However, the Food and Drug Administration (FDA) placed these studies on clinical hold in 2016 for safety after a patient treated with CRS-207 developed listeria bacteremia. 25 , 26 The hold was lifted after modifying the protocol and excluding patients with certain prostheses. Later, a pLADD platform-based clinical trial on colorectal cancer (CRC) was terminated due to business re-alignment. 27 Currently, the sole active study of CRS-207 is a Phase 2 study of epacadostat, pembrolizumab, and CRS-207, with or without cyclophosphamide and GVAX, which is a granulocyte-macrophage colony-stimulating factor (GM-CSF) – transduced autologous tumor-cell vaccine in patients with metastatic pancreas cancer. The study has recruited 41 patients with no active recruiting and pending results. 28 While patients who developed listeria infection as a consequence of treatment generally responded to antibiotics, the ongoing development of listeria-based products must consider strategies to minimize infectious toxicity (i.e., attenuated bacterial strains, incorporation of stronger antimicrobial prophylaxis into the study treatment). 25 This approach could enhance the risk/benefit profile and expand the potential approvability of these products.

A key innovation in Listeria-based vaccines is the fusion of the antigen with Listeriolysin O (LLO), a virulence factor of Listeria. 29 This fusion enhances the immunogenicity of the antigen and promotes stability and effective presentation to the immune system. 29 In vaccines targeting prostate cancer, prostate-specific antigen (PSA) is fused with LLO, resulting in a potent and targeted immune response against PSA-expressing tumor cells. 29 Advaxis Inc., now Ayala Pharmaceutics, had focused on developing Lm vaccines based on the XFL-7 and LmddA strains. These LLO-based vaccine strains typically express TAAs episomally, promoting robust antigen presentation and activation of the immune response. Among the vaccine strains, three specifically targeted the E7 protein from HPV-16, PSA, and Her2. 30–32 Similar to the LADD platform, one patient who received this vaccine in 2013 died in 2015 with trace amounts of bacteria in the blood leading to a brief hold by FDA. 26 However, as of date 15 th September 2024, all studies of this product have been terminated. Johnson & Johnson (JNJ) also evaluated vaccines targeting epidermal growth factor receptor variant III (EGFRvIII) and mesothelin (JNJ-61757) and multiple prostate cancer antigens (JNJ-61809). The Phase I trial evaluated the safety of JNJ-61809, targeting four relevant prostate cancer antigens with no reported dose-limiting toxicities (DLT). 33

Despite compelling pre-clinical and early clinical data, safety and efficacy concerns led to the termination of all clinical trials of this vaccine product except one ongoing trial of RT201 (Tumor Antigen-specific Macrophage Tumor Vaccine) in the Treatment of Advanced Cervical Cancer. 25–26 , 34–36 Lm vaccine products have been studied in combination with other agents to a limited extend. 34 , 37 Two promising platforms under research in pre-clinical studies, Lm-RIID (Lm recombinase-induced intracellular death) and KBMA (Killed but metabolically active) strains, achieve this goal primarily by deleting virulence factors. The anti-tumor KBMA Lm stimulates effective CD4+ and CD8+ T cell responses and an increase in the number of mature Dendritic Cells in colon cancer model without significantly side effects while Lm-RIID commits suicide intracellulary by inducing Cre recombinase in host cell cytosol to delete essential genes for bacterial viability. 38 , 39 As a result, Lm-RIID can elicit potent anti-tumor effects without normal tissue injury. Besides, Lm-RIID exhibits a higher clearance rate than double-deleted Lm strains. 37 , 40

Salmonella

Salmonella typhimurium (ST) strains have also been engineered to exert anticancer effects. 41 , 42 These strains colonize tumor tissues at a rate 1000 times higher than normal tissues, with preclinical murine studies demonstrating activity in multiple tumor models. 42 ST achieves tumor lysis by promoting apoptosis in tumor cells through nutritional deficiency and bacterial toxin release. Additionally, ST can induce autophagy, which downregulates the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathways, inhibits the expression of hypoxia-inducible factor 1-alpha (HIF-alpha) and vascular endothelial growth factor (VEGF), and eventually slows tumor growth. 41–44 ST also downregulates certain oncoproteins involved in drug resistance and tumor metastasis. 45 , 46

Several strains of ST have been engineered to enhance their safety and efficacy, the most prominent being VNP0009 and CHI4550. VNP0009 is a genetically modified strain designed to minimize virulence while retaining its ability to target and kill cancer cells. 42 This strain has been tested in multiple clinical trials, demonstrating a tolerable safety profile, though no objective tumor regression was reported. 47 , 48 Subsequent development explored the oral route with ST Ty21a expressing VEGF Receptor-2, leading to the first-in-kind oral V×M01vaccine against advanced pancreatic cancer, which showed safety, immunogenicity, and increased vaccine-specific T cell responses compared to placebo. 49 Another Phase I, dose-escalation, single-dose trial of oral, attenuated ST-containing human interleukin (IL)-2 demonstrated a statistically significant increase in circulating natural killer (NK) and NK-T cells, indicating an immunologic effect despite no observed survival benefit. 50 The current active study, NCT9234, is a Phase II trial with the primary objective to assess the efficacy of multiple-dose oral administration of Saltikva, an attenuated strain of ST expressing IL-2, in patients with metastatic pancreatic cancer on a standard of care (SOC) (either FOLFIRINOX or Gemcitabine/Abraxane, in combination with Saltikva). 51

A new modified strain of VNP0009, SGN1, is being investigated in an ongoing clinical trial, NCT3345. This strain overexpresses L-methionine, allowing it to target and preferentially replicate in tumors, depriving them of methionine. 52 Pre-clinical data in combination with PD-L1 (programmed death ligand-1) in murine melanoma models have shown that combination therapy with systemic anti-PD-L1 therapy results in better antitumor activity than individual monotherapies. 52 Currently, SGN1 studies are actively recruiting. 53 , 54 Another active trial for patients with multiple myeloma: NCT2291 is a Phase I dose escalation study of TXSVN, a weakened form of a live vaccine strain of Salmonella (also known as the CVD908ssb strain) that has been genetically modified to produce the protein survivin, a tumor antigen. Survivin, a member of the inhibitor of apoptosis protein family, serves a dual function by promoting cellular proliferation and inhibiting apoptosis. Its elevated expression in tumor cells represents an adaptation that enables the evasion of apoptosis. However, the overexpression of survivin in tumors compared to normal tissues presents an attractive target for immunotherapy, aiming to disrupt this tolerance mechanism. Widely recognized as a TAA, it has emerged as a significant target for investigation both in vitro and in clinical trials for cancer diagnosis and treatment. 55 , 56

A modified version of VNP0009 was engineered to express single-chain antibody fragments specific to carcinoembryonic antigen (CEA), a marker commonly found on human gastrointestinal tract carcinomas, pancreatic cancer, NSCLC, and breast cancer. Additionally, to boost antitumor activity, an extra copy of the sipB gene, which encodes the proapoptotic Salmonella invasion protein B, was inserted into the VNP0009 chromosome. While Salmonella naturally targets tumor tissues, these modifications enable recombinant vaccines to be specifically guided and concentrated within tumors, effectively delivering apoptotic proteins to eliminate cancer cells. 57

BCG

BCG, a live-attenuated form of Mycobacterium bovis , was first used successfully to treat NMIBC in 1976. 58 Since FDA approval in 1990, BCG has become SOC for intermediate- and high-risk NMIBC following transurethral bladder tumor resection. 59 , 60 Although the precise mechanisms of BCG’s antitumor effects remain unclear, several theories have emerged. It is believed to stimulate innate and adaptive immune responses through local inflammatory reactions. 61 Some studies suggest BCG activates TLR-7, inducing cytotoxic effects on tumor cells. 62 , 63 Preclinical evidence also indicates that BCG recruits macrophages and pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), IL-1β, and IL-6, into the tumor microenvironment (TME), potentially promoting tumor apoptosis and clearance. 64 Beyond bladder cancer, BCG has been evaluated in combination with other therapies for malignancies, though such studies have been limited. For example, a 2008 study explored using the New York esophageal squamous cell carcinoma 1 (NY-ESO-1) recombinant peptide vaccine with BCG and GM-CSF in urothelial carcinoma. This combination elicited antibody and T-cell responses, with BCG serving as an adjuvant. 65 These findings suggest that BCG may enhance immunological responses when used in combination therapies; however, its role in such settings remains largely exploratory. There are two ongoing trials Phase III trials evaluating the use of BCG in bladder cancer ( Table 1 ). The S1602 trial investigates different strains of BCG with or without a BCG vaccine in high-grade NMIBC. One of its primary objectives is to determine whether intradermal Tokyo-172 BCG vaccination before intravesical Tokyo-172 BCR strain instillation improves the time to high-grade recurrence for patients with BCG-naïve NMIBC. 66 Another trial is a multicenter study assessing the efficacy and safety of tislelizumab combined with BCG bladder instillation in preventing postoperative recurrence in intermediate- and high-risk NMIBC. 67 Table 1. Active clinical trials of bacterial vector-based cancer vaccines. Sr No. Vaccine Antigen Cancer Indication Drug Combination Phase Study Title Start Date Results Posted NCT Number Listeria 1 RT201 Tumor Antigen-specific Cervical Vaccine Monotherapy I Clinical Research on the Safety and Effectiveness of RT201 (Tumor Antigen-specific Macrophage Tumor Vaccine) in the Treatment of Advanced Cervical Cancer. 10/1/2021 No NCT0301 Salmonella 1 Modified Salmonella Typhimurium SGN1 No TSA Various cancers Vaccine Monotherapy I/IIa Phase I/IIa, Open-label Study to Evaluate Safety, Tolerability and Preliminary Efficacy of Modified Salmonella Typhimurium SGN1 in Patients With Advanced Solid Tumor 1/16/2023 No NCT8150 2 Various cancers Vaccine Monotherapy I A Phase I/IIa, Open-label, Dose Escalation and Dose-Expansion Study to Evaluate the Safety and Tolerability of Modified Salmonella Typhimurium SGN1 Administered Via Intratumoral Injection in Patients With Advanced Solid Tumors 4/19/2023 No NCT3345 3 Saltikva (Attenuated Salmonella Typhimurium Containing the Human Gene for Interleukin-2) IL-2 Pancreatic cancer Vaccine Monotherapy II A Phase 2 Study of Saltikva (Attenuated Salmonella Typhimurium Containing the Human Gene for Interleukin-2) in Patients With Metastatic Pancreatic Cancer 10/20/2020 No NCT9234 FOLFIRINOX + Vaccine Gemcitabine/Abraxane + Vaccine 4 Salmonella CVD908ssb strain (TXSVN Vaccine) Survivin Multiple myeloma Vaccine Monotherapy I Multiple Myeloma Trial of Orally Administered Salmonella Based Survivin Vaccine. 8/6/2921 No NCT2291 BCG 1 BCG Vaccine No TSA Bladder cancer Tislelizumab + Vaccine III Multicenter Clinical Trial on the Effectiveness and Safety of Instillation of BCG and Alternative BCG Protocols for Intermediate and High-risk Non-muscle Invasive Bladder Cancer 6/1/2023 No NCT1110 67 2 BCG Tokyo-172 Strain Vaccine No TSA Bladder cancer Vaccine Monotherapy III S1602, A Phase III Randomized Trial to Evaluate the Influence of BCG Strain Differences and T Cell Priming With Intradermal BCG Before Intravesical Therapy for BCG-Naive High-Grade Non-muscle Invasive Bladder Cancer 2/24/2017 No NCT1660 BCG: Bacillus Calmette-Guérin. TSA: Tumor-Specific Antigen. Sr. No: Serial Number.

