Advances in Adoptive Cell Therapies in Cancer: From Mechanistic Breakthroughs to Clinical Frontiers and Overcoming Barriers

This generation of cells comprises mainly four components: an extracellular domain for antigen binding, which is generally a single-chain variable fragment (scFv) made up of light or heavy chains of antibodies, a hinge (spacer) region, an intracellular signaling domain (CD3ζ), and a transmembrane. Structurally, first-generation CARs lack costimulatory molecules and cytokine-mediated signaling. These CARs are reported to have less T-cell proliferation, poor in vivo responses, and cytokine release, which result in reduced antitumor activity, hence making them obsolete [20,21].

This generation of cells is engineered with one/two costimulatory molecule (CM) domains, such as CD28, CD134, CD19, or CD137 (4-1BB), along with an intracellular signaling domain (CD3ζ), to enhance the proliferation of T cells, the activation of T cells, survival, and cytotoxicity. All of the FDA-approved therapies that are commercially available are only from the second-generation CAR-T cells category, composed of a costimulatory molecule (CM) with a CD3ζ signaling domain [22]. As reported in various studies, CAR-T-cell therapy with the CD137 domain possesses a durable response, with constant persistence, but weak tonic signaling. In contrast, CAR-T cells with the CD28 domain have greater survival, T-cell expansion, memory cell formation, signaling, and quick responses. CAR-T cell therapies with CD19 domains are under clinical development and reported to be potentially effective against B-cell malignancies. Obecabtagene autoleucel, also known as “obe-cel,” is a novel CD19 CAR-T cell that was genetically engineered with CAT-41BB-Z. It is now undergoing clinical trials and is exhibiting exceptional outcomes in certain adult patients with recurrent B-cell acute lymphoblastic lymphoma (ALL) [23]. Regardless of these remarkable improvements, the challenges of persistence and relapse with CAR-T-cell therapies that have a single costimulatory domain continue, which has led to the development of third-generation cells [24].

This generation of cells is constructed with a CD3ζ signaling domain combined with several CM domains, namely, CD27, CD28, CD134 (OX40), CD137, NKG2D, and TLR2, to create a CAR-T-cell integrated construct, such as CD3ζ-CD28-CD134, CD3ζ-CD28-CD137, and CD3ζ TLR2-CD28. Multiple additions of costimulatory molecules in the third generation help to overcome the constraint of a single costimulatory domain that is used in second-generation CARs; also, these additions provide the potent and faster elimination of tumors [25]. In some preclinical studies, it was found that third-generation CARs exhibited better antitumor activity and safety compared to second-generation CARs. However, the association of rapid CAR-T-cell exhaustion and severe side effects due to the overactivation of costimulatory molecules with third-generation CAR-T cells is common, which limits further application [26].

This generation is also called T cells that are redirected for universal cytokine-mediated killing (TRUCKS), or armed CAR-T cells structurally based on the construct similar to the second generation, with the addition of transgenic proteins such as IL2, IL5, and IL12, and cytokine costimulatory molecules, such as CD27, CD28, CD134, and CD137 [27]. The presence of cytokines activates the innate immune cells that help to kill cancer cells and alleviate cancer. This type of CAR-T cell also helps in reestablishing the immune system of the cancer patient post-infusion. Even with these advantages, fourth-generation CARs have the limitation of moderate efficacy against solid tumors and are associated with some adverse events due to cytokine release in healthy tissues [28,29].

Currently, the development of fifth-generation or next-generation CARs is in progress; structurally, they are like the construct of second-generation CARs, including a transcription-factor-binding motif and truncated cytoplasmic receptor [30]. This generation contains both advanced CARs, which outperform conventional CARs by adding extra structures to the standard CAR for the identification of numerous antigens or targets with low antigen density, and greatly enhanced conventional CARs, which are monovalent CARs that solely target one specific antigen [31]. The pros and cons of CAR-T cell generation are outlined in Table 1.

