The extracellular domain of a CAR is constructed by combining the variable regions of heavy (VH) and light chain (VL) of antibodies (Ab) through a flexible linker, forming a single-chain variable fragment (scFv) (40, 41). Since CAR is composed of the antibody’s variable portion, it retains the antigen-binding properties and binds specifically to a target antigen. Notably, the scFv can recognize and bind to an antigen in a major histocompatibility complex (MHC)-independent manner, leading to the activation of CAR-T cells and the induction of potent antitumor activity (41). Extracellular domain determines the CAR’s affinity for the target tumor-associated antigen; studies have shown that a higher affinity of the scFv for the target antigen can reduce on-tumor off-target effects. However, excessively higher affinity may compromise the tumor penetration ability and trigger unwanted toxicity (42). Thus, while designing a CAR, careful consideration of the affinity of the extracellular domain toward the target tumor antigen is essential to optimize its therapeutic efficacy while minimizing potential adverse effects.

The hinge domain is part of an extracellular region that links the transmembrane domain and the extracellular antigen-binding domain in a CAR. This region is usually derived from CD8, CD28, IgG1, or IgG4 (43, 44) and provides flexibility and helps to overcome steric hindrance, which is required to enhance the binding affinity of scFv with the target antigen (40, 43–45).

The extracellular region derived from IgG1, of the CAR bears a resemblance to the Fc region of antibodies. Consequently, immune cells with Fc gamma receptors (FcγR) can bind to the CAR’s extracellular region through Fc-FcγR interaction (46). This interaction has been observed to accelerate the aging process of CAR-T cells, resulting in a negative impact on CAR-T cell persistence and function (46). To mitigate this Fc-FcγR interaction, increasing the flexibility of the hinge domain becomes crucial. Therefore, when designing a CAR, careful consideration of the hinge region’s flexibility is essential to optimize CAR-T cell performance and longevity.

The transmembrane domain facilitates the connection between the intracellular and the extracellular domain through the hinge domain and transmembrane region is usually derived from proteins such as CD3ζ, CD28, CD4 and CD8α (41). CAR-T cells containing transmembrane domains derived from each of these proteins are known to release different levels and types of cytokines. Leah et al. (2017) evaluated the CD8α and CD28 transmembrane and hinge domain containing CAR-T cells, and they found that CAR-T cells containing CD8α released fewer cytokines (such as IFN-γ and TNF) as compared to CD28 containing CAR-T cells. They also found that the CAR containing transmembrane domain derived from CD8α showed lower activation induced cell death (AICD) as compared to a CAR containing the same regions derived from CD28 (47). Therefore, the choice of transmembrane domain plays an important role in determining the persistence of CAR-T cells.

The intracellular signaling domain is composed of the activation and costimulatory domains. The intracellular domain tries to replicate the typical sequence of events that triggers T-cell activation. The activation domain of CAR is usually derived from CD3ζ, which can activate CAR-T cells to some extent, but full activation requires extra costimulatory domains (48). These costimulatory domains are usually derived from CD28 or 4-1BB(CD137) (41, 43). CAR-T cells having either of these domains will have different distinct functions, CAR-T cells containing CD28 will differentiate into effector memory cells, while CAR-T cells containing 4-1BB (CD137) will differentiate into central memory T-cells (49). In general, upon binding with the target antigen, CAR-T cells containing activation and costimulatory domain produce IL-2, which helps in its proliferation and persistence (43, 50).

The first-generation CAR-T had a single intracellular signaling domain, such as CD3ζ or FcϵRIγ, but lacked costimulatory domains, such as CD27, CD28, CD134, and CD137 (55). These intracellular domains were responsible for transmitting signals from T-cell receptors, but CAR-T cells containing only intracellular domains had some limitations. The primary limitations were low cytotoxicity and proliferative responses. Thus, to overcome this limitation, exogenous IL-2 had to be supplemented to enhance their proliferative and cytotoxic capabilities. However, even after exogenous administration of IL-2, CAR-T showed limited proliferation and cytotoxicity, necessitating the development of second-generation CAR-T with additional costimulatory domains (55, 56).

The development of second-generation CAR-T cells aimed to overcome the limitations of first-generation, such as insufficient cytokine production such as IL-2 and a short lifespan. These challenges were effectively addressed by incorporating a dual signaling domain that promotes robust T-cell proliferation and differentiation into effector cells.

