The mismatch between exogenous and endogenous TCR chains has been a non-negligible problem for engineered TCR-T cell therapy. Exogenous TCR chains can have a competitive relationship with endogenous TCR chains, and exogenous TCR chains introduced after screening have a high affinity to dominate (Heemskerk et al., 2007). The introduction of exogenous TCRs into T cells has several effects, including the formation of mixed TCR dimers. However, the properties of different mixed TCR dimers are unpredictable and may affect the subsequent biological activity of T cells. These dimers might prevent transferring TCR-T cell therapy to clinical applications. It has been shown that TCR transfer leads to the generation of hybrid TCR dimers of unknown specificity, which may cause these T cells to cannibalize each other and exhibit novel deleterious reactivity, such as graft-versus-host response (GVHD) (van Loenen et al., 2010). Mixed TCR dimers may compete with engineered TCR heterodimers for binding of restricted CD3 components, resulting in off-target presentation of new antigenic peptides to the surface and triggering off-target effects. In the absence of the ability to eliminate mixed dimers, cysteines were used to design TCR constant regions of the exogenous chain, increasing the total surface expression of the introduced TCR chain, reducing the extent of mismatch, and decreasing the risk of developing graft-versus-host disease (Kuball et al., 2007). Enhanced TCR chain expression using hybridized human TCR chains containing mouse constant structural domains does not bind to endogenous TCR chains containing human constant structural domains (Chen et al., 2017). However, immunogenicity can limit T-cell viability to some extent (Cohen et al., 2006). In contrast to phage display methods, since the binding affinity of TCRs or mutants expressed on yeast cells can be directly assessed, yeast surface display technology screens for high-affinity TCRs and reduces the chance of TCR mismatch (Smith et al., 2015; Shafer et al., 2022). By introducing an additional stable disulfide bond between residue 48 of the TCR constant region α and residue 57 of the TCR constant region β through cysteine substitution, the inter stream binding affinity of the engineered TCR α/β chains is enhanced while simultaneously reducing their binding affinity to the endogenous TCR α/β chains (Krshnan et al., 2016). The stability of the engineered TCR α chain can be enhanced through the selection of hydrophobic substitutions in the transmembrane region (Haga-Friedman et al., 2012). Single-chain TCR (scTCR) entails the incorporation of TCR antigen recognition and signaling domains into a singular chain, thereby minimizing mismatches through spatial site-blocking (Knies et al., 2016).

For a considerable time, tumor-specific antigens, or TSAs, have been considered ideal targets for cellular immunotherapy. Nonetheless, the existing repertory of TSAs against solid tumors is still restricted, which reduces the effectiveness of traditional CAR-T cell therapy. On the other hand, the advantage of TCR-T treatment is that it targets both intracellular and cell surface antigens, greatly expanding the pool of potential targets. Notwithstanding this promise, very few peptide antigen targets have been shown to be both safe and efficacious for TCR-T immunotherapy. Research to detect immunogenic neoantigens in tumor cells may provide a significant discovery. Tumor cell-produced neoantigens need to be unique to tumor cells, not expressed in normal cells, and have high expression levels so that MHC molecules can recognize them (Wang and Cao, 2020). Not all people produce neoantigens in the same way, and even individuals with the same type of solid tumor have diverse tumor cells that express different antigens. Individual differences in neoantigens need the creation of customized TCR-T cell therapy regimens, which are closely related to the financial and schedule aspects of TCR-T cell therapy (Li et al., 2023a). Clear studies on the stability of neoantigens are lacking, posing challenges to mitigating the risk of neoantigen loss. Downregulation or loss of MHC class I molecules on tumor cells diminishes the sensitivity of TCR-T therapy and serves as a prominent pathway for tumor evasion (Dutta et al., 2006; Singh and Banerjee, 2020; Xu et al., 2022). The targets of TCR-T cell therapies are further constrained by MHC type. HLA genes encoded MHC molecules exhibit extensive diversity within the population, with over 20,000 HLA I human alleles identified to date (Gonzalez-Galarza et al., 2020). At present, the research field of TCR-T therapy is constantly exploring new targets to improve the therapeutic effect and expand the therapeutic scope. The research and development of novel targets mainly include tumor-specific neoantigens (Xie et al., 2023), virus-associated antigens and cancer-testis antigens (Gordeeva, 2018) (Table 1).