Hodgkin and non-Hodgkin are two major types of lymphoma that are traditionally treated with chemotherapy and monoclonal antibodies due to the limitation of disease deterioration that has been reported by patients in need of potential therapy. Both lymphomas have proven to be suitable to immunotherapy treatment because of their occurrence in immune-rich lymphoid tissues [32,33]. In various studies, CAR-T-cell therapy exhibited very promising results in B-cell lymphoma patients who were in a primary therapy setting [34]. In one study reported, CD19-antigen-directed CAR-T cells showed favorable outcomes in lymphoma, with patients achieving a 75% partial response. According to a clinical study by Kochenderfer et al., out of 15 patients with advanced B-cell malignancies who received CD19 CAR-T-cell therapy, 8 of them experienced complete remission, 4 showed partial remission, 2 were not responsive, and 1 had stable lymphoma [35]. In different studies, the treatment of lymphoma patients with CD19-targeted CAR-T cells has shown promising findings [36,37]. CD20-antigen-directed CAR-T cells have shown considerable results in the treatment of non-Hodgkin lymphoma patients [38,39]. Apart from the CD19 and CD20 antigens, CD30-targeted CAR-T cells have also been found to be vital in the treatment of lymphomas [40,41]. To understand and explore further the application of CAR-T cells, various clinical studies are in progress.

In recent times, various researchers have widely explored CAR-T-cell therapies in the treatment of patients with CLL, relapsed CLL, and relapsed or refractory (R/R) CLL. Among all CAR-T-cell therapies with CD19, antigens are extensively used. Some studies have demonstrated potential efficacy but limited proliferative responses and cell expansion abilities [42,43]. However, various toxic effects, such as tumor lysis and the depletion of B cells, are always associated with CAR-T-cell therapy. To overcome these various issues, researchers have explored the incorporation of interleukin and suicidal genes to improve antitumor efficacy and toxic effects, respectively [44,45].

Treatment with CAR-T-cell therapies has exhibited notable efficacy in patients with acute lymphoblastic leukemia (ALL), especially for curing fatal R/R B-ALL. Targeting the antigen CD19, which is a crucial marker of B cells, has become a productive approach, as the results have been satisfactory and the therapeutic effects have been in accordance with expectations. Kymirah was the first FDA-approved CD19-directed CAR-T-cell therapy used for the treatment of R/R ALL and B-cell lymphomas. However, due to the limitation of antigen escape, CD19-specific CAR-T cells are not responsive in some patients. To overcome this, researchers have explored CD20 and CD22 antigens for developing CAR-T cells and have found that both antigens can be a potential target in treating relapsed patients who are on CD19-directed CAR-T therapies [46,47,48].

Multiple myeloma (MM) is a plasma cell malignancy often associated with anemia, hypercalcemia, immunosuppression, bone lesions, and renal failure [49]. The treatment of MM with conventional chemotherapy is challenging and is considered largely incurable due to molecular abnormalities, cellular heterogeneity, mutations in relapse malignant plasma cells, and high resistance to standard therapies. Recently, B-cell maturation antigen (BCMA)-targeted CAR-T-cell therapies, such as idecabtagene vicleucel and ciltacabtagene autoleucel, have emerged as promising options, demonstrating high response rates in relapsed or refractory MM. These advances highlight CAR-T-cell therapy as a novel and effective approach to overcoming therapeutic resistance in MM [50,51].

CAR-T cells target 19 antigens for breast cancer, most of which are cell-surface proteins and members of the RTK family: HER2, AXL, EGFR, ROR1, HGFR/cMET, MUC1, CD70, MSLN, CD133, EpCAM, CD44v6, CSGP4, TEM8, ICAM1, TROP2, NKG2D, FRα, GD2, and CEA. While only 12 antigens have made it to clinical trials, all 19 have undergone considerable investigations and shown anticancer benefits in preclinical tests, such as tumor growth suppression and the release of pro-inflammatory cytokines. Although CAR-T-cell treatment for breast cancer has made significant progress thus far, individuals with the disease are still a long way from using it because of a lack of supporting data [52]. Key clinical outcomes of CAR-T cell therapies across different cancer types are illustrated in Figure 3.

