ICIs are a promising class of cancer immunotherapy with numerous pre-FDA-approved treatment strategies. Their mechanism of action is based on inhibiting negative regulatory markers from T-cells, which are checkpoints responsible for their regulation (Figure 1). Activated T cells induce the inhibitory receptor CTLA4, followed by activation of PD-1, which binds to the stimulatory ligands B7-1, B7-2, and PD-L1 (programmed death ligand 1) or PD-L2 (programmed death ligand 2). The ligands are next presented to CD4⁺, CD25⁺ regulatory T cells (Tregs), myeloid cells, and antigen-presenting cells (APCs), to suppress cytotoxic T-cell activation, which allows tumor cells to grow and mitigate immune system activation (Buchbinder and Desai, 2016). It was hypothesized that by targeting ICIs, cancer cell growth can be controlled via the activation of primed cytotoxic T cells (Wang et al., 2022). As a result of adaptive resistance, traditional targets have demonstrated limited efficacy thereby paving the path for the discovery and investigation of novel checkpoint molecules including NKG2A (Natural killer cell receptor 2A) ligands, TIM3, Galectin 3, B7-H6 ligands, and so on that can improve treatment outcomes.

ICIs against CTLA-4 and PD-1, and combination therapy for anti-CTLA4 and anti-PD-1 have been approved by FDA, while there are ongoing trials of other candidates such as TIGIT, Tim3, LAG-3, Ig T-cell activation suppressor (VISTA), and IDO1 (Indoleamine 2,3-Dioxygenase 1)/IDO2 (Indoleamine 2, 3-Dioxygenase 2)/TDO (Tryptophan 2,3-Dioxygenas), CD27/CD70, CD39/73, HVEM (Herpesvirus entry mediator)/BTLA and B7-H3 (B7 Homolog 3 Protein, CD276) (Ziogas et al., 2023; Peterson et al., 2022). Clinical trials data of immune checkpoint inhibitors targeting corresponding receptors has been reported and summarized in Table 1.

Adoptive cellular therapies encompass ex vivo activation of a patient’s immune cells, which are then transferred back to fight cancer. These include 3 major therapies, i.e., CAR-T cells, tumor-infiltrating lymphocytes (TILs), and genetically modified TCR therapies. Among them, Chimeric Antigen Receptor T-cell therapy (CAR-T) cell therapy has revolutionized cancer treatment, producing promising results in certain subtypes of B-cell leukemia, with over 5 products approved by the FDA (Fang et al., 2022). The treatment entails separation of a patient’s peripheral blood mononuclear cells (PBMCs) by leukapheresis or density-gradient centrifugation, following which CD8⁺ and CD4⁺ regulatory T cells are activated through exposure to nano-matrix-, plate- or beads bound anti CD3 and anti-CD28 antibodies (Vormittag et al., 2018). These isolated T cells are genetically engineered by DNA-encoded permanent impression into the T cell genome. The modified CAR-T cells replicate the typical T cell signaling pathways when they come into contact with cancer cells, proliferating and initiating an anti-tumor response. A recognition domain built into the CAR’s architecture aims to target tumor-associated antigens (TAAs) present on cancer cells. The CAR-T cell and the cancer cell form a non-traditional immunological synapse upon antigen identification, which triggers complex biological processes such as clonal expansion and receptor clustering. As a result, target cells are destroyed by processes like apoptosis and exocytosis (Ruf et al., 2024). Additionally, activated CAR-T cells release cytokines including IL-2, IL-6, and IFN-γ, which attract and activate immune cells like macrophages, natural killer (NK) cells, and more T cells, thus promoting tumor suppression (De Marco et al., 2023).