Clostridium

One of the key challenges in cancer treatment is that many tumors thrive in hypoxic, poorly vascularized environments, which hinders effective therapy delivery. Due to their growing ability in oxygen-poor conditions, Clostridium species can lyse tumor cells by colonizing and metabolizing within hypoxic TMEs. 68 Clostridial-directed enzyme prodrug therapy (CDEPT) is an innovative approach that utilizes clostridium’s preference for hypoxic environments. In CDEPT, Clostridium is genetically engineered to activate nontoxic prodrugs into active chemotherapeutic agents. 68 , 69 Although no ongoing clinical trials use CDEPT, preclinical studies have generated promising results. 69 Another area of interest involves using C. butyricum MIYAIRI 588 (CBM588) in combination with BCG in bladder cancer. A preclinical study found that CBM588 was as effective as BCG in enhancing the release of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from neutrophils, promoting apoptosis of cancer cells. 70 C. novyi-NT, a modified strain of C. novyi, has also shown potential. Preclinical single-dose administration of C. novyi generated tumor regression in small murine tumors, although responses in larger murine tumors were less impressive. A high percentage of animals treated with C. novyi experienced fatal toxicity, highlighting the need for additional safety development. 71 , 72 Attempts were made to reduce toxicity by removing a bacterial gene shown to correlate with toxicity, but safety concerns persisted. 73 , 74 In a Phase 1 trial completed in 2001, a melanoma vaccine incorporating epitopes from tetanus toxoid successfully induced CD4+ T-helper cell responses. 75 Recent studies on C. tetani-based products primarily focus on combination therapies, where the accompanying product also serves as an immune stimulant. One study evaluating the combination of the Human Papillomavirus (HPV) vaccine GARDASIL with REPEVAX (diphtheria, tetanus, acellular pertussis, and poliomyelitis vaccine) demonstrated favorable tolerability and evidence of immunological response. 76 Another trial evaluated the combination of the ALVAC-CEA/CD80 (B7.1) vaccine with chemotherapy and tetanus vaccine, finding an acceptable safety profile, although no significant clinical or T-cell response was observed. 77 Although Clostridium-based therapies demonstrated potential for targeting malignant cells, the discontinuation of earlier studies highlights significant challenges in translating this approach to clinical practice. Furthermore, there are no ongoing studies utilizing Clostridium as a vector for cancer vaccines, underscoring the need for renewed research efforts and innovations to overcome these limitations.

Yeast

Yeast-based cancer vaccines represent a cost effective, versatile and promising approach leveraging innate and adaptive immune responses. 78–80 Yeast is an effective platform for delivering TAAs due to its proven ability to generate robust immune activation. The immune system recognizes engineered yeast cells expressing TAAs as foreign, prompting DCs to phagocytose them. The TAAs are processed and presented on MHC molecules, activating CD4+ and CD8+ T-cells. Additionally, yeast cells contain pathogen-associated molecular patterns (PAMP), such as β-glucans, which bind to pattern recognition receptors (PRR) on immune cells. This interaction enhances immune activation and fosters a pro-inflammatory environment, leading to an effective anti-tumor response. 78 , 80 , 81 Also, heat-killed yeast, which is extremely stable and easy to transport and store, offers practical advantages over other vaccine platforms.

Preclinical studies have demonstrated the efficacy of yeast-based vaccines across multiple cancer models, including melanoma, breast cancer, and prostate cancer. 82–85 In one study using human CEA-transgenic mice, a recombinant S. cerevisiae expressing human CEA (yeast-CEA) was evaluated for its ability to elicit CEA-specific T-cell responses and antitumor activity. The results demonstrated that yeast-CEA vaccination could break immune tolerance and elicit both CEA-specific CD4+ and CD8+ T-cell responses. 85 , 86 In tumor-bearing mice, repeated administration of yeast-CEA significantly reduced tumor burden and increased overall survival, particularly in models of pulmonary metastasis and subcutaneous pancreatic tumors. 85

While there is no FDA-approved yeast-based therapeutic cancer vaccine, there are currently multiple investigational products under study ( Table 2 ). GI-6207 is a yeast-CEA vaccine, genetically modified to express the recombinant CEA protein using heat-killed Saccharomyces cerevisiae as a vector. In clinical trials, patients with metastatic CEA-expressing carcinomas received subcutaneous vaccine with minimal toxicity while inducing antigen-specific immune responses and stabilizing CEA levels. 87 GI-4000 is a yeast-based vaccine that targets mutations in the RAS oncogene. RAS plays a crucial role in many solid tumors, including pancreatic and NSCLC. A Phase-II trial showed that GI-4000 was well tolerated and immunogenic when used as consolidation therapy in patients with stage I-III KRAS-mutant lung cancer. 88 Immune responses were measured by interferon-γ (IFNγ) ELISpot assay and by regulatory T cell (Treg) frequencies on treatment. In pancreatic cancer, a phase II trial (NCT6406) combined GI-4000 with gemcitabine for patients with resected RAS-mutated tumors, showing trends toward improved survival in certain patient subgroups identified by proteomic signatures predictive of GI-4000 responsiveness. 89 However, despite the promising immune responses, many GI-4000 trials have failed (either terminated or withdrawn) secondary to poor accrual or lack of efficacy. 90 GI-6301 is a yeast-based vaccine that targets brachyury, a transcription factor overexpressed in various advanced cancers as well as universally in chordoma. A Phase I clinical trial 91 demonstrated the vaccine’s ability to induce brachyury-specific immune responses with minimal adverse effects. Early signs of clinical activity were observed. 92 However, a Phase II trial combining GI-6301 with radiation therapy for patients with unresectable chordoma was terminated early due to a lack of synergistic antitumor efficacy. 93 For the brachyury vaccine, recent efforts have shifted toward non-yeast-based platforms, such as the BN-Brachyury vaccine, which employs a heterologous prime-boost strategy using Modified Vaccinia Ankara (MVA) for priming and Fowlpox Virus (FPV) for boosting. The prime-boost strategy is used to enhance and sustain the immune response against a target, particularly when the body may quickly neutralize the initial vaccine. The prime dose introduces the immune system to the target antigen, while subsequent booster doses use a different vaccine platform to bypass immune neutralization and strengthen the response. Early trials of this vaccine in patients with metastatic solid tumors have shown a good safety profile and the induction of brachyury-specific T-cell responses. 94 Table 2. Active clinical trials of yeast vector-based cancer vaccines. Sr No. Vaccine Antigen Cancer Indication Drug Combination Phase Study Title Start Date Results Posted NCT Number 1 OPT-821 GD2L and GD3L Neuroblastoma Oral dietary supplement (Beta glucan) + Vaccine I/II Phase I/II Trial of a Bivalent Vaccine With Escalating Doses of the Immunological Adjuvant OPT-821, in Combination With Oral β-glucan for High-Risk Neuroblastoma 5/27/2009 No NCT1560 2 GI-4000 (Pancreatic Cancer Vaccine) KRAS Pancreatic Cancer ALT-803 I/II QUILT-3.080: Phase 1B/2 Trial Of The NANT Pancreatic Cancer Vaccine As Treatment For Subjects With Pancreatic Cancer Who Have Progressed on or After Standard-Of-Care Therapy. 7/17/2018 Yes NCT6869 ETBX-011 ETBX-021 ETBX-051 ETBX-061 GI-4000 GI-6207 GI-6301 haNK for infusion Avelumab Bevacizumab Capecitabine Cyclophosphamide Fluorouracil Leucovorin nab-Paclitaxel Oxaliplatin SBRT 3 GI-4000 (NANT Pancreatic Cancer Vaccine) KRAS Pancreatic Cancer ALT-803 I/II Molecularly Informed Integrated Immunotherapy Combining Innate haNK Cell Therapy With Adaptive T-cell Therapy (Adenovirus, Yeast, Fusion Protein Vaccine) in Subjects With Pancreatic Cancer Who Have Progressed on or After Standard-of-care Therapy. 11/6/2017 Yes NCT9248 ETBX-011 GI-4000 haNK for infusion Avelumab Bevacizumab Capecitabine Cyclophosphamide Fluorouracil Leucovorin nab-paclitaxel Omega-3 acid Oxalipatin SBRT 4 GI-4000 (NANT CRC Vaccine) KRAS CRC ETBX-011 I/II A Phase 1b/2 Trial of the NANT CRC Vaccine vs Regorafenib in Subjects With Metastatic CRC Who Have Been Previously Treated With Standard-of-Care Therapy 9/28/2018 Yes NCT3157 ETBX-021 ETBX-051 ETBX-061 GI-4000 GI-6207 GI-6301 haNK for infusion Avelumab Cetuximab Capecitabine Cyclophosphamide Fluorouracil Leucovorin nab-paclitaxel Omega-3 acid Oxaliplatin SBRT 5 GI-4000 (NANT TNBC Vaccine) KRAS Breast Cancer N -803 I/II Molecularly Informed Integrated Immunotherapy Combining Innate haNK Cell Therapy With Adenoviral and Yeast-based Vaccines to Induce T-cell Responses in Subjects With TNBC Who Have Progressed on or After Standard-of-care Therapy. 3/19/2018 Yes NCT7085 ETBX-011 ETBX-051 ETBX-061 GI-4000 GI-6207 GI-6301 haNK for infusion Avelumab Bevacizumab Capecitabine Cyclophosphamide Fluorouracil Leucovorin nab-paclitaxel Cisplatin SBRT 6 TLPO vaccine The patient’s TL Solid Tumor Vaccine monotherapy II A Prospective, Open-Label, All-Comers Phase IIa Trial of The TLPO Cancer Vaccine in Solid Tumor Malignancies 11/29/2023 No NCT5221 7 YE-NEO-001 Personalized targets Various Cancers Vaccine monotherapy I QUILT-2.025 NANT Neoepitope Yeast Vaccine (YE-NEO-001): Adjuvant Immunotherapy Using a Personalized Neoepitope Yeast-Based Vaccine To Induce T-Cell Responses In Subjects W/Previously Treated Cancers. 8/10/2018 No NCT2718 CRC: Colorectal Cancer, haNK: High-affinity Natural Killer Cells, YE-NEO-001: Neoepitope Yeast Vaccine, SBRT: Stereotactic body radiation therapy, Sr. No: Serial Number. TL: Tumor Lysate, TLPO: Tumor Lysate Particle Only, TNBC: Triple Negative Breast Cancer.