Recently, antigen-targeted CAR-T-cell therapies have displayed significant success in the treatment of various hematological malignancies, especially second-generation CARs, which have shown promising efficacy and an acceptable safety profile. CD19-targeted CAR-T cells are extensively explored for the indication of different lymphomas, myelomas, and B-cell leukemia [53,54,55]. Considering the success of CAR-T in hematological malignancies as a proof of concept, researchers have now focused on exploring the therapeutic potential of various surface proteins to treat solid tumors, namely, human epidermal growth factor receptors 1 and 2 (HER1 and 2), mesothelin (MSLN), fibroblast activation protein (FAP), diganlioside (GD2), L1 cell adhesion molecule (L1 CAM), mucin (MUC) 1, MUC 16, and vascular endothelial growth factor receptor 2 (VEGFR 2) [56,57]. Table 2 represents the clinical trials for anti-surface protein CAR-T-cell therapy against solid tumors.

It is defined as the partial or complete loss of the specific expression at targets by antigens, which results in tumor resistance. Antigen escape is considered one of the most challenging limitations associated with CAR-T-cell therapies and is generally observed in single-antigen-directed CAR structures. In various studies, it has been reported that patients with R/R ALL indications when treated with CD19-directed CAR-T-cell therapy showed durable response; however, the follow-up data revealed the downregulation of CD19 antigens, along with the development of disease resistance in 30–70% patients who had a recurrent disease post-therapy [58,59]. In multiple myeloma patients who have received BCMA-directed CAR-T-cell therapy, the loss of BCMA antigen expression was observed [60,61]. Table 3 outlines the key challenges encountered in CAR-T cell therapy and the strategies developed to address them.

To address this issue of antigen escape, several tactics have been used. Antigen-negative tumor variations are less likely to evade immune monitoring when CAR-T cells are engineered to identify many tumor-associated antigens at once. Approaches include pooled CAR-Ts (combining different CAR-T-cell populations), dual CAR-Ts (expressing two distinct CARs), and tandem CAR-Ts (single CAR constructs with dual antigen specificity). For instance, dual-targeting CAR-T cells against CD19 and CD22 has demonstrated enhanced efficacy in B-cell malignancies. The ability of dual-specific antibodies to bind to two distinct antigens makes it easier for immune effector cells to attach to malignant cells. Bispecific T-cell engager blinatumomab, which targets CD3 on T cells and CD19 on tumor cells, has shown promise in treating B-cell precursor acute lymphoblastic leukemia [63].

Integrating blinatumomab provides a parallel perspective on antigen-directed immunotherapies. While not genetically engineered like CAR-T cells, blinatumomab harnesses T-cell cytotoxicity through physical proximity between T cells and malignant cells, differing in manufacturing complexity, administration route, and in vivo persistence. This comparison situates CAR-T-cell therapy within a broader continuum of strategies aimed at overcoming antigen escapes and enhancing tumor-specific cytotoxicity [63].

T-cell exhaustion, a dysfunctional state induced by chronic antigen stimulation, is a major factor limiting CAR-T-cell persistence and potency, particularly in solid tumors with sustained antigen exposure. It is marked by reduced proliferation, impaired cytokine production, and the continuous expression of inhibitory receptors such as PD-1, TIM-3, and LAG-3. These markers not only signify functional decline but also actively suppress CAR-T activity through inhibitory signaling pathways. To counter exhaustion, strategies include checkpoint blockades, the genetic disruption of inhibitory receptors, the optimization of CAR costimulatory domains to fine-tune activation, and metabolic reprogramming to maintain cellular fitness. Advanced approaches—such as transient CAR expression, combinatorial checkpoint targeting, and epigenetic reprogramming—are being explored to restore effector functions, prolong persistence, and expand efficacy in both hematologic and solid malignancies [44].