Regarding the dynamics of therapy, TCR-T therapy is more sensitive than CAR-T therapy, making it more vulnerable to non-tumor-targeted toxicity (on-target off-tumor) and cross-reactivity (off-target off-tumor). The former is related to the expression of target antigen in normal tissues, especially in the case of TAA. The latter refers to the fact that the TCR recognizes different antigens on normal cells, especially when the affinity of the TCR sequence is enhanced. This vulnerability may cause damage to normal human tissues that have comparable antigenic epitopes. An illustrative instance involves the treatment of myeloma and melanoma patients with engineered T cells targeting the affinity-enhancing TCR of MAGE-A3, resulting in unforeseen occurrences of cardiogenic shock and mortality. In these cases of myocardial injury, histopathological analysis revealed the infiltration of T cells but no expression of MAGE-A3 was detected in cardiac autopsy tissue. Because TCR-T cells recognize an unrelated peptide from the rhabdomyosarcoma-specific protein titin, it is thought to be due to cross-reactivity (Linette et al., 2013). In a clinical investigation, highly reactive transferred TCR-T cells transported to melanoma tumors in patients and destroyed melanoma in their bodies, but patients showed damage to normal melanocytes in the skin, eyes, and ears (Johnson et al., 2009). Another study delineates instances of severe acute colitis induced in patients with metastatic colorectal cancer who were treated with TCR-T cells targeting carcinoembryonic antigen (CEA). This outcome arose from the expression of the target antigen on normal intestinal tissue as well (Parkhurst et al., 2011). The concept of TCR affinity embodies a paradox. It is widely acknowledged that robust affinity is imperative for sustaining T-cell expansion and facilitating the regression of human cancers. Excessive affinity, however, can cause cross-reactivity with self-antigens by targeting healthy cells across the body that carry homologous antigens and prematurely exhausting T cells.

Cytokine storm represents a spectrum of clinical disorders marked by significant health risks arising from the excessive production of inflammatory factors by cell therapy. This phenomenon encompasses manifestations such as fever, tachycardia, hypotension, rash, and respiratory distress, emerging as the predominant adverse reaction to T-cell immunotherapy. Key cytokines implicated in a cytokine storm, including Interferon-gamma, IL-1, IL-6, TNF, and IL-18, exhibit consistently elevated levels and are believed to orchestrate the central pathological mechanisms underlying cytokine release syndrome (CRS). Since TCR-T can recognize peptide epitopes derived from proteins in any subcellular (e.g., membrane, cytoplasmic, and nuclear) compartment (Chandran and Klebanoff, 2019), has a broader range of antigen selection, and the TCR chain has intrinsic signaling and regulates T cells, resulting in a lower rate of CRS as compared to CAR-T cells, T cell receptor (TCR)-T cells are also considered promising immunotherapy. Low incidence is not the same as complete avoidance. Mikiya Ishihara et al. utilized retroviral vectors to engineer precise silencing of endogenous TCRs and induce the forced expression of the affinity-enhancing NY-ESO-1-specific TCR in T cells. The system expresses small interfering RNAs (siRNAs) that are specific to endogenous TCR genes to enhance the expression of transduced tumor-specific TCR while minimizing potential TCR mispairing. In patients with tumors that highly express NY - ESO - 1, the infusion of TCR - T cells led to a significant tumor response as well as early-onset CRS (Ishihara et al., 2022). Similarly, a patient with fallopian tube cancer, unresponsive to adjuvant chemotherapy, participated in a clinical trial of MAGE-A4-targeted T-cell receptor T-cell therapy. Following T-cell infusion, the patient experienced CRS and pseudogouty arthritis accompanied by immune effector cell-associated neurotoxicity syndrome (ICANS) within 7 days. Notably, the drug tocilizumab was ultimately utilized to eliminate CRS and ICANS successfully (Kim et al., 2021). Hence, to enhance the safety and efficacy of TCR-T cell immunotherapy, it is imperative to prudently evaluate the significance of cytokine storm risk for patient prognosis.