First-generation CARs activated T cells independently of the major histocompatibility complex, presenting specific target antigen via the extracellular single-chain variable fragment antibody domain. The hinge region further connects the extracellular and intracellular domain through the transmembrane domain. Whilst the first-generation CARs comprise a CD3ζ signaling domain that mimics the T-cell receptor signaling pathway, facilitating low cytotoxicity and anti-tumor effectiveness, the second-generation CARs include both CD3ζ and co-stimulatory domain CD28, that enhances T cell proliferation (Tomasik et al., 2022). The third-generation CARs comprise of two co-stimulatory domains, whereas the fourth and fifth generation are enriched by additional signaling domains including 4-1BB or interleukin 12 cytokines. A key upgrade in fourth and beyond generation is the genetic manipulation via T cell redirected for antigen-unrestricted cytokine-initiated killing (TRUCKs), which mediates extensive cytokine release and modulation of immunological and vascular tumor environment, enhancing anti-tumor activity and minimizing off target toxicity (Chen T. et al., 2024).

CARs consist of antigen-binding domain associated with T cell receptor signaling domains and co-stimulatory molecules to acquire specificity through polymorphic α- and β-glycoprotein chains with antigen-binding and conserved domains to bind to CD3, CDγ, CDδ, CDε, and CDζ. Alternatively, lymphocytes derived from the patient’s peripheral blood mononuclear cells (PBMC) express the desired TCR or CAR (Chang and Chen, 2017). For example, ZUMA-1 (axicabtagene ciloleucel or axi-cel, which is the CD28 costimulatory domain) and JULIET (tisagenlecleucel or tisa-cel, which is the 4-1BB co-stimulatory domain) act as anti-CD19 CART (CAR19) in relapsed or refractory diffuse large B-cell lymphoma and multiple myeloma, as well as non-Hodgkin’s B-cell lymphoma (Frigault and Maus, 2020). Axicabtagene ciloleucel, brexucabtagene, autoleucel, lisocabtagene, maraleucel, and tisagenlecleucel have been approved by the FDA, while idecabtegene, vicleucel and ciltacabtagene, autoleucel are approved as B-cell maturation antigen (anti-BCMA) CAR-T (Chekol Abebe et al., 2022). Moreover, other immune cell therapies such as CAR-NK cells, or CAR-macrophages therapies are being developed but have not yet been approved by the FDA (Meng et al., 2021). Hematological malignancies are the most effective targets for CAR-T cell therapies compared to solid tumors due to the absence of a thick extracellular matrix (ECM).

Despite ground-breaking results in the clinical management of malignant tumors, CAR-T cell therapy faces obstacles concerning wider use and efficacy. The short half-life and less effectiveness of CAR-T cells are major concerns associated with CAR-T cell therapies, leading to eventual signalling worn out despite an initial strong stimulation. Low CAR-T cell persistence or the unregulated proliferation of certain subclones (antigen-negative cells) might lead to the development of resistant tumors or relapse (Bouziana and Bouzianas, 2021). Newer generations of CARs have been developed to counter this, as mentioned above. Vast evidence in clinical data, increased stability, and more mature technological processes have placed second-generation CARs at the forefront of cancer treatment. Solid tumors offer a variety of challenges, such as minimal T cell infiltration, unfavorable tumor microenvironment, and low levels of antigen expression in healthy tissues, which may lead to off-target effects for which some counter-strategies have been identified in recent times (Flugel et al., 2023). Significant hurdles to CAR-T cell entry in solid tumors include the extracellular matrix, a hostile milieu, and the variable expression of TAAs. Hostile environments like hypoxia, acidity, and immunosuppressive cells make it challenging for CAR-T cells to perform their job, especially in patients with solid tumors (Bot et al., 2015). Novel CAR-T armed with a synNotch receptor that upregulated IL-2 expression when in contact with specific antigens, reported greater tumor-infiltration and better efficacy (Allen et al., 2022). Development of diverse fourth-generation CARs is paving the way to improve control, specificity, and effectiveness in targeting cancer cells (Tang et al., 2023a). Different types of CAR-T cells and their mechanisms are as follows:

A. Logic-gated CAR-T Cells: Operated as Boolean cells. As the name suggests, operator techniques AND, IF-THEN, and OR increase efficacy by expanding the target cell pool while decreasing off-tumor damage. Cells that exhibit AND/OR mechanism are called Dual CAR-T cells and those that exhibit IF/THEN are Conditional expression CAR-T cells.