Efforts to develop yeast-based personalized cancer vaccines, such as neoepitope yeast vaccine YE-NEO-001, have also been underway. They have been explored for use in metronomic combination therapies for pancreatic cancer, 95 , 96 and CRC. 97 One of the Phase I trials of NANT vaccine for Triple Negative Breast Cancer (TNBC) showed safety and efficacy in combination with chemo-radiation. 98 However, it was prematurely terminated before the completion for Phase II. 99 These personalized neoepitope vaccines, which target cancer-specific mutations, represent a new frontier in cancer immunotherapy.

Viral vectors (including oncolytic viruses)

Over a century ago, tumor regression was observed in cancer patients infected with certain viruses. 100 Since then, multiple trials have evaluated the role of various viruses as vectors for cancer vaccines. The use of viruses in vaccines can be divided into two groups: oncolytic viruses (OV) and genetically engineered viruses. While viral-based therapeutic cancer vaccines are engineered to express tumor antigens via transgenes, OV do not target a specific tumor antigen. 101 Rather, OVs exert direct cytotoxic effects on cancer cells. Tumor cells lyse and spill their contents, offering a host of TSAs to the immune system to ultimately effect an antitumor response. 100 , 102 The FDA and European Medicines Agency (EMA) approved the first OV, Imlygic (talimogene laherparepvec, or T-VEC), in 2015. 103 , 104 Multiple OV products are now under investigation both as monotherapy and in combination. 103 As of September 2024, 114 active clinical trials are ongoing. 105

Amongst various virus platforms, adenovirus and poxvirus have been extensively studied historically and possess well-characterized genomes. Adenovirus was among the earliest viruses used as a vector for gene therapy. 106 Advances in genetic engineering have further enhanced the safety and efficacy of adenoviral vectors, increasing their use in experimental therapies. 107 Poxvirus, on the other hand, gained prominence due to its large genome, which allows for easy genetic manipulation. 108 Similarly, herpes simplex virus (HSV) is recognized for its ability to infect both dividing and non-dividing cells and its favorable safety profile, making it a suitable vector for cancer vaccine delivery systems. 109 At the same time retrovirus vectors, such as lenti- and gammaretroviruses, are the most common choice for the transformation of isolated patient cells ex vivo. 110 Lentiviral vectors, in particular, are extensively utilized in the manufacturing of CAR-T cells 110 An alphavirus vector, AVX701 (CEA(6D)-VRP), has also been evaluated in phase I/II clinical trial patients with metastatic cancer expressing CEA with vaccine successfully elicited CEA-specific T-cell and antibody responses in most patients. 111 Other platforms, utilizing measles virus, alphavirus, and retroviruses, have also been studied and described. 101 , 106 , 110 However, given that the majority of currently active clinical trials focus on adenovirus, poxvirus, and HSV, and considering the unique attributes of these vectors, this review will primarily focus on adenovirus, poxvirus, and HSV as leading candidates in ongoing clinical trials.

