In Situ Cancer Vaccines: Redefining Immune Activation in the Tumor Microenvironment

Abstract

Cancer is one of the leading causes of mortality worldwide. Nanomedicines have significantly improved life expectancy and survival rates for cancer patients in current standard care. However, recurrence of cancer due to metastasis remains a significant challenge. Vaccines can provide long-term protection and are ideal for preventing bacterial and viral infections. Cancer vaccines, however, have shown limited therapeutic efficacy and raised safety concerns despite extensive research. Cancer vaccines target and stimulate responses against tumor-specific antigens and have demonstrated great potential for cancer treatment in preclinical studies. However, tumor-associated immunosuppression and immune tolerance driven by immunoediting pose significant challenges for vaccine design. In situ vaccination represents an alternative approach to traditional cancer vaccines. This strategy involves the intratumoral administration of immunostimulants to modulate the growth and differentiation of innate immune cells, such as dendritic cells, macrophages, and neutrophils, and restore T-cell activity. Currently approved in situ vaccines, such as T-VEC, have demonstrated clinical promise, while ongoing clinical trials continue to explore novel strategies for broader efficacy. Despite these advancements, failures in vaccine research highlight the need to address tumor-associated immune suppression and immune escape mechanisms. In situ vaccination strategies combine innate and adaptive immune stimulation, leveraging tumor-associated antigens to activate dendritic cells and cross-prime CD8+ T cells. Various vaccine modalities, such as nucleotide-based vaccines (e.g., RNA and DNA vaccines), peptide-based vaccines, and cell-based vaccines (including dendritic, T-cell, and B-cell approaches), show significant potential. Plant-based viral approaches, including cowpea mosaic virus and Newcastle disease virus, further expand the toolkit for in situ vaccination. Therapeutic modalities such as chemotherapy, radiation, photodynamic therapy, photothermal therapy, and Checkpoint blockade inhibitors contribute to enhanced antigen presentation and immune activation. Adjuvants like CpG-ODN and PRR agonists further enhance immune modulation and vaccine efficacy. The advantages of in situ vaccination include patient specificity, personalization, minimized antigen immune escape, and reduced logistical costs. However, significant barriers such as tumor heterogeneity, immune evasion, and logistical challenges remain. This review explores strategies for developing potent cancer vaccines, examines ongoing clinical trials, evaluates immune stimulation methods, and discusses prospects for advancing in situ cancer vaccination.

Graphical Abstract

1. INTRODUCTION

Vaccines significantly impact global health by preventing infectious diseases and reducing their spread. WHO estimates that vaccines prevent 2–3 million deaths worldwide each year. By reducing the burden on healthcare budgets by over $500 billion, vaccinations have made a significant impact on the global economy.¹ Vaccines have significantly reduced the burden of bacterial and viral infectious diseases, increasing survival rates, reducing early childhood mortality, and protecting elderly patients from chronic infections.² However, clinical studies have shown limited success for conventional cancer vaccine. Harnessing host immunity for the prevention and eradication of cancer is being explored by immunotherapy. The tumor microenvironment plays an essential role in cancer treatment. An immune suppressive environment in tumors contributes to the evasion of immune response and resistance to therapy. Interactions between immune cells lead to insufficient antigen presentation due to low MHC expression, increased checkpoint protein expression, and decreased activities of immunostimulatory cytokines. These factors favor immune inactivation and evasion, ultimately reducing the ability of the immune system to fight tumors.³

Immunotherapy has emerged as a promising strategy for cancer treatment over the past few decades.⁴ Unlike traditional therapies, which directly target cancer cells, immunotherapy focuses on harnessing the body’s immune system to target and eliminate cancer cells. In situ vaccination is a novel strategy that generates a cancer vaccine directly from the tumor itself using chemotherapy, radiation, photothermal effect, photodynamic therapy, and intratumoral administration of the immunostimulatory agents for immune modulation of tumor environment by improving antigen-presentation, activating innate (dendritic cells, NK cell, macrophage, and neutrophils) and adaptive immune system (CD8+ T cells, CD4+ T cells) players. An American physician first used Coley’s toxin, as an in situ vaccine, over a century ago in clinics.⁵ The method is personalized, patient-specific, and off-the-shelf. Coley’s toxin inspired future researchers for further develop immunotherapy.

By utilizing tumors as a source of antigens, in situ vaccines result in the polarization of the immune suppressive environment into an immunostimulatory one, eliciting a systemic and immune memory response that prevents disease recurrence.^6,7 Treatment of metastatic cancer is a major scientific challenge in the oncology field. In situ vaccines can transform immune suppressive “cold” tumors to “hot” tumors by activating adaptive and innate immunity. The first step for in situ vaccination involves immunogenic cell death (ICD), activation, maturation of dendritic cells, and presentation of antigen followed by cross-priming of CD8+ T cells for cytotoxic effect against tumors. Figure 1 displays the principle of in situ vaccination. The first step for in situ vaccination involves generating in situ vaccines through primary tumor (T1) treatment utilizing radiotherapy, surgery, hormonal therapy, immunotherapy, or chemotherapy. These treatments convert the tumor into the damage-associated molecular pattern (tumor-specific, tumor-associated antigen, and immunogenic cell death), which is taken up by antigen-presenting cells such as dendritic cells. Dendritic cells present these antigens to the nearest lymph nodes, where most lymphocytes reside. After that, activated naïve CD8+ or CD4+ T cells recognize antigens from the primary tumor, transforming into cytotoxic CD8+ T cells. These cytotoxic T cells then attack tumors of the same antigenic origin (T2), preventing metastasis and cancer recurrence. CD4+ T cells play an essential role in coordinating the immune response. This process sustains the cancer immunity cycle, maintaining the normal immune function of the host.

Figure 1.

In situ vaccination principle. (Created with BioRender.)

In large tumors, various factors contribute to the inhibition of CD8+ T cells effect. First, tumor interstitial pressure restricts T-cell infiltration and impairs immune cell function by hindering circulation, rather than directly interfering with PD-1 and CTLA-4 coinhibitory signaling. Second, the immunosuppressive microenvironment contains regulatory T-cells (Tregs), myeloid-derived suppressor cells (MDSCs), regulatory CD8+ T cells, tumor-associated macrophages (TAMs), regulatory natural killer, and soluble immunosuppressive factors such as VEGF, TGF-β, IL-10, and indoleamine-pyrrole 2,3 dioxygenase (IDO).^8,9 Additionally, the therapeutic outcomes of cancer vaccination are also limited due to issues of preparation, isolation, and purification of personalized vaccines.¹⁰ The timeline for the use of cancer vaccines is shown in Figure 2.

Figure 2.

Key milestones in the use of vaccines for cancer. (Created with BioRender.)

The FDA approval of BCG, Sipuleucel-T, T-VEC, and imiquimod has demonstrated the success of immunotherapy and vaccination in cancer treatment. In contrast, the HPV vaccine is primarily used for cancer prevention, particularly in reducing the risk of cervical cancer.

Our comprehensive review explores a wide array of vaccination approaches, including mRNA, DNA, peptides, and cell-based vaccines. We have focused primarily on in situ vaccination strategies revolutionizing immune activation within the tumor microenvironment. Strategies involve the use of oncolytic viruses, Flt3L, GM-CSF, TLR agonists, chemotherapy, radiation, photodynamic therapy, combinations with checkpoint inhibitors, and various immunomodulators.

Throughout this review, we provide a detailed analysis of how these approaches are reshaping the landscape of in situ cancer vaccination. We aim to highlight both the known and unknown aspects, bridging the gap between current understanding and the emerging frontiers of in situ vaccination. Ultimately, the goal of in situ vaccination is to overcome the immunosuppressive environment and facilitate a sustained, robust immune response.

2. IN SITU VACCINES APPROVED FOR CANCER IN CLINICS

The primary purpose of in situ vaccines is to stimulate the immune system so that cancer cells can be recognized and attacked efficiently.¹¹ Local administration of these vaccines enhances antitumor effects, minimizes toxic side effects, and prevents antigen loss (helps retain the target antigen at the tumor site). In preclinical and early clinical trials, in situ vaccines have demonstrated effector function, immune cell proliferation, and tumor cell responsiveness.⁵ It is important to note, however, that there are only a few cancer vaccines available for the prevention and treatment of cancer under certain conditions. Compared to therapies like chemotherapy and checkpoint inhibitors, in situ vaccines have demonstrated lower clinical effectiveness, leading to their limited success. This lack of clinical efficacy has limited their ability to regress tumors effectively. The limited effectiveness of in situ vaccines is largely due to the immunosuppressive tumor microenvironment, which inhibits immune cell activation and function. This suppressive environment limits the vaccine’s ability to elicit a strong, sustained immune response. As a result, successful outcomes remain rare despite extensive research efforts. To improve clinical outcomes, researchers are combining in situ vaccines with immune checkpoint inhibitors or adoptive cell therapies to enhance immune activation and overcome tumor microenvironment suppression. These combinations have shown promise in preclinical studies, and ongoing clinical trials aim to evaluate their potential in various cancer types.

2.1. Vaccines for Cancer Treatment.

The immunosuppressive microenvironment and immunoediting present in tumors have always made developing vaccines difficult. Figure 3a shows the interaction between cancer and vaccines after administration. Vaccination boosts immunity against cancer and prevents metastasis. Therefore, vaccines containing tumor-specific antigens or tumor-associated antigens injected with or without adjuvant activate Langerhans cells beneath the epidermis layer.¹² This antigen is processed by dendritic cells to activate specific T and B cells. The tumor-specific T cell migrates to the tumor site and kills tumor cells by apoptosis via enzymes released (Figure 3b). The microenvironment of tumors and the heterogeneity of tumors compromise the efficiency of T-cell activation and proliferation. When tumor tissue lacks an antigen recognizable by T cells, metastases continue to cause life expectancy and survival rates to decline (Figure 3c).

Figure 3.

(a) Intradermal or subcutaneous therapeutic vaccine for cancer prevention and treatment. (b) Migration of T cells specific to tumor antigens to lymph nodes after antigen processing by dendritic cell. (c) Metastasis of cancer due to loss of antigens. (Created with BioRender.)

The vaccine is administered to patients who show signs of premalignant cancer or abnormal changes in normal tissues as a prophylactic measure.¹³ The prophylactic vaccine contains an antigen that has been overexpressed on cancer cells along with an immunostimulant for activating Langerhans cells and dendritic cells to stimulate the production of CD8+ T cells for future protection against cancer (Figure 4a). In future remissions of tumors, the tumor antigen migrates to draining lymph nodes, activating tumor-specific lymphocytes like T-cells and resulting in secondary immune responses (Figure 4b). This response activates dendritic cells in the tumor site and produces an amplification of the immune response. The immune memory effect prevents tumor growth and metastasis through tumor-induced immune activation. After killing cancer cells, activated T cells may differentiate into immunological memory cells. These memory cells retain information about the tumor antigens, enabling a faster and more effective immune response upon future encounters with cancer cells (Figure 4c).

Figure 4.

(a) Prophylactic vaccine for antimetastatic effect injected to patients with suitable routes of administration. (b) Development of immunological memory for future protection of metastasis, (c) Tumor-mediated immune boost for future protection. (Created with BioRender.)

The FDA approved the Sipuleucel-T dendritic cell-based vaccine in 2010 for metastatic prostate cancer that is asymptomatic or minimally symptomatic.¹⁴ This marks the first milestone in the development of cancer vaccines. Sipuleucel-T patients experienced common vaccine side effects such as fever, chills, and headaches. Compared to placebo, Sipuleucel-T improved survival in patients with MCRPC (metastatic castration-resistant prostate cancer).¹⁵ Sipuleucel-T treated patients had a median overall survival of 25.8 months compared to 21.7 months for placebo-treated patients. A significant reduction in mortality rate was seen in the patients who received Sipuleucel-T.¹⁶

As prostatic acid phosphatase antigen is overexpressed in nearly all prostate cancers, it is a promising target for development as a therapeutic prostate cancer vaccine.¹⁷ A recombinant fusion protein of prostatic acid phosphatase and granulocyte-macrophage colony-stimulating factor (GM-CSF), an immune cell activator, was used to generate Sipuleucel-T to enhance the potency of the immune response to prostatic acid phosphatase. A dose of Sipuleucel-T contains at least 50 million CD54+ cells activated with PAP-GM-CSF. The vaccine is administered intravenously every 2 weeks for three doses. This regimen successfully induced a specific immune response against the prostatic acid phosphatase antigen, which resulted in a survival benefit for patients. Clinical trials of Sipuleucel-T have demonstrated a survival benefit in men with metastatic castration-resistant prostate cancer, supporting its use as a therapeutic vaccine. It is currently approved in the U.S. and Europe for the treatment of asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer.

Bacillus Calmette-Guerin (BCG) has been used to treat nonmuscle invasive bladder cancer (NMIBC) for over 30 years.¹⁸ BCG helps to stimulate the body’s immune system to fight bladder cancer cells. BCG helps to stimulate the body’s immune system to fight bladder cancer cells. It is one of the most common treatments for early stage bladder cancer. CD4+ and CD8+ lymphocytes, natural killer cells, and granulocytes are important components of the cellular immune response to BCG. TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), IL-2, IL-8, IL-18, IL-12, interferon (IFN), and tumor necrosis factor (TNF) are crucial components of the humoral immune response to BCG. BCG also triggers an immune response by activating macrophages and dendritic cells by producing cytokines and other mediators. These mediators are responsible for the immunological effects of BCG in bladder cancer. Urothelial cells are likely to be involved in recognizing BCG by the immune system and subsequent processing to produce an antitumor effect. Cytokines are secreted due to the immune response and can lead to the destruction of tumor cells. Additionally, BCG can inhibit the growth of bladder cancer cells and stimulate the recruitment of immune cells to the tumor site.