The first obstacle genetically modified T cells must overcome while fighting solid tumors is to infiltrate the tumor’s hostile environment successfully. Solid tumors reinforce their conquered area by creating complex and formidable ecosystems. Cancer cells are remarkably adaptive as tumors progress, repurposing various non-tumor cell types to create an environment that supports their growth and survival (Heindl et al., 2015; Wang S. et al., 2020). The tumor microenvironment comprises three main categories: hypoxia, chronic inflammation, and immunosuppression (Policastro et al., 2013). Within the relatively constrained confines of solid tumors, hypoxia emerges as a pervasive condition, significantly impacting the rapid proliferation and metabolic activity of T cells. While CD8⁺ T cells serve as the cornerstone of tumor elimination, their vigor wanes, and exhaustion sets in when confronted with hypoxic conditions. Recent discoveries have unveiled a paradox: as this fundamental component becomes terminally exhausted, it transforms, upregulating CD39 expression to foster an immunosuppressive milieu that undermines the robust anti-tumor capabilities of other T cells (Vignali et al., 2023; Canale et al., 2018). Tumor-associated macrophages (TAMs) constitute pivotal constituents of the tumor microenvironment. Studies in hepatocellular carcinoma therapeutics have unveiled a robust correlation between downregulation of xanthine oxidoreductase (XOR) expression and specific tumor microenvironment traits, particularly hypoxia (Ullah et al., 2024b; Ullah et al., 2024c). Deletion or dysfunction of XOR in monocyte-derived macrophages fosters CD8+ T cell depletion by inducing the upregulation of immunosuppressive metabolites (Lu et al., 2023). Most tumor stromal cells within the TME exhibit numerous immunosuppressive signaling proteins, including programmed death ligand-1 (PD-L1). The interaction between PD-L1 and programmed cell death protein-1 (PD-1) (Noman et al., 2014) expressed in T cells can result in immune cell dysfunction and apoptosis (Dermani et al., 2019). Hypoxia induces elevated potassium levels and an acidic milieu, disrupting potassium homeostasis in tumor-infiltrating lymphocytes (TILs) within necrotic tumor microenvironment regions (Conforti, 2017). This severely suppresses T-cell effector function, impacting cytokine secretion capacity and activity (Eil et al., 2016).

The PD-1/PD-L1 inhibitory axis represents an immune checkpoint mechanism evolved by the body to regulate the magnitude and duration of immune responses, thereby preventing excessive inflammatory reactions and autoimmune diseases that may harm healthy tissues (Dai et al., 2014; Dyble et al., 2015). In the endeavor to corral tumor cells, the PD-1/PD-L1 inhibitory axis is a vulnerability. Tumor cells accomplish immune evasion by elevating PD-L1 ligand expression and inhibiting T cell function through PD-1 engagement. PD-L1, functioning as a natural ligand for PD-1 (Ji et al., 2019), assumes the role of a guardian for tumor cells, endowed with the capability to convey anti-apoptotic signals and foster tumor proliferation (Dong et al., 2018).

When this route is unchecked, T cells’ ability to fight cancer is compromised. To prevent the PD-1/PD-L1 pathway, scientists have developed a variety of PD-1/PD-L1 immune checkpoint inhibitors. These inhibitors have been successfully used to treat a variety of malignancies, including classical Hodgkin’s lymphoma, melanoma, non-small cell lung cancer, and squamous cell carcinoma of the head and neck. Moon EK et al. reported that the combination of TCR-T cell therapy with PD-1 antibody reduced hypofunctionality in tumor-infiltrating lymphocytes (TILs) and augmented the efficacy of overt metastatic T cells under conditions of chronic antigenic stimulation (Moon et al., 2016). At present, cellular immunotherapies engineered to produce autocrine PD-1 antibodies or to suppress PD-1 expression are available for treating solid tumors (Hamilton et al., 2023; Wu et al., 2023; Zhang et al., 2022). PD-1/PD-L1 immune checkpoint inhibitors provide advantages but also significant hazards. Studies reveal significant effects on the heart, resulting in serious illnesses such as myocarditis, stress cardiomyopathy, pericardial disease, and an increased risk of complications associated to the heart’s immune system (Varricchi et al., 2017; Costa et al., 2017; Khunger et al., 2020).

If playing hardball proves ineffective, why not transform adversaries into allies? The EtoE platform introduces an inherent 2-in-1 combination therapy (Schendel, 2023). The pivotal element of this therapy lies in the utilization of the PD1-41BB co-stimulatory switch protein (CPS) incorporated into TCR-T cells, enabling these cells to concurrently express recombinant TCR and PD1-41BB switch receptors (Sailer et al., 2022). The ingenuity of the concept resides in substituting the inhibitory signaling structural domain of PD-1 with the activating signaling structural domain of 4-1BB while retaining the external structural domain of PD-1 responsible for binding to PD-L1 on tumor cells. The intracellular signaling domain of the 4-1BB protein offers a comprehensively characterized pathway for co-stimulation, augmenting T-cell responses, and activating various immune cells (Vinay and Kwon, 2014). Therefore, by not impeding PD-1 binding to PD-L1, PD1-41BB-modified TCR-T cells exhibit enhanced tumor cell killing even in the presence of PD-L1-overexpressing tumor cells. This scenario converts potent inhibitory signals into secondary activating signals, prompting T-cell proliferation, cytokine expression, and bolstering T-cell effector function (Schlenker et al., 2017). The PD1-41BB CPS efficiently regulates the self-defense mechanism of tumor cells.