Pox virus

Pox viruses, including vaccinia, offer several characteristics that make them attractive vectors. A large viral genome facilitates the insertion of multiple therapeutic genes. 108 Vaccinia vaccines elicit robust immune responses by inducing interferon (IFN) production and activating both cellular and humoral immunity. 112 , 113 Studies have demonstrated that pox virus strains, including particularly MVA, are safe and well tolerated in humans. 114 , 115 While the anti-tumor efficacy of pox virus-based therapeutic cancer vaccines has not reached the threshold for regulatory approval, ongoing studies seek to enhance efficacy ( Table 3 ). One promising strategy involves a pox virus vaccine modified to express three costimulatory molecules or TRICOM: B7.1, ICAM-1 (Intracellular adhesion molecule-1), and LFA-3 (Lymphocyte function-associated antigen 3). These molecules enhance the immune response by promoting T-cell proliferation and cytokine production. 116 MVA-Bavarian Nordic (MVA-BN)-brachyury-TRICOM vaccine has been developed to target brachyury, the previously noted transcription factor associated with increased cytokine release, angiogenic factor production, and IL-8 signaling. 117 Elevated IL-8 levels drive epithelial-to-mesenchymal transition in malignant cells, which increases tumor cell motility and invasiveness. 117 A Phase 1 clinical trial with escalating doses of this vaccine in patients with solid tumors demonstrated no DLTs, with most patients showing a brachyury-specific T-cell response. 118 Another Phase 1 trial evaluating the PROSTVAC-V/F vaccine incorporating PSA and TRICOM demonstrated a favorable toxicity profile as well as evidence of vaccinia-specific immune response. 119 However, no studies of this product have met their primary endpoint for efficacy as of this publication. Table 3. Active clinical trials of viral vector-based cancer vaccines. Sr No. Vaccine Antigen Cancer Indication Drug Combination Phase Study Title NCT Number Adenovirus 1 Autologous dendritic cell adenovirus CCL21 vaccine CCL21 NSCLC Vaccine I A Phase I Trial of Intratumoral Administration of CCL21-Gene Modified Dendritic Cell (Ad-CCL21-DC) Combined With Intravenous Pembrolizumab for Advanced NSCLC 7/8/2019 No NCT6361 Pembrolizumab 2 Nous-209 vaccine GAd20-209-FSP and MVA-209-FSP CRC, gastric or GEJ tumors Vaccine I/II A Phase I/II, Multicenter, Open-Label Study of Nous-209 Genetic Vaccine for the Treatment of Microsatellite Unstable Solid Tumors. 10/21/2019 No NCT1310 Pembrolizumab 3 Lynch Syndrome Vaccine monotherapy I/II A Phase Ib/II Clinical Trial of Nous-209 for Recurrent Neoantigen Immunogenicity and Cancer Immune Interception in Lynch Syndrome No NCT8866 4 PRGN-2009 HPV 16/18 E6 and E7 Cervical Cancer Vaccine II A Randomized, Open-label, Two-arm, Multicenter Phase 2 Study to Evaluate Efficacy and Safety of PRGN-2009 in Combination With Pembrolizumab Versus Pembrolizumab Monotherapy in Patients With Recurrent or Metastatic Cervical Cancer. 11/2024 No NCT7151 Pembrolizumab 5 DCs vaccine combined with CIK MUC1 and Survivin AML Adenovirus-transfected DC + CIK I/II Safety and Therapeutic Efficacy of DCs Vaccine Combined With Cytokine-induced Killer Cells in Patients With AML: a Phase I/II Study 11/2024 No NCT8663 6 Combination of trivalent adenovirus-5 (Tri-Ad5) vaccines and IL-15 superagonist nogapendekin alfa inbakicept (N-803) vaccines MUC1, CEA and brachyury Lynch Syndrome Vaccine monotherapy II A Phase IIB Clinical Trial of the Multitargeted Recombinant Adenovirus 5 (CEA/MUC1/Brachyury) Vaccine (TRI-AD5) and IL-15 Superagonist N-803 in Lynch Syndrome 5/8/2023 No NCT9011 7 NANT Pancreatic Cancer Vaccine: multiple neoantigens Pancreatic Cancer ALT-803 I/II NANT Pancreatic Cancer Vaccine: Molecularly Informed Integrated Immunotherapy Combining Innate High-affinity Natural Killer (haNK) Cell Therapy With Adaptive T-cell Therapy (Adenovirus, Yeast, Fusion Protein Vaccine) in Subjects With Pancreatic Cancer Who Have Progressed on or After Standard-of-care Therapy 11/6/2017 Yes NCT9248 ETBX-011 GI-4000 haNK for infusion avelumab bevacizumab Capecitabine Cyclophosphamide Fluorouracil Leucovorin nab-Paclitaxel lovaza Oxaliplatin SBRT Oncolytic Adenovirus 1 LOAd703 No TSA Pancreatic Cancer LOAd703 I/II Phase I/IIa Trial Evaluating Safety of LOAd703, an Armed Oncolytic Adenovirus for Pancreatic Cancer 11/2016 No NCT5196 Gemcitabine Nab-paclitaxel atezolizumab 2 Pancreatic Adenocarcinoma Ovarian Cancer Biliary Carcinoma CRC LOAd703 monotherapy I/II Phase I/II Trial Investigating an Immunostimulatory Oncolytic Adenovirus for Cancer 3/1/2018 Yes NCT5989 3 Recombinant Human Adenovirus Type 5 No TSA Uterine Cervical Neoplasms Recombinant human adenovirus II Oncolytic Virus(H101) Combined With Camrelizumab for Recurrent Cervical Cancer: a Prospective, Multicenter Study 03/2022 No NCT4905 Camrelizumab 4 Cholangiocarcinoma Recombinant Human Adenovirus Type 5 IV Recombinant Human Adenovirus Type 5 Combined With Hepatic Artery Infusion Chemotherapy of FOLFOX in Patients With Intrahepatic Mass-forming Cholangiocarcinoma: a Single-site, Single-arm, Prospective Study 8/1/2022 No NCT4002 HAIC of FOLFOX 5 H101 No TSA Malignant Pleural Mesothelioma Oncolytic Adenovirus H101 Observational Observation of the Efficacy and Safety of Oncolytic Adenovirus Injection Combined With Programmed Death Receptor Inhibitors in Treatment of Advanced Malignant Pleural Mesothelioma: a Single Center, Prospective, Case Registration Study 7/20/2023 No NCT1636 pdL1- inhibitor 6 KD01(recombinant oncolytic adenovirus) No TSA Cervical Cancer KD01 monotherapy I The Safety, Tolerability, and Efficacy of Intratumoral Administration of the Recombinant Oncolytic Adenovirus Injection (KD01) in Patients With Cervical Malignancies 4/1/2024 No NCT2598 7 Bladder Cancer KD01 II Phase II Single Center Open-Label Single-Arm Study of the Safety and Efficacy of Oncolytic Adenovirus H101 Combined With PD-1 Inhibitor in Patients With Non-muscle-invasive Bladder Cancer Who Failed BCG Therapy 8/26/2022 No NCT4897 Camrelizumab 8 GM103 No TSA Head and Neck Cancer Malignant Melanoma CRC RCCCervical Cancer Breast Cancer GM103 vs GM103 I/II A Phase I/II, Open-label, Dose-escalation With Expansion Study of GM103 Via Intratumoral Injection, Alone and in Combination With Pembrolizumab in Adult Patients With Locally Advanced, Unresectable, Refractory and/or Metastatic Solid Tumors 2/20/2024 No NCT5025 Pembrolizumab 9 Recombinant Human Adenovirus Type 5 No TSA Cervical Cancer AK104 II Efficacy and Safety of AK104 Combined With Pemetrexed, Carboplatin and Recombinant Human Adenovirus 5 Injection in Advanced Recurrent Cervical Cancer: a Multicenter, Single-arm, Prospective Phase II Clinical Study 2/20/2024 No NCT5046 Recombinant Human Adenovirus 5 Injection 10 TILT-123 No TSA Melanoma Head and Neck SCC TILT-123 I A Phase I Open-Label, Dose-escalation Clinical Trial of Tumor Necrosis Factor Alpha and IL-2 Coding Oncolytic Adenovirus TILT-123 and Avelumab in Solid Tumor Patients (Melanoma and SCCHN) Refractory to or Progressing After Anti-PD(L)1 3/8/2023 No NCT2932 Avelumab 11 Ovarian and Fallopian tube tumor TILT-123 I A Two-part, Phase I/Ib, Open-Label, Dose-escalation Trial of Tumor Necrosis Factor Alpha and Interleukin-2 Coding Oncolytic Adenovirus (TILT-123) in Combination With Pembrolizumab (Phase I Part) and Pembrolizumab and Pegylated Liposomal Doxorubicin (Phase Ib Part) in Patients With Platinum Resistant or Refractory Ovarian Cancer. 5/17/2022 No NCT1318 pembrolizumab pegylated liposomal doxorubicin 12 Advanced Solid Tumors TILT-123 monotherapy I A Phase 1, Open-Label, Dose-escalation Clinical Trial of Tumor Necrosis Factor Alpha and Interleukin-2 Coding Oncolytic Adenovirus (TILT-123) in Patients With Injectable Solid Tumors 1/11/2021 No NCT5327 13 Metastatic Melanoma TILT-123 monotherapy I A Phase 1, Open-Label, Dose-Escalation Clinical Trial of Tumor Necrosis Factor Alpha and Interleukin 2 Coding Oncolytic Adenovirus TILT-123 in Melanoma Patients Receiving Adoptive Cell Therapy With Tumor Infiltrating Lymphocytes 2/26/2020 No NCT7473 14 ORCA-010 No TSA Adenocarcinoma of the Prostate ORCA-010 monotherapy I A Phase I/IIa Study Evaluating the Safety and Tolerability of Intratumoral Administration of ORCA-010 in Treatment-Naïve Patients With Localized Prostate Cancer. 11/12/2019 No NCT7002 15 DNX-2401 No TSA IDH1 wt (Wild Type) Allele Recurrent Anaplastic Astrocytoma GBM Gliosarcoma Glioma Oncolytic Adenovirus Ad5-DNX-2401 I Phase I Clinical Trial of Allogeneic Bone Marrow Human Mesenchymal Stem Cells Loaded With A Tumor Selective Oncolytic Adenovirus, DNX-2401, Administered Via Intra-Arterial Injection in Patients With Recurrent High-Grade Glioma 2/12/2019 No NCT6568 Therapeutic Conventional Surgery 16 AdMA3 No TSA NSCLC Cutaneous SCC Merkel Cell Carcinoma Melanoma Pancreatic Cancer TNBC Head and Neck Cancer MG1MA3 I/II A Phase I/II Study of MG1 Maraba/MAGE-A3 (MG1MA3), With and Without Adenovirus Vaccine, With Transgenic MAGE-A3 Insertion (AdMA3) in Patients With Incurable Advanced/Metastatic MAGE-A3-Expressing Solid Tumours 1/22/2015 No NCT5816 AdMA3 17 CAdVEC No TSA Bladder Cancer Head and Neck SCC Salivary Gland Cancer Lung Cancer Breast Cancer Gastric Cancer Esophageal Cancer CRC Pancreatic Adenocarcinoma CAdVEC monotherapy I A First in Human Phase I Trial of Binary Oncolytic Adenovirus in Combination With HER2-Specific Autologous CAR T Cells in Patients With Advanced HER2 Positive Solid Tumors 12/14/2020 No NCT0256 18 BioTTT001 No TSA Gastric Cancer BioTTT001 intraperitoneal infusion II A Clinical Study to Evaluate the Safety and Efficacy of Recombinant Human nsIL12 Oncolytic Adenovirus Injection (BioTTT001) in Combination With SOX and Toripalimab in Patients With Peritoneal Metastases From Gastric Cancer 4/2/2024 No NCT3121 SOX regimen Toripalimab 19 Advanced Solid Tumors BioTTT001 injection I Phase I Clinical Study on the Safety, Tolerability, and Pharmacokinetics of Recombinant Human nsIL12 Oncolytic Adenovirus Injection (BioTTT001) in Patients With Malignant Solid Tumors 5/7/2024 No NCT5846 20 CRC BioTTT001 hepatic artery infusion I A Clinical Study to Evaluate the Safety and Efficacy of Recombinant Human nsIL12 Oncolytic Adenovirus Injection (BioTTT001) in Combination With Toripalimab and Regorafenib in Patients With Liver Metastases From Colorectal Cancer 4/2/2024 No NCT3134 Toripalimab Regorafenib 21 AloCELY No TSA Diffuse Intrinsic Pontine Glioma AloCELYVIR monotherapy I/II Phase IB Clinical Trial to Assess the Safety, Tolerability, and Preliminary Efficacy of AloCELYVIR (Mesenchymal Allogenic Cells + ICOVIR-5) in Children, Adolescent and Young Adults With Newly Diagnosed Diffuse Intrinsic Pontine Glioma (DIPG) in Combination With Radiotherapy or Medulloblastoma in Relapse/Progression in Monotherapy 4/19/2021 No NCT8533 Medulloblastoma 22 Ad-TD-nsIL12 No TSA Diffuse Intrinsic Pontine Glioma Ad-TD-nsIL12 monotherapy I Oncolytic Virus Ad-TD-nsIL12 for Primary Pediatric Diffuse Intrinsic Pontine Glioma 1/4/2023 No NCT7712 23 Diffuse Intrinsic Pontine Glioma Ad-TD-nsIL12 monotherapy I Oncolytic Virus Ad-TD-nsIL12 for Progressive Pediatric Diffuse Intrinsic Pontine Glioma 1/4/2023 No NCT7699 24 AdAPT-001 No TSA Advanced Solid Tumors AdAPT-001 + ICI II An Adapted Phase 2a/2b, Study to Evaluate the Safety, Tolerability, and Efficacy of AdAPT-001 in Subjects With Refractory Solid Tumors 3/29/2021 No NCT3942 Sarcoma, Chondrosarcoma 25 TS-2021 No TSA GBM TS-2021 monotherapy I Clinical Study on the Safety and Efficacy of the Third-Generation Oncolytic Virus TS-2021 in the Treatment of Recurrent Malignant Glioma 8/29/2024 No NCT5527 26 JNJ-84916 No TSA Advanced Solid Tumors JNJ-84916 + Cetrelimab I Phase 1 Study of Intratumoral Administration of JNJ-84916, an Oncolytic Virus, as Monotherapy and in Combination for Advanced Solid Tumors 4/10/2024 No NCT1578 27 CG0070 No TSA Bladder cancer CG0070 + Nivolumab I A Phase 1 Study of CG0070 Combined With Nivolumab in Cisplatin Ineligible Patients With Muscle Invasive Bladder Cancer (MIBC) 10/26/2020 No NCT0671 Poxvirus (Including Vaccinia) 1 MVA-BN-Brachyury Brachyury Advanced Solid Tumors Vaccine, M7824, N-803 and Epacadostat I/II A Phase I/II Study of Immunotherapy Combination BN-Brachyury Vaccine, M7824, N-803 and Epacadostat (QuEST1) 5/1/2018 No NCT3945 2 CV301 vaccine CEA and MUC-1 and B7-1, ICAM-1 and LFA-3 Small Bowel and CRC Vaccine II Phase II Trial of Combination Immunotherapy in Subjects With Advanced Small Bowel and Colorectal Cancers 9/22/2020 Yes NCT1955 MSB1359C N-803 NHS-IL12 3 PANVAC-V, PANVAC-F CEA and MUC-1 and B7-1, ICAM-1 and LFA-3 Pancreatic Cancer Vaccine monotherapy + Sargramostim I Immunotherapy for Unresectable Pancreas Cancer: A Phase 1 Study of Intratumoral Recombinant Fowlpox PANVAC (PANVAC-F) Plus Subcutaneous Recombinant Vaccinia PANVAC (PANVAC-V), PANVAC-F and Recombinant Granulocyte-Macrophage Colony Stimulating Factor (rH-GM-CSF) 2/1/2010 No NCT9734 4 Multi-peptide CMV-MVAVaccine. CMV tumor-associated TAAs, including UL83 (pp65), UL123 (IE1) and UL122 (IE2) Hematologic malignancies Vaccine monotherapy +/- Letermovir II A Phase II Randomized, Placebo-Controlled, Multicenter Trial to Evaluate the Protective Function of CMV-MVA Triplex Vaccine in Adult Recipients of Haploidentical Hematopoietic Stem Cell Transplant 11/27/2019 Yes NCT0277 5 Hematologic malignancies Vaccine monotherapy A Phase II Randomized, Placebo-Controlled, Multicenter Trial to Evaluate the Protective Function of a CMV-MVA Triplex Vaccine in Recipients of an Allogeneic Hematopoietic Stem Cell Transplant 5/11/2025 Yes NCT6933 6 Hematologic malignancies Anti-CD19-CAR CMV-specific T-lymphocytes I A Pilot / Feasibility Study of Autologous CMV-Specific CD19-CAR T Cells Plus CMV-MVA Triplex Vaccine in Patients With Intermediate or High Grade B-Lineage Non-Hodgkin Lymphoma 9/29/2023 No NCT1913 Leukapheresis Lymphodepletion Therapy Multi-peptide CMV-MVA Vaccine 7 Hematologic malignancies Anti-CD19-CAR CMV-specific T-lymphocytes I Pilot/Feasibility Study of CMV-Specific CD19-CAR T Cells Plus CMV-MVA Triplex Following Autologous Hematopoietic Stem Cell Transplantation for Patients With Intermediate or High Grade B-Lineage Non-Hodgkin Lymphoma (B-NHL) 8/1/2023 No NCT2635 Autologous Hematopoietic Stem Cell Transplantation Multi-peptide CMV-Modified Vaccinia Ankara Vaccine Myeloablative Conditioning 8 Nous-209 GAd20-209-FSP and MVA-209-FSP CRC Vaccine I/II A Phase I/II, Multicenter, Open-Label Study of Nous-209 Genetic Vaccine for the Treatment of Microsatellite Unstable Solid Tumors 10/21/2019 No NCT1310 Pembrolizumab 9 GAd20-209-FSP and MVA-209-FSP Lynch Syndrome Vaccine monotherapy I/II A Phase Ib/II Clinical Trial of Nous-209 for Recurrent Neoantigen Immunogenicity and Cancer Immune Interception in Lynch Syndrome 11/10/2022 No NCT8866 10 MVA Expressing p53 p53 p53 expressing solid tumors Vaccine I A Phase I Study of a p53MVA Vaccine in Combination With Pembrolizumab 6/14/2016 No NCT2963 Pembrolizumab 11 PROSTVAC-V and PROSTVAC-F PSA and the three co-stimulatory molecule transgenes (TRICOM), B7.1, ICAM-1), and LFA-3. Prostate Cancer Vaccine II Phase II Trial of Combination Immunotherapy in Biochemically Recurrent Prostate Cancer 3/20/2018 No NCT5871 CV301 +/- MSB1359C (M7824) Oncolytic Vaccinia 1 hV01 No TSA Advanced Solid Tumors hV01 monotherapy I A Phase I Dose Escalation Study to Evaluate the Safety, Tolerance, Pharmacokinetics, and Biological Properties of Recombinant Human IL-21 Oncolytic Vaccinia Virus Injection (hV01) in Patients With Advanced Malignant Solid Tumors 7/5/2023 No NCT4376 2 GC001 No TSA Cervical Cancer CRC Lung Cancer Ovarian CancerPancreatic Cancer HCC Breast Cancer Gastric Cancer GC001 monotherapy I A Phase I Study Evaluating the Safety, Tolerability, Biodistribution and Shedding of the Virus, Pharmacodynamics, Immunogenicity, and Antitumor Activity of GC001 Oncolytic Vaccinia Virus Injection in Patient With Advanced Solid Tumors. 