Cancer vaccines constitute another modality of the immunotherapeutic approach, based on the principle of enhancing and building the patient’s T-cell immunity, which strengthens the immune response and destroys TAAs. Cancer vaccines are fabricated to target TAAs (neoantigens, or neoepitopes) that are generally expressed on cancer cells. The therapeutic pathway followed by most cancer vaccines is the stimulation of cell-mediated responses, like those from CTLs that can minimize tumor burden (Vigneron, 2015; Türeci et al., 2016). In this way, a durable immunity is created and directed strictly to target cancer cells (Le et al., 2022). Currently, there are 4 different categories of cancer vaccines, namely, cell-based, peptide-based, nucleic-acid-based, and viral-based vaccine (Elsheikh et al., 2023). In 2010, the FDA approved the first cancer vaccine sipuleucel-T (Provenge) for prostate cancer treatment. Sipuleucel-T is a dendritic cell (DC)-based vaccine that builds immunity against prostatic acid phosphatase and activates antigen-specific T cells (Cheever and Higano, 2011). The allogeneic tumor cell vaccine uses the patient’s cancer cells to produce the vaccine and injects irradiated tumor cells together with an adjuvant, as well as genetically alters the cancer cells in such a way as to incorporate additional functions, including the production of cytokines, costimulators or granulocyte-macrophage colony-stimulating factor (GM-CSF) (Hammerstrom et al., 2011).

DC vaccines are successful because cancerous DNA or RNA, viruses, proteins, or peptides can be loaded on DCs, while the DC surface expresses receptors for antigen binding (Igarashi and Sasada, 2020). These antigens are presented to immune cells, which activate the immune system. In contrast, protein-peptide vaccines stimulate the immune response against specific antigenic epitopes derived from proteins or peptides of cancer cells. APCs will recognize those peptides or proteins and capture them, and then T cells will induce an immune response (Purcell et al., 2007). Thus, antigenic epitopes derived from TAAs are capable of binding to HLA, while antigens derived from cancer-specific gene mutations are used, which will be more specific in detection (Zhao et al., 2021). Vaccines generally comprise short-chain peptides (SPs) restricted to MHC class I, which reduces the success in activation of MHC class II-restricted cells, such as helper T cells. Therefore, new generation vaccines consisting of long-chain peptides with epitopes for both CTL and helper T cells and capable of activating MHC I and MHC II are under development for more successful immune responses and clinical applications (Buonaguro and Tagliamonte, 2020).

Nucleic acid-based cancer vaccines have also shown activation of MHC class I-mediated CD8⁺ T-cell response through activation of humoral and cellular immunity. They activate the immune system through multiple epitopes (Shi et al., 2022). RNA-based vaccines have several advantages over DNA-based vaccines as they don't incorporate into the genome, prevent carcinogenicity, and are functional in the cytoplasm, while DNA vaccines need to be transferred to the nucleus. To transfer nucleic acids, viral vectors are used (Leitner et al., 1999).

Oncolytic virus immunotherapy attacks tumor cells directly and stimulates immune responses. Vectors for specific genes and tumor antigen expression, including adenovirus, poxvirus, alphavirus, and herpes simplex virus, are utilized abundantly, either naturally occurring or genetically engineered (Kaufman et al., 2015). Adenoviruses are preferred because of their ability to transduce dividing and nondividing cells, while herpes simplex virus type 1 is prominent due to its oncolytic properties and GM-CSF expression (Matsunaga and Gotoh, 2023). The virus is made benign and aims to kill cancer cells without affecting normal, healthy cells. The destruction of cancer cells occurs through the introduction of a modified virus that targets cancer in two ways: Direct and Indirect (Javid et al., 2023).

The direct mechanism causes oncolysis through cell infection with an oncolytic virus and death. In the indirect mechanism, the virus induces an anti-inflammatory response and the release of microorganism-associated molecular patterns, danger-associated molecular patterns (DAMPs), thus producing APCs, DCs, and macrophages in TME (Hofman et al., 2021). Moreover, captured tumor antigens are released through oncolysis and migrate to regional lymphoid structures to activate tumor-specific T cells to destroy cancer cells by production of perforin and granzyme B, and their recognition by T cells through expression of MHC molecules and presentation of tumor antigens on the surface of cancer cells (Del Prete et al., 2023). T-VEC (Imlygic), is an FDA-approved viral-based vaccine that uses herpes simplex virus as a vector and has displayed strong anti-cancer immunity (Conry et al., 2018). However, TAAs are expressed not only in tumor cells, but also in normal cells with gene mutations caused by carcinogenesis. Therefore, a vaccine targeting TAAs may induce autoreactive immune responses, high toxicity, and autoimmunity (Kaczmarek et al., 2023).

