Emerging strategies in cancer immunotherapy: Expanding horizons and future perspectives

CTLA-4 inhibitors

CTLA-4 inhibitors work by blocking the interaction between CTLA-4 and its ligands CD80 and CD86, thereby enhancing T-cell activation and triggering a stronger immune response against tumors. These inhibitors have been clinically approved for treating various cancers, including melanoma, RCC, bladder cancer, and advanced non-small-cell lung cancer.

Ipilimumab, the first anti-CTLA-4 antibody, was approved by the United States (US) Federal Drug Administration (FDA) in 2011 in 2011 for treating advanced melanoma.^[15] Since then, six additional ICIs have gained approval, including three anti-PD-1 and three anti-PD-L1 antibodies [Table 3]. Ipilimumab has significantly improved overall survival (OS) in patients with advanced melanoma. Phase III trials demonstrated its superiority over chemotherapy as a first-line treatment and over a less effective vaccine in second-line therapy.^[14] Ipilimumab works by lifting T-cell suppression, facilitating T-cell activation and proliferation, and increasing the diversity of T-cell populations by blocking the CD28-B7 costimulatory pathway, thereby broadening the T-cell repertoire.^[19]

PD-1 inhibitors and PD-L1 inhibitors

PD-1 inhibitors belong to a group of immunotherapy drugs that have demonstrated significant clinical advantages in treating different forms of cancer. These drugs function by obstructing the PD-1 receptor found on activated T-cells, thereby preventing its interaction with its ligands, PD-L1 and PD-L2. On the other hand, PD-L1 inhibitors represent another category of immunotherapy agents that target the PD-L1 protein. These inhibitors operate by impeding the connection between PD-L1 and its receptor, PD-1, on immune cells. This interaction between PD-1 and its ligands causes a suppression of T-cell activity, enabling cancer cells to evade detection by the immune system. Through the inhibition of the PD-1 pathway, PD-1 inhibitors not only boost the immune response against tumors but also reinstate the functionality of T-cells. The approved clinical uses for PD-1 inhibitors and PD-L1 inhibitors cover various cancer types, including melanoma, non-small-cell lung cancer, RCC, and Hodgkin’s lymphoma (HL). Furthermore, ongoing investigations explore their potential efficacy in addressing other malignancies, such as head-and-neck squamous cell carcinoma (HNSCC) and various solid tumors.

Nivolumab, a PD-1 inhibitor, was approved by the FDA in 2014 for treating metastatic or unresectable melanoma, particularly in patients who failed ipilimumab treatment or had B-Raf proto-oncogene (BRAF) V600 mutation-positive tumors or disease progression post-BRAF inhibitor therapy.^[20] Early trials of nivolumab showed durable tumor shrinkage in patients with advanced solid tumors, including melanoma.^[19] Building on these results, subsequent investigations into a multi-dose regimen of nivolumab revealed that around 20– 30% of patients with advanced, treatment-resistant melanoma, non-small-cell lung cancer, or kidney cancer experienced objective tumor shrinkage.^[21] In India, efforts to optimize nivolumab’s therapeutic benefits and cost-effectiveness have garnered attention. John et al. (2023) highlighted the advantages of weight-based dosing, showing that it maintains clinical efficacy while significantly reducing financial toxicity, thereby improving treatment accessibility.^[22] Similarly, Patel et al. (2024) conducted a retrospective analysis that found weight-based dosing achieved comparable outcomes to fixed-dose regimens but at a lower cost, making immunotherapy more feasible for patients in low- and middle-income countries.^[23] Further advancements in treatment strategies include combining low-dose nivolumab with metronomic chemotherapy. A study by Kate et al. (2024) demonstrated this combination improved OS in an Indian cohort of patients with advanced head-and-neck cancer, a population significantly affected by tobacco-related malignancies.^[24] Similarly, clinical trial results of comparing the OS of patients treated with traditional metronomic chemotherapy combined with a low dose (20 mg) of nivolumab in HNSCC demonstrated superiority in OS compared to the traditional metronomic chemotherapy.^[25] These findings underscore the potential of tailored dosing and combination therapies to expand access and improve outcomes for patients in resource-constrained settings. Pembrolizumab, a humanized monoclonal IgG4 kappa antibody that blocks the interaction between PD-1 and its ligands, allowing cytotoxic T cells to recognize and attack tumour cells more effectively.^[26] It received FDA approval in 2016 for treating recurrent or metastatic HNSCC in patients whose disease has progressed following platinum-containing chemotherapy.^[20] Initially administered as a weight-based dose of 2 mg/kg – since higher doses did not improve efficacy – Pembrolizumab later shifted to a flat dosing regimen of 200 mg every 3 weeks. While this fixed dosing approach was implemented for simplicity, it has faced criticism for potential cost inefficiencies without offering advantages in controlling pharmacokinetic (PK) variability. Current evidence indicates that both weight-based and fixed-dose regimens are viable options for pembrolizumab.^[27] However, PK modeling suggests that reverting to weight-based dosing of pembrolizumab could preserve therapeutic efficacy while significantly reducing overall treatment costs.