This achievement involved two types of signals. The first signal occurs when a foreign peptide binds to an MHC protein on the surface of an antigen-presenting cell, triggering recognition by a T-cell receptor and downstream signaling through CD3ζ. The second signal is initiated by the interaction between the CD28 co-receptor protein on the T-cell and the CD80/86 costimulatory protein on the antigen-presenting cell (57). Together, these signal 1 and signal 2 events stimulate T-cells to release IL2, which, in an autocrine manner, promotes their proliferation and differentiation (56–59).

Notably, a CAR with only CD3ζ is insufficient for full T-cell activation; the inclusion of a costimulatory domain is also required. Therefore, second-generation CARs incorporated CD28, 4-1BB, or OX-40 costimulatory domains. This design has been associated with improved T-cell proliferation, enhanced cytotoxicity, sustained response, and increased survivability of CAR-T cells (56, 60).

Third-generation CAR-T cells were developed by incorporating multiple costimulatory signaling domains along with the endodomain. Commonly utilized configurations included CD3ζ-CD28-OX40 or CD3ζ-CD28-41BB (61, 62). In a pilot clinical trial conducted by Brain et al. (2012) to assess the effectiveness of third-generation CAR-T cells containing CD28 and 4-1BB costimulatory domains, the study revealed improved persistence, proliferation, and safety (63). However, there were no significant enhancements in efficacy observed.

The development of fourth-generation CAR-T cells aimed to enhance the antitumor response of CAR-T by delivering transgenic products to the tumor site, thereby recruiting other immune cells (64). This advancement involved re-engineering third-generation CAR-T cells by incorporating an additional nuclear factor of the activated T-cell (NFAT) responsive cassette (65). This cassette allowed for the inducible expression of a transgenic cytokine, such as IL12. Commonly referred to as T-cell redirected for universal cytokine-mediated killing (TRUCK), the fourth-generation CAR-T exhibited improved activation and persistence (65).

To address challenges associated with fourth-generation CAR-T cells, the fifth-generation CAR-T cells were developed. Along with conventional CD3ζ domain fifth generation CAR-T has additional IL-2Rβ and STAT3 domain (46, 62, 66). These supplementary domains provide fifth-generation CAR-T cells with enhanced T-cell activation, proliferation, and improved properties for infiltrating solid tumors. Notably, unlike conventional monovalent CAR-T cells, fifth-generation CAR-T cells are designed to be multivalent, enabling them to recognize a broader range of tumor-associated antigens (62).

In the extrinsic method, the PD-1 blocking agents are administered separately before or after the CAR-T cell treatment. Currently, at least three such anti PD-1 monoclonal antibodies (mAb) are approved by FDA. Namely, Nivolumab (Opdivo), Pembrolizumab (Keytruda), and Cemiplimab (Libtayo), several anti PD-1 agents are under trial (118). PD-1 mAbs tend to promote the secretion of IL-2 and IFN-γ (119, 120). IL-2 acts as a T-cell growth factor; thus, it was amongst the first immunotherapeutic drugs to get FDA approval nearly three decades ago for cancer treatment (121). IFN-γ has shown to reduce the immune suppression that occurs at times and reduce tumor burden (122). Thus, combining these anti PD-1 agents with CAR-T cells can significantly increase the persistence and anti-tumor effect of CAR-T cells. In an in vivo study by John, L.B. et al. (2013) in an HER-2+ mice model, CAR-T cells combined with an anti PD-1 antibody were administered. PD-1 antibody significantly increased the level of IFN-γ, granzyme-B, and a proliferation marker of T-cell that is Ki-67; the CAR-T cell combined with anti PD-1 showed significantly higher anti-tumor response compared to only CAR-T cells and isotype control antibody (123). However, the optimal doses and frequencies of administration of anti PD-1 may not be generalized as different targets for immunotherapy and different agents show different dose response characteristics (124). Thus, in the case of cell extrinsic combination therapy, the doses of PD-1 blocking agents vary based on tumor type and grading, and there may be a need for repeated administration of anti PD-1 antibodies at a regular interval. For example, in a phase I study, Adusumilli et al. (2021) used Pembrolizumab as an extrinsic PD-1 blockade along with mesothelin-specific CAR-T cells in a group of 16 patients with malignant pleural mesothelioma (MPM). They observed a higher persistence of CAR-T cells in the combination group than in the group that received standalone CAR-T cell therapy. They have administered multiple Pembrolizumab doses, which varied from 1 to as high as 30 doses (125). Although extrinsic combination therapy shows promising results, there are some drawbacks associated with it. Kuah et al. (2023) suggested optimizing the doses of anti PD-1 drugs as current administration regimens may result in overtreatment with potentially important implications for cost, quality of life, and toxicity (124).