Microbes can potentially affect the safety and efficacy of cancer immunotherapy by regulating the host’s immune responses. They impact tumorigenesis by secreting certain toxins and creating an immunosuppressive ecosystem. Lipopolysaccharides (LPS) and short-chain fatty acids (SCFA) are microbe-derived components that expend anti-tumor activity. One such SCFA is butyrate, which inhibits histone deacetylases and subdues tumor progression. Emerging research proposes four mechanisms by which gut microbiota modulates responses to immunotherapy: 1) Enhanced anti-tumor immune response due to stimulated T cell activity in response to microbial antigens. 2) cross-interaction with TSA (Tumor-specific antigens). 3) Induction of immuno-stimulatory or anti-inflammatory response by attaching to Pattern recognition receptors (PRRs). 4) Through metabolites that regulate systemic reactions on the host. Diet is known to direct the intestinal microbiota towards modulating the host immune response to cancer immunotherapy. Fibre-rich diet flourishes the microbial ecosystem, thereby promoting SCFAs synthesis.

mRNA vaccines are an upcoming class of therapeutics broadly treating cancer, cardiovascular, cerebrovascular, infectious, metabolic genetic diseases, and autoimmune disorders. Almost two decades ago, the potential of mRNA to combat cancer was explored, and it was not just restricted to ideation but also backed with published proof-of-concept studies (Conry et al., 1995; Boczkowski et al., 1996). Boczkowski et al were the first ever research group to showcase the feasibility of electroporation of dendritic cells with ovalbumin encoding mRNA to initiate an immune response against tumor antigens in melanoma mice models (Boczkowski et al., 1996). A similar study proved the efficacy of a formulation containing Trimix, which is a fusion of mRNA-encoded adjuvants (CD70, CD40L, and constitutively active TLR4) electroporated with mRNA in pre-clinical studies (Bonehill et al., 2008). The reason for this enhanced efficacy was suspected to be improved activation of dendritic cells, and switching of CD4⁺ T cell phenotype from T regulatory cells to T helper 1-like cells (Bonehill et al., 2008; Wilgenhof et al., 2011). A related report published data showing tumor regression in 27% of stage III or IV melanoma patients immunized with the above-mentioned formulation (Wilgenhof et al., 2013). Surprisingly, a tailored mRNA vaccine for metastatic melanoma developed by BioNTech showed no identifiable lesions on radioactive imaging and was found to be recurrence-free even after 23 months of intra-nodal delivery. However, lack of sufficient data necessitates further research to consider mRNA vaccines for melanoma as trustworthy (Sahin et al., 2017).

These exciting results have paved the way for multiple clinical trials that exploited DC (dendritic cells) vaccines for treating various cancers including metastatic prostate cancer, metastatic lung cancer, renal cell carcinoma, brain cancers, melanoma, acute myeloid leukaemia, and pancreatic cancer (Mitchell et al., 2015; Batich et al., 2017). mRNA pulsed DC vaccines developed using mRNA copies of GBM (Glioblastoma) in patients increased progression-free survival up to 2.9 times greater than the control (Vik-Mo et al., 2013). The survival time was further prolonged due to increased bilateral DC migration on pre-conditioning the immunization site with potent recall antigen, namely, tetanus/diphtheria toxoid (Mitchell et al., 2015). Hanna J Khoury et al performed a study that resulted in encouraging outcomes to treat acute myeloid leukemia, where an intradermally injected mRNA encoding human telomerase reverse transcriptase ensured complete remission in 11 out of 19 treated patients (Khoury et al., 2017; de Lima et al., 2014). For renal cell carcinoma, anti RCC-DC based mRNA vaccine was intradermally administered to generate specific immune response that resulted in prolonged survival of RCC patients (Rittig et al., 2016).

