Current Trends and Innovative Approaches in Cancer Immunotherapy

Monoclonal Antibody (mAbs)

The development of mAbs began in the 1970s. By 1975, mAbs with a predetermined specificity were successfully generated in mice using the hybridoma technique [23]. There are three types of mAbs commonly used to treat cancer. The first is a common type known as naked mAbs. Naked mAbs function independently and operate through various mechanisms. These mechanisms involve attaching to cancer cells, enhancing their recognition and destruction by the immune system, binding to and obstructing proteins that facilitate the growth or spread of cancer cells, and targeting immune system checkpoints to amplify the immune response. Immune checkpoint inhibitors, a type of mAb that operates through the last mechanism, play a significant role in cancer immunotherapy [24]. One example of naked mAbs is trastuzumab, which blocks the overexpression of human epidermal growth factor receptor 2 (HER2) on breast and stomach cancer cells. In a phase 3 clinical trial to evaluate the efficacy and safety of adding trastuzumab to anthracycline-based and non-anthracycline-containing adjuvant chemotherapy, the addition of adjuvant trastuzumab significantly improved disease-free and overall survival (OS) among women with HER2-positive breast cancer. The estimated disease-free survival rates at 5 years were 75% in the AC-T group (doxorubicin and cyclophosphamide followed by docetaxel), 84% in the AC-T plus trastuzumab group, and 81% in the TCH group (docetaxel and carboplatin plus trastuzumab). Furthermore, estimated rates of OS were 87%, 92%, and 91%, respectively [25]. Another example, rituximab, is an anti-CD20 mAb for the treatment of non-Hodgkin’s lymphoma. Clinical trials have demonstrated that rituximab prolongs the time to disease progression and extends OS [26]. In the meta-analysis of seven randomized clinical trials in patients with follicular lymphoma, the OS rate of patients with rituximab maintenance was improved compared to patients without rituximab maintenance (hazard ratio 0.79) [27].

The second type is a conjugated mAb chemically conjugated with a chemotherapy agent or radioactive isotope to deliver one of these substances directly to the cancer cells [28, 29]. Examples include brentuximab vedotin and trastuzumab emtansine, both conjugated with monomethyl auristatin E (MMAE) or DM1, targeting CD30 or HER2 [30]. In the clinical trial about brentuximab vedotin, the 6-year OS estimates were 93.9% in the A + AVD (brentuximab vedotin plus doxorubicin, vinblastine, and dacarbazine) group and 89.4% in the ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) group. It showed the survival advantage of brentuximab vedotin for the treatment of stage III or IV Hodgkin’s lymphoma [31].

The third type, Bispecific mAbs is composed of parts of two different mAbs, enabling them to attach to two different proteins at the same time. Among the various forms of Bispecific mAbs, bispecific T-cell engagers (BiTEs) have one part that binds to a T cell-specific molecule (usually CD3) and another part that binds to a tumor-associated antigen (TAA). This leads to the recruitment of T cells to tumor cells, enabling them to initiate an effective immune response [32]. An example is Blinatumomab, a CD3/CD19 BiTE, which was approved by the US Food and Drug Administration (FDA) in 2014 [33, 34]. In a clinical trial comparing blinatumomab and standard-of-care chemotherapy, blinatumomab showed significantly longer OS than chemotherapy among patients with relapsed or refractory B-cell precursor acute lymphoblastic leukemia. The median OS was 7.7 months in the blinatumomab group and 4 months in the chemotherapy group [35].

Although mAb therapy has been successful in treating cancer and is widely used in clinical settings, it has side effects including chills, fever, headache, nausea, and infusion reactions. Additionally, the development of resistance to mAb in some patients also remains a major challenge [36, 37]. This resistance includes mutations of the antibody target, induction of alternative signaling pathways, and impaired effector cell responses. Because mAbs vary in their effectiveness depending on the expression of the target molecule to which the antibody binds, resistance may arise due to mutations in the target, and resistance may be acquired by activating alternative growth or survival signaling pathways associated with the target. Additionally, Antibody-dependent cellular cytotoxicity (ADCC) is thought to be a key therapeutic mechanism for mAb treatment, as changes in effector cell function may affect the ability of the antibody to eliminate target cells [37].