In the cell intrinsic method of combination therapy, the CAR-T cells are genetically engineered to have anti PD-1 activity. One of the methods to create cell intrinsic CAR-T cells is by knocking out genes that are responsible for PD-1 expression, such as PDCD1 on CAR-T cells using various genome editing tools such as CRISPR-Cas9 or TALEN and then the recombinant DNA is transduced using viral vectors (126) or electroporation (127) into the autologous T-cell. PD-1/PDL-1 axis is responsible for the modulation of immune tolerance (128). Genetic disruption of PD-1 may pose a chance of potential autoimmune response and higher chances of immune related adverse events. Wang, et al. (2021) studied the efficacy of intrinsic CAR-T cells by knocking out PDCD-1. To reduce the chances of GVHD, they also knocked out the T-cell receptor alpha constant (TRAC) using CRISPR-Cas9 and produced MSLN-directed 28ζ CAR-T cells with PD-1 and TCR disruption (MPTK-CAR-T cells). From the study it was found that using CRISPR-edited CAR-T cells with PD-1 disruption was viable and secure to be used.

Nonetheless, the enhanced endurance of CAR-T cells did not exhibit substantial improvement, even when combined with the natural T-cell receptor and lymphodepletion. They suggested that TCR may play a role in CAR-T cell activity (126). In an in vitro study by Guo, X. et al. (2018), they demonstrated that PD-1 disrupted GPC3-CAR-T cells showed stronger anti-tumor immune response compared to wild-type GPC3-CAR-T cells against PD-L1 Positive HCC cells; additionally, the disruption of PD-1 protected the CAR-T cells from exhaustion compared to wild type CAR-T cells while co-culturing (129). Another possible method of intrinsic combination is generating anti PD-1 CAR-T cells by inserting sequences that encode and secrete full-length antibodies or scFv against the PD-1 receptor (130). In a study by Fang et al. (2021) they modified autologous T-cells that contained sequences encoding scFv specific for MSLN and full-length antibody for PD-1, administered it along with small daily doses of Aptinib against advanced ovarian serous adenocarcinoma at stage IIIc and observed shrinkage in metastatic nodules, patient had progression-free survival (PFS) for five months and survived for 17 months. They found the combination therapy to be a feasible and promising treatment for advanced refractory ovarian cancer (130). Recently a novel method of cell intrinsic combination therapy is being studied that is expression of dominant negative receptor (DNR). The dominant negative receptor (DNR) has an extracellular domain of PD-1 but lacks the intracellular signaling domain of the PD-1 receptor; it acts like a decoy receptor. PD-1 DNR are prepared by inserting a sequence by viral transduction that lacks the signaling domain. CAR-T cells with PD-1 DNR show statistically significant higher proliferation rates and enhanced cytotoxicity (115). Figure 4A shows standalone CAR-T cell therapy Figure 4B shows the mechanism of cell extrinsic combination therapy and Figure 4C shows various types of cell intrinsic combination therapy of CAR-T cell and anti PD-1 agents.

Both types of combination therapy demonstrate encouraging efficacy, making it challenging to determine a definitive superior option. In the case of cell extrinsic combination therapy, as the anti PD-1 drugs are FDA approved, this can simplify logistics. Also, the efficacy of cell extrinsic combination therapy is encouraging. Despite this, there is a need for repeated administration of anti PD-1 drugs, and the doses and frequencies of doses still need to be generalized. In the cell intrinsic model, though, there is no need for separate dosing. However, a study found that the complete absence of PD-1 may produce terminally differentiated T-cell and can cause exhaustion of CD-8+ T-cell (131). CRISPR-Cas9 can also cause off-target mutations in Modified T-cell (132). In our opinion, using both intrinsic and extrinsic combination therapy simultaneously can show promising results, but further research and individual patient assessment are necessary to make informed treatment decisions.

Mesothelin, a glycoprotein with a molecular weight of 40 kDa, is encoded by the MSLN gene (141). Its expression in normal cells is considerably lower compared to specific cancer cells such as pancreatic cancer, mesothelioma, ovarian and extra bile duct tumors, lung adenocarcinomas, and stomach cancers (27) This differential mesothelin expression in healthy tissues makes it an attractive target for cancer immunotherapy specific to these tumors (142). Moreover, trace amounts of mesothelin can be detected in the bloodstream of patients with mesothelin-positive cancers, making it a valuable tool for diagnosis and monitoring (143). In mesothelin-positive patients, soluble mesothelin-related peptide (SMRP), a 42–44 kDa protein, is also detected (144).