Talimogene laherparepvec (T-VEC) is an in situ vaccination approach used in the United States, Europe, and Australia to treat metastatic melanoma stage III and IV.¹⁹ Talimogene laherparepvec (T-VEC) is an in situ vaccination approach used in the United States, Europe, and Australia to treat metastatic melanoma stage III and IV. In T-VEC, a virus is genetically engineered to produce a protein that can help the immune system recognize and attack cancer cells. It is administered directly into a melanoma tumor and stimulates the immune system to fight cancer cells. This approach contains genetically modified herpes viruses. T-VEC preferentially infects melanoma cells, multiplies, causes local bursts, and exerts antitumor activity by directly mediating cell death and enhancing local and even distal immune responses. It has been used in clinical trials to treat melanoma and has been shown to shrink tumors significantly. In some cases, the tumors have been eliminated. It is a promising treatment for melanoma and other types of cancer. Several factors limit T-VEC’s use. Medical oncologists are unfamiliar with the intratumoral route of administration, biosafety concerns regarding using a live virus in the clinic, and side effects. Additionally, T-VEC is expensive and not widely available, limiting its use. Furthermore, it may not be suitable for all patients, as it is not effective for all types of cancer. In addition to pain, fever, and chills, cytokine release syndrome and stroke have been reported among patients as serious health concerns. Despite these concerns, T-VEC therapy is a promising new treatment option for some cancer patients. Further research is needed to understand its safety and efficacy in different types of cancer. Figure 5 shows how T-VEC replication leads to tumor lysis and DAMP release, which enhances immune activation and may reduce metastasis; however, excessive DAMP release can also promote chronic inflammation and tumor progression, making the author’s statement overly simplified.

Figure 5.

T-VEC mechanism of action. (Created with BioRender.)

Toll-like receptor (TLR) agonists can be useful therapeutic targets for treating cancer. TLR agonists are proteins that recognize molecules associated with pathogens and initiate an immune response.²⁰ By utilizing TLR agonist, it is possible to create a targeted response to cancer cells while leaving healthy cells unharmed. This may provide a promising way to combat cancer without the side effects of other treatments. Combining TLR agonist with other therapies used to treat cancer has a synergistic effect. TLR agonist therapy can be used alone or in combination with other treatments, such as chemotherapy or radiation, to attack cancer cells more effectively. This combination therapy is more successful in treating cancer than either method used alone. The TLR agonist Imiquimod has been approved for treating genital/perianal warts, actinic keratosis, and superficial basal cell carcinoma. Imiquimod can also stimulate the immune system to attack tumors or enhance the effects of other therapies. Clinical trials have demonstrated the efficacy of TLR agonists in combination with other treatments in treating various types of cancers.

There are nine different Toll-like receptor agonists in clinical trials, but only three have been approved by the FDA for cancer treatment.²¹ These three ligands are effective in fighting a variety of cancers, including breast, prostate, and colorectal cancer.²² The other ligands are still in clinical trials and may be approved in the future. Researchers hope the remaining ligands will be just as effective, if not more so. These ligands may provide new hope for cancer patients with continued research and development. Further, clinical trials are underway for more TLR agonist, potentially increasing the range of cancer treatments available. For instance, TLR3 agonists have shown to be promising adjuvants for cancer vaccines, with various studies highlighting their ability to potentiate vaccine-mediated antitumor responses. If successful, these treatments could revolutionize cancer care and improve outcomes for patients globally.

2.2. Vaccination for Cancer Prevention.

Vaccination is the best way to prevent precancerous and cancerous lesions in patients.²³ Cancer prevention by vaccination has two main goals: (1) targeting the primary cause, such as microorganisms, and (2) preventing further infection by specific microorganisms. Vaccination can also reduce the risk of cancer by boosting the body’s natural defenses against cancer-causing agents. It also helps reduce the severity and duration of cancer symptoms and can even reduce the risk of recurrence. Vaccinations are safe and effective and can provide lifelong protection against certain types of cancer. Vaccines are available for many different types of cancer and are recommended for individuals of all ages. For instance, the human papillomavirus (HPV) vaccine is recommended for preteens and young adults to reduce their risk of HPV-associated cancer. Prophylactic vaccines have illustrated how vaccines can effectively intervene in cancer development by targeting viral infections that drive tumorigenesis. This has transformed cancer prevention efforts by offering safe, effective, and long-term protection against major cancer-causing viruses, underscoring the vital role of vaccination in global cancer control and public health.

As a source of antigens, the HPV vaccine contains virus-like particles without a viral genome.²⁴ The virus-like particles are created in the laboratory and contain only some of the virus’s genetic material. This means that the virus-like particles cannot replicate or cause infection but can still stimulate an immune response in the body. For example, the HPV vaccine contains virus-like particles of the capsid proteins of the virus, which are enough to stimulate an immune response without replicating the virus. If exposed, it triggers the body to make antibodies to recognize and fight the virus. For the vaccine to be most effective, it should be given before puberty. These vaccines are most effective when administered before age 15. Vaccination of both men and women is important for protection against HPV-related cancers. HPV vaccination program focuses on prepubertal girls and boys before sexual debut who are prone to HPV virus infection followed by cervical, vaginal, and penile cancer. Vaccination can also help protect against genital warts and other HPV-related diseases. HPV vaccine provides immunity for up to 10 years, so getting a booster dose after the initial vaccination.

In 2006, Gardasil was licensed against HPV strains 6/11/16/18 and for protection against HPV strains 31/33/45/52/58 in Europe. Gardasil 9, a nonavalent HPV vaccine was licensed in June 2015 against HPV strains 31/33/45/52/58.²⁵ HPV vaccine is used for its prophylactic effect to prevent vaginal, penile, survival, and genital cancers associated with HPV infection. HPV vaccine is recommended for men and women aged 9–45 years. Vaccination is recommended for immunocompromised individuals but is not advised for pregnant women. HPV Vaccination should be done before potential exposure to HPV.

Hepatitis infection contributes 60% to 90% of hepatocellular carcinomas (HCC) in adults and 100% to childhood.²⁶ HCC is a type of liver cancer that is caused by chronic inflammation of the liver. It is one of the leading causes of death in the world and is the fifth most common cancer in the world. Vaccines and early detection are the best ways to reduce the risk of hepatitis-related cancers. The first national campaign to immunize infants against Hepatitis B virus (HBV) demonstrated its ability to protect against cancer. Vaccination is especially important for all those at high risk for HBV, such as healthcare workers, people who inject drugs, and those who have had multiple sexual partners. It is also recommended for people with chronic liver conditions, such as cirrhosis. Vaccination can also be beneficial for those who have already had hepatitis B infection. In the general population under 20, the vaccination campaign has decreased the infection rate from 10%–17% to 0.7%–1.7%. For those at high risk of HBV, vaccination is crucial as it prevents the virus from spreading and provides immunity in the event of reinfection. Therefore, everyone of all ages must consider vaccination to help reduce the incidence of HBV and protect those who are at greater risk.

Overall, both HPV and HBV vaccines have proven to be valuable tools in the fight against cancer, demonstrating significant global impact by reducing the incidence of cervical, liver, and other HPV-associated cancers. Their success in preventing infections that lead to malignancies underscores the power of vaccination as a public health strategy, offering long-lasting protection and saving millions of lives. As vaccination efforts continue to expand, these vaccines hold the potential to further reduce the global cancer burden, making them essential components of cancer prevention and control strategies worldwide.

2.3. Ongoing Clinical Trials.

Advances in science continue to drive the development of new vaccines, and cancer vaccines represent one of the most challenging areas for researchers.²⁷ Research in this area is focused on understanding the mechanisms of cancer development to design effective vaccines. Scientists are also exploring gene therapy and other novel approaches to develop improved cancer vaccines. Multifaceted antitumor immune responses, immunosuppressive tumor microenvironments, and immunoediting challenges limit vaccine efficacy. Despite these challenges, researchers are optimistic about developing more effective cancer vaccines. They are currently investigating the use of combination therapies and personalized approaches to improve vaccine efficacy. For instance, a recent study suggested that combining checkpoint blockade immunotherapy with an in situ vaccination strategy to generate tumor-associated antigen vaccines can increase the effectiveness of cancer vaccines by enhancing the activity of the immune system. Vaccines for cancer have not been fully established due to incomplete understanding, limited research, and incorrect use of optimal combinations of antigens, adjuvants, delivery vehicles, and administration routes. Further research is required to better understand the immune system and optimize these components to enhance cancer vaccine efficacy. Additionally, more clinical trials are needed to evaluate the safety and efficacy of cancer vaccines.

The combination of tumor-specific antigens and the standard care of treatment was assessed in various clinical trials.²⁸ Currently, there are more than 300 active clinical trials of cancer vaccines, varying in their delivery vehicle, antigen type, and indication. Table 1 illustrates some of the clinical trials performed for vaccine design. This table contains peptide-based vaccines, dendritic cell-based vaccines, gene-based vaccines, carbohydrate-based vaccines targeting antigens, tumor-associated antigens, and tumor-specific antigen-based vaccines. These vaccines have demonstrated promising results in clinical trials for cancer treatment. Clinical trials have demonstrated that tumor-specific antigen-based vaccines can induce an immune response to the tumor and can effectively reduce tumor growth. For example, in a clinical trial of the GVAX vaccine for pancreatic cancer, patients on the vaccine arm had a median survival of 25.5 months, compared to 17.7 months in the placebo arm. Additionally, researchers have found that when combined with other treatments, such as chemotherapy and radiation, vaccines have the potential to further reduce tumor growth and improve patient outcomes. Some of these vaccines have completed clinical trials, while others remain under development.

Table 1.

Representative Clinical Trials of Therapeutic Cancer Vaccines

vaccine source	targeted antigen	adjuvant	combination strategy	cancer type	clinical trial
NeuVax E75	E75 peptide acetate	GM-CSF		Breast cancer	NCT01479244
EGF vaccine	Recombinant human rEGF-P64K	Montanide ISA 51	Low dose of cyclophosphamide	Stage IV biomarker-positive, wild-type EGF-R NSCLC	NCT0218736
CMB305	NY-ESO-1	TLR-4 agonist	Surgery	Synovial sarcoma	NCT03520959
Rindopepimut	EGFRvIII protein	GM-CSF	Temozolomide	EGFRvIII-positive glioblastoma	NCT01480479
Adagloxad simolenin/OBI-821	Globo H		Capecitabine or platinum	Triple-negative breast cancer	NCT03562637
IMA901	Aeptide antigens	GM-CSF	Sunitinib 01	Renal cell carcinoma	NCT01265901
Dendritic cell vaccine	Autologous tumor cells		Bevacizumab	Glioblastoma	NCT04277221
Dendritic cell vaccine	Autologous tumor mRNA		Temozolomide	Glioblastoma	NCT03548571
Antigen-pulsed dendritic cells	Autologous tumor lysates		FOLFOX6-chemotherapy	Metastatic colorectal cancer	NCT02503150
DC Vaccine	Autologous tumor		Chemotherapy	Pancreatic cancer	NCT02548169
T cell therapy + HER2-Pulsed Dendritic Cell Vaccine	Autologous tumor		Pepinemab Trastuzumab	Metastatic breast cancer	NCT05378464
MIDRIX4-Lung dendritic cell vaccine	Autologous tumor		Checkpoint Inhibitors	Nonsmall Cell Lung Cancer Metastatic	NCT04082182
DCVAC/OvCa	Autologous tumor lysis		Platinum and/or	Relapsed ovarian	NCT03905902
ProstAtak	Autologous tumor antigens		Valacyclovir and radiation	localized prostate cancer	NCT01436968
Ad-sig-hMUC-1/ecdCD40L	hMUC-1 epithelial antigen			Epithelial cancer	NCT02140996

The main challenge with the use of cancer vaccines in clinical trials is immune-related toxicity, particularly when cancer vaccines are combined with immune checkpoint inhibitors like anti-PD-1 or anti-CTLA-4 therapies.²⁹ While these combinations can enhance immune activation against tumors, they also raise the risk of autoimmune reactions, as the enhanced immune system can attack healthy tissues alongside tumor cells. Additionally, ensuring the optimum timing and treatment sequence is not yet fully understood. For example, chemotherapy and radiation can impair the immune system, potentially reducing the vaccine’s ability to prime an effective immune response if administered too early in the treatment sequence. Conversely, they can reduce tumor burden and enhance the immune response when used appropriately. The use of cancer vaccines is still in the early stages, but the potential for success is promising. As more research is conducted, researchers remain hopeful that cancer vaccines will become widely available and an effective standard of care.

3. CHALLENGES FOR DESIGN OF VACCINES FOR CANCER

Cancer vaccines pose greater challenges in development compared to conventional bacterial and viral vaccines.^30,31 One significant challenge is targeting tumor antigens, which often exhibit low immunogenicity within the tumor environment and may mutate to evade immune responses. Since cancer cells originate from the body’s cells, the immune system must be trained to differentiate and destroy them effectively. Furthermore, the rapid mutation rate of cancer cells complicates their recognition by the immune system. Unlike conventional vaccines that typically target a single pathogen, cancer vaccines must address the diversity and heterogeneity of cancer cells across and within patients.

Several factors contribute to the success of conventional vaccines, including the presence of universal antigens, the ability to target foreign infectious agents that the immune system can easily identify, and the availability of clinical and diagnostic tests to determine disease burden.^32,33 Cancer vaccines, however, must recognize the various mutations in cancer cells to be effective. This requires the development of personalized vaccines tailored to specific cancer types and mutations. Furthermore, cancer vaccines must effectively stimulate the immune system to recognize and attack cancer cells. According to an analysis of current clinical trial data, therapeutic vaccines are currently in development for 13 of the 22 cancer types specified by the NCI.

Despite these significant efforts, cancer vaccines remain in the early stages of development.³⁴ Further clinical trials are required to evaluate the safety and efficacy of these cancer vaccines. Additionally, research is needed to identify new targets and strategies for cancer vaccine development. Figure 6 illustrates the challenges involved in designing of cancer vaccines.

Figure 6.

Challenges of cancer vaccine design. (Created with BioRender.)

An additional major obstacle to vaccine success in elderly patients is the aging of their immune systems.³⁵ Vaccines are being tested in elderly cancer patients (65–80 years old) and those with advanced-stage cancer. Older people may have weaker immune systems, making it harder for them to respond effectively to vaccines. This can lead to lower levels of protection and reduced efficacy of the vaccine. The aging immune system must be considered when designing vaccines to ensure maximum effectiveness. Developers must use techniques such as adjuvants or altered dosing regimens to create effective vaccines for elderly populations. Additionally, more research is needed to understand how vaccines interact with aging immune systems. These patients often lack sufficient naive T cells to launch a robust immune response. Furthermore, the development of immunological memory and primary antibody responses is compromised in elderly people. Vaccines must be designed to account for these changes to be effective in elderly populations. In conclusion, elderly populations present unique challenges to vaccine development. A combination of advanced techniques and additional research is needed to develop effective vaccines for aging populations.