Immune Checkpoint Inhibitor (ICI)

ICI is a type of cancer treatment that involves targeting specific proteins on immune cells to enhance the body’s natural immune response against cancer cells. This approach has gained significant attention in recent years due to its effectiveness in treating certain types of cancer, particularly melanoma, non-small cell lung cancer, bladder cancer, head and neck cancer, kidney cancer, and colorectal cancer [38, 39].

Due to the intricate and specific nature of the immune system, it has major regulations and checkpoints designed to prevent autoimmunity and modulate immune responses. T cells are activated by antigen presentation involving the major histocompatibility complex (MHC), molecules of APCs, and the T cell receptor (TCR) of naive T cells. The full activation necessitates the interplay between costimulatory molecules CD28 and B7, a process that is carefully controlled or restrained by the inhibitory checkpoint. This regulation is crucial to prevent unintended harm resulting from autoimmunity [40]. However, tumors can bypass these checkpoints and diminish the body’s natural immune responses. Effector T cells in the tumor have reduced cytokine expression and effector capacity, and these T cells show increased expression of various inhibitory surface molecules, including CTLA-4, programmed cell death protein 1 (PD-1), and lymphocyte activation gene-3 (LAG-3) [41]. Overexpression of these inhibitory checkpoint molecules contributes to tumor-promoting immunosuppression, making the inhibition of these molecules a promising target for enhancing immune responses against tumors [39]. Thus, ICIs function by preventing the transmission of the “off” signal to T-cells. This enables T-cells to attack cancer cells by blocking the binding of checkpoint proteins to their partner proteins.

Among the many immune checkpoints, PD-1, CTLA-4, and the antibodies targeting them are the most studied in the laboratory and clinical trials [42]. CTLA-4 (also known as CD152) is the first co-inhibitory molecule identified and its expression on effector T cells and Treg cells was reported in 1987. CTLA-4 has an opposing effect on CD28 activity (costimulatory receptor) and has a competitive binding relationship with CD28, but has a 100-fold higher affinity for CD80/CD86 ligands [43]. CTLA-4 reduces T cell activity through binding to CD80 and CD86, activating intracellular signals that lead to IL-2 downregulation, apoptosis, and anergy, resulting in the suppression of T cell proliferation and IL-2 secretion [44]. Additionally, CTLA-4 is highly expressed on Treg cells, and CTLA-4 blockade can reduce Treg-mediated immune suppression [45]. Ipilimumab, a CTLA-4 inhibitor, is the first ICI approved by the FDA for advanced melanoma in 2011. PD-1 is mainly induced by activated T cells, monocytes, and DCs. PD-1 has two ligands, programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2), and interaction with the ligands results in dephosphorylation of TCR signaling molecules and inhibition of T cell activation [46, 47]. Tumors can escape immune surveillance by expressing PD-L1/L2 and thus can be suppressed through anti-PD-1 strategies. Additionally, since PD-1 is highly induced in T-reg cells, blocking the PD-1 pathway may enhance antitumor immune responses by reducing the function of Treg cells [48]. The first anti-PD-1 inhibitor, nivolumab, was approved by the FDA in 2014 and has proven effective against several cancers, such as melanoma [49–53]. In a phase 3 trial comparing nivolumab and dacarbazine in patients with melanoma, the median OS was 37.5 months and 11.2 months for the nivolumab and dacarbazine groups, respectively. Complete response and partial response were also higher in the nivolumab group [54]. Additionally, the 5-year OS rate for the combination therapy of nivolumab and ipilimumab was 52%, which was better than 44% for nivolumab alone or 26% for ipilimumab alone [55].