Adusumilli et al. (2021) conducted a phase I trial combining Pembrolizumab, an anti PD-1 agent, with regional mesothelin-targeted CAR-T cell therapy against patients with malignant pleural disease, including metastatic breast, lung, and malignant pleural mesothelioma (MPM) (145). In this study involving twenty-seven patients from the US, out of twenty-seven patients, twenty four had received cyclophosphamide earlier. They administered CAR-T cells intrapleurally at a dose of 0.3M - 60M CAR-T cells/kg body weight to all the patients. A significant median overall survival of 23.9 months was observed, with an average one-year survival rate of 83%. This combination therapy showed better results than the FDA-approved combination of nivolumab and ipilimumab. The combination immunotherapy also enhanced the persistence and function of CAR-T cells in peripheral blood and tumor tissue and induced new IgG responses (>3.5 fold than baseline) against tumor antigens. Sixteen patients of combination immunotherapy had measurable disease by mRECIST. The radiologic evaluation of these patients revealed partial response (PR) in two out of sixteen, stable disease (SD) in eight out of sixteen, and partial disease (PD) in five out of sixteen. The SD or better was sustained for more than six months in 8 patients; they were functionally well and did not require the subsequent treatment for a prolonged duration. They found this combination therapy safe, feasible, and effective. The study also noted reductions in serum SMRP levels post-treatment, enhanced CAR-T cell persistence and function, and induced IgG responses against tumor antigens (125).

In another phase I study led by Wang et al. (2021), PD-1 and TCR gene-disrupted CAR-T cells were assessed for their effectiveness against various solid tumors. The study included fifteen patients who underwent mesothelin-directed CAR-T cell therapy. Specifically, a mesothelin-directed 28ζ CAR-T cell was chosen, and its PDCD-1 gene and TRAC gene were knocked out using CRISPR Cas9 genome editing tools. Among the fifteen patients, six had pancreatic cancer, three had biliary tract cancer, and one each had gastric cancer, tubal cancer, esophageal cancer, ovarian cancer, cervical cancer, and triple-negative breast cancer. The treatment was found to be safe, with no observed neurotoxicity or autoimmune reactions. However, out of the fifteen patients, only seven exhibited stable disease (SD) three to four weeks after the infusion. Unfortunately, this response was sustained in only two patients until the subsequent follow-up after eight to twelve weeks. The median overall survival for all fifteen patients was 3.0 months, while for the seven patients showing stable disease at weeks 3–4 post-infusion, it was 4.9 months. The study attributed the relatively poor outcome to the disruption of the TCR, which had a negative impact on CAR-T cell proliferation (126).

Furthermore, Fang J et al. presented a case study involving a 54-year-old female diagnosed with advanced ovarian serous adenocarcinoma. The immunohistochemical staining revealed increased mesothelin expression in tumor cells (MSLN +++84%). To address advanced ovarian serous adenocarcinoma, T-cells were co-cultured with mesothelin antigen, anti-CD28, and recombinant human IL-2 to prepare αPD-1-meso CAR-T cells. These cells were then administered to the patient via intravenous drip on day 0 and day 26. Additionally, the patient received daily aptinib at a dose of 250 mg, known for its antiangiogenic properties and ability to enhance the efficacy of immune checkpoint inhibitors (ICI).

Flow cytometry analysis showed no change in total leukocyte count but a shift in lymphocyte count. Specifically, CD8+ T-cell count increased to 522 and 612 cells/µl at months one and three, respectively, compared to 352 cells/µl before CAR-T therapy. Conversely, CD4+ T-cell count decreased to 512 and 522 cells/µl at months one and three, respectively, compared to 800 cells/µl before CAR-T therapy. These findings suggested that αPD-1-meso CAR-T cells contributed to the activation of CD8+ T-cells. Notably, one tumor nodule decreased in size from 51.9 mm to 39.1 mm, while another nodule became undetectable. Based on Response Evaluation Criteria in Solid Tumors V.1.1, the patient exhibited partial response (PR) and showed shrinkage of nodules and progression-free survival for five months, demonstrating the effectiveness and feasibility of this immunotherapy against mesothelin-positive refractory breast cancer (130).