Recently, lipid and polymeric nanoparticle technology have emerged as robust delivery systems owing to their versatile nature. Lipid nanoparticles have garnered attention as convenient mRNA delivery agents. This wide usage is attributed to its invaluable characteristics like improved stability, safety, and transfection efficiency (Qin et al., 2022). They are comprised of 4 elements, namely, cholesterol acting as a stabilizer, phospholipids that support the lipid bilayer geometry, a lipid-linked PEG that increases half-life of the delivery systems, an ionizable cationic lipid (e.g., 1,2-di-O-octadecenyl-3-trimethylammonium propane [DOTMA]) that causes self-assembly into particles of size as small as 100 nm, and initiates endosomal mRNA release into the cytosol (Qin et al., 2022). Some commonly used lipids include N, N-Dimethyl-2,3- bis [(9Z, 12Z)-octadeca-9,12-dienyloxy]propan-1-amine (DLinDMA) and N1, N3, N5 - tris(3-(didodecylamino) propyl)benzene-1,3,5-tricarboxamide (TT3).

The positive charge carried by the lipid at a particular pH is responsible for the encapsulation of negatively charged RNA through electrostatic interactions, and forms something called lipoplex (Zeng et al., 2022; Liang et al., 2021). Dr. Pieter Cullis, a pioneer in this field, discovered that encapsulation of antigen in liposome nanoparticles showed greater immunogenicity (Liang et al., 2021; de Jong et al., 2007).

Yang et al., synthesized lipid nanoparticles containing cholesterol and a modified cationic peptide DP7. This resulted in enhanced in vivo cellular uptake of mRNA, and also induced activation of dendritic cells (Qin et al., 2022). Phua et al., fabricated a mesoporous-silica nanoparticle to condense both mRNA and RNA-activated protein kinase inhibitor C16. This vaccine was found to inhibit tumor growth significantly. As of June 2021, it was reported that every SARS-CoV-2 mRNA vaccine that received approval for clinical use was delivered using lipid nanoparticles (Chaudhary et al., 2021).

Another functional material-based mRNA delivery approach leverages the potential of polymeric materials such as polyamines (e.g., polyethyleneimine) and dendrimers (e.g., polyamidoamine), which protect mRNA from degradation and promote intracellular uptake. One plausible mechanism by which polyplexes (complex of cationic polymers and mRNA) evade endosomal uptake is proposed to be the proton buffering by the cationic polymer ensuing osmotic swelling, followed by endosomal rupture, also known as the proton sponge effect (Chaudhary et al., 2021).

Back in 1987, polylysine (PLL) came into limelight as the first ever cationic polymeric vector to effectively transfect plasmid DNA. Later, polymeric carriers like spermine, polyurethane, etc., were scouted for their ability to safely deliver mRNA. The use of PEI is restricted due to its toxicity profile and non-biodegradable nature, thereby making room for further developments. Jere et al., have performed some studies, to deduce that introducing polysaccharides, and encapsulation of mRNA with PEI into neutrally charged liposomes has diminished non-specific adhesion (Jere et al., 2009). Xiyu et al., designed PEI-g-PEG using multiple PEG terminal groups with varying PEG grafting degrees to deliver mRNA with adequate potency (Ke et al., 2020). This mode of delivery has been further advanced by incorporating some additional components, such as biodegradable units and lipid chains to aid in improving the stability of the formulation. For instance, Kaczmarek et al., successfully synthesized a biocompatible polymer, poly (β-amino esters), capable of delivering mRNA that could target lung endothelial cells and immune cells (Kaczmarek et al., 2018). A slight modification to this was made by Kowalski et al, where mRNA was injected intravenously into the spleen using a novel PCL-PBAE construct (Kowalski et al., 2018).