LAG-3 (also known as CD223) is structurally homologous to CD4 and interacts with MHC class II molecules [56]. LAG-3 has a higher affinity than CD4 and can negatively regulate CD4 + T cells [57, 58]. In addition to effector CD4 + T cells, LAG-3 is also expressed on Treg cells, where it helps increase the immunosuppressive activity of Treg cells [59, 60]. However, despite long-term research, much remains unknown about the structure and function of LAG-3 [61]. The FDA approved combination therapy with LAG-3 monoclonal antibody in 2022, making LAG-3 the third immune checkpoint to be clinically used, following PD-1 and CTLA-4. In a clinical trial conducted in combination with relatlimab, a LAG-3 blocking antibody, and nivolumab, the median progression-free survival was 10.1 months in relatlimab-nivolumab compared with 4.6 months in nivolumab alone. It shows that the inhibition of two immune checkpoints, LAG-3 and PD-1, has benefits in patients with previously untreated metastatic or unresectable melanoma [62].

As indicated in Table I, ICI targeting CTLA-4, PD-1/PD-L1, and LAG3 have been approved by the FDA. Additionally, several preclinical and clinical trials are underway to study the efficacy of ICIs, either alone or in combination with chemotherapy, targeted therapies, radiation therapy, and other approaches, across various types of cancer [63–65]. Moreover, various immune checkpoints, including T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TGIT), T cell immunoglobulin-3 (TIM3), and NKG2A, have been discovered, and ongoing clinical trials are assessing the efficacy and safety of ICIs targeting these checkpoints [66–68]. However, some patients treated with ICIs do not respond to the treatment, and there are a variety of mechanisms contributing to this resistance [69, 70]. Therefore, ICI may not be universally effective, and it can be associated with side effects, including inflammation and autoimmune reactions [71].

Overall, ICIs represent an exciting and innovative approach to cancer treatment that has the potential to significantly improve the lives of individuals with cancer. Further research is needed to fully understand the mechanisms of action and optimal use of this therapy and to identify additional indications for its use.

Adoptive cell Therapy (ACT)

ACT, also known as T-cell transfer therapy, is a promising immunotherapy method. In ACT, tumor-specific T cells are isolated and stimulated ex vivo, and then the activated T cells are subsequently reintroduced into the patient. The primary sources of T cells for this therapy are resected tumors and the peripheral blood of cancer patients [72].

Tumor-infiltrating lymphocyte (TIL) therapy utilizes T cells obtained from the tumor. This approach leverages the inherent ability of lymphocytes within or near the tumor to recognize and target tumor cells. TIL therapy has shown promise in patients with metastatic melanoma in various clinical trials [73–75]. However, TIL therapy has limitations due to the difficulty in expanding viable TILs and selectively displaying specific effector functions. Moreover, TILs have not demonstrated benefits for certain types of cancer. To achieve a robust antitumor response, the presence of effector T cells with antitumor activity in the tumor is essential, but this is not consistently observed in solid tumors. Additionally, in contrast to melanoma, TIL cultures capable of specific antitumor recognition were not consistently reproducible in other cancers [76, 77]. Moreover, there is the issue of IL-2 toxicity, which is used to promote TIL growth and activity [78]. To overcome these limitations and apply ACT more broadly, there has been an attempt to genetically modify T cells. By harvesting peripheral blood T cells and enabling them to express specific anti-tumor receptors, ACT can potentially be applied to cancer types where specific TILs cannot be obtained. This strategy uses two approaches: one using TCR-based receptors and the other employing chimeric antigen receptors (CARs) that contain antibody fragments linked to T cell signaling domains [79–81].

TCR-T cell therapy engineered T cells from patients to express a specific TCR, which can also recognize antigens inside tumor cells. Early TCR-T cell therapy targeted metastatic melanoma and used TCRs that bind to a restricted peptide from a melanocytic differentiation antigen. Later methods targeting higher avidity TCRs showed improved response rates, but normal melanocytes were also targeted. The challenge of on-target and off-tumor toxicity in numerous patients has been a concern with TCR-T cell therapy targeting shared antigens [82, 83]. As a result, there is ongoing development of TCR-T cell therapy that utilizes TCRs designed for specific tumor neoantigens, which is expected to be a safer approach compared to targeting shared antigens [84].