GD-2, also referred to as disialoganglioside GD2, is a type of glycosphingolipid found abundantly on the surfaces of tumor cells but with limited expression in normal cells. This characteristic makes it an appealing target for cancer immunotherapy (28), particularly in the treatment of neuroblastoma (NB), a prevalent extracranial solid tumor in children (146). Monoclonal antibodies designed to target GD2 have become part of standard care for high-risk neuroblastoma patients, despite the significant toxicities they may cause (135). As an alternative approach, researchers are exploring GD2-targeted CAR-T cell therapy.

Heczey et al. (2017) conducted a Phase I trial involving eleven patients with relapsed or refractory neuroblastoma, with a median age of 6.5 years. All patients received third-generation GD2-CAR-T cells incorporating CD28 and OX40 costimulatory endodomains. Ten out of eleven patients had stage 4 (metastatic) NB, while one had stage 3 NB located in the pelvis region; all patients had active disease at the time of CAR-T infusions. The patients were divided into three cohorts for comparison.

In Cohort 1, four patients received only GD-2 CAR3-T. Cohort 2 comprised four patients who received GD-2 CAR3-T along with cyclophosphamide and fludarabine (Cy/Flu) preconditioning to enhance CAR-T expansion and persistence. In Cohort 3, three patients received both Cy/Flu and GD-2 CAR3-T, along with pembrolizumab, an anti PD-1 agent. The doses of GD2-CAR3-T Cells were calculated based on body surface area according to the INSS International Neuroblastoma Staging System. Cyclophosphamide was administered at 500 mg/m2/dose on days -4, −3, and −2, and fludarabine at 30 mg/m2/dose on days −4 and −3 intravenously, while pembrolizumab was given at 2 mg/kg/dose intravenously on the -1st and 21st day of CAR-T administration. Among the eleven patients, only one patient in Cohort 1 experienced cytokine release syndrome, which did not exceed grade 2.

While there was no significant difference observed between Cy/Flu, GD-2 CAR3-T patients and anti PD-1, Cy/Flu, GD-2 CAR3-T patients in terms of T-cell persistence and expansion, the best response was noted in Cohort 3. In this cohort, which involved anti PD-1 administration, two out of three patients exhibited complete response, while one showed partial disease response. In Cohort 1, out of four patients, two had stable disease, and two showed progressive disease. In Cohort 2, out of four patients, three had partial disease, and only one showed stable disease. These findings suggest that combining checkpoint inhibitors with CAR-T cells might result in improved patient responses, although further studies with larger patient cohorts are warranted to validate these observations (135).

CD-19, a protein weighing 95 kDa, is part of the immunoglobulin superfamily and is primarily located on the surface of B lymphocytes. It acts as a co-receptor for the B cell antigen receptor (BCR) and plays a crucial role in B-cell signaling by enhancing cell proliferation, mitogen-activated protein kinase activity, and calcium release synergistically in B-cells when co-ligated with the BCR (147).B-cell lymphoma, cancer affecting lymphocytes, can present as a solid tumor in lymph nodes or other tissues, involving the bone marrow and circulating in the blood (29).

In a phase Ib study by Liu et al. (2021), six PD-L1 positive B-cell R/R DLBCL lymphoma patients were treated with CD19-PD-1/CD28-CAR-T cells derived from autologous blood via leukapheresis (148). Among these patients, three achieved complete response (CR), one had stable disease, while two succumbed to progressive disease by day 60. Of the three patients with CR, two maintained remissions up to the cutoff date, and one showed remission after 12 months. Although anti-CD19-CAR-T cell therapy showed promising results, the main contributor to CAR-T remission against DLBCL was found to be the expression of PD-L1 on tumor cells. Consequently, in subsequent research, authors incorporated a chimeric switch-receptor (CSR) where the truncated extracellular domain of PD-1 binds to cytoplasmic signaling domains of CD28, facilitating T-cell activation. When PD-L1 binds to the PD-1 of CSR, it activates downstream CD28 signaling (149).

This study involved seventeen Chinese patients with a median age of 55, including thirteen with DLBCL, two with mantle cell lymphoma (MCL), and two with transformed follicular lymphoma (tFL). All patients received chemotherapy as conditioning before administration of CD19-PD-1/CD28-CAR-T cells at doses ranging from 0.5 to 4.0 × 10^6 CAR-T cells/kg. During the initial four weeks, all patients experienced one or more adverse events (AE) of grade 3 or 4, with leukopenia (88.23%), thrombocytopenia (35.29%), anemia (23.53%), and pyrexia (23.53%) being the most frequent. Thirteen patients (88.24%) had moderate CRS symptoms, managed with supportive therapy. Seven patients (41.17%) experienced hypotension, and one patient (5.88%) had hypoxia, all treated with appropriate measures.