Pidilizumab (CT-011), a humanized IgG-1κ mAb targeting PD-1, has shown preclinical efficacy in inhibiting tumor growth in various cancers, including melanoma, lymphoma, lung, colon, and breast cancers, as well as prolonging survival in mouse models.^[28]

Atezolizumab, a modified humanized monoclonal immunoglobulin G1 antibody, specifically binds to PD-L1, preventing its interaction with PD-1 and B7-1, while leaving the interaction between PD-L2 and PD-1 intact.^[29] This alteration eliminates antibody-dependent cell-mediated cytotoxicity, thus avoiding potential loss of PD-L1-expressing T-effector cells and reducing anticancer immunity.^[23] In clinical trials, atezolizumab has demonstrated enduring responses in a group of patients with metastatic bladder cancer, particularly in those with higher levels of PD-L1 expression on tumor-infiltrating immune cells, as observed in a Phase I study. Moreover, in Phase II trials, atezolizumab has elicited persistent anti-tumor responses in patients with advanced urothelial carcinoma whose tumors progressed during or after treatment with platinum-based chemotherapy.^[30] Atezolizumab, combined with chemotherapy, represents a significant breakthrough as the first immunotherapy approved for the first-line treatment of extensive small-cell lung cancer (SCLC).^[31] Real-world studies from countries such as Japan, Canada, and Turkey have validated its effectiveness in SCLC. In addition, clinical trials such as IMpower110 and IMpower132 demonstrated the efficacy of atezolizumab, either as monotherapy or in combination with chemotherapy, for non-squamous non-SCLC (NSCLC). Notably, IMpower110 revealed a significantly longer OS with atezolizumab compared to chemotherapy in NSCLC patients with high PD-L1 expression, regardless of histological type.^[31]

Durvalumab, a fully human IgG1k mAb against PD-L1, is under development by AstraZeneca for the treatment of advanced or metastatic urothelial cancer, non-small-cell lung cancer, and extensive-stage small cell lung cancer.^[32] By blocking the PD-L1/PD-1 and PD-L1/CD80 interactions, durvalumab prevents the inhibition of T-cell activity, leading to enhanced recognition and destruction of tumor cells without causing antibody-dependent cytotoxicity.^[33]

Breast cancer

CAR-T-cell therapy has demonstrated promising outcomes in treating triple-negative breast cancer (TNBC), particularly by targeting Tumor-associated MUC 1 with high specificity. Mucin-2 8 CD3 zeta chain (MUC28z) CAR-T-cells, which incorporate CD3ζ and CD28 signaling domains, have been shown to increase cytokine production, including IFN -γ and Granzyme B, leading to the suppression of TNBC cell proliferation and improved survival in xenograft models.^[38]

In addition, researchers have explored natural killer (NK) NK group 2D ligands (NKG2DL) as potential immunotherapy targets for TNBC. CAR-T-cells engineered by combining full-length NKG2DL with the CD3ζ cytoplasmic domain, along with endogenous DNAX-activating protein 10 (DAP10) costimulation, have demonstrated cytokine secretion, cytotoxicity, and in vivo tumor suppression. A Phase I clinical trial (NCT04107142) is currently investigating the safety and tolerability of these CAR-T-cells in patients with R/R solid tumors, including TNBC.^[39]

Human epidermal growth factor receptor 2 (HER2) is overexpressed in a significant percentage of breast cancers and represents a promising target for CAR-T therapy. Preclinical studies with HER2-targeted CAR-T cells have shown tumor growth inhibition, metastasis regression, and potential efficacy against trastuzumab resistance.^[40]

Moreover, mesothelin (MSLN) is gaining attention as a biomarker in breast cancers, particularly TNBC. CAR-T therapies directed toward MSLN have demonstrated potent antitumor activity in preclinical models. Ongoing clinical trials (NCT02792114 and NCT02414269) are assessing the safety and efficacy of these therapies in patients with MSLN-positive breast cancers.^[30-32]

Pancreatic tumor

CAR-T-cell therapy has shown promise in treating pancreatic cancer, with significant efficacy observed in vitro and in xenograft models. Enhancing CAR-T-cells with the chemokine receptor CXC chemokine receptor 2 (CXCR2) has improved their migration toward Interleukin 8 (IL-8), resulting in notable antitumor activity against αvβ6-expressing pancreatic tumors in animal models.^[38]