Another co-stimulatory switch protein targets the CD40L/CD40 pathway and the CD28 co-stimulatory signal. The CD40 receptor and its ligand, CD40L, constitute one of the most crucial molecular pairs of stimulatory immune checkpoints. CD40 binds to its ligand, CD40L, which is transiently expressed in T cells and other non-immune cells under inflammatory conditions (Elgueta et al., 2009). Initially characterized on B cells (Sacco et al., 2023), CD40 is expressed on professional antigen-presenting cells as well as non-immune cells and tumors (van Kooten and Banchereau, 1997). The CD40L-CD28 CPS is composed of an extracellular CD40L structural domain combined with an intracellular signaling structural domain of the CD28 co-stimulatory receptor (Olguín-Contreras et al., 2021). The principles related to the chimeric design of this CPS are not yet clear, and it is still unknown whether it can successfully achieve biological effects (Oda et al., 2017). CD40L-CD28 co-stimulatory switching protein-modified TCR-T cells provide a variety of new attributes that can contribute to the enhancement of cellular immune responses. As multiple cells in the tumor microenvironment express CD40, CD40L-CD28 engineers enhanced T-cell infiltration, and CD40L activates CD40-expressing immune cells to amplify anti-tumor responses. The presence of the CD28 signaling structural domain in the CSP provides further stimulation of TCR-T cells, improving cytokine secretion and antigen-specific cytotoxicity. This intrinsic combination enhances the function of TCR-T cells, alters the strong inhibitory nature of the tumor microenvironment against T cells, and mobilizes immune cells in the tumor microenvironment to participate in the battle against solid tumors (Schendel, 2023).

Although TCR-T cell therapy has great promise in treating solid tumors, it still confronts several obstacles and frequently produces unsatisfactory clinical results. TCR-T therapy faces significant challenges in efficiently treating solid tumors due to factors such low T-cell infiltration capacity, hostile tumor milieu for T-cell activation, and limited in vivo durability of infused TCR-T cells. Permissive T cells can be genetically engineered to express different pro-inflammatory molecules, such as IL-12 (Liu et al., 2019; Pegram et al., 2012), IL-15 (Nguyen et al., 2023), IL-18 (Avanzi et al., 2018), CD40L (Kuhn et al., 2019), or DC growth factor FLT3 (Zhu et al., 2024; Lai et al., 2020). These modified T cells have the ability to self-supply pro-inflammatory chemicals, promote epitope dissemination, and enhance T cells’ anti-tumor activity in vivo. These tactics are supported by the fact that transformed T cells are viable and abundant in vivo and that T-cell death reduces the release of pro-inflammatory chemicals. However, because the in vitro production method takes time, there is a chance that genetically altered T cells will not be easily accessible for subsequent infusion. In contrast, the simplicity of tumor vaccine preparation and the ability to control the infusion timing outweigh these strategies. Consequently, the combination of vaccines with TCR-T cell therapy holds promise in overcoming the lackluster outcomes associated with TCR-T cell therapy in the treatment of solid tumors.

While co-stimulatory signaling plays a crucial role in T cell activation, the downregulation of co-stimulatory ligand expression within the tumor microenvironment presents a challenge by restricting T cell activation. Attempts to enhance TCR-T efficacy through combined vaccination in early TCR-T trials did not yield the anticipated results in providing both TCR stimulation and co-stimulation in vivo (Nowicki et al., 2019; Kageyama et al., 2015). Significantly, the integration of vaccines with overt cellular therapies effectively triggers the activation of the endogenous immune system, facilitating the generation of host T cells targeting additional tumor antigens, thus preventing antigen-negative tumor escapes (Author anonymous, 2023; Mateus-Tique and Brown, 2023; Corbière et al., 2011). The phenomenon, termed “Antigen Spreading” (AS), has the potential to enhance the efficacy of TCR-T cell therapy for solid tumors. Recent studies conducted by Elicio Therapeutics have demonstrated that the combination of amphiphilic (AMP) vaccination, which selectively targets homologous peptides and adjuvants to lymph nodes, induces dendritic cell activation. This activation not only enhances T-cell activation through endogenous co-stimulation and cytokine-supported delivery of TCR-stimulants but also has the potential to trigger the collaboration of newly generated tumor-specific T cells with TCR-T cells, thus collectively combating tumors (Drakes et al., 2024).

In order to facilitate the targeted growth of T cells in vivo, customized vaccinations can also be made to target certain antigens of interest called neoantigens (Hellmann and Snyder, 2017). Personalized vaccinations can undoubtedly enhance antigen-specific T cells in vivo, as evidenced by research done on human HLA transgenic mice and cancer vaccine trial subjects (Sahin et al., 2017; Kunert et al., 2016). However, the combination of TCR-T cell treatments and cancer vaccines has only been studied in a small number of clinical trials, thus more study is needed to determine their therapeutic efficacy and develop a thorough and well-defined rationale.