Despite patient 17’s unfortunate death due to lymphoma progression on day 30, without severe CRS or CAR-T related neurotoxicity, the study found no significant correlation between the degree of adverse events and PD-L1 tumor expression. Ten of seventeen patients (58.8%) achieved an objective response (CR or PR) at three months, with seven (41.2%) reaching CR, including one patient each with MCL and tFL. Among those with CR or PR at three months, 80% had progression-free survival (PFS) at eighteen months. While these findings are promising, further research is necessary to mitigate adverse events (136).

Wang et al. (2021) investigated a study involving five patients with relapsed/refractory (R/R) DLBCL, with a median age of 41 years, who had previously undergone CD19/20-CAR-T therapy but experienced disease progression afterward (126). In an effort to enhance efficacy, a combination therapy of CD19/20-CAR-T and PD-1 blockade therapy was administered to these patients. Participants received 200 mg of PD-1-blocking antibodies, such as sintilimab or camrelizumab, every two weeks until disease progression or intolerable toxicity occurred. The anti-tumor response was monitored using PET/CT imaging. Notably, no CRS or toxicities ≥ grade 3 were observed. Among the five patients, three achieved objective responses following PD-1 therapy, with two achieving complete response (CR) and one achieving partial response (PR). Unfortunately, the remaining two patients experienced disease progression and succumbed to their conditions between 15.6 and 16.2 months after treatment. Patient 4 maintained CR up to the cutoff date, which was 21.3 months post-treatment, with a progression-free survival (PFS) of 21.8 months. Additionally, one patient exhibited complete elimination of the baseline tumor, although a new lesion emerged ten months later. The study suggests that PD-1 blockade therapy may be effective for patients with relapsed or refractory DLBCL who did not respond adequately to CAR-T cell therapy and who exhibit high levels of PD-1 in T-cells infiltrated by tumors (126).

A case report by Zhang et al. (2022) examined a 38-year-old female patient with relapsed DLBCL of the CNS and TP53 mutation. Initially diagnosed with stage IIIA GCB-DLBCL, the patient underwent chemotherapy alongside antiviral therapy due to a history of hepatitis B and detectable HBV DNA levels. Four months into chemotherapy, the patient experienced her first relapse, diagnosed as stage IVA GCB-DLBCL, prompting immediate chemotherapy. However, after two months, the disease relapsed again. Given the circumstances, the patient was enrolled for CD19 CAR-T therapy, receiving lymphodepleting therapy in addition to CAR-T therapy.

Following infusion, monitoring of CD-19 CAR-T cell DNA copy levels revealed counts of 4.6×10 copies/µg on day 0 and 5.65×10 copies/µg on day 7. On day 8, due to the patient’s symptoms, a Bruton Tyrosine Kinase (BTK) inhibitor, Zanubrutinib (160 mg), was administered alongside CAR-T therapy. Additionally, on day 15, the PD-1 inhibitor Tislelizumab (200 mg) was introduced to improve the patient’s condition. However, the patient experienced menorrhagia and severe anemia, necessitating an RBC transfusion.

One month later, further complications arose as the patient was diagnosed with stage II endometrial carcinoma. Subsequent radiation treatment for endometrial cancer was delayed due to the Covid-19 pandemic. Nonetheless, follow-up examinations indicated no recurrence of symptoms, and complete remission (CR) was achieved approximately 18 months following CD19 CAR-T cell treatment (137).

Mu et al. (2022) conducted a study involving forty-four patients with relapsed/refractory (R/R) DLBCL to assess the safety and efficacy of combining PD-1 inhibitors with anti-CD19 CAR-T cell therapy, followed by PD-1 inhibitor maintenance therapy in DLBCL patients with high tumor burden (138). These patients were divided into two groups: a combination group of twenty-six patients and a control group of eighteen patients. Lymphodepleting chemotherapy was administered to all patients, consisting of fludarabine (30 mg/m2/day) and cyclophosphamide (400 mg/m2/day) between days 4 and 2. On day 1, patients in the combination group received PD-1 inhibitors (Sintilimab, 200 mg). The anti-CD19 CAR-T cell infusion dose on day 0 for all forty-four patients with R/R DLBCL was 2 × 10^6 cells/kg. The efficacy of these therapies was evaluated at one- and two-months post-infusion.