3.1. Failure in Vaccine Research.

Clinical trials of therapeutic vaccines failed to demonstrate therapeutic benefits over existing vaccines in most cases.^36,37 However, some of the trials did show promising results, and further research should be conducted to determine the potential for therapeutic vaccines. Greater investment is needed to develop vaccines with enhanced capabilities for disease prevention and treatment. While some vaccines have shown promising results, others have demonstrated poor outcomes and limited efficacy for their intended purpose. These results highlight the challenges of therapeutic vaccine development and potential pathways for improvement. Research should focus on finding ways to improve vaccine efficacy and making them more cost-effective. To do this, researchers need more data to better understand the underlying mechanisms that cause vaccine failure. They also need to develop more effective strategies to prevent it.

The limitations faced by cancer vaccines are similar to broader challenges in the field of immunotherapy.³⁸ One major difficulty for antigen-specific immune therapies is identifying antigens that can elicit a robust immune response. Tumors can evade immune detection by downregulating MHC class I expression or undergoing immune editing, leading to the loss of targeted antigens and reducing the efficacy of cancer vaccines. Another challenge in immunotherapy is tumor heterogeneity. Factors such as mutations, antigen expression, stromal composition, and metabolic properties can vary significantly within a single tumor and among patients, impacting therapeutic outcomes. The use of patient-specific neoantigens as cancer vaccine targets offers a promising approach to address this variability. Furthermore, immunosuppression, both systemic and tumor-specific, is a significant challenge in immunotherapy. Although vaccines can generate CD8+ T cells against tumor antigens, their effectiveness is often limited by poor infiltration of the tumor and reduced cytotoxic activity in the immunosuppressive tumor microenvironment. This challenge makes combination therapies essential, as they can enhance immune responses by encouraging antibody production, antigen spreading, and increased effector cell infiltration.

The preparation of biomarkers for selecting patients and antigens may be useful in preventing vaccine-specific side effects in the future. Tools should be designed to identify high-risk individuals and select the most suitable vaccines.^39,40 For this, more studies are needed to determine the most accurate and reliable biomarkers. The use of biomarkers in vaccine selection has the potential to reduce the burden of vaccine-associated adverse events. This will improve the safety and efficacy of vaccines and support the development of personalized vaccines for individual patients. In addition to biomarkers, addressing adjuvant failures, associated needs, and concerns is crucial for the future development of vaccine adjuvants. Adjuvants play a key role in enhancing antigen-specific immunity and may also help reduce the number of doses needed for effective vaccination.

Adjuvants can target specific populations, including the elderly or immunocompromised individuals.⁴¹ A better understanding of adjuvants could enhance vaccine efficacy and accessibility for a broader population. Adjuvants can also be used to extend the duration of immunity, allowing for longer-lasting protection against pathogens. Therefore, researchers need to focus on developing new and improved adjuvants to meet the needs of the changing global population. Table 2 illustrates the status of unsuccessful clinical trials for therapeutic vaccines. During phase-III clinical trials, these vaccines showed tremendous promise but ultimately failed to reach clinical application due to various challenges. A detailed analysis of these randomized trials highlights the strategies employed, the primary obstacles to clinical cancer elimination, and the current status of therapeutic vaccines. Key reasons for halting patient recruitment in clinical trials include late-stage patients’ death, limited clinical benefits, and flaws in study design. In addition, poor safety profile and lack of financial gain were also major reasons for stopping the use of therapeutic cancer vaccines in clinical trials. These issues need to be addressed to make therapeutic vaccines an effective option for cancer treatment.

Table 2.

Failures in Randomized Clinical Trials of Therapeutic Cancer

vaccine	immunogen	target cancer	disease status	clinical trial
Melacine	Allogeneic cell lysate	Melanoma	Adjuvant	NCT00002767
Canvaxin	Allogeneic cells	Melanoma	Metastasis, adjuvant	NCT00052156
PANVAC-VF	CEA, MUC-1	Pancreas cancer	Metastasis	NCT00088660
Oncophage	Vitespen, heat-shock protein	Renal cell cancer	Metastasis, adjuvant	NCT01147536
Oncophage	Vitespen, heat-shock protein	Melanoma	Metastasis	NCT00039000
GM2-KLH21	GM2-KLH21	Melanoma	Adjuvant	NCT00005052
TroVax	MVA-5T4	Renal cell cancer	Adjuvant	NCT00397345
MyVax	Id-KLH + GM-CSF	Non-Hodgkin’s lymphoma	Adjuvant	NCT00017290
FavId	Autologous immunoglobulin idiotype-KLH conjugate vaccine	Lymphosarcoma B-cell Lymphoma Diffuse Large B-cell lymphoma	Adjuvant	NCT00324831
FavId	Id-KLH + GM-CSF	Non-Hodgkin’s lymphoma	Adjuvant	NCT00041730
Listeria/GVAX	GVAX pancreas vaccine	Pancreatic Cancer	Metastasis, refractory	NCT01417000
Theratope	sTn-KLH	Breast cancer	Metastasis	NCT00046371

In the future, novel vaccination approaches will be developed based on the findings of these failed trials. Tailored vaccines will offer individuals greater opportunities for personalized and effective treatments. Additionally, these vaccines could be combined with other therapies to achieve greater efficacy. As a result, they will play an increasingly pivotal role in combating cancers and saving lives.

4. INNATE AND ADAPTIVE IMMUNE STIMULATION BY IN SITU VACCINATION

In situ vaccination stimulates innate immunity immediately, followed by adaptive immunity and immunological memory within one to 3 weeks. A “hot tumor” exhibits an in situ vaccination effect, unlike a “cold tumor”, which is less responsive to this effect.⁴² Cold tumors lack T-lymphocyte infiltration and cannot produce a durable immune response, unlike hot tumors. Checkpoint inhibitors enhance lymphocyte infiltration into immunologically cold tumors through an in situ vaccination effect. Similarly, other immunomodulatory agents help sustain the immune cycle in cancer. This in situ vaccination effect improves the immune response to tumors, leading to better clinical outcomes. It also opens new avenues for developing novel cancer therapies. For example, combining checkpoint antibodies with vaccines can increase the immune system’s sensitivity to the presence of cancer cells, thereby improving the likelihood of treatment success.

4.1. Innate Immune Stimulation.

Neutrophils, dendritic cells, natural killer cells, and macrophages are key players in initiating the innate immune response.⁴³ Therapeutic modulation of these cells can activate the innate immune system. Innate immune cells serve as the primary force of the immune system, and the activation of the adaptive immune system is dependent on them. The innate immune system produces cytokines and chemokines, attracting other immune cells to fight off infections, forming the first line of defense against pathogens. To optimize the immune response, the body must respond quickly and effectively to pathogens. Currently, many researchers are employing various strategies to treat cancer with immunotherapy, aiming to stimulate innate immune responses by targeting pattern recognition receptors (PRRs). Examples include toll-like receptor (TLR) agonists, such as imiquimod and resiquimod. The FDA has approved imiquimod for treating skin cancer, and other TRL agonists are undergoing the clinical trials. Imiquimod can be used alone or in combination with standard treatments and is being investigated for its potential to treat other cancers, including breast, colon, lung, and ovarian cancer. Studies have also suggested that imiquimod may be effective in treating other types of conditions, such as viral infections and autoimmune diseases.

Furthermore, DNA sensing through cGAS/STING agonists, nucleotide-binding oligomerization domain-like receptors (NLRs) such as NLRP3 agonists, and retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) agonists has been shown to play a significant role in initiating inflammation and activating innate immunity.⁴⁴ These agonists are being explored for their potential to target and activate the immune system to combat diseases like cancer and infections. The use of cGAS/STING agonists, NLRs, and RLR agonists has the potential to revolutionize disease treatment.

Several therapies, including chemotherapy, photodynamic therapy, radiation, and photothermal effect, activate the innate immune system and approved for various cancers.⁴⁵ These therapies release tumor-associated antigens from dying tumor cells, which in turn activate the innate immune system. This activation helps eliminate tumor cells or inhibit their growth. Agent that modulates the growth and differentiation of immune cells contribute to a durable immune response. The antitumor effect of intratumoral injection of Flt3L combined with chemotherapy is synergistic. Combining intratumoral Flt3L with chemotherapy has been shown to reduce tumor volume and increase survival rates in animal models. This suggests that intratumoral Flt3L can enhance the efficacy of chemotherapy by promoting dendritic cell maturation.

4.2. Adaptive Immune Stimulation.

As a result of in situ vaccination, innate immune cells such as neutrophils and dendritic cells are activated, contributing to the infiltration of CD8+ T cells into the tumor microenvironment, thereby reducing tumor growth.⁴⁶ Additionally, in situ vaccination increases cytokine production, which further stimulates the immune response. A cross-priming procedure activates lymphocytes specific to the antigens released by dendritic cells. This process, known as immunomodulation, is a key factor in the success of in situ vaccination adaptive immunity is more specific and selective, requiring one to 3 weeks to mount a response. CD4+ T cells, CD8+ T cells, and cytokines such as IFN-γ and IL-12 are key players in modulating adaptive immunity for the treatment of various cancers. Adaptive immunity can be further enhanced by immunotherapy drugs targeting cancer cells. Vaccines that induce an adaptive immune response can both prevent and treat cancer.

In response to cellular stress and danger signals, dendritic cells mature, migrate, and present tumor antigens in lymph nodes, stimulating effector cells.⁴⁷ These effector cells then recognize tumor cells, enabling cytotoxic killing by CD8+ T cells. Cytotoxic T cells secrete cytokines and chemokines to recruit additional effector cells for tumor destruction. Vaccines and immunotherapy drugs activate the adaptive immune system, enabling it to fight cancer effectively. Figure 7 illustrates immune mechanism of classical vaccines for B-cell and T-cell mediated immune responses.

Figure 7.

Immune mechanism of classical vaccines for B-cell and T-cell mediated immune response. This figure represents how classical vaccines activate B cells and T cells. Upon activation, these cells recognize antigens and initiate an immune response, ultimately resulting in the development of protective immunity against the disease. (Created with BioRender.)

5. CANCER VACCINES

Conventional cancer vaccines are not yet approved for the treatment of cancer.⁴⁸ Unlike bacterial and viral vaccines, these vaccines have shown limited promise in clinical trials. Traditional vaccination strategies, initially designed for bacterial and viral infection, have been tested against cancer but demonstrated poor responses, limited efficacy, and a lower success rate in translating these approaches for cancer treatment and prevention. The conventional vaccination faces several challenges in its development identifying appropriate therapeutic targets, designing efficient delivery methods, and ensuring its safety. Addressing these challenges is critical to unlocking the full potential of immunotherapy in combating cancer.

Several cancer vaccines, including cell-based vaccines, RNA vaccines, peptide vaccines, and tumor-specific antigen-based vaccines, have been extensively studied but have achieved limited success.^49,50 These approaches have yet to demonstrate a significant improvement in therapeutic outcomes. Many obstacles must be addressed before cancer vaccines can be widely adopted as a cancer treatment. These include the need to develop more robust immune responses and to identify reliable biomarkers for predicting the efficacy of immunotherapy. Despite promising immune responses in preclinical studies, many cancer vaccines have failed to demonstrate effectiveness in clinical trials. Additionally, these treatments remain prohibitively expensive for patients. Concerns about safety and efficacy have also prevented these vaccines from gaining approval for clinical use. Furthermore, many uncertainties remain regarding the effectiveness of cancer vaccines. A lack of standardization in approaches can render vaccines ineffectiveness, and safety concern. Researchers are actively exploring new strategies to develop safe and effective cancer vaccines. There is growing interest in in situ vaccination approaches due to their unique features. These strategies include the development of novel vaccine platforms and the combination of adjuvants to enhance the efficacy of cancer vaccines. Here, we have summarized an overview of conventional vaccines tested against cancer and vaccines used as immunotherapy to combat cancer.

5.1. Nucleotide-Based Cancer Vaccines.

A nucleotide-based cancer vaccine uses DNA or RNA to target and activate the immune system to attack cancer cells.⁵¹ This approach minimizes damage to healthy cells while simultaneously stimulating the immune system. Clinical trials have demonstrated that these vaccines target cancer-specific mutations, activate the immune system, and produce effective antitumor responses. Unlike normal cells, cancer cells express tumor-specific antigens encoded by nucleotide sequences, such as RNA and DNA, which are the basis of these vaccines. There are two primary types of nucleotide-based vaccines: DNA vaccines and RNA vaccines. While nucleotide-based immunization has been extensively studied in preclinical and clinical research, it has yet to achieve significant clinical success. Despite these setbacks, this approach has provided valuable insights for advancing future research in cancer immunotherapy.

5.1.1. RNA-Based Vaccines.

In these vaccines, mRNA encoding the tumor-specific protein is introduced into the body. In response, the body produces the tumor-specific protein, which stimulates an immune response. Antibodies are subsequently produced to target and combat the tumor, enabling the recognition and destruction of tumor cells. The use of mRNA vaccine was first studied in 1989 with synthetic cationic lipids (DOTMA) and liposomal formulations (lipofection).⁵² For the development of bridging vaccines, live attenuated vaccines expressing endogenous antigens are combined with T cell-mediated immunity. This platform has become increasingly popular due to its ability to induce both humoral and cellular immune responses. Moreover, this type of vaccine is safer and more effective than traditional protein-based vaccines.⁵³

RNAs are promising vaccine vectors due to their ability to induce robust immune responses and their adaptability for encoding various antigens.⁵⁴ However, they pose significant challenges related to translational efficiency and inherent instability, requiring innovative delivery systems and stabilization strategies. RNA vaccines work through pattern recognition receptors, in which cancer and chronic inflammation can be exploited by CD8+ T cell activation via mRNA vaccines. However, RNA vaccines are unstable and do not penetrate cells very well. The prostate cancer mRNA vaccine stimulates innate immunity by increasing DC maturation and cytokine production. This strategy involves plasmid DNA encoding tumor-associated antigens to produce cellular and humoral immunity. Vaccination based on nucleotides is the most effective way to treat prostate cancer, which has a lifetime rate of 5–20% in Western countries.

Over time, there has been growing interest in various nanocarrier-based delivery methods to improve mRNA stability.⁵⁵ Here, we have highlighted a few successful nanoformulations used for mRNA delivery. CureVac, a biopharmaceutical company, manufactures Reactive vaccines (mRNA-containing vaccines) for prostate cancer treatment, including CV9103 and CV9104. As part of phase I and II clinical trials, CV9103 and CV9104 are currently being investigated for men at high prostate cancer risk. RNActive is an m-RNA-based technology developed by CureVac (Tubingen) for men at high prostate cancer risk. The 5-year relative survival rate for most people with local or regional prostate cancer is nearly 100%. For people diagnosed with prostate cancer that has spread to other parts of the body, the 5-year relative survival rate is 32%. RNActive molecules are derived from mRNA and formulated for use in human cells for translational activity and immune stimulation.