4/26/2023 No NCT8307 3 RGV004 No TSA Relapsed or Refractory B-cell Lymphoma RGV004 monotherapy I Exploratory Study of a Novel Oncolytic Vaccinia Virus Expressing Bispecific Antibody in the Treatment of Refractory/Relapsed B-cell Lymphoma 2/8/2022 No NCT7025 4 IDOV-SAFE No TSA Advanced Malignant Solid Tumor of Digestive System IDOV-SAFE monotherapy I An Open Label, Dose-escalation and Extension, Phase I Clinical Study on Evaluating Safety, Tolerability and Pharmacodynamics of Intravenous Administration of IDOV-SAFE in Patients With Advanced Malignant Solid Tumors Who Have Failed in Standard Treatment. 5/6/2024 No NCT0309 5 Pexa-Vec No TSA RCC Pexa-Vec I/II A Phase 1b/2a Dose-escalation and Safety/Efficacy Evaluation Study of Pexa-Vec (Thymidine Kinase-Deactivated Vaccinia Virus Plus GM-CSF) in Combination With Cemiplimab (REGN2810; Anti-PD-1) in Patients With Metastatic or Unresectable Renal Cell Carcinoma (RCC) 6/7/2018 Yes NCT4083 Cemipilimab 6 KM1 No TSA Ovarian Cancer KM1 + Chemotherapy I A Single-Arm, Open-Label Clinical Study to Evaluate the Safety, Tolerance and Preliminary Efficacy of KM1 Oncolytic Vaccinia Virus Injection Combined With Chemotherapy in Subjects With Recurrent or Refractory Ovarian Cancer 2/1/2023 No NCT4731 7 Olvimulogene nanivacirepvec No TSA NSCLC Olvimulogene nanivacirepvec II A Randomized Phase 2 Study Assessing the Efficacy and Safety of Olvimulogene Nanivacirepvec Followed by Platinum-doublet Chemotherapy + Physician’s Choice of Immune Checkpoint Inhibitor Compared With Docetaxel in Patients With NSCL Cancer After First Progression While on Front-line Immune Checkpoint Inhibitor-based Maintenance 9/26/2024 No NCT3665 Platinum chemotherapy: carboplatin or cisplatin Non-platinum chemotherapy: paclitaxel or nab-paclitaxel for squamous cell NSCLC or pemetrexed for Nonsquamous cell NSCLC ICI: pembrolizumab, nivolumab, cemiplimab, atezolizumab, durvalumab Docetaxel 8 TG6050 No TSA NSCLC TG6050 monotherapy I A Phase I Dose-escalation Trial of TG6050 Administered by Intravenous Infusion in Patients With Advanced Non-small Cell Lung Cancer (NSCLC) 4/5/2023 No NCT8926 9 VET3-TGI No TSA CRCHead and Neck SCC Cervical CancerRCC Melanoma Merkel Cell Carcinoma of SkinMesothelioma NSCLC VET3-TGI I A Phase 1/1b Study of VET3-TGI Administered Alone and in Combination With Pembrolizumab in Patients With Advanced Solid Tumors 9/16/2024 No NCT4815 Pembrolizumab 10 Olvi-Vec No TSA Ovarian Cancer Fallopian Tube Cancer Primary Peritoneal Cancer Olvi-Vec III A Randomized Phase 3 Study Assessing the Efficacy and Safety of Olvi-Vec Followed by Platinum-doublet Chemotherapy and Bevacizumab Compared With Physician’s Choice of Chemotherapy and Bevacizumab in Women With Platinum-Resistant/Refractory Ovarian Cancer (PRROC) (OnPrime, GOG-3076) 8/31/2022 No NCT1471 Platinum chemotherapy: carboplatin (preferred) or cisplatin. Non-platinum chemotherapy: Physician’s Choice of gemcitabine, Taxane (paclitaxel, docetaxel or nab-paclitaxel) or pegylated liposomal doxorubicin. Bevacizumab (or biosimilar) 11 BT-001 No TSA Soft Tissue Sarcoma Merkel Cell Carcinoma MelanomaTNBC NSCLC BT-001 I/II A Phase I/IIa Study of Intra-tumoral BT-001 (TG6030) Administered Alone and in Combination With Pembrolizumab in Patients With Cutaneous or, Subcutaneous Lesions or Easily Injectable Lymph Nodes of Metastatic/Advanced Solid Tumors. 2/25/2021 No NCT5331 Pembrolizumab Oncolytic HSV 1 BS-006 No TSA Uterine Cervical Neoplasms BS-006 monotherapy I A First-in-human Phase I Two-stage Clinical Trial for Intratumoral Injection of Recombinant Oncolytic Type II Herpes Simplex Virus (BS-006) in Patients With Recurrent Cervical Cancer 9/16/2022 No NCT3440 2 C134 No TSA GBM of Brain Anaplastic Astrocytoma of Brain Gliosarcoma of Brain C134 monotherapy I A Phase I Trial of IRS-1 HSV C134 Administered Intratumorally in Patients With Recurrent Malignant Glioma 9/23/2019 No NCT7576 3 HSV G207 No TSA Primary CNS malignancy G207 monotherapy II Phase II Clinical Trial of HSV G207 with a Single 5 Gy Radiation Dose in Children with Recurrent High-Grade Glioma 12/2024 No NCT2933 4 GBM G207 monotherapy I Phase 1 Trial of Engineered HSV G207 in Children With Recurrent or Refractory Cerebellar Brain Tumors 9/12/2019 No NCT1388 5 M032 No TSA GBMAnaplastic Astrocytoma Gliosarcoma M032 monotherapy I A Phase 1 Study of M032 (NSC 73972), a Genetically Engineered HSV-1 Expressing IL-12, in Patients With Recurrent/Progressive Glioblastoma Multiforme, Anaplastic Astrocytoma, or Gliosarcoma 11/25/2013 Yes NCT2827 6 GBM Anaplastic Astrocytoma Gliosarcoma M032 I/II A Phase I/II Study of Pembrolizumab and M032 (NSC 73972), a Genetically Engineered HSV-1 Expressing IL-12, in Patients With Recurrent/Progressive and Newly Diagnosed Glioblastoma Multiforme, Anaplastic Astrocytoma, or Gliosarcoma 2/25/2022 No NCT4430 Pembrolizumab 7 MVR-C5252 No TSA High-Grade Glioma MVR-C5252 monotherapy I The PuMP Trial: “A Multistage Phase 1 Open-Label Study to Evaluate the Safety, Tolerability and Efficacy of the Oncolytic HSV1 MVR-C5252 in Patients With Recurrent High-Grade Glioma” 6/11/2024 No NCT6744 8 OH2 No TSA Solid Tumor Gastrointestinal Cancer OH2 injection, with or without irinotecan or HX008 I/II Phase I/II Study of OH2 Injection, an Oncolytic Type 2 Herpes Simplex Virus Expressing Granulocyte Macrophage Colony-Stimulating Factor, in Malignant Solid Tumors 4/2/2019 No NCT6525 9 Bladder Carcinoma Monotherapy OH2 injection II Efficacy and Safety Study of Oncolytic Virus (OH2) Intratumoral Injection in Locally Advanced or Metastatic Bladder Cancer a Phase II Clinical Trial 5/25/2022 No NCT8789 10 Non-muscle-invasive Bladder Cancer Monotherapy OH2 injection I/II Oncolytic Virus (OH2) Adjuvant Therapy After Transurethral Resection of Bladder Tumor in Non-Muscle-Invasive Bladder Cancer Who Have Failed First-line Prophylactic Intravesical Instillation Therapy: a Phase Ib/II Clinical Trial 7/11/2022 No NCT2136 11 CNS Tumors Monotherapy OH2 injection I/II A Clinical Study of Oncolytic Virus (OH2) Injection in the Treatment of Patients Undergoing Surgery After Recurrence of Central Nervous System Tumors 11/16/2021 No NCT5074 12 Melanoma OH2 I/II Phase Ib Study of the Combination Use of Recombinant Human GM-CSF Type II Herpes Simplex Virus (OH2) Injection (Vero Cells) and HX008 Injection in the Treatment of Melanoma 12/1/2020 No NCT6443 HX008 injection 13 Melanoma OH2 I/II Open and Incremental Phase I Clinical Trial of Recombinant Human GM-CSF Type II Herpes Simplex Virus (OH2) Injection (Vero Cells) in the Treatment of Advanced Solid Tumors 11/22/2018 No NCT6967 Pembrolizumab 14 Melanoma OH2 III To Evaluate a Phase III Study of OH2 Versus Investigator-selected Salvage Chemotherapy or Best Supportive Care in Melanoma Patients Who Had Failed Standard Therapy 3/8/2023 No NCT8707 Salvage chemotherapy or best supportive care 15 R130 No TSA Sarcoma Carcinoma Breast Cancer Pancreatic Cancer CRCGastric Cancer Liver Cancer Lung Cancer Gynecologic Cancer R130 Monotherapy I A Clinical Safety and Efficacy Study on Oncolytic Virus Injection (R130) for the Treatment of Advanced Solid Tumors 3/2/2023 No NCT0374 16 Digestive Cancer Breast Cancer Lung Cancer Brain CancerMelanoma Gynecologic Cancer Head and Neck Cancer Kidney Cancer R130 Monotherapy I An Open, Single-armed, Clinical Safety and Efficacy Study on Oncolytic Virus Injection (R130 OV) for the Treatment of Advanced Solid Tumors 6/24/2023 No NCT1111 17 Head and Neck Cancer Esophageal Cancer Otorhinolaryngologic Neoplasms Pharyngeal Cancer R130 Monotherapy I A Clinical Safety and Efficacy Study on Oncolytic Virus Injection (R130) for the Treatment of Relapsed/Refractory Head and Neck Cancer 3/27/2023 No NCT0240 18 Lung Cancer (NSCLC. SCLC) Bronchial Cancer Sarcoma CRC Gastric Cancer Liver Cancer Breast Cancer Pancreatic Cancer Head and Neck Cancer Ovarian Cancer R130 Monotherapy I A Clinical Safety and Efficacy Study on Oncolytic Virus Injection (R130) for the Treatment of Relapsed/Refractory Advanced Solid Tumors 3/30/2023 No NCT6075 19 Cervical Cancer Endometrial Cancer R130 Monotherapy I A Clinical Safety and Efficacy Study on Oncolytic Virus Injection (R130) for the Treatment of Relapsed/Refractory Cervical and Endometrial Cancer 2/6/2023 No NCT2677 20 Osteosarcoma Sarcoma,Soft Tissue R130 Monotherapy I A Clinical Safety and Efficacy Study on Oncolytic Virus Injection (R130) for the Treatment of Advanced Bone and Soft Tissue Tumors 7/12/2023 No NCT1282 21 Osteosarcoma Sarcoma,Soft Tissue R130 Monotherapy I A Clinical Study on Oncolytic Virus Injection (R130) for the Treatment of Relapsed/Refractory Bone and Soft Tissue Tumors 4/10/2023 No NCT1456 22 Ovarian Cancer Peritoneal Carcinomatosis Fallopian Tube Cancer R130 Monotherapy I A Clinical Safety and Efficacy Study on Oncolytic Virus Injection (R130) for the Treatment of Relapsed/Refractory Ovarian Cancer 12/2/2022 No NCT1783 23 RP1 No TSA Cutaneous SCC Merkel Cell Carcinoma BCC Melanoma RP1 (intra-tumoral injection) I/II An Open-Label, Multicenter, Phase 1B/2 Study of RP1 in Solid Organ and Hematopoietic Cell Transplant Recipients With Advanced Cutaneous Malignancies 5/15/2020 No NCT9436 24 Cutaneous SCC RP1 II A Randomized, Controlled, Open-Label, Phase 2 Study of Cemiplimab as a Single Agent and in Combination With RP1 in Patients With Advanced Cutaneous Squamous Cell Carcinoma 10/8/2019 No NCT0436 Cemipilimab 25 Melanoma RP1 monotherapy I Phase I Pilot Study of RP1 in Primary Melanoma to Reduce the Risk of Sentinel Lymph Node Metastasis 2/2/2024 No NCT6938 26 MelanomaNon-melanoma Skin Cancer NSCLC RP1 II An Open-Label, Multicenter, Phase 1/2 Study of RP1 as a Single Agent and in Combination With PD1 Blockade in Patients With Solid Tumors 9/20/2017 No NCT7348 Nivolumab 27 RP2 No TSA Metastatic Uveal Melanoma RP2 II / III A Randomized, Phase 2/3, Open-Label Study to Investigate the Efficacy and Safety of RP2 in Combination With Nivolumab Versus Ipilimumab in Combination With Nivolumab in Immune Checkpoint Inhibitor-Naïve Adult Patients With Metastatic Uveal Melanoma 1/27/2025 No NCT1406 Ipilimumab / Nivolumab 28 RP2/RP3 No TSA mCRC RP2 II A Phase 2 Clinical Trial Investigating Oncolytic Immunotherapy in Combination With Atezolizumab and Bevacizumab for the Treatment of Patients With Advanced Microsatellite Stable and Mismatch Repair Proficient Colorectal Carcinoma 6/29/2023 No NCT3611 RP3 Atezolizumab Bevacizumab 29 RP3 No TSA Advanced Solid Tumor RP3 I An Open-Label, Multicenter, Phase 1 Study of RP3 as a Single Agent and in Combination With PD-1 Blockade in Patients With Solid Tumors 12/29/2020 No NCT5978 Nivolumab 30 HCC RP3 II A Phase 2, Open-label, Multicenter Study Investigating RP2 Oncolytic Immunotherapy in Combination With Second-line Therapy in Patients With Locally Advanced Unresectable, Recurrent and/or Metastatic Hepatocellular Carcinoma 8/1/2024 No NCT3598 Atezolizumab Bevacizumab 31 T3011 No TSA mCRC T3011 hepatic artery infusion I A Clinical Study to Evaluate the Safety and Efficacy of Herpes Virus T3011 Injection in Combination With Toripalimab and Regorafenib in Patients With Liver Metastases From Colorectal Cancer 4/2/2024 No NCT3303 Toripalimab Regorafenib 32 Advanced Solid Tumor T3011 Monotherapy I/II A Phase I/IIa Study to Assess the Safety, Tolerability, Biodistribution and Pharmacodynamic of T3011 Herpes Virus Administered Via Intravenously in Patients With Advanced Solid Tumors 3/1/2022 No NCT8268 33 Advanced Solid Tumor T3011 Monotherapy I/II A Phase I/IIa Study to Assess the Safety, Tolerability, Biodistribution and Pharmacodynamic of T3011 Herpes Virus Administered Via Intratumoral Injection in Patients With Advanced Solid Tumors 4/21/2020 No NCT2792 34 T-VEC No TSA TNBC T-VEC I/II A Phase 1/2 Study of Talimogene Laherparepvec in Combination With Neoadjuvant Chemotherapy in Triple Negative Breast Cancer 5/2/2017 Yes NCT9855 Paclitaxel 35 VG161 No TSA Gastric Cancer oHSV-1 (Vero Cell) I/II A Dose Ascending, Open Phase Ib/IIa Clinical Study to Evaluate the Safety, Tolerability, Pharmacokinetic Characteristics and Preliminary Efficacy of VG161 Combined With Nivolumab Injection in Subjects With Metastatic Gastric Cancer 11/17/2022 No NCT8925 Nivolumab 36 HCC oHSV-1 (Vero Cell) I/II An Open-label Phase Ib/IIa Clinical Trial of VG161 Combined With Camrelizumab in the Treatment of Advanced Primary Hepatocellular Carcinoma 11/30/2023 No NCT4001 Camrelizumab 37 VG2025 No TSA Advanced Solid Tumor VG2025 Monotherapy I A Dose Escalation, Open-label Phase I Clinical Study to Evaluate the Safety, Tolerability, Pharmacokinetics and Biologic Effect of VG2025 in the Treatment of Patients With Advanced Malignant Solid Tumors 8/23/2022 No NCT7849 AML: Acute Myeloid Leukemia, B7.1: CD80, BCC: Basal Cell Carcinoma, CAR: chimeric antigen receptor, CCL21: C-C motif chemokine ligand 21, CEA: carcinoembryonic antigen, CRC: Colorectal Cancer, CIK: cytokine-induced killer, CMV: Cytomegalovirus, CNS: central nervous system, DIPG: Diagnosed Diffuse Intrinsic Pontine Glioma, dMMR: deficient mismatch repair, DCs: Dendritic Cells, Hank: High-affinity Natural Killer, HCC: Hepatocellular Carcinoma, HER-2: human epidermal growth factor receptor 2, HPV: human papillomavirus, HSV: herpes simplex virus, GBM: Glio-Blastoma Multiforme, GEJ: Gastro Esophageal Junction, ICAM-1: intercellular adhesion molecule-1, IL: interleukin, LFA-3: lymphocyte function-associated antigen-3, mCRC: metastatic colorectal cancer, MSI-H: microsatellite instability-high, MUC-1: mucin 1, MVA: Modified Vaccine Ankara, NSCLC: Non-Small Cell Lung Cancer, oAV: Oncolytic Adenovirus, oHSV: Oncolytic HSV virus, OV: Oncolytic Virus, oVV: Oncolytic Vaccinia Virus, Pd-1: Programmed Cell Death Protein 1, pMMR: proficient mismatch repair, MSS: microsatellite stability, PRROC: Platinum-Resistant/Refractory Ovarian Cancer, PSA: Prostate Specific Antigen, RCC: Renal Cell Carcinoma, rH-GM-CSF: Recombinant Granulocyte-Macrophage Colony Stimulating Factor, RP1: Vusolimogene oderparepvec, SCC: Squamous Cell Carcinoma, SCCHN: Squamous Cell Carcinoma of head and neck, SCLC: Small Cell Lung Cancer, Sr. No: Serial Number. TAA: Tumor-Associated Antigen, TNBC: Triple Negative Breast Cancer, TRICOM: TRIad of COstimulatory Molecules, TSA: Tumor-Specific Antigen, T-VEC: Talimogene Laherparepvec, WT: Wild Type.