Dual-targeting strategies have emerged as a potential approach to improve CAR-T-cell efficacy in pancreatic cancer. By targeting both carcinoembryonic antigen (CEA) and MSLN, researchers have achieved precise tumor targeting and a reduced tumor burden in pancreatic cancer models.^[33] In addition, modifying IL-8 receptors in CARs, in combination with CD7 0 enhancement, has been shown to enhance CAR T-cell efficacy in pancreatic cancer therapy. Moreover, the production of IL-7 and chemokine (C-C motif ligand 19) in 7 × 19 CAR T-cells demonstrated superior antitumor activity against pancreatic cancer compared to standard CAR-T cell therapy, as reported in preclinical studies.^[41]

Thyroid cancer

Thyroid cancer incidence is rising rapidly, and the thyroid-stimulating hormone receptor (TSHR) is a highly expressed glycoprotein receptor in most thyroid cancers.^[34] Consequently, CAR T-cells engineered with two co-stimulatory domains and targeting the TSHR antigen have shown both safety and potent efficacy in treating differentiated thyroid cancer, releasing elevated levels of IL-2, IFN -γ, tumor necrosis factor-alpha (TNF-α), and Granzyme-B compared to conventional T-cells.^[42]

In addition, there exists a positive correlation between the expression levels of Intercellular Adhesion Molecule 1 (ICAM-1) RNA and the aggressiveness of papillary thyroid cancer, often driven by mutually exclusive somatic mutations, BRAFV600E or mutated Rat Sarcoma (RAS) is a promising therapeutic target in thyroid cancer.^[43] To specifically target ICAM-1, a scFv derived from the ICAM-1–specific R6.5 mAb was integrated into a third-generation CAR construct comprising intracellular CD3ζ, CD28, and 4–1BB (CD13 7) signal transduction domains. This approach significantly reduced tumor burden in metastatic and aggressive thyroid cancer, prolonging OS in animal models xenografted with autologous anaplastic thyroid carcinomas (ATC) tumors.^[43] AIC100, a CAR-T therapy developed by AffyImmune Therapeutics, is being tested in Phase I clinical trials (NCT04420754) for treating ATC and poorly differentiated thyroid cancers.^[44]

Brain cancer

Epidermal growth factor receptor variant III (EGFRvIII), a mutated variant of the epidermal growth factor receptor (EGFR) resulting from an in-frame deletion spanning exons 2–7, is prevalent in various cancers. In glioblastomas (GBMs), approximately 40% of newly diagnosed patients exhibit EGFR gene amplification, with around 50% of EGFR-amplified GBM cases featuring the constitutively oncogenic EGFRvIII.^[37] The mutation-induced alteration in the extracellular domain structure presents a unique epitope targeted by specific mAbs with minimal risk of on-target/off-tumor toxicity.^[45] Consequently, both vaccine and CAR-T cell therapies directed at EGFRvIII have been meticulously developed. In preclinical studies, EGFRvIII CAR T-cells demonstrated significant efficacy in reducing tumor growth. However, translating this success to GBM patients has been somewhat limited, with EGFRvIII-specific CAR-T-cells showing restricted efficacy.

Advancing CAR-T-cell therapy requires identifying a stable and specific tumor-associated antigen (TAA) exhibiting heterogeneity throughout the tumor region. Meeting these criteria, a suitable target has been identified. A study demonstrated the in vivo therapeutic effects of intracranial delivery of chondroitin sulfate proteoglycan 4 (CSPG4)-CART-cells in nude mice transplanted with CSPG4-expressing glioma cells or GBM neurosphere models.^[46] This research marks a significant step forward in the pursuit of effective CAR-T-cell therapies in the complex landscape of brain cancer.

Interleukin 13 receptor alpha 2 (IL13Rα2), a receptor involved in regulating inflammation, binds to IL-1 3 and is found in more than 75% of GBMs, which correlates with the aggressiveness of the tumor and poor prognosis. Its limited presence in normal brain tissue suggests that IL13Rα2 could be a promising target for CAR-T-cell therapy in treating GBM.^[47] In a pioneering human trial, the intracranial administration of IL13Rα2 CAR–T-cells demonstrated acceptable tolerance, remarkable anti-tumor responses in approximately two-thirds of treated patients, and manageable side effects.^[47] Meanwhile, HER2, which is overexpressed in 80% of GBMs, initially showed potential with third-generation CAR-T-cells. However, safety concerns prompted a modified approach using second-generation CAR-T-cells, which exhibited persistence for up to 1 year without adverse effects.