Combined with protamine, RNActive derived from mRNA increases Th1 T cell responses against antigens activated by TLR7/TLR8. Cytosolic delivery of the mRNA vaccine induces immunological effects.⁵⁶ Various carriers improved mRNA delivery to the cytosol via endocytic, micropinocytosis, and diffusion-mediated mechanisms. Hydrodynamic pressure plays a major role in transfecting localized cells. This delivery of mRNA leads to increased expression of the T cells, increasing the vaccine’s effectiveness. The use of carriers and hydrodynamic pressure further improves the speed and efficiency of the vaccine delivery.

5.1.2. DNA Vaccines.

DNA-based cancer vaccines work by delivering plasmids that carry genetic material encoding tumor antigens.⁵⁷ These vaccines depend on the body’s cells to absorb the plasmids and produce the corresponding antigens. Unlike peptide-based vaccines, DNA vaccines rely less on adjuvants because introducing foreign genetic material naturally triggers an immune response. DNA plasmids (genes) encoding tumor-specific antigens are the main components of these vaccines. DNA plasmids are transcribed into mRNA in the host machinery. mRNA is translated into tumor antigens—proteins or peptides—which then stimulate the immune system. When these antigens are recognized as foreign invaders, the immune system launches a strong immune response by activating cytotoxic CD8+ T cells to eliminate cancer cells and combat cancer growth. In the early 1990s, DNA vaccines gained considerable attention for cancer immunotherapy. Conventional DNA vaccines were ineffective due to their low potency and immunogenicity. The traditional methods of plasmid DNA (pDNA) delivery, including intramuscular, intradermal, and subcutaneous injections with hypodermal needles, are remarkably ineffective for immunization despite extensive research. Due to its rapid clearance from the injection site, pDNA is ineffective for immunization.

The DNA vaccine can be administered through various routes, including intramuscular, subcutaneous, and intradermal injection.⁵⁸ These methods rely on the host’s cellular machinery to insert the plasmid into the nucleus of transfected antigen-presenting cells (APCs), keratinocytes, and monocytes. Once the host synthesizes the antigen, macrophages and dendritic cells process and present it to nearby draining lymph nodes using major histocompatibility complex (MHC) class I and II molecules. This activates naive CD8+ T cells, transforming them into cytotoxic CD8+ T cells, and naive CD4+ T cells, which become helper T cells that stimulate B cells to produce antibodies. In cancer, CD8+ T cells are activated to exert cytotoxic effects.

DNA vaccines are an effective and versatile method of delivering antigen-coding genes to activate both cellular and humoral immune responses.⁵⁹ To be effective in vivo, DNA vaccines should have excellent thermal stability. These factors facilitate technology transfer, cost savings, and large-scale manufacturing. One of the main disadvantages of DNA vaccines is that they cannot cross cell membranes to elicit an immune response, which results in compromised immunity.⁶⁰

5.1.2.1. Human Papillomavirus (HPV) DNA Vaccines.

The HPV vaccine is designed to prevent anogenital tumors that can result from infection with these viruses.⁶¹ As a preventive vaccination strategy, HPV-16 E7 DNA vaccines have shown promise in controlling cervical cancers. HPV-16 E7 DNA vaccines are specific for tumor antigens related to cervical cancer (HPV). The HPV vaccine contains a live vector, a protein/peptide, and a DNA vaccine. This whole-cell approach protects against animal and human tumors by the T-cell response. Antigen-specific immunity, stability, safety, and ease of manufacturing make DNA vaccines an attractive treatment option for cervical cancer. At early and advanced stages of cancer, a DNA vaccine used for prevention caused cytotoxic T-cell responses against infections associated with HPV.⁶² Figure 8 illustrates the immune mechanism of HPV vaccines.

Figure 8.

Dual roles of the human papillomavirus (HPV) vaccine in prevention and therapeutic effects. On the left, the preventive aspect demonstrates how the vaccine induces B-cell activation, leading to the production of plasma cells and antibodies that neutralize HPV. On the right, the therapeutic effect involves the activation of CD4+ T-helper cells and their role in assisting CD8+ cytotoxic T cells. These cytotoxic T cells recognize and kill HPV-infected tumor cells, promoting an immune response for tumor eradication. The interplay between immune cells highlights the vaccine’s potential in both preventing HPV infection and addressing HPV-related malignancies. (Created with BioRender.)

Nucleotide-based cancer vaccines provide both cellular and humoral immunity against cancer, yet they come with certain limitations. While these vaccines are relatively easier to develop for different cancer mutations, their efficient delivery into cells I hindered by immune evasion mechanisms and immunosuppressive tumor microenvironment. These challenges are being addressed to improve nucleotide-based cancer vaccines.

5.2. Peptide-Based Vaccines.

Mutated neoantigens have been utilized to develop innovative cancer peptide vaccine strategies.^63–65 Synthetic polypeptide-based vaccines show promise as a treatment option for cancer. Peptide vaccines stimulate T-cells more effectively than whole protein vaccines, promoting tumor-specific CD8+ T cell response. Single-peptide vaccines are safer, well-tolerated, and more effective than complete protein vaccines. Currently, no peptide vaccines have been approved for clinical use. However, they are in various stages of preclinical and clinical development. Personalized peptide vaccines target multiple melanoma neoantigens using a combination of long-chain peptides. These vaccines have demonstrated safety, efficacy, and immunogenicity in Phase I trials involving melanoma patients. This personalized approach shows great premise for melanoma treatment, with clinical trials underway to further evaluate its safety and effectiveness. Additional research is needed to explore its potential for broader application.

A Phase I/Ib trial of GAPVAC for glioblastoma demonstrated durable CD8+ and CD4+ T cell responses, along with a favorable safety profile and potential for clinical efficacy.⁶⁶ These findings suggest that GAPVAC could be a promising treatment option for glioblastoma patients. Peptide vaccines have been extensively studied in the literature for their ability to elicit antigen-specific immunity.⁶⁷ However, a significant challenge for peptide-based vaccines lies in their stability in vivo, susceptibility to degradation, size, formulation, and routes of administration. These factors collectively impact the effectiveness of peptide delivery of peptides.

For patients with nonsmall cell lung cancer (NSCLC) who have not responded to immune checkpoint blockade therapy, only one peptide-based Phase 3 trial is currently underway.⁶⁸ The vaccine, Tedopi (OSE2101, OSE Immunotherapeutics), targets five tumor-associated antigens and the CD4 helper epitope PADRE. In this trial, treated patients showed a significant improvement in overall survival, with a hazard ratio of 0.59 and a p-value of 0.017, resulting in compassionate use authorization in the European Union. However, the trial was prematurely halted due to the COVID-19 pandemic, and a confirmatory Phase 3 trial remains pending.

In the Hit and Run vaccine design strategy, T cells are primed with exogenous peptide neoantigens for effective tumor targeting.⁶⁹ Peptide neoantigens are decorated on nanocarriers to elicit a T cell-specific immune response against the same antigens. The unique feature of this strategy is its ability to rapidly prime T cells with peptide neoantigens while ensuring a durable immune response for effective tumor control. A literature case study demonstrated this strategy using a cancer vaccine in which the OVA peptide was efficiently conjugated to poly(2-oxazoline)s polymer. This design triggered an OVA-specific T cell response upon cleavage of the matrix metalloprotease 2 (MMP-2)-sensitive linker connecting the polymer and peptide vaccine. This approach has proven to be a uniform vaccine strategy against tumors with a low mutational burden.

In another study, researchers addressed the challenge of the lymph node’s paracortex barrier by designing nanovaccines that utilize Retro-Diels–Alder linkers to attach various oxazoline monomers to peptide vaccines.⁷⁰ The distinctive feature of these linkers is their time-dependent cleavage, enabling a sustained and durable release of peptide neoantigens within the lymph node. This strategy led to a significant improvement in antitumor efficacy (p < 0.001) in the B16-OVA model. Notably, these delivery vehicles remained within the lymph node for 415 days, indicating that prolonged retention at the target site is crucial for eliciting an essential T-cell immune response in peptide cancer vaccination.

Poly(2-oxazoline)-based polymeric nanocarriers have demonstrated significant potential in enhancing the targeting and efficacy of peptide neoantigens.⁷¹ These conjugate self-assemble into uniform nanocarriers approximately 50 nm in size, facilitating the accumulation and infiltration of antigens into lymph nodes. In the MC38 tumor cell line model, these nanocarriers exhibited superior tumor clearance compared to free peptide antigens. This efficacy is attributed to the nanocarriers’ ability to retain antigens without loss and penetrate lymph nodes effectively, thereby inducing a durable T-cell immune response.

5.3. Cell-Based Vaccine.

Cell-based vaccines can be broadly classified into two categories: tumor cell vaccines and immune cell vaccines.⁷² Tumor cell vaccines contain tumor-associated antigens, along with CD4+ T, and CD8+ T cell epitopes, while immune cell vaccines involves immune cells and their specific role. Dendritic cells, as professional antigen-presenting cells, are commonly used in cancer vaccines. They are capable of inducing both CD4+ and CD8+ T cell-mediated immunity and play a critical role in activating antigen-specific T cells. Clinical trials have shown that dendritic cell vaccines hold promise for treating various types of cancer. Most dendritic cell vaccines used monocyte-derived dendritic cells to load antigens, improving vaccine efficacy and tolerance. Ongoing research is exploring their potential to reduce immunogenicity (reduce the immune system’s overreaction) and enhance safety. These vaccines have the potential to become a significant tool in cancer immunotherapy.

5.3.1. Dendritic-Cell-Based Vaccines.

Dendritic cell (DC)-based vaccines play critical role in initiating and regulating both adaptive and innate immune responses.⁷³ They are essential for presenting cancer antigens to MHC I molecules and activating CD8+ T cells. Currently, Sipuleucel-T is the only FDA-approved DC-based vaccine, used for the treatment of prostate cancer. It stimulates an immune response against cancer by leveraging the patient’s own immune cells, including dendritic cells. Despite its promise, clinical trials have demonstrated limited success with this approach. The challenges include the functional limitations of in vitro-generated DCs and the immunosuppressive microenvironment, which hinders the vaccine’s effectiveness Some DC-based vaccines are used in cancer treatment, such as those involving DC-pulsed peptides of mutated cancers were administered to patients with stage III melanoma. In the future, DCs are expected to play a broader role in cancer vaccines, addressing various cancer types and stages and combining with immunotherapy, chemotherapy, and radiation. DC vaccines have proven safe, effective, and durable in early- and late-stage cancer patients.

Several clinical trials have investigated the use of these vaccines to harness the ability of DCs to present tumor antigens and stimulate immune responses.⁷⁴ For example, ongoing trial NCT03400917 is evaluating a DC-based vaccine is being evaluated with anti-PD-1 therapy for patients with glioblastoma, aiming to enhance immune activation in the tumor microenvironment. Similarly, the NCT04277221 trial explores a personalized DC vaccine combined with chemotherapy for patients with pancreatic cancer, targeting patient-specific tumor antigens to improve outcomes. An earlier trial, NCT00045968, investigated DC-based vaccines in patients with metastatic prostate cancer, reporting a modest improvement in overall survival. These findings highlight the potential of DC vaccines while emphasizing the need for combination strategies to improve efficacy. Another trial, NCT01983748, evaluated a DC vaccine in patients with malignant pleural mesothelioma, reporting promising immune responses and disease stabilization in some patients. However, further studies are required to confirm long-term benefits. Together, these trials demonstrate the potential of DC-based vaccines to enhance antitumor immunity across a range of cancers. They also suggest that combining these vaccines with therapies like checkpoint inhibitors or chemotherapy could optimize clinical outcomes. Future research should focus on identifying the most effective DC subsets and universal antigens for therapeutic use

5.3.2. T-Cell-Based Vaccine.

Activating antigen-presenting cells (APCs) is a crucial step in T-cell priming for T-cell-based cancer vaccines.⁷⁵ APCs process and present antigens via major histocompatibility complexes (MHC), activating T-cells and initiating tumor cell destruction. Adjuvants enhance APC activation and support T-cell priming, fostering a more durable immune response. This process is fundamental for effective cancer immunotherapy.

Several clinical trials have investigated T-cell-based vaccines in cancer immunotherapy to boost the body’s immune response against tumors.⁷⁶ However, only a few T-cell-based vaccines have advanced to Phase 3 trials. Therapies like Sipuleucel-T and DC Vax-L have demonstrated survival benefits in certain cancers, such as prostate cancer and glioblastoma. Despite these successes, challenges remain in achieving consistent and durable clinical responses, particularly in aggressive tumor types. Ongoing Phase 3 trials aim to evaluate the broader applicability of these vaccines across various cancer types. In contrast to the limited number of phase 3 studies, numerous phase 1 clinical trials are exploring T-cell-based vaccines. For example, the NCT03629080 trial demonstrated that personalized neoantigen vaccines represent a promising strategy for enhancing antitumor immunity in patients with advanced solid tumors. This vaccine proved safe, immunogenic, and capable of inducing durable T-cell responses when combined with checkpoint inhibitors. While these early results are encouraging, they lay a groundwork for further research to fully unlock the potential of personalized neoantigen vaccines in combination with other immunotherapies. Future studies involving larger patient populations and longer follow-up periods will be critical to confirm the clinical benefits observed in this Phase 1 trial.

5.3.4. B-Cell-Based Vaccine.

In B-cell-based vaccines, B-cell activation by CD4+ T cells is sufficient to generate antibodies without requiring peptide-bound major histocompatibility complexes.^77,78 This activation leads to indirect tumor cell death. The antitumor action mediated by B cells involves tumor-infiltrating B cells and antigen processing, which activate Th cells to secrete cytokines, promoting sustained cytotoxicity.