Unlike TCRs, CARs recognize MHC-nonrestricted structures on the surfaces of target cells. This is an advantage to the anti-tumor activity of CAR T cells because the primary mechanism of immune evasion by cancer involves the loss of MHC-related antigen presentation by tumor cells [85]. The typical structure of CARs contains the signaling domains of the CD3ζ chain, CD28, OX40, and/or 4–1BB, an antigen-binding single chain variable fragment (scFv) domain, and a transmembrane domain. The structure of CAR has now evolved into its fifth generation [86]. Early CARs had limited antitumor effects due to their lack of costimulatory signals, with only specific antigen recognition domains and CD3ζ signaling domains. Subsequently, second-generation CARs included additional costimulatory domains such as CD134, CD28, CD137, or 4–1BB to improve T cell function, and third-generation CARs contain multiple costimulatory molecules to increase T cell activity [87]. The fourth generation CAR, also known as TRUCK (T cells redirected for antigen unrestricted cytokine initiated killing), involves further incorporation of a nuclear factor of the activated T-cell (NFAT)-responsive cassette to express functional transgenic protein [88]. The 5th Generation CAR, also known as the next generation, integrates an additional membrane receptor such as the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling domain. This generation demonstrated antigen-dependent activation of the JAK-STAT3/5 signaling pathway through this cytoplasmic domain [89]. Tisagenlecleucel is the first CAR T-cell therapy approved by the FDA to treat ALL patients up to 25 years of age, receiving approval in 2017. A clinical trial of tisagenlecleucel in pediatric and young adult patients with refractory or relapsed ALL demonstrated an 81% overall remission rate within 3 months [90]. Subsequently, six more CAR T-cell therapies have been approved by the FDA. All of them target B-cell surface markers (four target CD19, two target B-cell maturation antigen (BCMA)) and are approved for the treatment of blood cancers, including lymphoma, some forms of leukemia, and most recently multiple myeloma (Table II).

While CAR-T therapy has shown effectiveness in treating B-cell malignancies, it has had limited success in solid tumors. This is due to the lack of specific targetable antigens, the complex TME, and the difficulty of infiltrating solid tumors [91, 92]. Serious side effects such as cytokine release syndrome and neurotoxicity have also been found [93]. To address these limitations, strategies such as the development of a more precise targeting system, including the identification and optimization of specific T cells for effective tumor destruction, are currently under consideration [77, 94].

Cancer Vaccines

Cancer vaccines are a type of immunotherapy that helps the immune system fight against cancer by stimulating or restoring the immune system’s capabilities. Cancer vaccines are designed to induce tumor-specific immune responses against TAAs or tumor-specific antigens (TSAs) [95]. A cancer vaccine can be classified as a preventive vaccine or as a therapeutic vaccine [96]. The main goal of the preventive vaccine is to prevent the development of cancer, and it is given to people who are at high risk of developing a specific type of cancer. These vaccines are based on infectious agents carrying antigens, making it easy for the immune system to recognize them as foreign invaders [5]. FDA-approved preventive cancer vaccines include hepatitis B virus (HBV) vaccines and human papillomavirus (HPV) vaccines [97]. Therapeutic cancer vaccines, on the other hand, are given to individuals who have already been diagnosed with cancer to help the body’s immune system fight cancer cells and aim to activate tumor-specific CD8 + T cells based on the interaction of MHC-I epitopes with TAAs [98]. As a therapeutic cancer vaccine, Sipuleucel-T was approved by the FDA in 2010 for patients with advanced prostate cancer, especially that refractory to hormone treatment. Sipuleucel-T is a DC-contained APC vaccine derived from patient blood and pulsed with a fusion protein of prostatic acid phosphatase (PAP) and Granulocyte-macrophage colony-stimulating factor (GM-CSF) [99]. In a phase 3 trial with metastatic castration-resistant prostate cancer (mCRPC), the risk of death in the sipuleucel-T group was reduced by 22% compared to the placebo group. This reduction represents an improvement in median survival of 4.1 months [100].