Similarly, polyamidoamines, the hyperbranched spherical dendrimers possessing high charge density, allow satisfactory complexation with mRNA, nonetheless, it causes serum aggregation. The incorporation of disulfide linkages into the dendrimer core attenuates their toxicity issue (Chaudhary et al., 2021). Another biodegradable positively-charged polymer, charge-altering releasable transporters (CARTs), was synthesized to improve mRNA loading efficiency and certain physical properties by the phenomena of charge-neutralization intramolecular rearrangement, which aids in the release of functional mRNA and promotes protein translation in cells.

Wang et al constructed an injectable hydrogel consisting of polyethylene imine and graphene oxide to carry mRNA-encoding ovalbumin alongside adjuvant R848 that reported inhibition of tumor progression in the B16-OVA melanoma model. Cationic peptides composed of amino acids such as lysine and arginine present amino groups that electrostatically interact with phosphate structures of mRNA. A cell-penetrating peptide RALA, was explored to deliver mRNA to the immune cells. Another widely accepted delivery strategy is cationic nanoemulsions, which integrates cationic lipids like DOTAP and nanoemulsions. Hydrophobic and hydrophilic surfactants are combined to stabilize the oil phase in the aqueous environment, leading to particle generation. It is tested and proven that the addition of cationic lipids facilitates complexation with nucleic acids via electrostatic forces and thereby imparts stability to mRNA (Teixeira et al., 2017).

Researchers have designed creative mRNA delivery strategies, which include, but not limited to reactive astrocyte-derived exosomes, tetrasulfide-embedded dendritic mesoporous organosilicon nanoparticles, lipid-decorated calcium phosphate nanoparticles, nucleoside lipids, and so on (O'Brien et al., 2020; Schumann et al., 2018; Wang et al., 2020). Immunologic adjuvants play a key role in enhancing the efficacy of cancer vaccines by boosting the immune system’s response to tumor-associated antigens. The mechanism involved is activation of the innate immune system, which triggers the production of cytokines that further activate the adaptive immune response. These adjuvants, through stimulation of antigen-presenting cells (APCs) like dendritic cells, promote the tumor antigen presentation to T-cells, thereby instigating an immune response. Few examples of immunoadjuvants are Toll-Like Receptor (TLR) agonists such as Polyinosinic-polycytidylic acid that activates TLR3, CpG Oligodeoxynucleotides that stimulate TLR9, and Imiquimod (TLR7 agonist), which are responsible for eliciting Th1-biased immune responses. Other pivotal adjuvants include DDA-Saponin Complexes (saponin-based adjuvants) that promote antigen presentation and foster balanced Th1/Th2 responses. Interleukin-15 (IL-15) is a cytokine adjuvant that prolongs anti-tumor activity via activation of natural killer (NK) cells and CD8⁺ T cells (Desai et al., 2025). Overall, adjuvants are crucial in driving the progress of strong cytotoxic T lymphocytes (CTLs) responses (Ren et al., 2024).

Cytokines are soluble proteins required for intercellular communication, cell differentiation and growth, and mediating innate and humoral immunity. Cytokines are activated in response to the stimulus. However, their circulation is short-term and have a limited half-life. Treatment with cytokines is based on either synergistic combination with approved drugs or their improved pharmacokinetics to increase half-life in the circulation, as well as topical administration of cytokines in the TME (Conlon et al., 2019). However, a conjugate with polyethylene glycol or a fusion protein with antibodies, Fc-domains, apolipoprotein A-I, albumin, or TGF-β (transforming growth factor-β) should be created to increase the half-life (Berraondo et al., 2019).

Cytokines upregulate TGF-β, triosephosphate isomerase, and IL-10, activation of Tregs, myeloid-derived suppressor cells, intracellular suppressors of cytokine signaling activation, and resulting in CD4⁺ activation. However, treatment with cytokines cannot directly affect the immune response to cancer cells, but only activate the immune response (Conlon et al., 2019). Therefore, “superkines” have been created using structural engineering. Moreover, applications of cytokines with cancer vaccines, ICIs, and monoclonal antibodies are being developed to enhance antibody-dependent cellular cytotoxicity, improve affinity binding, and enhance anticancer properties (Holder et al., 2022). Currently, cytokine structural engineering is focusing on the immunotherapeutic cytokines IL-2, IL-4, IL-15, and IFN (Aung et al., 2023).