Several clinical trials have investigated the potential of B-cell vaccines in cancer treatment. A notable example is the IMU-131 (HER-Vaxx) trial, a B-cell epitope vaccine targeting the HER2/neu protein in patients with HER2-positive gastric cancer (NCT02795988).⁷⁹ This vaccine elicited strong immunogenic responses, generating robust polyclonal antibody production against HER2 and demonstrated antitumor effects in preclinical models. In the Phase Ib trial, patients receiving IMU-131 in combination with chemotherapy exhibited favorable safety profiles and immune responses, paving the way for further studies. Another promising candidate is PD1-Vaxx (NCT04202003), a first-in-class B-cell vaccine targeting PD-1. Currently being evaluated in a Phase 1 trial for nonsmall cell lung cancer, this vaccine aims to generate antibodies against PD-1, potentially offering a novel therapeutic alternative to monoclonal antibody therapies like pembrolizumab. Early preclinical data demonstrated the vaccine’s capacity to inhibit tumor growth by activating immune responses against the PD-1 protein. These trials highlight the growing potential of B-cell-based vaccines in cancer immunotherapy, especially in scenarios where B-cell-derived antibodies can enhance tumor targeting or complement existing immune mechanisms. Future research is likely to focus on combining B-cell vaccines with other immunotherapies to improve patient outcomes and maximize therapeutic efficacy.

5.3.5. Combining B-Cell and T-Cell Vaccines.

Long-term vaccination effects can be achieved through a combination of B-cell and T-cell vaccines, which target different mechanism and act synergistically.⁸⁰ These vaccines work together to control tumor growth and metastasis, enhancing antitumor immunity. By simultaneously activating B-cells and T-cells, the immune system is comprehensively stimulate, minimizing the risk of tumor escape and improving efficacy. Despite their potential, no vaccine targeting either B-cells or T-cells has yet been approved for clinical use. Figure 9 illustrates the B- and T-cell-mediated antitumor mechanisms of these vaccines. B- and T-cells recognize tumor cells expressing tumor antigens and work to eliminate them. This process leads to the generation of tumor-specific memory B- and T-cells, which can recognize and eliminate tumor cells upon recurrence. Vaccines, therefore, play a critical role in stimulating the immune system to recognize and destroy tumor cells effectively. Table 3 summarizes the conventional vaccine strategies currently employed in preclinical studies and clinical trials, highlighting the progress and potential of these approaches.

Figure 9.

Schematic illustration of basic T-cell- and B-cell-based antitumor mechanisms and key focused strategies for cancer vaccine design. (Created with BioRender.)

Table 3.

Vaccine Strategies Currently Utilized in Preclinical Studies and Clinical Trials⁸¹

vaccine strategy	mechanism of action	benefit	limitation
DNA	CTL response induced by transfection of plasmids with immunogenic genes	Multiantigen specificity, long half-life, and scalable	High off-target delivery and low efficiency, Low immunogenicity requires high doses, Large size and negative charge, Mutagenic potential, Risk of autoimmunity and toxicity, Prolonged response monitoring, High cell manipulation required for immunogenicity
mRNA	Synthetic mRNA is delivered via a carrier molecule that generates specific immunogenic antigens	It is scalable, nonvirulent, Nonintegrating, and degrades naturally. Stimulates innate and adaptive responses	The formulation is unstable and inefficient, requires encapsulation or nanocarriers, High allergy risk with delivery systems, paradoxical suppression of innate immunity
Peptide	Short antigenic peptide fragments are used to induce a targeted response. Liposomal formulations used as delivery vehicles	Produced and delivered easily, minimal toxicity, Easy monitoring of immune response, less allergenic and reactogenic, Induce immunity against multiple strains by utilizing conserved peptides	Carriers are required for chemical stability, elicit mainly one type of immune response (cytotoxic or humoral), booster shots may be required, the antigen is HLA-restricted, epitope-specific, and challenging to formulate multivalent antigens, each new antigen requires new production and stability protocols.
DC	Activate adaptive immunity through cellular antigen uptake of antigen, either through ex vivo activation and readministration or in situ phagocytosis.	Scalable, can elicit both humoral and cytotoxic responses, Generates memory responses, HLA-independent, Antigen type (allogeneic vs autologous, whole-cell vs neoantigen)	DCs must be expanded, matured, and activated ex vivo, requiring GMP facilities, technically challenging, Regulatory challenges due to variability in antigen selection and activity.

5.4. In Situ Vaccination (ISV) for Cancer.

In situ vaccines offer a promising alternative to conventional cancer vaccines.^82–84 This approach enables tumor-associated antigens (TAAs) to be released directly from tumors in vivo, eliminating the need for their prior isolation, identification, or purification of antigens. The released TAAs are taken up by dendritic cells, which then activate antitumor immune responses. ISVs essentially transform immunologically “cold” tumors into “hot” tumors, priming the patient’s immune system to recognize and attack the tumor. This strategy allows cancer vaccines to be personalized for each patient, overcoming many of the challenges associated with conventional vaccines. By converting an optimal repertoire of tumor antigens into tumor-associated antigens, ISVs effectively activate dendritic cells and cross-prime CD8+ T cells. This unique mechanism provides significant advantages over conventional vaccinations, which are often limited by tumor heterogeneity. Figure 10 illustrates the divers mechanisms of in situ vaccination, highlighting its ability to harness the patient’s immune system to generate robust antitumor responses.

Figure 10.

Different mechanisms involved in in situ vaccination. (Created with BioRender.)

For successful ISV, therapies such as chemotherapy, immunostimulants, and ablative treatments play a crucial role in transforming the tumor into a vaccine.⁸⁵ The use of immunogenic cell death (ICD)-inducing agent is essential in this process. These agents—such as chemotherapy, radiation, phototherapy, photodynamic therapy, and immunostimulants—trigger immunogenic cell death, facilitating the release of TAAs and activating the immune system. Figure 11 highlight the unique features of in situ vaccines, showcasing their ability to harness the tumor microenvironment for effective immune responses.

Figure 11.

Unique features of in situ vaccines. (Created with BioRender.)

In situ vaccines work by inducing immunogenic cell death and triggering the release of pro-inflammatory cytokines, such as IL-12 and IL-6, from treated tumors.⁸⁶ These cytokines enhance dendritic cell activity, promoting antigen uptake and cross-priming of CD8+ T cells, thereby strengthening immune responses. Growth and differentiation factors further support dendritic cell function, improving their ability to process and present tumor antigens. Similar to TLR agonists, IL-12 plays a key role in stimulating dendritic cell maturation during immune responses. The following sections of this article provide a detailed overview of the various mechanisms of in situ vaccination and their unique characteristics.

5.4.1. Plant-Based Viral Agent for In Situ Vaccination.

Various plant-based viral agents have demonstrated immunomodulatory effects and are being explored as in situ vaccines.⁸⁷ These agents effectively overcome local immunosuppression in tumors by activating the innate, adaptive, and complement immune systems. Among the most studied plant viruses for improving antitumor immunity are cowpea mosaic virus, papaya mosaic virus, and tobacco mosaic viruses. Since these viruses are not derived from human pathogens, they are considered safe for therapeutic use. The systemic antitumor effects of plant-based viruses are achieved by introducing viral particles into tumor microenvironments. Examples include papaya mosaic virus, cowpea mosaic virus and their nanoparticles, which induce immunomodulation and stimulate systemic immune responses. Plant-based virus containing nanocarriers have proven effective in stimulating the immune system, with their effectiveness influenced by factors such as size, shape, and molecular weight. This innovative strategy paved the way for future clinical trials, although no plant-based virus has yet been approved for in situ vaccination. Figure 12 illustrates the antitumor mechanism of oncolytic virus. After infecting tumor cells and replicating within them, these viruses release tumor antigens and cytokines to activate innate and adaptive immunity, contributing to antitumor effects. Once tumor cells are destroyed, the released viruses can infect and target additional tumor cells, amplifying the therapeutic responses. The following sections describe individual plant viruses in detail regarding their role in in situ vaccination

Figure 12.

Antitumor mechanism of oncolytic virus. (Created with BioRender.)

5.4.1.1. Cowpea Mosaic Virus (CPMV).

Cowpea mosaic virus (CPMV), a plant virus that does not infect mammalian cells, has shown promise in cancer immunotherapy by acting as a potent immunostimulatory agent. CPMV vaccine is employed in immunotherapy to shift the immune environment from immunosuppressive to immunostimulatory.⁸⁸ CPMV exhibits inherent immunogenicity, as demonstrated through studies using bone marrow-derived dendritic cells and primary macrophages isolated from C57BL/6 mice. When cultured with CPMV, these cells exhibited elevated levels of the pro-inflammatory cytokines, as illustrated in Figure 13. This approach demonstrated a significant antitumor effect in preclinical tumor models the antitumor activity of CPMV has been studied in C57BL/6 mice inoculated with B16F10 dermal melanoma. Infectious CPMV activates innate immunity by recruiting monocytes and natural killer (NK) cells. This activation ultimately led to the stimulation of CD4+ and CD8+ T cells are activated along with immune memory.

Figure 13.

(a) Elevated levels of pro-inflammatory cytokines with eCPMV generated in Escherichia coli after exposure to bone marrow-derived dendritic cells. (b) Elevated level of pro-inflammatory cytokines with eCPMV after exposure to thioglycolate-elicited primary macrophages. Reproduced with permission from ref 88. Copyright 2015 Springer Nature.

Another study found that intratumoral injection of CPMV was shown to modulate the tumor microenvironment, alleviate immunosuppression (e.g., reducing MDSCs, FOXP3, inflammation pathways), and produced systemic antitumor effects.⁸⁹ Mouse and canine models of metastatic melanoma were used to evaluate CMPV as an in situ vaccine for its immunotherapeutic efficacy. Additionally, it was demonstrated that CPMV nucleocapsid induced robust antitumor immunity, enhancing therapeutic outcomes when combined with repeated treatment using anti-CPMV antibodies.

Oncolytic viruses restore systemic antitumor responses by reversing immune suppression within TME.⁹⁰ In the literature, CPMV and nucleic acid-free virus-like particles have been studied for their potential as in situ vaccines. In syngeneic mouse tumor models, including melanoma, breast, ovarian, glioma, and colon cancer, as well as in companion dogs with oral melanoma, oncolytic viruses showed strong in situ vaccination effects.

A combination of radiation therapy and CPMV nanoparticles used as an in situ vaccine showed an enhanced antitumor effect.⁹¹ In this study, C57BL/6 female mice bearing ID8-Defb29/Vegf-A-Luc tumors (100–150 mm³ in size) were treated with a combination of radiation and CPMV nanoparticles. The tumor growth delay observed with this combination was statistically significant (p < 0.05) compared to radiation alone. Furthermore, the treatment led to significant infiltration of CD4+ T cell and CD8+ T cell into tumors, underscoring the immune activation induced by the combination therapy. This study confirmed that combining CPMV with radiation effectively overcame immune suppression and highlighted the limitation of monotherapy for large tumors. Figure 14 shows a detailed overview of the study plan, the tumor growth curves for various treatment groups, and the total luciferase count after the treatment.

Figure 14.

(A) Treatment plan for after radiation therapy plus CPMV. (B) Tumor growth curve for different treatment groups. (C) Total luciferase counts on day 29. Reproduced from ref 91. Copyright 2018 American Chemical Society.

5.4.1.2. Tobacco Mosaic Virus (TMV).

TMV is another type of antiviral agent studied for in situ vaccine. Unlike CMPV, which does activate innate or adaptive immunity, TMV stimulates pro-inflammatory cytokines, particularly, IL-6.⁹² This indicates that TMV is more likely to trigger an immune response, making it a promising candidate for therapeutic applications aimed at stimulating antitumor immunity. TMV was encapsulated into nanoparticles and nanorods, and their morphology, their mechanisms, and efficacy as in situ vaccines were studied. This study revealed that intratumorally injection of TMV nanocarriers elicited antitumor immunity in a dermal melanoma model. Interestingly, the morphology (spherical vs rod-shaped) and aspect ratio of TMV nanocarriers did not significantly affect the in situ vaccine efficacy, which was primarily driven by the induction of strong pro-inflammatory cytokines, especially IL-6. Figure 15 compares the efficacy study of CPMV and TMV in the B16F10 melanoma model, demonstrating that CMPV induces a stronger antitumor response than TMV.

Figure 15.

Efficacy study for B16F10 melanoma model. (A) Tumor growth curves with overall growth. Arrows show the vaccination schedule. (B) Tumor growth curves by treatment group. (C) Tumor growth curves compared at individual time points. (D) Survival curve of mice post first in situ vaccination (time 0). (E) SNP tumor growth curve [left] and survival curve [right]. (F) TMV dose escalation experiment tumor growth curve (100 vs 500 μg). Reproduced from ref 92. Copyright 2018 American Chemical Society.

5.4.2. Oncolytic Virus.

5.4.2.1. Tamilogene Laherparepvee.

In melanoma, T-VEC is an oncolytic herpes virus engineered to produce the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF).^93,94 T-VEC is designed to recognize and destroy cancer cells while simultaneously stimulating the immune system to recognize and attack them. It is effective in treating certain types of advanced melanoma and has been approved for use in the U.S., Europe, and other countries. A study involving 80 patients treated with T-VEC showed that 39% of patients experienced complete local responses, while 18% had partial responses.

T-VEC is the first viral immunotherapy to be approved for unresectable stage III–IV melanoma.⁹⁵ It selectively replicates in tumors through genetic modification to the herpes simplex virus type 1 (HSV-1), inducing cell lysis and producing antitumor effects. When administered intramurally, it causes tumor cell lysis and releases tumor-associated antigens, which activate CD8+ T cells and stimulate tumor-specific effector T-cells. Preclinical studies have shown that combining T-VEC with immunotherapy agents, such as immune checkpoint inhibitors (e.g., a-CTLA-4, a-PD-1), induces T-cell reactivation and synergistically enhance therapeutic efficacy. This combination has proven effective for controlling cutaneous and subcutaneous metastases, although the systemic effect of T-VEC alon can be inconsistent. Through the production of GM-CSF, T-VEC generates a systemic antitumor response. Preclinical studies show that T-VEC effectively infects melanoma cells, controlling both local and metastatic melanoma spread to distant organs. Phase II and III clinical trials of T-VEC-based monotherapy have showed a favorable safety profile with minimum side effects and efficacy in treated and untreated lesions. However, the limited systemic effect highlights the necessity of combining of T-VEC with immunotherapy, such as checkpoint inhibitor like ipilimumab and pembrolizumab, for a sustained and durable response.