Cancer vaccines can be categorized based on the introduced antigen, including tumor cells or their lysates, peptides, and RNA or DNA. Cancer cell vaccines include tumor cell vaccines and DC vaccines. Tumor cell vaccines consist of freshly isolated primary tumor cells through syngeneic, allogeneic, or autologous irradiation. These are often genetically modified because irradiated tumor cells alone do not provide sufficient efficacy. One example is GVAX, genetically modified with the cytokine genome GM-CSF [98, 101, 102].

Other cancer cell vaccines are based on DCs. DCs are key cells of innate immunity, play an important role in inducing adaptive immune responses, and have been recognized as powerful APCs. In the development of DC-based vaccines, it is recommended to use DCs that present highly specific tumor antigens. Various methods can be employed to convey targeting information to DCs, and the most common approach involves culturing with peptides, proteins, tumor cells, or their lysates [103–105]. Another method involves transfecting DNA or mRNA encoding TAA using viral transduction and electroporation [106, 107]. Sipuleucel-T, described above, is a DC-based therapeutic vaccine.

Peptide-based vaccines are designed based on epitope peptides that can induce both CD4 + and CD8 + immune responses against TAAs or TSAs [108, 109]. Peptide vaccines are simple to manufacture, low cost, and have excellent safety. However, tumors have a complex microenvironment, so single peptide vaccines are not clinically effective. Additionally, due to the diversity of human MHC alleles, it may not be suitable for all patients. Therefore, a more personalized selection of antigens and adjuvants as well as a combination with other immunotherapy is needed [98, 110].

The various types of cancer vaccines in clinical use are illustrated in Table III. In addition, various vaccines are being developed, including therapeutic vaccines using induced pluripotent stem cells (iPSCs) and customized vaccines using genetic sequences from patient tumor samples [111, 112].

Cancer vaccines are still a relatively new area of research, and there are many challenges to overcome in their development and use. The key challenges include Immune-suppressive TME and tumor heterogeneity. Because tumors have various genetic mutations and protein expression profiles, there is great individual heterogeneity among patients, and tumors create a suppressive microenvironment that dampens the immune response, allowing them to evade immune surveillance and destruction. To overcome these challenges, personalized vaccine strategies for individual patients and combination therapies that target both the tumor cells and the immunosuppressive microenvironment are being studied [117, 118]. Despite these challenges, they have significant promise as a treatment option for individuals with cancer, and ongoing research continues to explore and enhance their efficacy.

Cytokine Therapy

Cytokines are small proteins that play a key role in the immune system’s response to infection and disease. Cytokines are produced by a variety of cells, including immune cells such as T cells and B cells, and contribute to the regulation of the immune response by signaling to other immune cells to either activate or inhibit their activity [119].

These chemical messengers operate by binding to their respective receptors on target cells, exerting their effects in both an autocrine and paracrine manner. This intricate signaling mechanism helps coordinate the functions of cells in both the innate and adaptive immune systems. The two major cytokines being used in immunotherapy are IL-2 and IFN-α [120–122]. IL-2 is produced naturally by the body to fight infections and prevent autoimmune diseases. In cancer therapy, IL-2 plays a critical role in boosting anti-tumor effects by enhancing the targeting capabilities of adaptive immune cells. This includes a boost in T-cell activity and the promotion of NK-cell activity [4]. Similarly, in cancer therapy, IFN-α contributes to the body’s ability to generate innate immune cells such as macrophages and DCs [42].

The FDA approved IL-2 for the treatment of metastatic renal cell carcinoma in 1992 and for the treatment of metastatic melanoma in 1998. Additionally, IFN-α2b received FDA approval in 1986 for the treatment of hairy cell leukemia [123]. The various types of cytokines such as IL-2, IL-8, IL-10, IL-12, IL-15, TNF-α, transforming growth factor-β (TGF-β), CSF-1 have been demonstrated to be effective in preclinical studies. Currently, they are in ongoing clinical trials [124, 125]. Table IV illustrates some of the clinical studies of cytokines in various types of cancer. In cytokine therapy, a significant challenge lies in achieving notable anti-tumor responses without triggering treatment-limiting toxicities. This difficulty arises from the dual nature of many cytokines, which can function both in immune activation and immune suppression [125]. Moreover, cytokines are generally administered via the parenteral route, making them unable to reach the tumor site with significant concentration due to their rapid clearance from the body. A further threat comes from the extensive pleiotropism and redundancy of cytokine signaling [125–127].