Current clinical and preclinical investigations are exploring CAR-T-cells targeting TAAs, including B7-H3, CD147, and Ganglioside D2 (GD2). B7-H3 CAR-T-cells, which are highly prevalent in solid cancers, aim to disrupt the stroma and hinder neo-angiogenesis. CD147, associated with tumor progression, invasion, and metastasis, is being evaluated as a target in a dose-escalation clinical study for recurrent GBM patients. In addition, GD2, abundantly expressed in various cancers, is the subject of investigation in a Phase I clinical study for CAR T therapy against brain tumors, including GBM. These studies represent significant progress in the development of effective CAR-T-cell therapies targeting diverse TAAs in the complex landscape of brain cancer treatment.^[45]

Hematologic malignancies

Hematologic cancers, or blood cancers, account for approximately 10% of all cancer cases in the US, resulting from uncontrolled growth of abnormal blood cells.^[38] The introduction of CAR-T-cell therapy has revolutionized the treatment of these cancers, particularly in cases of R/R B-cell acute lymphocytic leukemia NHL, and multiple myeloma (MM), showing impressive outcomes in recent years

Cancer-preventive and treatment vaccines

Cancer vaccines are generally divided into two types: Preventive and therapeutic. Preventive vaccines aim to stop cancer from developing in healthy individuals, while therapeutic vaccines are designed to treat existing cancers by enhancing the immune system’s response to tumors.^[69] In the U.S., there are currently three preventive vaccines and one therapeutic vaccine approved.^[61]

Preventive cancer vaccines focus on stimulating the immune system to either prevent oncogenic viral infections or target pre-malignant and latent cancer cells that have not yet become clinically apparent.^[70] For example, vaccines against human PAPillomavirus (HPV) are expected to greatly reduce the incidence and mortality of cervical and other HPV-related cancers.^[71] Studies have shown that HPV vaccination can nearly eliminate persistent infections and precancerous cervical lesions caused by the HPV types included in the vaccine, though longer follow-up is necessary to confirm its effect on cancer incidence.^[71]

Since their introduction in 2007–08, two HPV vaccines, Gardasil (by Merck) and Cervarix (by GlaxoSmithKline), have been widely used. Gardasil targets HPV types 16 and 18 (which cause cancer), as well as types 6 and 11 (linked to genital warts), while Cervarix targets types 16 and 18 only. Both vaccines have received FDA approval for preventing HPV infections, particularly types 16 and 18, which are responsible for about 70% of cervical cancers.^[69,70] In addition, the FDA has approved a preventive vaccine against hepatitis B virus (HBV) infection, which can lead to hepatocellular carcinoma. Since 1981, HBV vaccines have evolved from plasma-derived to recombinant formulations, providing broad protection and significantly reducing HBV prevalence and hepatocellular carcinoma incidence, especially in infants.^[70]

In contrast to preventive vaccines, therapeutic cancer vaccines are administered to individuals already diagnosed with cancer to stimulate the immune system to fight the disease.^[72] These vaccines face the challenge of overcoming immune suppression mechanisms that cancers use to evade detection. Nevertheless, recent advancements show promising clinical outcomes for therapeutic vaccination.^[65] Notable examples include sipuleucel-T (Provenge®) and rilimogene galvacirepvec/rilimogene glafolivec (PROSTVAC-VF), both of which have demonstrated survival benefits in patients with hormone-resistant prostate cancer.^[73] Neoantigen-based vaccines, targeting mutation-derived epitopes, have shown promise in eliciting strong antitumor responses, with positive results in preclinical and clinical studies for various cancers, including melanoma. Initial trials of mRNA-based neoantigen vaccines and personalized cancer vaccines targeting predicted neo-epitopes have yielded encouraging results, leading to sustained progression-free survival and the induction of multifunctional antigen-specific T-cells in high-risk melanoma patients.^[74] Furthermore, combining neoantigen vaccines with ICIs has enhanced their efficacy. For instance, Personalized neoantigen vaccine (NEO-PV-01), a long peptide cancer vaccine combined with nivolumab, has induced cytotoxic neoantigen-specific T-cells in patients with NSCLC, melanoma, or bladder cancer.^[74]

Peptide- and protein-based vaccines

Peptide-based vaccines use short chains of amino acids derived from cancer-associated proteins to stimulate an immune response. These vaccines target specific TAAs within the TME, encouraging the immune system to attack the cancer cells.^[75] In contrast, protein-based vaccines use whole or modified proteins to stimulate a broad immune response, potentially providing greater efficacy by targeting multiple antigens. The rational design and clinical status of peptide- and protein-based cancer vaccines have been extensively studied but designing effective peptide-based vaccines remains challenging due to the complexity of the involved interactions.