5.4.2.2. Newcastle Disease Virus (NDV).

The Newcastle disease virus (NDV) has been explored as an ISV approach due to its potential to overcome resistance to therapy.⁹⁶ In murine melanoma models and human tumors, NDV showed enhanced antitumor activity through multiple independent mechanisms that bolster the host’s antitumor response. NDV is immunogenic and can modulate the host’s immune response. Beyond directly killing injected tumors, NDV can induce the regression of distant, noninjected tumors by generating tumor-specific antitumor immune responses, including responses to tumor neoantigens. These properties enable the targeting of hard-to-access tumors. Studies of NDV in murine melanoma tumor models and human tumors show delayed growth in NDV-injected tumors and rejection of untreated distant tumors. This contrasts with treatments like IFN-α, which only delay growth in injected tumor lesions. These findings confirm that NDV induces rejection of uninfected distant tumors through the expansion and recruitment of tumor-infiltrating lymphocytes (TILs).

Another report highlights NDV studies in an orthotopic syngeneic murine GL261 glioblastoma model.⁹⁷ Mice treated with intratumoral NDV injections showed a statistically significant prolongation of median survival, with 50% surviving long-term. This effect is associated with immunogenic cell death (ICD), characterized by the release of calreticulin and HMGB1 and increased PMEL17 cancer antigen expression in the NDV-treated mice. NDV treatment also enhanced infiltration of IFN-γ(+) T cells and reduced MDSCs due to its immunomodulatory effects. The ICD from treated tumors and subsequent priming of adaptive antitumor immunity contributed to the therapeutic benefits. Figure 16a shows that immunocompetent C57BL/6J mice treated with NDV survived longer than the control mice, whereas Rag2+ mice, due to their immunodeficiency, did not respond to NDV. Figure 16b demonstrates that wild-type NDV-treated mice had improved survival similar to immunocompetent C57BL/6J mice, while CD8+ T cell-depleted mice showed no efficacy from NDV treatment. This highlights the critical role of CD8+ T cells in regulating immune responses.

Figure 16.

Efficacy study for NDV. Kaplan–Meier curves showing immunocompetent (black line) and immunodeficient (dotted lines) cells are presented. Reproduced with permission from ref 97. Copyright 2014 UICC.

Overall, oncolytic viruses demonstrated significant potential for ISV, with T-VEC already approved for the treatment of metastatic melanoma. However, these technologies are not universally effective against all cancer types. The clinical translation of oncolytic viruses remains limited due to challenges in delivery, regulatory hurdles, ethical considerations, and immunological side effects.

5.5. Chemotherapy.

Clinically, chemotherapy is primarily used to inhibit tumor cell growth. Strong evidence suggests that chemotherapy not only reduces tumor growth but also prevents regression and recurrences by activating both the innate and adaptive immune systems.⁹⁸ However, the immunosuppressive effects of cytotoxic agents are not yet fully understood. It has been proposed that lymphodepletion activates homeostatic mechanisms to counteract immunosuppression by stimulating tumor-reactive T-cells.

Additionally, studies indicate that certain anticancer agents induce immunogenic cell death (ICD) and apoptosis, which generate tumor-specific antigens and effectively function as in situ vaccines.⁹⁹ These antigens, when presented to T cells, trigger a systemic antitumor effect by activating CD8+ T cells, ultimately halting tumor growth. Chemotherapy-induced ICD involves the release of ATP, calreticulin (CRT), and high mobility group box (HMGB1) from tumors, as shown in Figure 17. This process promotes DC maturation, activation of toll-like receptors, and the release of “eat-me” signals, all of which contribute to the activation of a tumor-specific adaptive immune response.

Figure 17.

Molecular mechanism of immunogenic cell death induced by photodynamic therapy. The process involves the activation of caspases, leading to apoptosis or autophagy, resulting in the release of antigens and danger signals to activate immune response. This results in the recruitment of immune cells, leading to tumor destruction. (Created with BioRender.)

Paclitaxel enhanced the antitumor effects at ultralow doses by modulating immunosuppressive regulatory T cells (Tregs) and MDSCs in the tumor microenvironment, as well as by activating DCs and CD8+ T cells.¹⁰⁰ This resulted in increased CD8+ T-cell infiltration, tumor growth inhibition, and improved overall survival. Ultralow doses of paclitaxel present a promising new therapeutic strategy for cancer treatment.

In situ vaccination to control cancer growth and prevent metastasis is achievable using chemotherapeutic agents that induce immunomodulation, immunogenic cell death (ICD), and release of tumor-specific antigens.¹⁰¹ This approach can be applied to treat various cancer types, reduce recurrence after surgery, and potentially enhance the efficacy of existing cancer treatments. Although only a limited number of chemotherapeutic drugs have demonstrated ICD effects, several ICD-inducing agents have been used in clinically for years. Table 4 highlights FDA-approved ICD-inducing chemotherapeutic agents for different cancer types.¹⁰²

Table 4.

Clinical Relevance of ICD Inducers Used for Cancer Chemotherapy

chemotherapeutic drugs	cancer types
Doxorubicin and mitoxantrone	Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), small cell lung carcinoma, breast carcinoma, neuroblastoma, lymphoma, thyroid carcinoma, soft tissue, and bone
Epirubicin	Breast carcinoma
Bleomycin	Bleomycin Hodgkin’s lymphoma, NHL, penile cancer, and testicular cancer
Idarubicin	Acute Myeloid Leukemia (AML)
Cyclophosphamide	Non-Hodgkin Lymphoma (ML), Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), chronic lymphocytic leukemia, MM, ovarian carcinoma, breast carcinoma, mycosis fungoides, lymphoma, neuroblastoma, and retinoblastoma;
Oxaliplatin	Advanced colorectal carcinoma
Bortezomib	Multiple Myeloma (MM), Mantle Cell Lymphoma (MCL)

The combination of chemotherapeutic agents with immune checkpoint inhibitors has shown synergistic antitumor responses.¹⁰³ For example, when paclitaxel is combined with checkpoint inhibitors in clinical trials for triple-negative breast cancer, overall survival is significantly improved compared to paclitaxel alone.

Chemotherapy is used for ISV to produce a robust systemic antitumor effect when combined with checkpoint inhibitors and immunostimulants. However, its efficacy remains limited, highlighting the urgent need for adjuvant to enhance this effect.

5.6. Radiation Therapy.

The use of radiation therapy in clinical settings has significantly improved survival and life expectancy for cancer patients.¹⁰⁴ Radiation therapy works by locally damaging tumors while minimizing damage to surrounding normal tissues. By releasing antigens, proinflammatory cytokines, and activating both the innate and adaptive immune systems, ionizing radiation therapy has been shown to convert tumors into in situ vaccines. Radiation increases T-cell infiltration into tumor microenvironments and promotes the development of immunological memory responses, which not only kills cancer cells but also helps prevent recurrences.

To initiate the ISV effect, radiation induces ICD by activating its mediators.¹⁰⁵ This results in an immunological response against distant metastases, including areas not directly targeted by radiation. Radiation therapy activates naive T cells by releasing damage-associated molecular patterns (DAMPs), which cross-present antigens to DCs. DNA fragments generated from radiation-induced damage in the nucleus are transferred to the cytoplasm, where act as cGAS/STING agonists, further activating innate and adaptive immunity. These DNA fragments also stimulate DCs present within the tumor microenvironment.

The in situ vaccine effect of radiation therapy plays a significant role in treating primary tumors, controlling metastatic tumors, and inducing systemic antitumor responses in irradiated tumors.¹⁰⁶ This effect has been studied in patients with malignant melanoma, where combining radiation therapy with immunological modulators enhanced ethe systemic antitumor response.^107–111 Radiation-induced DNA helix damage leads to cell death through apoptosis, necrosis, mitotic catastrophe, autophagy, and senescence. When combined with immune checkpoint inhibitors, radiation therapy enhanced the systemic effects, facilitating metastatic tumor regression Radiation also increased MHC class I expression on cell membranes, enabling antigen presentation and DC maturation, which contribute to ICD and in situ vaccination mediated by CD8+ T cells. Furthermore, ionizing radiation damages tumor cells by inducing DNA double-strand breaks, resulting in systemic antitumor effect.

Combining radiation therapy with immunostimulants has demonstrated improved efficacy and stronger immunological effects.¹¹² For example, patients with refractory nonsmall cell lung cancer treated with a combination of radiation and checkpoint inhibitor (e.g., ipilimumab) showed CD8+ T-cell infiltration, resulting in sustained and durable systemic anticancer effects.

Antigen capture following radiation therapy has also been shown to improve cancer immunotherapy, as illustrated in Figure 18. Antigen-capturing nanoparticles deliver antigen-specific proteins to DCs for cross-priming, significantly enhancing the antitumor efficacy of a-PD-1 therapy in the B16F10 melanoma model. Mechanistic studies confirm that this process expands CD8+ T cells and increases in both CD4+ T/Treg and CD8+ T/Treg ratios.¹¹³

Figure 18.

Antigen capturing nanocarriers for improved immunotherapy of cancer. Reproduced with permission from ref 113. Copyright 2017 Springer Nature.

Researchers have confirmed that radiation-induced systemic antitumor effects are immune-mediated.¹¹⁴ In a study using a bilateral tumor model with mammary carcinoma cells (67NR), the immunological effects of radiation were investigated. Primary tumors exposed to irradiation exhibited a significant delay in tumor growth. Additionally, the growth of secondary, nonirradiated tumors was impaired through the synergistic effect of radiation and Flt3-L, a growth and differentiation factor for DCs. In contrast, control and Flt3-L-treated mice without radiation showed no impact on the growth of secondary tumors, confirming the immunological role of radiation. To further validate these findings, a similar combination of radiation and Flt3-L was tested in nude (lacking T cells), which showed no effect on the growth pattern of nonirradiated tumors, as illustrated in Figure 19. This study provides strong evidence that T-cell-mediated immune responses are critical for controlling the growth of distant nonirradiated tumors

Figure 19.

Local RT and Flt3-L trigger systemic anticancer effects. This suggests that local RT induces a systemic antitumor reaction in the presence of Flt3-L. Furthermore, the combination of local RT and Flt3-L can be used to improve the efficacy of cancer treatment. Reproduced with permission from ref 114. Copyright 2004 Elsevier.

Achieving a systemic antitumor effect in the poorly immunogenic mice tumor models remains challenging, and radiation-induced in situ vaccinations have shown limited efficacy in patients.¹¹⁵ To address radiation’s poor immunological effects, it is crucial to identify and understand the factors within the tumor microenvironment that contributed to this limitation. The immunosuppressive effect of radiation, such as inhibition of dendritic cells and impaired immune function through reduced CD8+ T cell levels, must be thoroughly examined. Radiation can be effective in patients lacking prior antigen exposure to the tumor, but a successful antitumor response depends on the interplay between preexisting immunosuppressive factors, radiation-induced effects, and immune activities.

5.7. Photodynamic Therapy (PDT).

PDT is approved for the treatment of skin diseases, bladder, lung, esophageal cancer, and metastatic melanoma in a noninvasive and spatiotemporal manner. Following the injection of a photosensitizer into the patient, a specific wavelength of light is used to illuminate the tumor. At this wavelength, the photosensitizer transfers energy to molecular oxygen, generating singlet oxygen (a reactive oxygen species) in the process. PDT induces apoptosis and necrosis in tumor tissue, leading to the production of in situ vaccines, oxidative stress, and the release of pro-inflammatory cytokines. The resulting acute inflammatory response generates chemokines and cytokines, activates the innate immune system (including NK cells, neutrophils, macrophages, and dendritic cells), and triggers the adaptive immune response (via CD4+ T cells and CD8+ T cells). These responses collectively contribute to cell-mediated damage to secondary tumor, potentially aiding in the prevention of metastasis and recurrences. In addition to inducing ICD, PDT also generates damage-associated molecular patterns (DAMPs), further activating both innate and adaptive immunity.¹¹⁶ Figure 20 demonstrates that PDT induces apoptosis and necrosis, stimulating both innate and adaptive arms of immune system for a robust antitumor effect.

Figure 20.

PDT activates both adaptive and innate arms for antitumor immunity. Reproduced with permission from ref 116. Copyright 2020 American Society for Photobiology.

PDT may activate the immune system by increasing cytokine production and promoting the infiltration of immune cells into tumors following treatment.¹¹⁷ In a phase I clinical trial, perforin sodium PDT using 639 nm laser at a low fluence rate demonstrated control of distant tumor metastases in 2 out of 9 breast cancer patients. These results suggest that PDT holds promise as a therapy for metastatic breast cancer, though further clinical trials are needed to assess its long-term efficacy and safety. In patients with invasive esophageal squamous cell carcinoma, PDT shifted the tumor microenvironment from immunosuppressive to immunostimulatory by reducing Treg and causing damage to FoxP3, highlighting the role of PDT in immunomodulation.¹¹⁸

Preclinical studies have shown that the combination of porphyrin lipoprotein-based PDT and CBIs can effectively control tumor growth by activating both the innate and adaptive immune systems.¹¹⁹ In one study, AE17-OVA+ mesothelioma cells were injected into C57BL/6 mice 24 h after porphyrin lipoprotein administration, followed by 5 min of 671 nm illumination and 12 mg/kg CBI on days 0,3,6, or 10. This combination produced a synergistic effect on nontreated tumors (p < 0.05). A 60-fold increase in interleukin-6 levels activated the innate immune system, while a significantly higher presence of CD4+ T cells and CD8+ T cells in nontreated tumors confirmed their effector function. These results demonstrate that repeated PDT combined with a-PD-1 can generate in situ photovaccination effects. Figure 21 shows the in situ photo vaccination effects of PDT on both illuminated and nonilluminated tumors.

Figure 21.

Effect of repeated PDT of porphyrin and a-PD-1 for illuminated (left tumor) and nonilluminated tumor (right tumor) shows a delay in tumor growth for both tumors (p < 0.05), and the Kaplan–Meier curve confirms increased % survival for a synergistic effect. Reproduced with permission from ref 119. Copyright 2022 Elsevier.

PDT is used as a tool to enhance antitumor immunity, with multiple immune cells contributing to its immunological effects. However, the antitumor effects of PDT are limited and inconsistent. Combining PDT with an adjuvant is necessary to achieve a more durable response.

5.8. Photothermal Therapy (PTT).

Photothermal therapy uses photothermal agents like gold, indocyanine dye (ICG), and other agents with a laser or light source.¹²⁰ Those agents absorb light and generate heat to ablate cancer cells. This approach shows promise in medical treatment, including cancer. As a result of PTT treatment, cancer cells release tumor-associated antigens, such as damage-associated molecular patterns and proinflammatory cytokines, to activate an antitumor immune response specific to the tumor antigens. Targeting photodynamic and photothermal therapy to the endoplasmic reticulum also enhances ICD.