The treatment of hematological malignancies has seen significant progress due to CAR-T-cell therapy, but a significant concern with this strategy is the possibility of potentially fatal side effects. Among the most common adverse effects are cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). The cytokines IL-1 and IL-6, which mediate CRS, can result in fever, hypotension, and other systemic symptoms. For CAR-T-cell therapy, the FDA authorized the use of tocilizumab, a humanized anti-IL6 receptor antibody, to reduce the risk of CRS and neurotoxicity. The anti-IL6 chimeric antibody siltuximab and the IL-1 receptor antagonist anakinra are further possible therapies for these side effects [13]. Most cases of toxicities are reported in patients who were on second-generation CD19-directed CAR-T-cell therapy [70,71]. Besides these toxicities, renal failure, macrophage activation syndrome (MAS), and splenomegaly are other associated toxicities reported with CAR-T-cell therapies [72]. To counter the toxicity of CRS, IL-6 receptor blockade, along with corticosteroid and tocilizumab, which are approved by the FDA for the management of CRS, is used. Surprisingly, tocilizumab is associated with severe CRS and fatality [72,73,74]. To ameliorate toxicities associated with CAR-T-cell therapy, strategies such as altering the structure of CAR-T cells and engineering CAR-T cells are being explored.

This type of toxicity occurs due to the direct attack on normal tissues; it happens because of the shared expression of the target antigens. This occurs in the case of solid tumor antigens when they are exposed or expressed on normal tissues at different levels. Hence, during CAR construction, the selection of specific antigen targets is crucial to ensure efficacy and avoid toxicities that arise due to on-target off-tumor effects. CAR-T-cell targets such as B7H3 [75,76], MUC1 [64], MUC16 [77,78], and TAG72 [79] are some major targets that are under investigation. A Phase I clinical trial (NCT05225363↗) of second-generation TAG72-targeted CAR-T cells is ongoing for the treatment of patients with platinum-resistant recurrent epithelial ovarian cancer [65]. Further developments and investigations with specific, targeted antigens are necessary to minimize toxicity and improve efficacy and therapeutic outcomes for CAR-T-cell therapies.

According to research, immunosuppressive elements in the TME, including regulatory T cells, immunological checkpoint proteins, and suppressor cytokines, restrict the activation and growth of CAR-T cells. Furthermore, solid tumors typically have low vascular densities and high cell densities, which limit CAR-T-cell infiltration and spread and may lessen the tumor’s ability to fight it [80]. A study found that in a solid tumor mouse model, the combination of CAR-T-cell treatment and programmed cell death protein-1 (PD-1) inhibitors produced better therapeutic effects, effectively enhancing the CAR-T cells’ ability to survive and integrate. Furthermore, modifying CAR-T cells using gene-editing techniques to get beyond the TME’s limitations is another possible remedy. It has been discovered that even in the presence of large quantities of chemotherapeutic drugs, the cytotoxic capabilities of CAR-T cells can be improved by introducing genes that increase sensitivity towards antitumor drugs. Some researchers have employed the Siglec15 protein to test its therapeutic efficacy against lung cancer in nude mice. An anti-Siglec15 antibody was reported to improve the treatment of lung adenocarcinoma by increasing tumor cell death, presumably due to macrophage polarization altering the TME. cMet/Siglec15 CAR-T cells demonstrated clear cytotoxicity against lung cancer cells, both in vitro and in vivo. They could also dramatically reduce the development of lung adenocarcinoma cells in situ and enhance T-cell infiltration in TME.