While early clinical trials for peptide-based vaccines (such as NCT00088660, NCT00089856, and NCT00052130) showed limited success, advances in bioinformatics now allow researchers to better predict and design peptide vaccines.^[66,76] Peptide vaccines offer benefits such as faster production, easier storage, lower cost, and fewer side effects compared to conventional vaccines. However, successfully designing a peptide-based vaccine involves several tasks: Identifying potential antigens, predicting T-cell and B-cell epitopes, analyzing epitope immunogenicity, antigenicity, allergenicity, and toxicity, selecting linkers and adjuvant peptides, constructing and optimizing the final vaccine, and analyzing the characteristics of the final vaccines.^[76]

Peptides with medium-to-high major histocompatibility complex (MHC) binding affinity have typically been used in vaccinations but have shown limited success due to immune tolerance.^[77] Recently, low-to-medium affinity peptides have been explored for their immunogenic potential. Enhancing their MHC binding affinity through “anchor” residue modification, while maintaining or improving TCR binding, has shown promise.^[77] When peptide cancer vaccines are introduced into the TME, they are processed by APCs, such as dendritic cells (DCs). The APCs internalize the peptides and present them on their surface in complex with MHC molecules.^[75] This presentation allows the peptides to be recognized by T-cells, specifically CD8 + cytotoxic T lymphocytes (CTLs), which can directly kill cancer cells expressing the targeted TAAs.^[75] The interaction between the presented peptide-MHC complex and the TCR activates the CTLs. This activation leads to the expansion of a population of tumor-specific CTLs that can specifically recognize and target cancer cells expressing the TAAs. These CTLs infiltrate the tumor and exert their cytotoxic effects, thereby combating the cancer cells within the TME.^[75]

DNA and RNA vaccines

Catumaxomab (Removab^®)

Catumaxomab, marketed as Removab^®, is a trifunctional bsAb approved for treating malignant ascites, a common complication in cancer patients. It targets the TA epithelial cell adhesion molecule (EpCAM) (CD32 6), found on many human adenocarcinomas, squamous cell carcinomas, retinoblastoma, and hepatocellular carcinoma. While EpCAM is also present on normal cells, its location in intercellular spaces of epithelial cells makes it less accessible to antibodies, unlike its homogenous distribution on cancer cell surfaces. In addition, EpCAM is often present on cancer stem cells, further establishing its value as a tumor marker.^[92]

Catumaxomab destroys tumor cells primarily through T-cell-mediated lysis, ADCC, and phagocytosis. It binds to CD3 on T-cells and EpCAM on tumor cells, facilitating the recruitment of T-cells to kill EpCAM-positive cancer cells and treat malignant ascites.^[93] The trifunctional nature of catumaxomab allows its Fc region to bind Fcγ receptors on immune accessory cells, activating effector cells to release perforins and granzymes that accelerate tumor cell destruction. In addition, cytokines such as IFN -γ and TNF-α, secreted by T-cells, enhance its anti-tumor activity by stimulating both innate and adaptive immune responses.^[93]

Catumaxomab’s pharmacology has been well-studied. A Phase II PK study showed that intraperitoneal administration resulted in both local and systemic antibody concentrations, enabling it to target the primary tumor and potential metastatic lesions.^[94] Studies have demonstrated catumaxomab’s ability to stimulate an immune response in patients, with the most common side effects being manageable symptoms such as fever, nausea, and vomiting.^[95]

Blinatumomab (MT103)

Blinatumomab, also known as MT103 or bscCD19 × CD3, is a 55-kDa bispecific T-cell engager antibody that targets CD19 on B-cells and CD3 on T-cells. It is produced using a cDNA expression vector in Chinese hamster ovary cells.^[90]

On administration, blinatumomab brings B-cells and T-cells into close proximity, forming a cytolytic immune synapse. This interaction prompts T-cells to release perforins and granzymes, leading to apoptosis in CD19-positive cells such as NALM- 6 cells which is a human pre-B acute lymphoblastic leukemia (ALL) cell line. The drug’s efficacy depends on the perforin-mediated killing of tumor cells, as CD19-negative malignancies show no T-cell activation. Notably, Blinatumomab’s T-cell activation bypasses the need for MHC presentation, making it effective against tumors with downregulated MHC molecules. In addition, the drug enhances T-cell activation markers and adhesion molecules, boosting the immune response.^[96,97]

At present, blinatumomab is being evaluated in a Phase II trial for patients with B-precursor acute lymphoblastic leukemia (B-ALL) who have minimal residual disease (MRD). Early findings indicate that the drug can locate and eliminate rare disseminated tumor cells, achieving MRD negativity in 81% of patients, with responses lasting up to 47 weeks. The trial also noted significantly reduced toxicity compared to NHL patients at the same dose, suggesting that toxicity may be related to the number of target cells. Moreover, a Phase III trial comparing blinatumomab to standard chemotherapy in heavily pre-treated B-ALL patients showed superior survival outcomes and remission rates with blinatumomab.^[86,92]