Pexa-Vec is one of the most extensively studied oncolytic Vaccinia Virus (oVV). JX-594 (Pexa-Vec) is an engineered vaccinia virus, used initially as a vaccine for smallpox, now repurposed for cancer therapy. It selectively infects and lyses tumor cells due to their defective antiviral responses. Pexa-Vec also expresses GM-CSF, promoting immune cell recruitment. Strong immune-stimulating properties make it suitable for combination with immunotherapies, especially in advanced liver cancer. 103 , 120 However, phase III trials in combination with sorafenib have failed to improve outcomes as compared to SOC. 103 , 121 A Phase I/II clinical trial of Pexa-Vec in combination with cemiplimab in RCC is ongoing. 122

Several other oVVs are also being investigated. Double deleted vaccinia virus (vvDD), derived from the most potent strain of vaccinia virus (Western Reserve Strain), has shown promising outcomes. 123 vvDD was engineered to co-express somatostatin receptor and cytosine deaminase, generating a vvDD-CDSR strain, also known as JX-929, which has demonstrated tolerable safety as well as tumor specificity. 103 , 123 , 124 Various oVVs currently undergoing phase I clinical study include hV01 (recombinant human IL-21 oVV), IDOV-SAFE, GC001, and TG6050. 125–128 Based on previous trials showing a lower effect on eliciting an immune response by systemic administration, most of these trials are now evaluating intra-tumoral drug route. One exception is IDOV-SAFE, which remains a systemically-administered product. 103 , 126 While the majority of development is occurring in advanced solid tumors, RGV004 is also being investigated in B-cell lymphoma. 129