Thus, the alterations of IL-2 reduced stimulation of Tregs, and reduced interaction with IL-2Rα, induced augmented expression of cytotoxic T cells, and improved antitumor responses (Xue et al., 2021). While IL-15 fused with truncated IL-15Rα activates IL-15Rα-deficient cells and forms a signaling complex to trigger proliferation and anti-apoptosis (Guo et al., 2017).

The first indications that radiation treatment (RT) can boost immunity against cancer originated from case studies in which inconceivable, untreated cancers diminished after local RT. With the development of ICI treatments, the fascinating potential of RT in eliciting an anti-cancer immune response has attracted a great deal of attention. Various approaches to cancer immunotherapy that can be combined to improve treatment outcomes have been reviewed (Figure 3).

Two essential elements of the immune response, i.e., enhancing both adjuvanticity and antigenicity were made possible by RT (Demaria et al., 2005). Through several mechanisms, RT can alter the local TME and increase antigenicity. RT, just like chemotherapy, promotes tumor antigen presentation by inducing MHC-1 expression (Liu et al., 2023). RT causes immunogenic cell death (ICD), in which APCs are guided to dying cancer cells by molecules such as Annexin A1, and antigen-uptake and presentation to T cells is promoted by HSP70, HSP90, and HMGB1 (Yamazaki et al., 2020). The release of HMGB1 exposes that radiation causes calreticulin (a protein that marks the cell for immune destruction) to translocate within the plasma membrane. RT increases the ability of APCs to recognize and absorb cancer cells by downregulating the “do not eat me” signal (CD47) on these cells (Yamazaki et al., 2020). Macromolecules are modified by reactive oxygen species (ROS) produced by RT, which increases their antigenicity. Also, these ROS can cause direct tissue destruction of cancer cells. RT increases adjuvanticity, which raises anti-cancer immunity even more.

RT has been shown to enhance anti-cancer immunity, but there is also strong evidence that it can induce an immunosuppressive TME (Demaria et al., 2015). In addition to killing cancer cells, radiation, especially broad-field radiation can harm normal immune cells. This can cause the TME to shift towards immunosuppression rather than increasing anti-cancer immunity. Additionally, radiation may raise the expression of activin A and TGF-β, which can draw in regulatory T cells (Tregs) and decrease the invasion of CD8⁺ T cells (Jarosz-Biej et al., 2019). Radiation also has additional immunosuppressive effects due to hypoxia, alterations in the stroma, damage of tumor blood vessels, and the influence of cytokines, TAMs, cancer-associated fibroblasts (CAFs), etc (Dancea et al., 2009).

Most chemotherapeutic drugs were created with the immune system’s impact contemplated rather than their immediate cytotoxic effects. Studies highlighting the combination of immunotherapy and chemotherapy have shown enhanced responses to anthracyclines in tumors, especially in mice models with intact immune systems, where notable outcomes have been obtained (Sordo-Bahamonde et al., 2023).

Numerous studies that have been conducted to date have demonstrated the potential of cytotoxic chemotherapy to enhance anti-cancer immunity. Harnessing anti-tumor immunity by adding one or more immunotherapeutic agents is one of the leading strategies currently being applied in clinical trials. Among the chemotherapeutics, alkylating agents (cyclophosphamide, carboplatin, cisplatin, and dacarbazine) and tubulin inhibitors are the major players that are being investigated in combination with Immunotherapeutics (Tsvetkova and Ivanova, 2022).

Targeting the genomic alterations harbored by oncogenes can induce more anti-tumor responses than cytotoxic chemotherapy. Among numerous ongoing clinical trials of combined immunotherapy and targeted therapies, the most efficient immunomodulatory responses are ascertained with anti-angiogenesis agents affecting almost all subpopulations of the immune system. Some of the FDA-approved combinations that target VEGF and other angiogenic proteins like platelet-derived growth factor receptor (PDGFR), c-kit, fibroblast growth factor receptors, etc., are Axitinib, Cabozantinib, Lenvatinib, and Bevacizumab (Ansari et al., 2022). The only combination therapy that doesn’t target angiogenesis directly is given in BRAF-mutated advanced melanoma, i.e., a combination of Vemurafenib (a BRAF inhibitor), cobimetinib (a mitogen-activated extracellular kinase (MEK) inhibitor), and atezolizumab (Morante et al., 2022). Other combinations like axitinib with pembrolizumab, axitinib with avelumab, and nivolumab with cabozantinib are prescribed in advanced renal cancer (Chen X. et al., 2024).