In the combination group, eleven out of twenty-six patients achieved complete response (CR) (42.31%), while six achieved partial response (PR) (23.08%), resulting in an objective response rate (ORR) of 65.39%. In comparison, in the control group, seven patients (38.89%) achieved CR, four (22.22%) had PR, four (22.22%) had stable disease (SD), and three (16.67%) had progressive disease (PD). The ORR in the control group was 61.11%. The study suggests that combination therapy yields promising results; however, it may entail some side effects, particularly in the case of lymphomas (138).

CD-22 is a transmembrane protein weighing 140 kDa, primarily found in mature B-cell lineages, where it acts as a coreceptor of the B cell receptor (BCR) (30). The CD-22 gene, located on q13.1 of chromosome 19, spans around 22kb of DNA and consists of 13 exons (150).

Zou et al. (2023) reported a case involving a 49-year-old male patient with primary central nervous system lymphoma (PCNSL). Initially treated with a combination of 375 mg/m2 rituximab and 3.5 g/m2 methotrexate (MTX), the patient exhibited no improvement. Subsequent MRI scans revealed new lesions, prompting a shift to cytarabine (2 g/m2 q12h on days 1 and 2), with no significant response. Treatment was then transitioned to targeted therapies, including ibrutinib and lenalidomide, followed by three doses of the PD-1 inhibitor camrelizumab, yet still without improvement. Finally, the patient underwent CAR-T therapy.

Immunohistochemical staining confirmed the patient’s positivity for CD19/CD22. Peripheral blood mononuclear cells were collected and utilized to prepare second-generation CAR-T cells. Before CAR-T administration, the patient underwent chemotherapy with the DCF regimen (consisting of intravenous administration of 100 mg/m2 Decitabine for three consecutive days, 300 mg/m2 cyclophosphamide on days 1 to 3, and 30 mg/m2 fludarabine on days 1 to 3) to reduce lymphocytes. Subsequently, CD19 and CD22-specific CAR-T cells were infused at a total dose of 1 million CAR-T cells per kilogram of the patient’s body weight (1×10^7/kg), with three dose escalations of 10%, 30%, and 60% post-infusion. The patient experienced grade 2 cytokine release syndrome, which was managed without posing any imminent threat. One month post-CAR-T infusion, consistent levels of CAR-T cells ranging from 987 to 2,340,000 copies per microgram of DNA were observed in the patient’s peripheral blood. An MRI scan at this juncture revealed complete remission (CR). To prevent relapse, the patient received 200 mg of camrelizumab (a PD-1 inhibitor) every three weeks, along with daily administration of 560 mg of ibrutinib. Thankfully, the patient remains alive, demonstrating the efficacy and potential of CAR-T therapy in such cases (139).

CD133, a transmembrane glycoprotein weighing 97 kDa, is encoded by the prominin 1 (PROM1) gene located on chromosome 4 in humans (31). It has been identified as a marker for cancer stem cell (CSC) populations across various solid tumor types, including brain cancer (151), prostate cancer (152), colon cancer (153), lung cancer (154), hepatocellular carcinoma (HCC) (155), and ovarian cancer (156, 157).

Hepatocellular carcinoma (HCC) stands as the most prevalent form of liver cancer and ranks as the third leading cause of cancer-related mortality globally (2). Current therapies for advanced HCC, such as sorafenib, regorafenib, and lenvatinib, offer only modest improvements in overall survival (158). Notably, recently licensed PD-1 inhibitors such as pembrolizumab and nivolumab have exhibited inadequate response rates, underscoring the pressing need for more effective treatment options. CD133, associated with HCC stem cell-like cells, is linked to advanced tumors and poor prognosis, rendering it an appealing target for therapy. Chimeric antigen receptor-specific T (CAR-T) cells specifically targeting CD133-positive CSCs have emerged as a promising treatment modality for advanced HCC, offering potential advancements in the therapeutic landscape for this challenging disease.