Despite the side effects, many strategies have been developed to mitigate off-target activity and improve the efficacy of cytokine therapy. The combination of cytokines at naturally observed ratios has shown synergistic effects [127]. The successful application of polyethylene glycol to IFN-α2a has resulted in a more favorable safety profile and greater convenience associated with lesser dosing frequency. The cells have been genetically engineered to deliver cytokines into the TME to prime host anti-tumor immunity against natural tumor antigens [125]. The efficacy of cytokine therapy can be enhanced by combination with other immunotherapeutic agents such as immune checkpoint blockers, anti-cancer vaccines, and cell therapy [63, 134–136].

Cytokine therapy emerges as a promising approach for treating cancer and other diseases, with ongoing research fueling advancements. As our understanding of the role of cytokines in the immune system continues to improve, it is likely that new and innovative therapies based on cytokines will be developed in the future.

Gene Therapy

Gene therapy is a type of treatment that involves the introduction of new genetic material into cells in order to correct or modify the function of a specific gene [137]. In the context of cancer immunotherapy, gene therapy involves the use of genetically modified immune cells to enhance the body’s natural immune response against cancer cells.

Gene delivery has been increasingly used to engineer immune cells for immunotherapy through delivery of various types of nucleic acid [138–141]. However, the gene vector or carrier plays an important role in delivering the gene of interest effectively to the desired cell [142]. So, the ideal carrier should protect the gene from the immune system, rapid clearance, non-specific reactions, and endosomes to ensure successful delivery [140, 142]. The vectors for gene delivery can be generally divided into viral and non-viral vectors. Viral vectors include lentiviruses, adenoviruses, and adeno-associated viruses which have been extensively used for gene delivery due to their high transfection efficacy [139, 141, 142] but may elicit strong immune responses. On the other hand, non-viral vectors such as polymers, lipids, and peptides do not cause immunogenicity but exhibit lower transfection efficacy [142]. Several studies using both viral and non-viral vectors have been conducted to deliver the gene for cancer immunotherapy, some of the studies are listed in Table V.

Overall, gene therapy represents a promising approach to cancer immunotherapy and is an active area of research and development. However, there are still limitations, including specific delivery at target tissue and identification of genes to maximize efficacy while minimizing toxicity [147]. As research progresses, such as the development of safe and effective vectors for gene delivery and tumor genome analysis, it may show great potential in cancer immunotherapy in the future.

Combinations with Cancer Immunotherapy

To date, combination therapies such as ICI + chemotherapy, ICI + targeted therapy, and dual ICI therapy have been approved by the FDA across a variety of cancer types and various combination therapies are being studied. Chemotherapy offers the advantage of reducing tumor volume. Moreover, given that tumor cells contribute significantly to the immunosuppressive TME, chemotherapy may decrease the number of cancer cells requiring removal and mitigate the production of immunosuppressive factors they generate [153, 154]. Additionally, some chemotherapy can stimulate antitumor immune responses in addition to direct cytotoxicity. For example, doxorubicin integrates into DNA and generates excessive reactive oxygen species (ROS) through inhibition of topoisomerase II, leading to immunogenic cell death (ICD). Recent studies showed that the combination therapy with cyclophosphamide, ICI, and vinorelbine activates APCs, and decreases local and metastatic growth in triple-negative breast cancer models by T-cell-related effect. It may alter the balance between the terminally and progenitor exhausted T cells by favoring the latter [155]. Moreover, a combined regimen with an anti-PD-L1 antibody and gemcitabine showed activated antitumor responses in the gastrointestinal tumor model. It reduces circulating splenic and intratumoral MDSCs, as well as M2 macrophages. Additionally, infiltrating cytotoxic T-cells was increased in the tumor sample [156]. Tsutsumi et al. analyzed the relationship between ICI-chemo combination therapy and damage-associated molecular pattern (DAMP) levels and found a correlation between calreticulin levels and progression-free survival. Therefore, it has the potential to predict the efficacy of combination therapy through monitoring of plasma calreticulin levels [157].