Among chemotherapy combination trials, KM1 and Olvi-Vec (Olvimulogene nanivacirepvec) are currently being evaluated. KM1 is a genetically modified recombinant vaccinia virus that has demonstrated therapeutic efficacy in various solid tumors, including ovarian cancer. In the ongoing phase 1 study, KM1 is administered via intraperitoneal infusion in combination with chemotherapy. The physician’s choice of agents includes carboplatin or cisplatin, gemcitabine, taxanes, or pegylated liposomal doxorubicin, with or without bevacizumab. 130 Olvimulogene nanivacirepvec (Olvi-Vec), currently in phase 2 testing, has shown broad infectivity and therapeutic potential across a variety of tumor types, including NSCLC and platinum-resistant/refractory ovarian cancer (PRROC). In the ongoing phase 2 trial (VIRO-25), Olvi-Vec is being evaluated in combination with platinum-doublet chemotherapy and ICI. 131 Building on the efficacy and safety results from the phase 2 VIRO-15 trial, a phase 3 trial (OnPrime GOG-3076) is now assessing its role in combination with chemotherapy – including carboplatin, cisplatin, gemcitabine, taxanes, or pegylated liposomal doxorubicin – in conjunction with bevacizumab for PRROC. 132 Another vaccine, VET3-TGI, is oncolytic immunotherapy currently in phase 1 evaluation (STEALTH-001 trial) to treat advanced cancers. Although it has not yet been administered to human patients, it has been designed to selectively infect and kill cancer cells while sparing healthy tissue. The phase 1 study consists of dose escalation and expansion groups. Group A evaluates the highest tolerated dose (HTD) of VET3-TGI administered intratumorally, while Group C determines the HTD when administered intravenously. Groups B and D assess the combination of VET3-TGI with pembrolizumab, starting at the HTD identified in Groups A and C, respectively. 133 Additionally, BT-001, an oncolytic vaccinia virus containing genes encoding the 4-E03 human recombinant anti-hCTLA4 antibody and human GM-CSF, is under investigation in a phase 1/2 trial. This trial is evaluating its combination with pembrolizumab for metastatic melanoma, TNBC, and Merkel cell carcinoma. 134

Adenovirus

Adenovirus, a non-enveloped virus with a double-stranded DNA genome, has more than 50 known serotypes that can cause human illness. 135 They are highly efficient in transducing both dividing and non-dividing cells, making them a promising tool in cancer vaccine development. 106 , 136 Key advantages include their well-characterized genome, ease of genetic manipulation, and broad tropism. 135 Advances in genetic engineering have further improved their safety and efficacy. 107 This has paved the way for various vaccine platforms targeting specific TAAs, including CEA, Mucin 1 (MUC1), and PSA.

One notable example is ETBX-011, an engineered adenoviral vaccine that encodes a modified CEA ( Table 3 ). This vaccine includes the highly immunogenic epitope CAP1-6D, which has been shown to induce CEA-specific, cell-mediated immune responses with potent antitumor activity. 137 Initial Phase 1/2 trials in patients with metastatic CRC (mCRC) demonstrated favorable safety and immunogenicity profiles. 138 , 139 However, the Phase II trial evaluating (Adenovirus type 5) Ad5-CEA combination with avelumab vs SOC (FOLFOX + bevacizumab) in mCRC did not show sufficient improvement in progression-free survival, although it did generate multifunctional CD4+/CD8+ T cells specific to MUC1 and brachyury. 140 An ongoing trial now compares the vaccine with regorafenib in mCRC. 97

To address the challenge of tumor heterogeneity, recombinant adenoviral vaccines targeting a broader range of TAAs have shown promise in preclinical models. 137 , 141 An ongoing Phase 2 trial 142 is further evaluating the Tri-Ad5 vaccine regimen in combination with the IL-15 superagonist nogapendekin alfa inbakicept ( N -803) in Lynch Syndrome. The goal is to assess its effectiveness in preventing cancer in this high-risk population. Ad5-PSA, an adenoviral vaccine encoding PSA and prostate stem cell antigen, has also demonstrated strong preclinical efficacy. 143 This was followed by a Phase 1 trial, which confirmed the vaccine’s safety. 137 , 144 Future studies will likely evaluate the vaccine’s clinical benefits when combined with other immunotherapy agents or palliative radiation therapy. 145

A related trial 146 investigates Maraba’s product, MG1-MAGEA3, both as monotherapy and with a prime/boost strategy incorporating an adenovirus vaccine also targeting MAGEA3 (Melanoma-associated antigen). Early results suggest that the Ad-MAGEA3 prime followed by the MG1-MAGEA3 boost is tolerable and determined a recommended Phase 2 dose. 147 Trial NCT9760 is evaluating the combination of MG1-MAGEA3 with Ad-MAGEA3 with pembrolizumab in NSCLC; accrual is complete, and results are pending. 148 In addition, novel platforms are being developed to further enhance immune responses in hard-to-treat cancers. One such platform is the PRGN-2009 therapeutic gorilla adenovirus-based vaccine, which targets HPV-associated malignancies. PRGN-2009 contains multiple cytotoxic T cell epitopes targeting the viral oncoproteins HPV 16/18 E6 and E7, including T cell enhancer agonist epitopes. Preclinical studies have shown a significant reduction in tumor volume in humanized mouse models. 149 A Phase I study demonstrated that PRGN-2009, both as monotherapy and in combination with bintrafusp alfa (BA) – a bifunctional Tumor Growth Factor (TGF)-β trap/anti-PD-L1 fusion protein – was safe, well-tolerated, and induced HPV-specific T-cell immune responses in patients with recurrent/metastatic HPV-associated cancers. The combination of PRGN-2009 and BA exhibited clinical activity, in both patients naïve or resistant to ICI. 150 A Phase II study is currently evaluating the combination of PRGN-2009 with pembrolizumab, with biological correlative assays underway. 151

Another innovative approach is the development of Nous-209, a cancer vaccine aimed at targeting multiple neoantigens shared across both sporadic and hereditary microsatellite instability (MSI)-high tumors. 152 Nous-209 uses a heterologous prime/boost strategy, combining two multivalent viral vector platforms: GAd-209, which consists of four great ape adenovirus (GAd) vectors, and MVA-209, which contains four MVA vectors. Each vector encodes a synthetic polypeptide, allowing for broad neoantigen coverage. 153 Currently, Nous-209 is being tested in a Phase 1/2 trial 154 in patients with metastatic mismatch repair-deficient (dMMR) gastric, CRC, and gastroesophageal junction (GEJ) tumors, in combination with pembrolizumab. Interim results have demonstrated that the vaccine, when combined with pembrolizumab, is safe, immunogenic, and shows early signs of clinical efficacy. 155 Moreover, Nous-209 is also being explored as a preventive vaccine for Lynch syndrome carriers without any existing malignancies. 156

Adenoviral vectors have also been widely utilized in gene therapy applications, particularly in DC vaccines. These vectors are highly effective at transducing DCs, which are crucial in initiating adaptive immune responses. Several trials have explored autologous DC adenovirus CCL21 vaccines in NSCLC. While one trial 157 was terminated, another 158 demonstrated promising immune responses in advanced NSCLC, although detailed results are yet to be reported. An active but non-recruiting trial 159 evaluates the combination of the CCL21 adenovirus vaccine with pembrolizumab in Stage IV NSCLC. Ongoing trials, such as NCT8663, are investigating adenoviral-transfected DC vaccines combined with cytokine-induced killer (CIK) cells in patients with acute myeloid leukemia (AML), targeting antigens like MUC1 and survivin. 160

In addition, adenoviruses have emerged as powerful tools in developing OVs. One of the earliest OVs, Oncorine, was approved for clinical use in China in 2005 103 . However, as monotherapy, it failed to demonstrate sufficient therapeutic efficacy in patients with refractory solid tumors. 103 ONYX-015, one of the first attenuated adenoviruses, was designed to selectively replicate in p53-deficient tumor cells, a common mutation in many cancers, while leaving normal tissue unaffected. Since then, newer oncolytic adenoviruses (oAV) have been developed, including ONCOS-102, LoAd703, TILT-123, ORCA-010, and CG0070 (Cretostimogene grenadenorepvec). These viruses incorporate the intact E1B 55 kDa gene, which enhances viral replication and has been engineered to replicate in cancer cells preferentially. 103 Additionally, many of these oAVs feature modified fibers on their capsids, improving their ability to infect cancer cells independently of the coxsackie and adenovirus receptors, further enhancing their therapeutic potential. 161 , 162

Several oAVs are undergoing clinical trials ( Table 3 ). LOAd703 is an immunostimulatory gene therapy utilizing a selectively replicating adenovirus as a gene vehicle. The virus expresses the transgenes trimerized membrane-bound isoleucine zipper (TMZ)-CD40L and 41BBL under the control of a cytomegalovirus (CMV) promoter. The LOKON002 trial, a Phase I/II study, evaluated LOAd703 in patients with pancreatic, biliary, ovarian, and CRC cancers. 163 Results from LOKON002 showed that combining LOAd703 with nab-paclitaxel plus gemcitabine in patients with advanced pancreatic ductal adenocarcinoma was feasible and safe. 164 It also showed that LOAd703 has the ability to generate an inflamed tumor microenvironment in immunologically “cold” tumors, thereby priming them for ICIs or other immunotherapies, such as adoptive T or NK cell transfer. 165 Additionally, the LOKON001 trial is assessing LOAd703 administered via intratumoral injections for pancreatic cancer, combined with SOC treatment, including gemcitabine and nab-paclitaxel, with or without the anti-PD-L1 antibody atezolizumab. 166 Another oncolytic adenovirus, H101—a recombinant human Ad5—is being evaluated in various trials. In unresectable intrahepatic cholangiocarcinoma, H101 is combined with FOLFOX chemotherapy, while in advanced mesothelioma, it is being studied in combination with PD-1 inhibitors to reverse immune resistance. 167 , 168 In recurrent cervical cancer, it is being investigated along the anti-PD-1 antibody camrelizumab via intratumoral injections. 169

TILT-123, another oncolytic Ad5 engineered to express TNF-α and IL-2, has emerged as a key therapeutic agent, with trials in melanoma, ovarian carcinoma, and head and neck cancers, where it is being tested in combination with ICIs. 170–173 The TUNIMO trial demonstrated that TILT-123 was safe and capable of inducing antitumor effects in both local and distant lesions in heavily pre-treated patients. 174 Across three trials – TUNIMO, TUNINTIL, and PROTA – TILT-123 was well-tolerated, with no DLTs reported, and it showed significant intratumoral immunomodulation following a single intravenous administration. 175 Similarly, CG0070, another oAV has shown encouraging results. This conditionally replicating dsDNA-based Ad5 is genetically engineered with the essential E1a gene under the control of the human E2F1 promoter. A Phase 1 trial combining CG0070 with nivolumab for cisplatin-ineligible muscle-invasive bladder cancer (MIBC) reported no dose-limiting toxicities, achieving a 42.1% pathologic complete response rate and a 70.4% 1-year recurrence-free survival, highlighting the potential of this combination regimen to enhance therapeutic efficacy in cisplatin-ineligible patients with muscle-invasive bladder cancer. 176

Another notable viral vector is Seneca Valley Virus (SVV-001), a replication-competent oncolytic picornavirus initially discovered as a contaminant in laboratory adenovirus preparation. 177 SVV-001 was evaluated in a Phase 1 clinical trial, where a single IV dose was well tolerated in patients with advanced solid tumors exhibiting neuroendocrine features. The study reported a clinical benefit rate of 85% of neuroendocrine neoplasms. 178 Future trials are planned in combination with ICI, focusing on solid tumors with enhanced patient selection guided by biomarker-driven approaches. Other promising trials include ORCA-010, a novel oAV being evaluated for intratumoral administration in treatment-naïve patients with localized prostate cancer 179 and DNX-2401, 180 designed to be delivered intra-arterially for recurrent glioblastoma. Newer OVs, such as MEM-288 181 and Ixovex-1, 182 are currently under investigation for use in NSCLC and head and neck cancers, often in combination with ICI. Additional trials are exploring the efficacy of oAV such as CAdVEC 183 and BioTTT001 184–186 in HER2-positive solid tumors. Further details of ongoing trials are available in Table 3 .