PD1 upregulation after CAR-T cell infusion can optimize the co-stimulatory signaling of CD28, resulting in dysfunctional CAR-T cells. This suggests that CAR-T combined with PD1/PD-L1 blockade can achieve synergistic anti-tumor activity, as observed in many trials. Increased antitumor activity along with anti-PD-1 antibodies is noticed by modified CAR-T cells, i.e., PD1-deficient CAR-T cells, which cause no systemic toxicity. Along with PD1/PD-L1, other inhibitory immunosuppressive pathways have also been investigated with ACT, such as knockout of the TGF-β signaling, which is a major immunosuppressive regulator in CAR-T cells that increases CAR-T cell count, thereby showing efficient antitumor activity (Poorebrahim et al., 2021).

Relapsed malignancies show no improvement with previously administered CAR-T cells, as patients might have developed an inhibitory immune response to them. To prevent such lymphodepletion, pre-CAR-T cell infusion has been employed that elevates APC activation by reducing the effect of Tregs and other immune cells, thereby facilitating increased CAR-T cells’ effectiveness (Davies and Maher, 2021).

Combining two different structured CAR-T cells is an alternative strategy for zoning out the same target molecules instead of lymphodepletion. This can bypass the immune rejection developed against one type of CAR-T cells. Empirical evidence indicates that anti-CD19 CAR-T therapy (murine-derived) immune rejection can be overcome by humanized anti-CD19 CAR-T therapy (Nguyen et al., 2022). Another strategy to increase anti-tumor efficacy and avoid relapse is using CAR-T cells (containing a tandem build vector) targeting two different antigens on the same tumor cells.

Immunostimulatory molecules, such as cytokines and their receptors, and co-stimulatory molecules can be given in combination with the fourth generation of CAR-T cells. The fourth generation of CAR-T TRUCK cells expressing IL-12 is expected to treat solid tumors, which has not been possible with conventional CARs (Chmielewski and Abken, 2015), because IL-12 cytokines counteract the CAR-T suppression caused by Treg and MDSCs, and thereby improve the CD8 + T cells and NK cells cytotoxic activity, and stimulate the Th1 cell response against tumor cells.

Cancer vaccines activate immunity against cancer via antigen presentation, priming, and activation of immune cells. Activated immune cells identify and eliminate cancer cells, mobilize and invade tumors, and elicit cytotoxicity. Dampened vaccine effectiveness can be caused by resistance mechanisms, particularly in the TME, for which researchers are still examining different treatment pathways. Priming T cells for anti-cancer immunity is the key intent of cancer vaccines (Fan et al., 2023), because many malignancies are not infiltrated by immune cells (“cold tumors”), and immune checkpoint blockers (ICBs) do not work on them (Rezaei et al., 2022). Vaccines are frequently used in conjunction with adjuvants like the TLR-3 agonist poly-ICLC to increase efficacy. For instance, when combined with poly-ICLC, the NY-ESO-1 vaccination exhibits an enhanced immune response (Zhou et al., 2023). Also, compared to IL-2 alone, the combination of IL-2 with gp100 (a tumor-associated antigen) showed significant improvement and progression-free survival (Choudhry et al., 2018). To increase the efficacy of cancer vaccines, researchers are investigating immunological co-inhibitory and co-stimulatory substances in addition to anti-PD1/PD-L1 and anti-CTLA-4 antibodies. A Dendritic cell MART-1 peptide vaccination and IMP321 (LAG-3Ig fusion protein) were administered together in a Phase I study. This combination showed potential for improving vaccination effectiveness as it enhanced MART-1-specific CD8 T cells and decreased the regulatory T cells (Zanotta et al., 2024).