ICG, a photothermal agent approved clinically, and imiquimod, a TLR-7 agonist, were selected as the components of the nanoparticles (loaded into PLGA, poly (lactide-co-glycolide)).¹²¹ The nanoparticles eliminated the primary tumor and showed an antimetastatic effect in combination with CBIs. Figure 22 shows the detailed description of this approach utilizing PTT combined with CBIs and immunostimulants. Figure 22 indicated o/w emulsion method for loading ICG and R837 into PLGA nanoparticles. After sufficient absorption into the tumor and illuminated with NIR laser, these nanoparticles eliminated the primary tumor and released tumor-associated antigens for systemic antitumor effects, such as rejection of rechallenge and antimetastatic effects. The addition of a-CTLA-4 improved the local systemic antitumor effect significantly in CT26 and 4T1 orthotopic syngeneic mice models

Figure 22.

Efficacy study. (a) Schedule for cell inoculation, PTT, and a-CTLA administration. (b, c) Tumor growth curves of different groups of treatment 4T1 and CT26 tumor model. (d) In vivo bioluminescence images. (e, f) Morbidity-free survival of different groups of mice bearing metastatic 4T1 and orthotopic 4T1 tumors. Reproduced from ref 121. Copyright Nature portfolio 2021.

PTT-induced ISV elicits robust antitumor immunity against local, disseminated, and metastatic cancers.¹²² This approach was tested using novel amphiphilic polyTLRa-conjugated gold nanorods (AuNRs-IMDQ-R9-PEG), which incorporate gold as a photothermal agent and Imiquimod as a TLR7/8 agonist to promote dendritic cells maturation, as shown in Figure 23a. The multifunctional nanocarriers were evaluated as a proof of concept in 4T1 tumor-bearing balb/c mice treated with PTT at 808 nm (NIR laser). Antigen release from PTT-treated tumors serves as an in situ nanovaccine, inducing a vaccination effect. The study results confirm that the approach activates and matures endogenous dendritic cells, enabling innate immune cells to present antigens for CD8+ T-cell activation and immunological memory development. This immunological response demonstrated an antimetastatic effect, evidenced by a reduced number of lung nodules, delayed growth of nontreated distant tumors, and improved survival rates. Overall, the findings validate that AuNRs-IMDQ-R9-PEG nanocarriers combined with PTT at 808 nm (NIR laser) generate an effective in situ vaccine for metastatic tumor control, enhancing antitumor immunity and providing protection against cancer, as shown in Figure 23b.

Figure 23.

(a) Design of AuNRs-IMDQ-R9-PEG multifaceted nanocarriers. (b) In situ vaccination effect of AuNRs-IMDQ-R9-PEG multifaceted nanocarriers for antitumor immunity. Reproduced with permission from ref 122. Copyright 2021 Elsevier.

In another report, an alum-tuned hydrogel composed of dopamine grafted onto hyaluronic acid was used as a carrier for delivering Indocyanine green (ICG) for PTT-mediated in situ vaccination, aiming at treating local tumors and developing immunological memory.¹²³ In this system, alum controlled the degree of hydrogel cross-linking through complexation and enhanced the durability of the immune response via adjuvant effect. Intratumor injection of the ICG-loaded hydrogel into CT26 tumor-bearing balb/c mice, followed by illumination with an 808 nm NIR laser at 1 W/cm² for 3 min, resulted in significant antitumor effects. This treatment, combined with checkpoint blockade inhibitors (CBIs), led to complete rejection of nontreated secondary tumor upon rechallenge, demonstrating an in situ vaccination effect via the release of tumor-associated antigen from the primary tumor. Immunohistochemistry and TUNEL assay confirmed apoptosis in tumors treated with PPT and CBIs, while additional analysis revealed significant infiltration of CD8+ T cells into the tumors. Supporting these results, flow cytometry analysis showed a significantly higher presence (p ≤ 0.05) of CD8+ T cells, including effector and naïve subsets, in treated tumors. Furthermore, the results indicated lower levels of MDSCs, including CD11b+ Ly6C+ (monocytic) and CD11b+ Ly6C+ Ly6G+ (granulocytic) MDSCs. Overall, this study confirmed that ICG-loaded hydrogel carriers, in combination with PTT and CBIs, effectively induce an in situ vaccination effect against CT26 tumors. Figure 24 shows a detailed schematic overview of this approach of photothermal therapy approach using ICG with an alum-tuned dopamine-grafted hyaluronic acid copolymer hydrogel.

Figure 24.

In situ vaccination effect in a distant tumor model. (A) Tumor inoculation and therapy schedule for treated and nontreated tumor. (B) Nontreated secondary tumor growth curve. (C) Body weight change data following secondary tumor challenge. (D) Secondary tumor incidence rate profiles. (E) Tumor weight data of secondary tumor. Reproduced with permission from ref 123. Copyright 2023 Elsevier.

PTT shows promise as a vaccination approach when combined with other strategies such as immunostimulants, checkpoint inhibitors, and chemotherapeutic agents, offering a new combination strategy for cancer treatment.¹²¹ Clinical studies have demonstrated the efficacy of phototherapy in metastatic melanoma. In these clinical trials, in situ photoimmunotherapy (ISPI) combines indocyanine green as a photothermal agent for the photothermal effect and imiquimod as an immune response modifier. The patient response rate is promising, and this approach can be easily applied on an outpatient basis. It can also be combined with other modalities to improve the therapeutic response in metastatic melanoma. Based on these findings, in situ photoimmunotherapy is a palliative therapy with limited toxicity when compared to other methods.¹²⁴

5.9. Immune Checkpoint Blockage Inhibitors (CBIs).

The discovery of CBIs for cancer treatment has shown promising results for melanoma. In 2011, ipilimumab (CTLA-4), the first class of CBIs targeting antibodies, became the first FDA-approved treatment for metastatic melanoma.¹²⁵ Later, in 2014, another class of CBIs (PD-1), including pembrolizumab (Keytruda) and nivolumab (Opdivo), was approved for advanced melanomas and lung cancer, renal cell carcinoma, and other cancers. These CBIs are now used for treating cancers such as breast, cervical, bladder, colon, head and neck, liver, lung, and skin cancers. Pembrolizumab (a PD-1 inhibitor) was further approved in 2016 for nonsmall cell lung cancer and melanoma.¹²⁶ T-effector cell infiltration into tumors and immune-supportive microenvironments serve as markers for the effectiveness of CBIs.

Checkpoint proteins are inhibitory proteins on immune cells like T cells.¹²⁷ They prevent collateral damage to normal cells by controlling T-cell overactivation and are essential for normal immune function. However, in cancer, these checkpoints are hijacked by the tumor cells, allowing the tumors to evade the immune system and grow unchecked. Immune checkpoint inhibitors block these checkpoints, enabling the immune system to recognize and attack tumor cells. Up to 12.5% of patients respond to checkpoint inhibitor therapy.¹²⁸ Cancer cells exploit immune checkpoint activation to keep T cells engaged to overcome immunosurveillance, and promote tumor growth and disease progression, leading to a poor response to therapy.

T-cell activity is restored after inhibition of CTLA-4 and PD-1 ligands on T cell surfaces. Checkpoint inhibitors prevent cancer cells from evading immunosurveillance by activating cytotoxic T cells (CD4+ and CD8+ T cells).^129–131 Preclinical and clinical trials have shown that CBIs restore dysfunctional effector T cells, inhibit tumor growth through in situ vaccine effects, and prevent metastasis. Therapeutically, checkpoint inhibitors reactivate anticancer immunity and convert immunologically resistant tumors into “hot tumors” by increasing the immune infiltration into tumors, thereby improving antitumor effects. CBIs are administered in combination with standard care treatments such as chemotherapy, surgery, radiation therapy, and photodynamic therapy in sequential dosage regimens. This combination results in better antitumor effects and reduces recurrence of tumor metastases by enhancing cytotoxic T-cell function. Combining CBIs with standard treatments improves the systemic antitumor effects. The major challenges with checkpoint inhibitors include poor patient response, adverse events, and cytokine release syndrome. Figure 25 illustrates the role of checkpoint inhibitors in restoring T-cell activation. The combination of CBIs with PDT, radiation, PTT, chemotherapy, and other immunostimulants significantly enhances the in situ vaccine effect. Table 5 summarizes the results and discussions of in situ vaccination strategies in preclinical studies.

Figure 25.

Role of checkpoint inhibitors in restoring T-cell activation. Checkpoint inhibitors are molecules that allow T cells to recognize and attack cancer cells. They help restore normal T cell activation and function, increasing antitumor immunity. This helps to reduce the risk of cancer recurrence and increases the effectiveness of cancer treatments. (Created with BioRender.)

Table 5.

In Situ Vaccine Approach Studied in the Preclinical Study

Therapy	Combination	Immune marker	Observation	Animal model	ref.
Radiation	Flt3-Ligand	DCs, CD8+ T cells	Dendritic cells’ activation of tumor-specific T cells inhibits distant tumor growth	Mice bearing a syngeneic mammary carcinoma, 67NR	114
	CTLA-4 blockade	CD8+ but not CD4+ T cells.	Antitumor immunity inhibiting the metastases	4T1 model	132
	Chemotherapy	IFNα, memory T cells	Antitumor immunity in mice and humans	Hemagglutinin (HA)-transfected AB1 tumors in BALB/c mice	133
	CpG polyplex	DCs and effector T cells, cytokines	Antitumor immune memory effect	B78 tumor model	134
	Anti-CD 40	CD8+ T cells	Systemic antitumor effect on distant tumor	Panc02 tumors model	135
	a-CTLA-4	Expansion of CD8+ cytotoxic T cells	Improve cancer immunotherapy effect	B16F10 model	113
Interleukin-12	Interleukin-12	CD8+ T cell, NK cell	Antitumor efficacy	Different mice model	136
Chemotherapy	CpG	IFNα,IL-6, and CD8+ T cell	Programmable immune activation nanomedicine (PIAN) for immune activation and tumor inhibition.	CT26 model	137
	cGAMP (STING agonist	Tumor-associated macrophages, proinflammatory cytokines, and CD8+ T cell	Antitumor efficacy, Rejection of rechallenge	B16F10 model	138
	CpG	Dendritic cells (DCs), B cells, and Natural killer cells.	Antigen-specific antitumor immune responses improved survival in mice	Rhabdomyosarcoma tumor	139
Oncolytic viruses	Cowpea Mosaic Virus	IL-6, IFNβ and IFNγ, Inate immune cells	Priming of systemic antitumor immunity	B16F10 model	140
	Herpes Simplex Virus	(HSV-1) mutant G207,	Systemic antitumor immune response	CT26 and M3 melanoma model	141
	Cowpea Mosaic Virus	Il–12, Ifn-γ, adaptive immunity, and neutrophils	Systemic antitumor immunity for metastatic effect	B16F10 model	88
	Cowpea mosaic virus (CPMV) virus-like particles	Pro-inflammatory cytokines (e.g., IFN-γ, IL-6), cytotoxic CD8+ T cells	Antitumor immunity	B16F10 model	90
Dendritic Cell Vaccine	Chimeric cross-linked polymersome	DCs, CD8+ T cells	Priming of systemic antitumor immunity	MC38 model	142
	ELANE + Hiltonol (TLR3 agonist)	Activation of cDC Is, CD8+ T cell	Potent tumor inhibition in a poorly immunogenic triple-negative breast cancer (TNBC)	MDA-MB-231/luc cells Orthotopic TNBC model	143
Photothermal therapy (PTT)	MT@OMV-Mal	Dendritic cells (DCs), Tumor-infiltrating effector T cells,	Eliminating the residual lesions and distant metastases	CT26 model	144
	Vincristine, Adriamycin, Ifosfamide, and Actinomycin D (VAIA)	NK cells, Effector CD8+ and CD4+ T cells	Immune effects outside of the heated target	A female diagnosis of disseminated rhabdomyosarcoma	145
	TLR-7 agonist,) YM155 (survivin inhibitor); + (survivin inhibitor) + anti- CD47	IL-6, IL-12, TNF-α, CD4+ and CD8+ cells in distant tumors, and mature DC	Control primary and distant tumor growth	B16F10 model	146
	Imiquimod (R837), TLRs agonist + (a-CTLA4)	IL-6, TNF-α,	Cytotoxic T lymphocytes (CD3+CD4-CD8+) could directly kill targeted cancer cells, helper T cells (CD3+CD4+CD8-)	Orthotopic 4T1 models	121
	LR-7 agonist), ± aCTLA-4	DC maturation, IL-6, IL-12, TNFα	Inhibit the growth of distant tumors	4T1 and CT26 models	147
	TLR-7/8 agonist	CD3+ infiltration ↑, Granzyme B↑, Hsp70↑, IL-6↑, IL-10↓, apoptosis ↑, MDSCs	Long-lasting immunologic memory, inhibited tumor growth and prevented recurrence and metastasis	CT26 models	148
CBIs	aOX40	CD8+ T-cells, IFN-γ	Enhanced therapeutic efficacy, and increased immunological memory.	B16F10 and 4T1 models	149
Photodynamic therapy (PDT)	Oxygen-boosted immunogenic PDT	CD8+ T cells, Dendritic cells (DCs), activated T lymphocytes, and natural killer (NK) cells	Destroy primary tumors and effectively suppress distant tumors and lung metastasis in a metastatic triple-negative breast cancer	4T1 model	150
	Microneedle	Dendritic cells (DCs) maturation, T-cell-mediated immune response	Antitumor immune responses on distant tumor	B16F10 model	151
	Microneedle	IFN-γ, TNF-α, IL-12p70, IL-6, and T-cell-mediated immune response	Antitumor immunotherapy	B16 model	152
	NK cells Coated Nanoparticles	Ml-macrophage, anti-inflammatory cytokine, T-cell-mediated immune response	Antitumor immunotherapies eliminate primary tumors and effect to inhibit distant tumors	4T1 model	153
	a-PD-L1	CD8+ T cells activity	Systematic Antitumor Immune Response, antimetastatic effect	4T1 and TUBO models	154
	Metformin	CD8+ T cells activity	Control primary and abscopal tumor growth in bladder and colon cancers,	CT26 and MB49 model	155

5.10. Adjuvants.

In preclinical and clinical settings, tumor-ablative therapies such as radiation, photodynamic therapy, photothermal therapy, microwave, ultrasound, and radiofrequency are used to destroy tumors without removing them. Ablative therapy leaves residual tumor antigens (tumor-associated antigens) in the body after destroying the tumor.^156,157 Combining this ablative therapy with adjuvants, known as immunostimulants, can enhance the in situ vaccination effect. Immunostimulants modulate the intensity and duration of immune responses, resulting in a sustained immune response that develops immunological memory for future protection.