Herpes viruses

HSV is a promising vector for the delivery of TAAs due to its ability to infect a broad range of dividing and non-dividing host cells, accept large DNA inserts and a good safety profile. 109 Deleting the glycoprotein H gene from HSV results in a “disabled” virus that is only capable of one round of infection. 109 , 187 , 188 HSV has also been utilized as a vector for oncolysis. Like adenovirus, oncolytic HSV (oHSV) can promote antitumor immune response. 103 , 189 , 190 Derived from HSV-1, T-VEC is the first FDA-approved OV for melanoma. 191 T-VEC has been modified to preferentially replicate in tumor cells and produce GM-CSF. T-VEC’s ability to generate tumor-specific immune responses and induce systemic immunity has made it a promising candidate for combination with ICI. 191 Imlygic (T-VEC oHSV) remains the only oHSV to be approved by the US FDA and EMA to date. 103 T-VEC is also under investigation in new indications (i.e., in combination with paclitaxel for TNBC. 192

Multiple trials are currently underway to evaluate the efficacy and safety of oHSV across various malignancies ( Table 3 ). CAN-3110,a replication-competent HSV-1, is being developed for adult patients with recurrent high-grade glioma. 193 A Phase I trial in recurrent GBM demonstrated that CAN-3110 preferentially replicates in tumor cells due to its association with nestin, a protein overexpressed in GBM but absent in healthy brain cells. 194 Similarly, C134, a second-generation chimeric Human CMV/HSV-1 oncolytic virus, has been engineered for late viral protein synthesis and enhanced intra-tumor replication. It has shown a favorable safety profile in preclinical models and has advanced to a Phase I trial for primary CNS malignancies. 195 , 196 HSV-1 G207 is another genetically engineered oHSV lacking genes essential for replication in normal brain tissue. Phase I trial showed acceptable safety and responses in patients with recurrent or progressive pediatric high-grade gliomas. 196 , 197 M032 is also being evaluated in recurrent glioblastoma and anaplastic astrocytoma, both as a standalone therapy and in combination with ICIs. 198 , 199 Beyond its oncolytic activity, M032 functions as a gene therapy vector, delivering IL-12 directly into tumor cells, thereby enhancing the anti-tumor immune response. 200

Multiple recombinant oHSVs have also been developed. 201 These recombinant oHSVs are mutated or multimutated conditionally replicating recombinants, restricting the viral replication and final cell lysis to actively replicating cancer cells. 202 Amongst ongoing trials, RP1 is being tested for cutaneous squamous cell carcinoma, Merkel cell carcinoma, and melanoma in combination with ICI, 203 , 204 while RP2 and RP3 are in trials for uveal melanoma and mCRC, paired with ICI. 205 , 206 The recombinant OV R130 is under early-phase investigation for a range of advanced solid tumors. 207–209

Amongst other oHSV with ongoing trials, BS-006 is under investigation in the Phase I trial for intratumoral injection in cervical cancer. 210 OH2, is being explored across several cancers, including bladder cancer, CNS tumors, and melanoma, with combination approaches involving pembrolizumab in certain cases. 211–214 OH2, a genetically engineered oHSV type 2, was designed to selectively amplify tumor cells and express GM CSF to enhance antitumor immune responses. 215 T3011 is another promising candidate being tested for advanced solid tumors such as CRC and lung cancer, administered either intratumorally or via hepatic artery infusion 216 , 217 T3011 is a replication-competent, genetically modified, next-generation oHSV. Deletion of one ICP34.5 copy and retention of ICP47 maximizes its replication activity and virulence attenuation. Moreover, T3011 was inserted with biologically active IL-12 and anti-PD-1 antibody genes. Therefore, while lysing tumor cells, T3011 can significantly improve the TME and elicit the body’s specific anti-tumor immunity. 218

Conclusion

Cancer vaccines represent a rapidly evolving frontier in immunotherapy, stimulating the immune system to recognize and attack TAAs or TSAs. Each bacterial, viral, or yeast-based vector platform has unique advantages and limitations, and at present, there is no clear superior platform. Bacterial platforms such as LM and Clostridium excel in targeting hypoxic tumor environments. Viral vectors like adenovirus and HSV are highly effective in generating strong immune responses and direct oncolytic effects. Yeast-based vaccines offer stability, scalability, and the ability to present multiple tumor antigens to the immune system. Studies to date show that while cancer vaccines are active products, monotherapy administration is insufficient to generate adequate efficacy data necessary for regulatory approval. As such, sponsors of investigational therapeutic vaccines routinely incorporate other immune-stimulating products (whether approved or investigational) into their development programs. Beyond synergistic strategies and immunogenic intensification, there is the potential to enhance efficacy by targeting tumor neoantigens (i.e., personalized vaccines). While therapeutic vaccines as a class have been shown to be generally well tolerated and often have a more favorable safety profile relative to that of cytotoxics or other immuno-oncology products, further efficacy data will support making broad claims about the future role of therapeutic vaccines in the treatment of cancer.

Acknowledgments

The authors acknowledge Sylvester Comprehensive Cancer Center and Adam Weiss for their support in producing this article.

Marijo Bilusic’s current research interests focus on tumor immunology and the development of novel immunotherapy approaches for prostate cancer and other genitourinary tumors using therapeutic cancer vaccines, antibodies, immune modulators or immune checkpoint inhibitors, not only as monotherapies but in combination with other therapies as part of an immuno-oncology programmatic effort. He received his M.D. from the University of Zagreb School of Medicine and completed his Ph.D. training at the University of Split School of Medicine. In addition, he completed postdoctoral research fellowship in physiological genomics at the Medical College of Wisconsin’s Human and Molecular Genetics Center. He completed a clinical fellowship in medical oncology and hematology at the National Cancer Institute (NCI) and the National Heart, Lung, and Blood Institute. He served as an assistant professor at the Fox Chase Cancer Center and then joined the NCI Genitourinary Malignancy Branch as an Associate Research Physician. At the NCI, he also served as fellowship Program Director from July 2018 – July 2021. His passion for teaching and mentoring has earned multiple awards at NIH, including the National Institutes of Health (NIH) Director’s Award. In 2021, Dr. Bilusic joined Sylvester Comprehensive Cancer Center/University of Miami as Professor of Medicine and GU Medical Oncology Team Lead.

Disclosure statement

CJ: None; AM: None; PD: Pyxis Oncology - Consultant; MB: Hookipa Pharma, Inc. Research funding – institutional.

Author contributions

CJ, PD, AM and MB wrote the manuscript jointly and contributed equally to this work. All authors were involved in the conception, design, analysis, and interpretation of data, drafting the paper, and revising the paper for critical content. All authors have read and approved the final version of the manuscript.

Ethical approval

Ethical approval was not required for this study, as it is a review article.

Abbreviations

Ad5 Adenovirus 5 AE Adverse event AKT/mTOR Protein kinase B/mammalian target of rapamycin AML Acute myeloid leukemia APC Antigen-presenting cell B7.1 CD80 BA Bintrafusp alfa BC Breast cancer BCG Bacillus Calmette-Guérin CAR Chimeric antigen receptor CBM588 C. butyricum MIYAIRI 588 CDEPT Clostridial-directed enzyme prodrug therapy CEA Carcinoembryonic antigen CG0070 Cretostimogene grenadenorepvec CIK Cytokine-induced killer CMV Cytomegalovirus CRC Colorectal cancer DC Dendritic cell DLT Dose-limiting toxicities dMMR Mismatch repair-deficient DNA Deoxyribonucleic acid EMA European Medicines Agency FDA Food and Drug Administration GAd Great ape adenovirus GBM Glioblastoma multiforme GEJ Gastro-esophageal junction GI-6207 Yeast-CEA vaccine GM-CSF Granulocyte-macrophage colony-stimulating factor GVAX GM-CSF – transduced autologous tumor-cell vaccine HERV Human endogenous retroviral element HIF Hypoxia-inducible factor HPV Human papillomavirus HSV Herpes simplex virus HTD Highest Tolerated Dose ICAM-1 Intercellular adhesion molecule 1 ICI Immune checkpoint inhibitor IFN Interferon IL Interleukin JNJ Johnson & Johnson KBMA Killed but metabolically active LADD Live attenuated double deleted LFA-3 Lymphocyte function-associated antigen 3 Lm Listeria monocytogenes Lm-RIID Lm recombinase-induced intracellular death MAGE Melanoma-associated antigen mCRC Metastatic CRC MHC Major histocompatibility complex MIBC Muscle Invasive Bladder Cancer MSI Microsatellite instabillity MUC1 Mucin 1 MVA Modified Vaccinia ankara MVA-BN MVA-Bavarian Nordic NK Natural killer NMIBC Non-muscle-invasive bladder cancer NY-ESO-1 New York esophageal carcinoma antigen1 NSCLC Non-small cell lung cancer oAV Oncolytic adenovirus oHSV Oncolytic HSV virus Olvi-Vec Olvimulogene nanivacirepvec OV Oncolytic virus oVV Oncolytic vaccinia virus PAMP Pathogen-associated molecular pattern PD-L1 Programmed death ligand 1 PK Pharmaco-kinetics pLADD Personalized live attenuated double deleted PRR pattern recognition receptor PSA Prostate-specific antigen RCC Renal cell carcinoma RNA Ribonucleic acid SOC Standard of care ST Salmonella typhimurium TAA Tumor-associated antigen TGF Transforming growth factor TLR Toll-like receptor TNF- α Tumor-necrosis factor-alpha TME Tumor microenvironment TNBC Triple negative breast cancer TRAIL Tumor necrosis factor-related apoptosis-inducing ligand T-VEC Talimogene laherparepvec TSA Tumor-specific antigen TURBT Transurethral bladder tumor resection VEGF Vascular endothelial growth factor VLP Virus-like particle vvDD Double deleted vaccinia virus YE-NEO Neoepitope yeast vaccine

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