Immunotherapy against the oncolytic virus (OV) is a new and exciting treatment approach. Oncolytic viruses, whether natural or recombinant, have the ability to preferentially infect tumor cells, multiply within the tumor cells, and lyse tumor cells directly while sparing healthy cells. T-cell treatment with an oncolytic virus combination shows a synergistic impact. Targeting by lyssaviruses can compensate for the deficiency in T-cell trafficking (Kaufman et al., 2015), converting the “cold” tumor microenvironment into a “hot” one (Bommareddy et al., 2018). Moreover, it inhibits tumor immune evasion mechanisms and angiogenesis (Streby et al., 2017), achieving multifaceted tumor eradication. In addition to eliciting classical apoptosis and pyroptosis by targeting tumor antigens, it can induce autosis, a previously undocumented form of cancer cell demise. A study report delineates the synergy between an oncolytic virus and adoptive cellular therapies (CAR-T, TCR-T), effectively directing its attack toward drug-resistant cancer cells, ushering in new optimism for combating treatment-resistant cancers (Zheng et al., 2022).

Tumor-homing cell-penetrating peptides (CPPs) constitute small amino acid sequences, typically short peptides (comprising less than 30 residues) (Ye et al., 2016), capable of traversing cell membranes. These peptides serve as effective vehicles for intracellular delivery, both in vitro and in vivo, facilitating the transportation of diverse biologically active cargoes, ranging from nucleic acids to large proteins and other compounds, with molecular weights extending up to 120 kDa. CPP enhances the penetration of small molecule drugs and nanoparticles into tumor tissues (Silva et al., 2019). Naiqing Ding et al. achieved enhanced infiltration of tumor-specific T cells through the rapid modification of T cells with the tumor-penetrating peptide iRGD using a lipid insertion approach (Ding et al., 2019). This method circumvents the need for intricate gene editing procedures. A novel bifunctional drug, iRGD-anti-CD3, tightly bridges internalizing RGD (Arg-Gly-Asp) peptide (iRGD) and T cells by binding to CD3. iRGD-anti-CD3-modified T cells not only alleviate the challenging infiltration of T cells in the tumor microenvironment but also induce T-cell activation and cytotoxicity against cancer cells (Zhou et al., 2021). Using CPP in conjunction with TCR-T cell treatment is a novel approach that has the potential to eventually improve the therapeutic efficacy of off-the-shelf cell therapy.

A common and serious side effect of cell treatment is called CRS, which is caused by immune cells releasing cytokines out of control. Cytokine storm is characterized by its intensity and presents serious dangers, including organ failure and death. Researchers have started looking into the use of genetically altered exosomes made from altered cells in cancer treatment as a way to lessen this difficulty. Exosomes provide a significant benefit over using genetically changed post-modified cells directly because of their nanoscale size and innate ability to cross biological barriers, especially when it comes to solid tumor therapy. Exosomes have been shown in numerous studies to be able to be tailored for target specificity, which allows them to act as nanocarriers for anti-tumor medicines and antigen-specific anti-tumor immune responses (Roma-Rodrigues et al., 2014; Cheng and Hill, 2022). Therefore, exosome-based treatments show great therapeutic promise for cancer patients. Several examples of these therapies have been reported in preclinical models or clinical trials, including dendritic cell exosomes (DC-Exos) (Li et al., 2023b) and natural killer exosomes (NK-Exos) (Sun et al., 2020; Sun et al., 2019). Exosomes have advantages over cell-based therapies, including higher yield, increased safety, easier storage, and lower costs (Nag et al., 2023; Xu et al., 2020; Wang et al., 2016). Vδ2-T cells are known for their potential in anti-tumor and anti-infective capabilities, and they play a crucial role in recognizing and attacking both tumor and infected cells. Additionally, they are essential in controlling inflammatory pathways and immunological responses. A method of treating EBV-associated tumors with Vδ2-T-Exos, which possesses the cytotoxic and immunostimulatory characteristics of Vδ2-T cells, was suggested by Xiwei Wang et al. This strategy effectively controls EBV-associated tumors by combining the benefits of merging NK-Exos and DC-Exos (Wang X. et al., 2020). Exosomes produced from TCR-T cell culture can be used as a unique and potentially very effective cancer therapy approach. By acting as direct attackers and taking the place of TCR-T cells in vivo, they improve the safety and controllability of treatment.