Targeted therapies not only have direct antitumor activity but can also affect the immune context of the TME by influencing signaling pathways [158]. According to a recent study, the combination therapy of ICI and tyrosine kinase inhibitors provided a survival benefit compared to sunitinib monotherapy in patients with metastatic renal cell carcinoma [159]. Additionally, the combination therapy of nivolumab and trastuzumab successfully controlled cancers and improved the quality of life in patients with both gastric cancer and bladder cancer [160]. The study with patients who received chemotherapy alone, anti-PD-1 plus chemotherapy, anti-PD-1 plus targeted therapy, or anti-PD-1 plus targeted therapy and chemotherapy for the treatment of unresectable intrahepatic cholangiocarcinoma showed that ICI with chemotherapy and targeted therapy achieved better clinical outcomes than chemotherapy alone. The case of ICI with chemotherapy and targeted therapy, showed similar outcomes but more adverse events [161].

Radiotherapy (RT) not only induces the apoptosis of local tumor cells but also ICD, which promotes the release of TAAs, thereby activating the immune system to kill tumor cells. So, RT may be advantageous when combined with immunotherapy, especially in poorly immunogenic tumors [162]. Combination with RT and ICIs reduced the growth of abscopal tumor unlike RT or ICI alone in the osteosarcoma mice model [163]. In the meta-analysis of randomized trials that compared combination therapy with ICI and RT with RT or ICI alone for advanced non-small-cell lung cancer (NSCLC) and prostate cancer, combination therapy improved OS, PFS, and disease control rate but the risk of side effects such as dyspnea and pneumonia increased [164]. Zhao et al. demonstrate that ICIs combined with radiotherapy/chemotherapy are safe and effective in patients with locally advanced or recurrent/metastatic esophageal squamous cell carcinoma [165].

Additionally, research on combination treatments using other immunotherapies is actively underway [158, 166]. Combination therapy with mesothelin-targeted CAR-T cell and PD-1 inhibitor exhibited safety and efficacy in malignant pleural diseases. It supports the investigation of combination therapy with ACT and ICI [167]. In the study about the combination therapy with sipuleucel-T and androgen receptor-targeting agents (ARTA) in patients with metastatic castration-resistant prostate cancer, the patients receiving combination therapy had a longer median OS than ARTA alone and there was no impact of sequential or concurrent administration of ARTA [168, 169]. Nemvaleukin alfa, an engineered cytokine that selectively binds to the intermediate-affinity IL-2 receptor, improves outcomes in patients with advanced tumors when used alone or in combination with pembrolizumab [170].

Although combination therapy can overcome the limitations of conventional monotherapy, it has some challenges. When designing clinical trials targeting patients who do not respond to treatments, a unified definition of resistance is needed [171]. Additionally, when ICIs are added to other treatments, immune-related adverse events (irAEs) may occur [172]. Compared with anti-PD-1/L1 or CTLA-4 monotherapy, ICI-based combination therapy was associated with more frequent cutaneous adverse events [173]. Studying predictive biomarkers for ICI response may lead to ideal outcomes with manageable irAEs [172, 174].

Nanoparticles and Cancer Immunotherapy

In addressing the limitations inherent in cancer immunotherapy such as its low response rate and the onset of immune-related side effects, an alternative strategy involves the application of nanotechnology [175]. Specifically, NPs emerge as an effective treatment option when combined with cancer immunotherapy [176]. NPs can efficiently deliver adjuvants and cancer antigens including peptides, DNA, and RNA to APC [177, 178]. For example, mRNA-based antigens are easily degraded in the blood but can be delivered more stably to immune cells using NPs [179]. Additionally, since various cellular components in the TME interact to influence cancer progression and treatment response, NPs can reprogram the immunosuppressive TME into an immune-supportive environment by targeting immune-suppressive cells in the TME including tumor-associated macrophages (TAM), Treg, MDSC and cancer-associated fibroblast (CAF). Understanding the roles and interactions of these cells and developing NPs targeting them can effectively modulate the TME [180, 181]. Therefore, the combination of NPs and cancer immunotherapy can produce a longer lasting and broader immune response. Additionally, NPs can encapsulate various therapeutic agents such as vaccines and chemotherapy agents, which can increase antitumor response compared to monotherapy [182].