To counteract these suppressive signals, combination therapies have been devised that integrate CAR-T-cell strategies with checkpoint inhibitors. For example, in B-cell lymphomas and hematological malignancies, the combination of CAR-T cells with anti-PD1 therapy is under clinical investigation (NCT05659628↗) [81]. Similarly, a Phase I/II study is exploring the safety and efficacy of MUC1 CAR-T cells combined with PD1-engineered T cells in patients with advanced esophageal cancer (NCT03706326↗) [82]. Despite the potential of these combination techniques, more investigations are required to completely evaluate their capacities to improve tumor infiltration and T-cell activity, especially when it comes to solid tumors and other complex malignancies [83,84,85]. Figure 4 represents the immunosuppressive features of the TME along with the mechanistic basis of combination immunotherapy.

Addressing the limitations of CAR-T-cell therapy in solid tumors requires a multipronged approach that integrates biological, engineering, and translational innovations. Armoring CAR-T cells with cytokine-secreting payloads such as IL-12 or IL-18 can reprogram the immunosuppressive tumor microenvironment, while the co-expression of dominant-negative receptors or checkpoint inhibitors (e.g., PD-1 decoys) mitigates T-cell exhaustion. Engineering dual- or multi-target CARs enhances antigen coverage and reduces the risk of tumor antigen escape. Modifying chemokine receptors to match tumor-secreted chemokines (e.g., CCR2 for CCL2-rich tumors) improves homing and infiltration. Incorporating logic-gated CAR designs and switchable safety modules enhances precision and minimizes off-tumor toxicity. Beyond cell-intrinsic engineering, combining CAR-T therapy with oncolytic viruses, radiotherapy, or immune checkpoint blockades may synergistically improve efficacy against solid tumors. Together, these strategies aim to transform the hostile architecture of solid tumors into an accessible and immunologically favorable battlefield for CAR-T cells [57,85].

Recent advances in artificial intelligence (AI) and machine learning (ML) are increasingly being leveraged to optimize CAR-T-cell therapy design and clinical application. AI-driven algorithms can analyze high-dimensional omics datasets to identify optimal tumor-associated antigens with minimal off-target risks, enabling more precise target selection. Machine-learning models are also being used to predict CAR–antigen-binding affinities, optimize single-chain variable fragment (scFv) sequences, and model intracellular signaling domains for enhanced therapeutic efficacy and persistence. Furthermore, AI-enabled image analysis and real-time patient monitoring tools can assist in early detection and management of cytokine release syndrome (CRS) and neurotoxicity, improving patient safety. By integrating AI/ML across target discovery, construct engineering, and clinical monitoring, CAR-T-cell therapies can be made more effective, personalized, and adaptable to tumor evolution—potentially overcoming key limitations such as antigen escape and heterogeneous tumor expression [82,83].

CAR-T-cell therapy is constrained by prohibitively high costs—often driven by individualized autologous manufacturing, GMP-grade facilities, and complex cold-chain logistics. Limited availability of specialized centers, intensive monitoring requirements for toxicity management, and long vein-to-vein times further restrict access, especially in low-resource settings. Emerging solutions such as decentralized point-of-care manufacturing, automation, and off-the-shelf allogeneic CAR-T products aim to improve affordability and scalability [80,81].

In 1988, the first in-human TIL therapy was performed for the treatment of patients with metastatic melanoma; lymphocytes were extracted from resected melanoma, and after in vitro expansion, they were infused back into the melanoma patients, followed by interleukin 2 (IL-2) as an adjuvant therapy and cyclophosphamide as chemotherapy [86,87]. The results obtained were promising, with an OR rate of 55% and a median overall survival (OS) of approximately 11 months. The findings of this study demonstrated the potency of the TIL therapy in the elimination of tumor cells, along with an acceptable safety profile [88,89,90]. As of now, many researchers have explored the application of TIL therapies for the treatment of advanced melanoma (NCT06703398↗, NCT06120712↗, NCT05098184↗) [91], advance malignant solid tumors, pediatric solid tumors (NCT06047977↗) [92], advanced gynecological tumors (NCT05098171↗, NCT05468307↗, NCT04766320↗), R/R Gynecologic tumors (NCT04766320↗) [93], and non-small-cell lung cancer (NCT06473961↗, NCT06455917↗) [94].