Ertumaxomab (rexomun)

Ertumaxomab, marketed as Rexomun, is a trifunctional bsAb designed to treat cancers by linking T-lymphocytes and macrophages to cancer cells. Developed by Fresenius Biotech (Trion), it is currently used for metastatic breast cancer (MBC).^[98]

Ertumaxomab has two recognition sites: one for CD3 on T-cells and another for HER2/neu, a TAA. By targeting both HER2 + tumor cells and CD3-expressing T-cells, Ertumaxomab facilitates T-cell activation and macrophage-mediated phagocytosis. Phase I trials demonstrated tumor responses even in patients with low HER2 antigen density, suggesting its potential for patients ineligible for trastuzumab.^[98]

In a Phase I study involving 17 HER2 + MBC patients, ertumaxomab triggered strong immune responses, resulting in two partial responses, one complete response, and two cases of stable disease. The drug forms complexes between tumor cells, T-cells, and macrophages or DCs, leading to cytokine release and tumor cell phagocytosis, which may offer long-lasting anti-tumor immunity. Ertumaxomab is currently undergoing Phase II trials for MBC patients, irrespective of HER2 gene amplification.^[92,99]

Adenovirus

Adenoviral vectors were among the first viral-based therapies developed for cancer treatment. One example is ONYX-015 (CI-1042), designed to replicate selectively in tumor protein p53 (p53)-deficient cells by deleting the adenoviral early region 1B 55-kDa protein (E1B-55K) gene. This deletion reduces the virus’s activity in healthy cells, allowing replication in cancer cells that lack p53.^[101] While later studies suggested that the E1B-55k gene was not solely responsible for p53 inactivation, ONYX-015 showed antitumor efficacy in preclinical models.^[109]

In a Phase I trial, ONYX-015 was administered to 22 patients with recurrent HNSCC that was resistant to radiation or chemotherapy. The primary goal was to assess safety, and the trial reported mild flu-like symptoms as the most common side effect, with minimal toxicity. Some patients experienced moderate pain during injection, but it subsided quickly without additional treatment. The maximum injection dose reached 10¹¹ pfu, with no major side effects, suggesting the possibility of higher doses in future studies.^[110]

A Phase II trial evaluating multiple intratumoral injections of ONYX-015 in patients with recurrent squamous cell carcinoma of the head and neck demonstrated that approximately 14% of patients exhibited significant tumor destruction.^[111] ONYX-015 mouthwash therapy also showed promise for treating oral dysplasia when applied topically.^[112]

Tasadenoturev (DNX-2401), developed by DNAtrix, incorporates an Arginylglycylaspartic acid motif in its fiber knob HI-loop and features a 24 bp deletion in the early region 1A (E1A) region. A study showed that using convection-enhanced delivery of Delta24-RGD was safe for treating GBM.^[113] Another adenovirus, ONCOS-102 (Ad5/3-D24-GMCSF), which expresses GM-CSF, demonstrated safety and the ability to trigger anti-tumor immune responses in Phase I trials involving patients with advanced melanoma and solid tumors.^[114,115]

VCN-01(NCT05673811), an oncolytic adenovirus developed by VCN Biosciences, is armed with hyaluronidase and targets the Rb pathway. It is currently being evaluated in clinical trials for advanced solid tumors, refractory retinoblastoma, and in combination with durvalumab for HNSCC.^[114] VCN-01 has shown cytotoxicity against glioma cells in both in vitro and in vivo models.^[116] Adenovirus-based oncolytic therapies rely on the inactivation of p53 in cancer cells to sustain viral replication, with various studies affirming their safety and feasibility.^[114,117]

Oncolytic HERpes simplex virus (oHSV)

oHSV is engineered to specifically target and destroy tumor cells while also stimulating an anti-tumor immune response.^[118] The well-characterized viral proteins of HSV allow for the deletion of multiple genes to prevent neurotoxicity and increase cancer specificity. Modifications often include the removal of genes essential for viral replication in normal cells, thus confining replication to cancer cells with dysregulated signaling pathways. Additional genetic changes can enhance cancer cell targeting or stimulate the immune system to attack tumors.^[119]

Talimogene laherparepvec (T-VEC), or Imlygic, became the first FDA- and European medicines agency (EMA)-approved oncolytic virus. T-VEC expresses GM-CSF and has demonstrated notable efficacy in melanoma patients.^[118] Administered intratumorally, T-VEC kills tumor cells and provides a source of antigens that stimulate the immune system to generate a systemic anticancer response.^[120] Primed T-cells then generate a systemic polyclonal anticancer response, addressing intra- and intertumoral heterogeneity.^[121] T-VEC enters cancer cells through herpes virus glycoproteins, proliferates, and promotes cell lysis.^[122]