Yang et al. (2023) conducted a preliminary study to assess the feasibility of utilizing a CD133-specific CAR-T cell system to deliver locally PD-1 blocking scFv as a monotherapy for advanced hepatocellular carcinoma (HCC). The study included 67 HCC patient samples, categorized into stages, and twenty-six cases of stage IV lung metastasis. Higher CD133 expression was detected in nineteen out of sixty-seven primary HCC tissues, correlating significantly with portal vein invasion and AFP levels. However, there was no statistically significant relationship between CD133 expression and overall survival (OS) or progression-free survival (PFS) in the entire cohort. Notably, in late-stage (II and III) male patients, CD133 expression was significantly associated with poorer PFS and OS, consistent with findings from online TCGA data.

To evaluate the effectiveness of CD133 as a target, CAR-T cells were engineered with a custom plasmid containing an anti-CD133 scFv gene segment derived from HW350341.1, a c-Myc tag gene segment, a CD8TM gene segment, and a secretory PD-1 blocking scFv. These engineered CAR-T cells were then transduced using sleeping beauty-mediated transposition.

In vivo, experiments were conducted using NCG mice injected with Hep-3B-luc tumor cells to establish subcutaneous and in situ xenograft tumor models. Mice were treated with Mock T-cells, CD133 CAR-T cells alone, or PD-1 inhibitor-secreting CD133 CAR-T cells once tumors reached a size of 50–100 mm3. Intraperitoneal injections were administered, and tumor progression was monitored using bioluminescence imaging (BLI).

The study demonstrated that PD-1 inhibitor-secreting CD133 CAR-T cells significantly extended the survival of mice compared to CD133 CAR-T cells and Mock T-cells. Tumor BLI showed that PD-1 inhibitor-secreting CD133 CAR-T cells exhibited more pronounced antitumor activity compared to Mock T-cells and CD133 CAR-T cells alone. Additionally, a higher proportion of human T-cells was observed within the CD133 CAR-T tumor microenvironment and the PD-1 inhibitor-secreting CD133 CAR-T group compared to the Mock T group. Overall, these findings suggest that combining PD-1 blockade with CD133-specific CAR-T cell therapy holds promise as a feasible treatment approach for advanced HCC (159).

CD30 is a type 1 transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily 8 (TNFRSF), with a molecular weight ranging from 105 to 120 kDa. It is encoded by the CD30 gene located on chromosome 1p36.2-26.3. Initially identified on Hedgehog and Reed-Sternberg cells of Hodgkin lymphoma, it was termed Ki-1 (160–162). The high expression of CD30 is commonly observed in Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL) (163, 164). In addition to HL and ALCL, CD30 expression is also observed in a subset of diffuse large B cell lymphoma (DLBCL), albeit infrequently, as well as in follicular lymphomas (FL), primary cutaneous anaplastic lymphomas, and the lymphomatoid papulosis form of cutaneous T-cell lymphoma. Furthermore, certain non-hematological malignancies, such as embryonal carcinoma and specific seminomas, also express CD30. These diverse expressions of CD30 highlight its significance as a diagnostic and therapeutic target across various lymphoproliferative disorders and certain non-hematological malignancies (32, 165–167).

Sang et al. (2022) conducted phase II trials to assess the efficacy of combination therapy involving anti-CD30 directed CAR-T cells and anti PD-1 antibodies in cases of relapsed and refractory CD30+ lymphomas, including Hodgkin lymphoma (HL), gray zone lymphoma (GZL), and angioimmunoblastic T-cell lymphoma (AITL) of various grades. Three cohorts comprising a total of twelve patients were included in the study. In Cohort 1, patients received 10^6/kg CAR-T cells, while in Cohort 2, patients received 10^7/kg CAR-T cells. Cohort 3 patients received additional anti PD-1 antibodies starting 14 days after infusion of 10^7/kg CAR-T cells, administered every three weeks after that. The results showed that three out of four patients in Cohort 1 achieved partial response (PR), while two out of three patients in Cohort 2 achieved complete response (CR), with one patient achieving PR. In contrast, all patients in Cohort 3 showed a 100% objective response rate (ORR), with 80% achieving CR and exhibiting low toxicity levels. Furthermore, of the eleven patients who responded to CAR-T therapy, seven remained responsive until October 31, 2021, and four out of the six patients who achieved CR maintained their CR status. Notably, among HL patients receiving 10^7/kg CAR-T cells plus PD-1-blocking antibody, five out of six (83.3%) showed CR, while patients receiving 10^6/kg CAR-T cells (Cohort 1) did not achieve CR. In conclusion, the study demonstrated that CAR-T therapy and PD-1 blockade yielded better outcomes than CAR-T therapy alone, with a higher rate of complete responses observed, particularly in HL patients (140).