NSCLC has shown the most potential for TIL therapy among cancer types. In a Phase I pilot study at Moffett Cancer Center, 20 patients with NSCLC had their tumors harvested for TIL generation before being exposed to PD-1. At least two months of nivolumab (four cycles of 240 mg every two weeks) was then administered to them while their TILs were being produced. Preliminary outcomes showed an ORR of 25%, a disease control rate (DCR) of 60%, and a median progression-free survival (PFS) of 5.8 months. This was the first TIL encounter in NSCLC that was recorded, suggesting that TIL treatment may result in notable and durable outcomes for NSCLC [95].

The NCI’s Phase II study of TIL therapy for cervical cancers included a group of 18 patients whose lesions tested positive for HPV 16 or HPV 18. The trial reported an ORR of 28%, including complete responses (CR) in two patients and partial responses (PR) in three, with a median duration of response (DoR) exceeding 15 months. TIL treatment was examined in C-145-04, a different Phase II multicenter clinical trial from Iovance, in patients with advanced cervical cancer that was ICI-naïve and had advanced following at least one previous line of chemotherapy. This study showed an ORR of 44%, with CR in 11%, and a median OS of approximately 12 months. In order to accelerate the development of TIL therapy, the FDA granted breakthrough therapy designation status based on these findings [96]. The stepwise process of tumor-infiltrating lymphocyte (TIL) therapy production is illustrated in Figure 5.

TIL treatment is unlikely to target breast cancer. A total of 42 breast cancer patients who had not responded to previous treatments were involved in Phase II research undertaken by the NCI; the clinical benefit was limited, with an ORR below 5%, and a median progression-free survival (PFS) of 3 months. All patients’ tumor harvests were used to create TIL cultures, which were subsequently further chosen based on their reactivity to distinct neoantigens found in the patients’ tumors. Enhancing TIL for neoantigen-reactive populations to boost potency is of great interest [97].

There is a significant comparison between CAR-T-cell therapy and TIL therapy. TIL therapy is, basically, more favorable for the treatment of solid tumors that have more mutational burdens, like melanoma or certain types of lung cancer, where native immune responses exist but require amplification [98]. On the other hand, CAR-T-cell therapy is more effective and widely used in hematological malignancies with known and consistent antigen targets. The decision to use one approach over the other depends on multiple factors, including the tumor type, antigen expression, patient-specific immune profiles, and resource availability [99]. While CAR-T-cell therapy continues to dominate the hematologic cancer space, advances in TIL therapy are helping extend cellular immunotherapy to a broader range of solid tumors. Table 4 outlines the key ongoing clinical trials investigating CAR-T cell and T-cell therapies.

Although tumor cells express TAAs, at least some healthy tissues also do. Treatments that target TAAs must therefore deal with the possibility of T-cell-mediated on-target off-tumor toxicity. Both normal cells of the same tissue origin and malignant cells display differentiation antigens. Melanoma-differentiation antigens, which include the extensively researched melanoma-associated antigen recognized by T lymphocytes (MART-1) [113] and glycoprotein 100 (gp100) [114], were among the first tumor antigens to be identified. Cancer cells express overexpressed antigens in high quantities, whereas healthy cells express them at low levels. Wilms’ Tumor Antigen 1 (WT1), a transcription factor that is produced 10–1000 times more frequently in leukemic cells than in normal cells, is an example of an extensively researched overexpressed antigen [115,116].

Since CGAs are only expressed in germline cells, such as testis cells, which do not express HLA-class I, they significantly lower the chance of on-target off-tumor damage. In contrast, CGAs are aberrantly expressed in cancer cells. NY-ESO-1 and MAGE-A4, which are found to be expressed at high levels in a variety of solid and hematological malignancies, are examples of CGAs with high clinical significance [117].