Clinical studies on T-VEC have demonstrated its safety and efficacy. A Phase I study reported that T-VEC monotherapy was well-tolerated, with mild toxicity characterized by fever, myalgia, shivering, and local responses.^[123] In a Phase II study of 50 patients with locally advanced or metastatic melanoma, T-VEC achieved a 26% ORR, including eight complete responses and five partial responses, with most responses lasting nearly 3 years. The OS rates were 58% at 12 months and 52% at 24 months.^[124] Real-world data further support T-VEC’s efficacy. A multi-institutional study on 80 patients with stage IIIB-IV melanoma reported a 57% ORR, with 31 complete responses and 14 partial responses after a median follow-up of 9 months. Adverse events were mainly moderate, with flu-like symptoms occurring in 28% of participants.^[125] Another single-institution study treated 26 patients with stage IIIB-IVM1a melanoma using the OPTiM protocol, achieving an ORR of 88.5% and a disease control rate of 92.3% without additional toxicity compared to the OPTiM trial results.^[126]

NV1020 is another recombinant HSV virus with deletions of HBV, Immediate Early Protein 0 (ICP0), Immediate Early Protein 4 (ICP4), and latency-associated transcripts. It also includes an additional copy of the HSV -1 thymidine kinase gene, making it highly sensitive to the antiviral medication acyclovir.^[109] NV1020 was tested in a Phase I trial for intrahepatic arterial infusion in patients with colorectal cancer and liver metastases.^[127] Twelve participants received a single dose of NV1020 at four dose levels, followed by intra-arterial chemotherapy. The treatment was generally safe, with no significant adverse effects, and two patients showed an objective tumor response, although the impact of NV1020 was unclear due to concurrent chemotherapy.^[109]

Oncolytic vaccinia virus (OVV)

Vaccinia virus, a double-stranded DNA virus originally used in smallpox vaccines, has emerged as a promising candidate for oncolytic therapy.^[101] Its ability to replicate in the cytoplasm and encapsulate itself within a host cell-derived envelope enables it to evade the immune system and circulate to metastatic tumor sites.^[109]

OVV induces tumor destruction through direct oncolysis, disruption of tumor vasculature, and activation of anti-tumor immunity. Pexa-Vec and GL-ONC1 (olvimulogene nanivacirepvec), two prominent OVVs, are currently in Phase III clinical trials.^[120] While Pexa-Vec’s Phase III trial for hepatocellular carcinoma was unsuccessful in improving patient survival, ongoing research into combination therapies holds promise for future treatments.^[128]

Research by Chen et al. highlighted the potential of vaccinia virus to target various hematologic cancers, with constructs from the lister institute virus prague (LIVP) strain showing the greatest efficacy in eliminating leukemic cells.^[129] The LIVP-based constructs were the most successful at infecting leukemic cells, followed by WR-based constructs.^[129] In another study investigating the effects of different vaccinia virus strains on murine mesothelioma cell lines, the Western Reserve virus strain genelux (GLV-0b347) was found to be the most effective in oncolysis. At 96 h’ post-infection, GLV-0b347 significantly reduced cell death in AB12 cells, achieving an 80% reduction at a multiplicity of infection (MOI) of 0.1 and over 90% at an MOI of 1. Although no cell death was detected at lower MOIs, GLV-0b347 therapy significantly improved OS, reduced tumor load, and suppressed ascite development in mice with low tumor burden at the time of virus application.^[130]

Passive targeting

Passive targeting relies on the virus’s ability to naturally accumulate within tumors due to the tumor’s leaky vasculature and reduced lymphatic drainage – hallmarks of malignant tissues. Despite this, issues such as poor tumor tropism and inefficient transduction have limited the success of oncolytic viruses in cancer therapy. For example, T-VEC has demonstrated therapeutic benefits in melanoma, but its utility is constrained by inadequate tumor cell tropism and transduction.^[135] To address these challenges, viral modifications, such as introducing an RGD motif into the HI loop of the adenoviral fiber knob, have improved infection efficiency.^[136] This enhancement has been particularly beneficial in cancer models that lack coxsackievirus and adenovirus receptor. Furthermore, combining oncolytic viruses with conventional therapies, such as chemotherapy or ICIs, is being explored as a way to boost efficacy and overcome resistance.^[137]