The FDA has approved a limited number of adjuvants, while many others are under preclinical investigation. Adjuvants have the ability to stimulate the maturation and differentiation of dendritic cells and activate toll-like receptors (TLRs), NOD-like receptors, and RIG receptors.^158,159 Using ablative therapies (such as cryoablation, laser, radiation, ultrasound, chemotherapy, and surgery) followed by local administration of alum, saponin, or monophosphorylate lipid has been shown to improve the in situ vaccination effect. However, the safety and efficacy of saponin-derived adjuvants are limited due to their hemolytic effects, toxicity, and instability in the aqueous solutions. The literature reports that saponin-based classical adjuvants, when injected peritumorally and administered postablation, show a synergistic advantage with superior antitumor immune responses. Below, we describe the use of different adjuvants in combination with other treatments and their important roles in cancer therapy.¹⁶⁰

5.10.1. CpG-ODN.

CpG-ODN adjuvants activate toll-like receptor 9 (TLR9) on the endosomal surface of immune cells, stimulating humoral and cellular immune responses.¹⁶¹ They modulate immune responses to control tumor growth, and the in situ vaccine effect of intratumor CpG injections has been shown to produce sustained immune responses. Several types of CpG-ODN exhibit different properties in preclinical models. In the literature, five types of CpG-ODN are reported (Types A, B, C, P, and S), categorized based on their effects on immune cells, sequences, and structures. Among these, Types A, B, and C have been shown to stimulate immunity in humansCombining CpG with other ablative therapies has significantly enhanced the in situ vaccine effect significantly.

Several clinical trials have evaluated the use of CpG-ODN as a vaccine adjuvant. For example, CpG 7909, a TLR9 agonist, has been assessed in combination with cancer vaccines for multiple cancer types, including non-Hodgkin’s lymphoma and cutaneous T-cell lymphoma.¹⁶² In these trials, CpG 7909 enhanced antitumor immune responses by increasing the activation of T cells and antigen-presenting cells, leading to improved immune recognition of tumor cells. Additionally, CpG 1018, a CpG-ODN, has been used as an adjuvant in the HEPLISAV-B vaccine for hepatitis B. In clinical trials, CpG 1018 significantly enhanced the immune responses compared to traditional alum-adjuvanted hepatitis B vaccines. This adjuvant stimulated higher antibody titers with fewer doses, demonstrating its potency in improving the vaccine’s effectiveness, particularly in populations that typically respond poorly to hepatitis B vaccination, such as older adults. These examples highlight the potential of CpG-ODNs as versatile and effective adjuvants for both infectious disease and cancer vaccines, enhancing immunogenicity and potentially improving clinical outcomes across various treatments. However, further research is needed to ensure their safety and address the adverse events associated with their use.

5.10.2. Pattern Recognition Receptors (PRRs).

Pattern recognition receptors (PPRs) are costimulatory molecules expressed on myeloid immune cells, such as dendritic cells and macrophages.¹⁶³ They are activated and recruited in response to stress signals and immunogenic cell death (e.g., ATP, HMGB1). Synthetic PRR agonists stimulate phagocytosis and antigen presentation by myeloid cells within the tumor microenvironment. Preclinical data suggest that PRR agonists can overcome resistance to T-cell-targeted CBIs.

Vaccines can achieve improved efficacy and immunogenicity with this class of adjuvants.¹⁶⁴ A recently discovered pathway involving cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) has gained attention in this category for its role in modulating immune responses. As part of the in situ vaccine effect for cancer immunotherapy, activating the STING pathway through cyclic dinucleotides enhances the immunogenicity of immunologically “cold” tumors. However, enzymatic degradation and rapid clearance can impact toxicity and efficacy, necessitating the use of various nanocarriers is essential to overcome these challenges.

In clinical trials, PRR agonists have shown both promise and limitations as vaccine adjuvants.¹⁶⁵ Agonists targeting TLRs, such as TLR9 agonists, have demonstrated the ability to enhance both cellular and humoral immune responses. For instance, CpG 1018, used in the HEPLISAV-B hepatitis B vaccine, was approved for its superior immunogenicity compared to alum-adjuvanted vaccines. Similarly, TLR4 agonists, such as monophosphoryl lipid A, are also widely used in vaccines like in Cervarix (an HPV vaccine), where they increase efficacy and durability of the immune response. Additionally, RIG-I-like receptors, which detect viral RNA, have been explored in early stage cancer and viral infection vaccines. These agonists enhance antiviral immune responses by inducing type I interferons, which prime the adaptive immune system and strengthen immune responses. Despite promising preclinical results, PRR agonists face challenges in clinical trials. Excessive immune activation can lead to autoimmunity or severe inflammatory responses, limiting their efficacy. For instance, TLR agonists may trigger systemic inflammation, causing adverse effects such as fever, fatigue, or, in rare cases, autoimmune reactions. While some TLR4 agonists have shown moderate success in enhancing immune responses in cancer immunotherapy trials, achieving consistent therapeutic outcomes remains challenging.

5.10.2.1. Toll-like Receptors (TLRs).

TLRs are discussed in detail in section 2.1. These adjuvants show synergistic effects when combined with PDT, PTT, radiation, and chemotherapy as immunotherapy approaches to modulate the immune response via in situ vaccination.¹⁶⁶ A TLR9 ligand, K3-SPG, has demonstrated a synergistic effect with systemic and local immunotherapy. In one study, researchers investigated the antitumor effects of the TRL9 ligand K3-SPG as in situ vaccine in preclinical animal models of colon cancer and pancreatic ductal adenocarcinoma (PDA). In situ vaccination with K3-SPG, in combination with a-PD-1, enhanced the antitumor effect significantly (p ≤ 0.01) in the PDA model, leading to complete rejection upon rechallenge and a significant improvement in significant survival. This effect was attributed to an increase in the immune populations, including CD8+ Tnaïve, CD8+ T memory, and CD8+ T effector cells, through the synergistic combination of K3-SPG and combination with a-PD-1. TLRs play a critical role in activating innate immune cells.

5.10.2.2. Retinoic Acid-Inducible Gene I (RIG-I)-like Receptors (RLRs).

Another class of PRRs is the retinoic acid-induced gene I (RIG-I)-like receptors (RLRs), which act as sensors for viral infections and generate type I interferons along with other cytokines.¹⁶⁷ RLRs detect dsRNA and recognize viral RNA, activating the immune response to produce antitumor effects.

In addition to playing a crucial role in antiviral immunity, retinal acid-inducible gene I (RIG-I)-like receptors (RLRs) also regulate antitumor immune responses.^168,169 It has been demonstrated that RIG-I, melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2) are capable of recognizing single-stranded RNAs and triggering type I interferons and proinflammatory mediators. As a result, signaling cascades enhance the recruitment and activation of antigen-presenting cells (APCs), such as dendritic cells (DCs), which prime antigen-specific cytotoxic T lymphocytes (CTLs) that attack tumors. In addition, activation of RLRs in tumor cells can initiate immunogenic cell death, which further enhances antitumor immunity by exposing the immune system to tumor antigens. RLR agonists have been found to enhance tumor immunogenicity in preclinical and clinical studies, making them promising candidates for cancer immunotherapy combination therapies.

RLRs have gained attention in immunotherapy, particularly in the development of cancer and antiviral vaccines, due to their ability to stimulate robust innate immune responses. Preclinical studies have demonstrated that activating RLRs, particularly RIG-I, can induce potent immune responses that help control viral infections and cancer progression. In early clinical trials, RLR agonists have shown promise as vaccine adjuvants and as direct therapeutic agents for melanoma and hepatocellular carcinoma. However, while RLR agonists have shown potential in preclinical studies, challenges remain in their clinical application. The risk of excessive immune activation leading to inflammation or autoimmunity is a concern, as overstimulation of RLR pathways can cause unwanted side effects such as systemic inflammation. Additionally, tumor evasion mechanisms in certain cancers may limit the efficacy of RLR-targeted therapies.

5.10.2.3. NOD-like Receptors (NLRs).

NLRs are another class of pro-inflammatory cytosolic PRRs. They are classified into different types based on the N-terminal domain and apoptosis inhibitory proteins.¹⁷⁰ PRRs stimulate the secretion of pro-inflammatory signals and activate NF-kB, leading to inflammation followed by an immune response.

Liposomal muramyl tripeptide, a NOD-2 agonist known as mifamurtide, is approved in the EU for the treatment of nonmetastatic osteosarcoma in combination with chemotherapy (doxorubicin, cisplatin, and high-dose methotrexate with, or without, ifosfamide).¹⁷¹ Mifamurtide is an immunomodifier that activates macrophages and monocytes to produce antitumor effects. Results demonstrate that mifamurtide is well-tolerated, with common side effects such as chills and fever, and significantly improves survival in newly diagnosed, high-grade, nonmetastatic cancers and respectable osteosarcomas.

These adjuvants have the potential to increase vaccine efficacy of the vaccine and reduce the risk of cancer recurrence. PRR agonists can also be administered intratumorally to stimulate an immune response and reduce tumor growth. Table 6 lists the adjuvants of the PPR class used for cancer vaccines and intratumoral delivery in various stages of clinical trials.

Table 6.

Adjuvants for Cancer Vaccines and Intratumoral Delivery of PRR Agonists in Clinical Trials

PRRs class	PRR agonist	tumors	phase	objective	clinical trial
TLR7/8	Imiquimod	Metastatic melanoma	1/2	Advanced metastatic melanoma patients treated with peptide vaccination combined with tumor immunomodulation (ADJ)	NCT01191034
		Breast cancer	2	An agonist for TLR7 and radiotherapy for breast cancer with skin metastases	NCT01421017
		Breast cancer	2	Treatment of advanced breast cancer with topical imiquimod and abraxane	NCT00821964
		Vulvar neoplasia/anogenital condyloma	2	A study of the effectiveness of imiquimod for the treatment of vulvar intraepithelial neoplasias 2/3 and anogenital warts assessed by immunoevasion of HPV	NCT00941811
		Nonmelanomatous skin cancer	3	Treatment of basal cell skin cancer with topical imiquimod compared to surgery	NCT00066872
	Resiquimod	Metastatic melanoma	2	Toll-like receptor (TLR) agonists at tumors and vaccine sites	NCT00960752
STING	MIW815 (ADU-S100)	Solid tumors and lymphomas	1	An investigation of the safety and efficacy of MIW815 (ADU-S100) in patients with advanced/metastatic solid tumors and lymphomas	NCT02675439
	MK-1454	Solid tumors and lymphomas	1	Use of MK-1454 alone or in combination with pembrolizumab in patients with advanced/metastatic solid tumors or lymphomas	NCT03010176
TLR8	Motolimod	Ovarian cancer	1	Treatment of ovarian epithelial, fallopian tube, or peritoneal cavity cancer with VTX-2337 and liposomal doxorubicin hydrochloride	NCT01294293
		Ovarian, fallopian tube or primary peritoneal cancer	2	Pegylated liposomal doxorubicin (PLD) and VTX-2337 in patients with recurrent or persistent epithelial ovarian, fallopian tube, or primary peritoneal cancer	NCT01666444
		HNSCC	1	The combination of TLR8 agonist VTX-2337 with cetuximab in the treatment of locally advanced, recurrent, or metastatic HNSCC	NCT01334177

6. BARRIERS TO IN SITU VACCINATION

A significant number of immunostimulants, including chemotherapeutic agents, oncolytic viruses, phototherapy, and photothermal therapy, have been reported to exhibit in situ vaccine (ISV) effects. However, these effects remain relatively rare in clinical settings. In situ vaccines targeting “cold” tumors, poorly immunogenic mouse tumors, often fail to induce an effective antitumor immune response, demonstrating the limited efficacy of ISVs. Improving the efficacy of ISVs remains a significant scientific endeavor. To achieve better outcomes with ISVs, it is essential to review all published preclinical literature to identify, understand, and address technical and scientific barriers within the tumor microenvironment that need to be overcome.

The molecular mechanisms underlying the ability of ISVs to induce antitumor effects are not fully understood. Tumor-associated antigens, CSF1, ICDs, cytokines, and other factors influence the relevant signaling pathways. These mechanisms must be supported by a solid scientific foundation. The effectiveness of ISVs in inducing antitumor immunity depends on the balance between preexisting immunosuppressive factors and immune-activating signals. This balance must be maintained for optimal therapeutics effects. The immunogenicity of ISVs can be enhanced by targeting several novel actionable pathways enabled by these vaccines. As a new tool for boosting antitumor T-cell activity, ISVs warrant further investigation.

7. FUTURE PROSPECTS

In situ vaccination for cancer has the potential to revolutionize treatment by leveraging the patient’s own immune system to fight the disease. Despite the challenges, several promising avenues for future progress exist. One approach is to combine ISV with other immunotherapy strategies, such as checkpoint inhibitors or CAR-T cell therapy, to enhance the immune response and improve clinical outcomes. Another promising direction is to develop new technologies to address the technical and logistical challenges of personalized cancer vaccine production and administration. Advances in genomic profiling and immune monitoring techniques could also help identify patients most likely to benefit from ISV and provide insights into the mechanisms underlying the immune response to the vaccine. Additionally, expanding the use of ISV beyond melanoma to other cancer types, such as breast and lung cancer, could significantly increase its impact. Overall, the future of ISV for cancer is promising, with ongoing research and innovation offering the potential for improved patient outcomes and a new era of personalized cancer therapy.

8. CONCLUSION

ISV, which use patient’s own tumor to create a personalized cancer vaccine, has shown promising results in preclinical and clinical studies. This approach aims to activate patients immune system to recognize and attack cancer cells while minimizing damage to healthy tissues. However, challenges remain in developing effective in situ vaccination strategies. One major obstacle is tumor heterogeneity, which can impact the vaccine’s ability to generate an immune response that targets all cancer cells. Additionally, there are technical and logistical challenges associated with the manufacturing and administration of personalized cancer vaccines. Overall, in situ vaccination for cancer is a promising approach that has demonstrated encouraging results in early studies, but further research is necessary to optimize the strategies and techniques, and to address the remaining challenges.