TSAs are not found in the genomes of any normal cells, but they are genetically encoded in cancer cells. TSAs can also be categorized as neoantigens or viral antigens. Since the expression of viral oncogenes is almost nonexistent in normal cells and frequently homogenously expressed in virus-driven malignancies, they make very appealing targets for tumor antigens. Clinically significant viral antigens include EBV viral oncogenes LMP1 and LMP2, which are expressed in several solid and hematological malignancies [117,118], and the HPV viral oncogenes E6 and E7, which are expressed in several forms of epithelial carcinoma [106].

Immunopeptidomics, the bioinformatics analysis of transcriptomic and proteomic databases, and in vitro or ex vivo tests are used to assess TCR-T-cell-related on-target off-tumor toxicity and ascertain whether TCR-T cells are able to recognize normal cells or tissues. TCR recognition of an antigen other than the target antigen on normal cells is associated with off-target off-tumor toxicities or cross-reactivity. Clinical experiments employing affinity-enhanced TCR-T cells have documented cross-reactivities [119].

The main concern is whether TCR-T cells acquire secondary resistance mechanisms. By activating immune checkpoint receptors, immune checkpoint ligands on tumor cell surfaces may be overexpressed, which could impair transplanted T-cell proliferation and function and ultimately cause fatigue. The main mechanism that causes tumor cells to evade TCR-T-cell therapy is by losing or reducing MHC class I molecules, which prevent TCR-T cells from identifying the target epitope. A relapse occurred six months following TCR-T-cell therapy in a clinical study that targeted P53 mutations in a patient with breast cancer. The tumor cells displayed LOH of chromosome 6, with the HLA-A*02:01 locus and expressed intact P53 [119].

The three primary components of the tumor microenvironment are immunosuppression, persistent inflammation, and hypoxia. Although CD8+ T lymphocytes are essential for the removal of tumors, they become weaker and eventually fatigued when exposed to hypoxic environments. Recent findings have revealed a paradox: as this essential component runs out, it changes, increasing the expression of CD39 to create an immunosuppressive environment that weakens other T cells’ potent anticancer properties. Programmed death ligand-1 (PD-L1) is one of many immunosuppressive signaling proteins present in the majority of tumor stromal cells in the TME. Immune cell malfunction and apoptosis may arise from the interaction of PD-L1 with T-cell-expressed programmed cell death protein-1 (PD-1). In necrotic tumor microenvironment regions, hypoxia causes an acidic environment and increased potassium levels, which upset potassium homeostasis in tumor-infiltrating lymphocytes (TILs). This significantly reduces T-cell effector function, which affects the ability and activity of cytokine release [103].

TCR-engineered T-cell therapy faces similar barriers, with high production costs from patient-specific cell isolation, genetic modification, and expansion under GMP conditions. Additional complexity arises from mandatory HLA typing and antigen validation, limiting timely access in aggressive disease. In low- and middle-income regions, there is inadequate infrastructure, and trained personnel exacerbate inequities, highlighting the need for streamlined manufacturing, shared international production hubs, and cost-efficient vector generation strategies [101,102].

Deep-learning platforms such as NeoaPred have achieved >80% accuracy in predicting peptide–HLA binding and immunogenicity, streamlining the identification of viable TCR targets from vast genomic datasets.

ML-based structural prediction tools are enhancing in silico screening of TCRs against predicted epitopes, enabling the early detection of potential off-target cross-reactivity and improving therapeutic safety.

By integrating patient-specific tumor sequencing data with AI algorithms, novel neoantigen targets can be identified and prioritized, particularly for rare HLA alleles, where traditional databases are sparse [99].

These computational advancements shorten the discovery pipeline, improve the precision of TCR design, and lay the foundation for next-generation TCR therapies that are more personalized, effective, and safe.