Immune response

Pre-existing immunity, either from prior virus exposure or infection, and the body’s innate immune response to viral infections present significant hurdles to OVT.^[131,138] When injected into the body, the oncolytic virus triggers the immune system, which is designed to detect and eliminate viral infections.^[139] This immune response may hinder the virus’s ability to propagate and infect enough cancer cells, thereby reducing its efficacy.^[140] In addition, some patients may have pre-existing immunity to the virus used in therapy, rendering the treatment ineffective from the start. Tumors often create immunosuppressive environments that further dampen both antiviral and anticancer immune responses.^[141] Researchers are investigating methods to overcome these barriers, including “stealthing” viruses with polymers to evade immune detection and using cellular carriers such as Mesenchymal Stem Cells to protect viruses and enhance their tumor-targeting capabilities.^[142-144]

Other challenges

Additional issues include the effects of hypoxic conditions within tumors and difficulties in selecting suitable patients for OVT. Delivery and safety concerns also pose significant obstacles, as highlighted by Chen et al.,^[129] with ongoing research exploring improved methods for administration and risk mitigation.

Next-generation sequencing (NGS)

NGS allows for rapid and comprehensive analysis of a patient’s entire genome or select genes.^[148] This technique helps identify mutations or genetic alterations that drive cancer progression,^[149] enabling clinicians to tailor treatments to target the specific vulnerabilities of cancer cells.^[150] NGS provides a detailed study of cancer genomes, facilitating more accurate diagnosis, prognosis, and the identification of drug-responsive mutations.^[140] Pilot programs are currently assessing the clinical use of NGS for mutation-targeted therapies.^[148]

Liquid biopsies

Liquid biopsies offer a non-invasive approach for monitoring disease progression, detecting therapy resistance, and identifying actionable mutations in real time. By analyzing circulating tumor DNA (ctDNA), they provide a dynamic snapshot of tumor evolution.^[151] A decrease in ctDNA after treatment signals a positive response, whereas an increase may indicate resistance or recurrence, prompting early intervention.^[152] Unlike single tissue biopsies, liquid biopsies capture tumor-derived components from the bloodstream, offering a comprehensive view of the tumor’s genetic profile and guiding the selection of effective therapies.^[153] Even when imaging fails to detect residual cancer, liquid biopsies can identify it, enabling early relapse detection.^[154]

Machine learning and artificial intelligence

Machine learning algorithms analyze vast amounts of genetic, clinical, and imaging data to identify patterns related to treatment response, disease progression, and patient outcomes. These models can predict survival, stratify patients into risk categories, and inform treatment decisions. By analyzing individual tumor characteristics, machine learning can forecast a patient’s response to specific therapies. Furthermore, it helps identify distinct tumor subtypes with unique molecular profiles,^[155] supporting the development of tailored therapies for each subtype.

Multi-omics integration

Multi-omics integration combines data from multiple molecular layers, providing a holistic view of tumor biology. This approach enables the discovery of molecular subtypes, dysregulated pathways, and therapeutic vulnerabilities, ultimately driving personalized cancer treatments. Omics layers include genomics, proteomics, metabolomics, and transcriptomics. For instance, metabolomics investigates small molecules within cells, offering insights into cancer classification and patient prognosis through unique metabolic fingerprints.^[156,157]

Multimodal biomarkers

Combining biomarkers from multiple sources, such as genomes, metabolomics, and proteomics, provides a comprehensive view of a patient’s cancer. This approach can improve patient classification and guide treatment decisions. Multimodal biomarkers enhance accuracy by integrating data from genetics, metabolites, and proteins, offering a thorough picture of cancer for more accurate diagnoses, risk stratification, and treatment options. They address tumor heterogeneity, ensuring treatment targets the most relevant components of the malignancy, and identify resistance mechanisms, facilitating the development of strategies to overcome them. Researchers can develop targeted therapies addressing specific anomalies identified by multimodal biomarkers. For instance, Cancer antigen 19-9 (CA19-9) is a pancreatic cancer biomarker, but recent studies on non-coding RNAs such as microRNAs (miRNAs), circular RNAs (circRNAs), and long non-coding RNAs (lncRNAs) show significant promise as biomarkers and for understanding pancreatic regulatory network components.^[158]

Liquid biopsy advancements

Liquid biopsies are relatively painless and yield a wealth of data. Ongoing research focuses on isolating and analyzing distinct subpopulations of circulating tumor cells (CTCs) or ctDNA to better understand tumor heterogeneity and treatment resistance. As liquid biopsy technology evolves, it holds the potential to revolutionize cancer diagnosis and treatment management, allowing real-time monitoring of tumor dynamics and facilitating personalized treatment strategies.^[159]

Microbiome analysis

The gut microbiome plays a critical role in immune function and may influence cancer development.^[160] Future research is expected to further explore the relationship between the microbiome and cancer, with the goal of identifying microbiome-based biomarkers that can guide treatment decisions and potentially enhance responses to immunotherapy.