NPs for cancer immunotherapy can be divided into two types: synthetic and naturally derived nanoparticles. A range of synthetic NPs, including polymer nanoparticles, liposomes, and inorganic nanoparticles, can be tailored to meet specific requirements by manipulating physicochemical properties such as size, shape, composition, and surface modification to modulate immune responses [183–185]. An example of a nature-derived NP is an exosome, a cell-derived nano-sized particle (40–160 nm). Exosomes can be released by a variety of cells and be used as carriers, given their many advantages including small size, bio-compatibility, low immunogenicity, and specific homing to target [186]. There are two types of exosomes currently under investigation for cancer immunotherapy: Immune cell-derived exosomes containing specific proteins and endosome-associated peptides and tumor-derived exosomes containing specific tumor antigens [183, 187]. Recently, incorporating NPs with cancer immunotherapy such as ICI has been actively researched, and this strategy will present a new direction for cancer immunotherapy [188–190]. There are some recent studies applying nanoparticles to cancer immunotherapy. Reda et al. developed a PLK1 inhibitor-loaded nanoparticle and conjugated it into PD-L1 antibody. It reduced the effective doses of PLK1 inhibitor and PD-L1 antibody by 5-fold in a metastatic lung tumor model and showed efficacy in the tumor model, which does not respond to CTLA-4 and PD-1 inhibitor combination [191]. Zhang et al. reported a bispecific nano-immunoengager, in which the peptidic nanoparticles can bind tumor cell surface α3β1 integrin and transform to nanofibrillar network at the TME captures Teff cells. This bispecific nano-immunoengager demonstrated efficacy in a cancer model in mice when administered together with anti-PD-1 antibody [192]. Bahreyni et al. applied the nanoparticle strategy for combining the doxorubicin and melittin, with PD-L1 DsiRNA (Dicer-substrate short-interfering RNA), an immune checkpoint inhibitor. The surface of the nanoparticles was then modified with hyaluronic acid to increase their stability and distribution. It significantly enhanced its specificity towards breast cancer cells and reduction in PD-L1 expression with a synergistic effect of chemotherapeutic agents on killing cancer cells [193]. Lai et al. developed a novel nanoparticle by attaching PD-L1 aptamers to nanoparticles loaded with fexofenadine, known to reduce the M2-like macrophages in the TME. This nanoparticle can attach to the PD-L1 expressing tumor cell and enhance antitumor activity [194]. This strategy also applied to the CTLA-4 aptamer and showed enhanced selectivity and efficacy [195].

NPs are also being studied as a platform for delivering genes such as mRNA and siRNA. Silva et al. developed an ionizable lipid nanoparticle (LNP) to deliver tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA directly to the TME. TRAIL is an innate immune cytokine that can suppress tumor initiation and metastasis. This LNP induced cancer cell death in vitro and effectively inhibited cancer progression in vivo [196]. Lin et al. developed a nanoparticle for siRNA delivery utilizing mPEG-b-PLGA and lipids. They optimized the formulation of the nanoparticles for better gene silencing efficiency, and this optimized nanoparticle encapsulating siRNA targeting CD47 could efficiently suppress melanoma tumor progression [197].

To overcome the limitations of conventional immunotherapy, NPs are being studied as a promising approach. NPs have various advantages, such as improving drug permeability into tumors, improving drug solubility, targeting, and achieving the combination of treatments, but their clinical application is still limited. To promote the clinical application of immunotherapy using NPs, it is necessary to solve the problems of effective delivery to the target site and non-selective removal of NPs from the body. In addition, optimization and standardization of manufacturing protocols are necessary, as well as evaluation of the safety of nanomaterials, including their irritant, ROS generation, or allergic properties [198].