Chimeric antigen receptor (CAR) T-cell therapy represents a breakthrough immunotherapy by adoptive transfer of T cells genetically engineered to express a CAR, which is a synthetic receptor with binding specificity for tumor antigen in a major histocompatibility complex (MHC)-independent fashion. The original concept of CAR was proposed in 1989. The CAR molecule was designed to mimic the structure of T cell receptor (TCR), enabling similar antigen recognition strength but without MHC restriction. Accordingly, CAR T cells can overcome immune escape due to the down-regulation or mutation of MHC molecules in cancer cells.[2,3] CARs can recognize antigens derived from proteins, carbohydrates, or glycolipids; and targets such as CD19 and epidermal growth factor receptor are widely expressed in multiple tumor types. Therefore, CAR T therapy can be universally applied for the treatment of various cancers.
The first-generation CAR with the CD3ζ signaling domain only was limited by inadequate cell expansion and poor anti-tumor efficacy in vivo.[4,5] In light of the essential role of co-stimulation in T cell activation, a co-stimulatory domain was integrated upstream of the CD3ζ signaling domain in the second-generation CAR, which greatly improves CAR T cell proliferation and persistence.[6-9] As a result, the second-generation CAR T therapy has been widely used and has shown remarkable clinical efficacies, especially in treating hematological malignancies.[10-15] At present, the Food and Drug Administration (FDA) has approved six CAR T cell products in the United States, and the National Medical Products Administration has approved two in China.
Despite the substantial responses observed early after CAR T cell infusion, nearly half of the patients who achieve complete response (CR) will relapse within 6 months.[16-18] The low CAR T cell persistence and loss of target antigen may account for the lack of durable disease control in these patients.[19-21] In addition, many patients show primary resistance to CAR T therapy, likely owing to CAR T cell intrinsic defects or immunosuppressive tumor microenvironment.[22-26] Many studies have explored different strategies to address these problems. In this review, we will summarize the current status and major progress in CAR T therapy, describe crucial factors related to CAR T resistance, such as CAR T cell exhaustion and antigen escape, and discuss the potential optimization strategies to enhance the efficacy of CAR T therapy [Figure 1].
Overview of CAR T Therapy in Hematological Malignancies
Recently, CAR T therapy has shown significant efficacy in relapsed and refractory (R/R) B cell non-Hodgkin's lymphoma (B-NHL) patients and has gradually become the officially admitted protocol for the rescue treatment of R/R patients. CD19 expression is restricted to the surface of B cells throughout the development,[27,28] and B cell aplasia can be tolerable, supporting CD19 as an ideal target of CAR T therapy in B cell lymphoma and leukemia. The first clinical trial of CD19-targeting CAR T (CAR T-19) therapy in B-NHL dates back to 2011, when a group from M.D. Anderson Cancer Center reported that two patients achieved partial remission and four patients achieved stable disease (SD) after CAR T-19 cell infusion. In Phase II clinical trial of ZUMA-1, 101 patients with B-NHL received CAR T-19 treatment, and the best overall response rate (ORR) was 83%, with CR at 58%. At a median follow-up of 27.1 months, the median overall survival (OS) was not reached, the median progression-free survival (PFS) was 5.9 months, and median duration of response (DOR) was 11.1 months. In the JULIET trial, the best ORR was 52% including 40% CR in 115 patients. At a follow-up of 32.6 months, the median OS was 11.1 months and the median PFS was not reached for patients who had a complete remission.[14,30,31] In the TRANSCEND trial, 256 patients were enrolled and the best ORR was 73%, with a CR rate of 53%. With 12 months of follow-up, the median OS and PFS were 21.1 and 6.8 months, respectively.
CD20 and CD22 are also potential targets for B-NHL CAR T therapy. In the first CAR T-20 clinical trial, the ORR was 81.8% in diffuse large B-cell lymphoma (DLBCL) patients. Subsequently, 17 patients with R/R B-NHL were treated with CAR T-20, 54.5% of patients achieved CR without severe toxicities. The median PFS was 10 months and 2-year PFS was 41.7%. Additionally, anti-CD22 CAR T therapy resulted in high efficiency in patients with R/R acute lymphoblastic leukemia (ALL) and DLBCL.[35-38]
The success rate of CAR T cell therapy for B-ALL was demonstrated in a recent meta-analysis, in which patients with R/R B-ALL achieved a CR rate of 80% after anti-CD19 CAR T cell therapy. In subgroup analyses, 195 (74%) of 263 adult patients and 242 (70%) of 346 pediatric patients achieved CR. More than 30% of patients had PFS of ≥1 year. As for side effects, 242 (28%) of 854 patients developed grade 3 or worse cytokine release syndrome (CRS) and 97 (18%) of 532 developed grade 3 or worse neurotoxicity. Central nervous system (CNS) involvement is a difficult-to-treat condition in ALL. A study suggested that patients with CNS-involved ALL responded well to CD19 CAR T therapy, with a CR rate of 91.7% and 6-month PFS rate of 81.8%. However, four of five patients with cerebrospinal fluid containing >5/μL blasts or solid mass before CAR T-cell expansion developed severe (grade 3–4) neurotoxicity.
Unlike B-NHL and ALL, acute myeloid leukemia (AML) has not achieved significant clinical responses following CAR T therapy, mainly due to the high heterogeneity of AML and the lack of ideal targets like CD19. Moreover, concomitant expression of AML targets in hematopoietic stem cells, even at low levels, increases the risk of bone marrow (BM) failure.[41,42] Early attempts of CAR T cells targeting LeY or CD33 only led to modest responses in a small number of AML patients.[43,44] A recent study summarized the results of 65 AML patients receiving CAR T therapy and reported a CR rate of 26%, comparable to those achieved by salvage chemotherapy and targeted therapy in R/R AML, ranging from 19% to 73%. Recently, C-type lectin-like molecule-1 (CLL-1)[46-48] and Siglec-6 have attracted more attention, mainly because these two antigens show little expression in hematopoietic stem cells. For CLL-1, a recent CAR T clinical trial has achieved promising success. Among 11 patients with AML, eight patients obtained CR, of which five were minimal residual disease (MRD) negative, after CAR T CLL-1 treatment.
Wang et al first reported a trial of anti-CD30 CAR T therapy for R/R Hodgkin's lymphoma (HL), 7 of 18 patients achieved PR and six achieved SD with a median PFS of 6 months. Based on these results, Ramos et al initiated a phase I/II trial to further test the efficacy of CAR T-30 therapy in R/R HL. A total 41 patients were enrolled and ORR was 72% with a CR of 59%. The 1-year PFS rate was 36% and 1-year OS rate was 94%.
In T cell ALL, the development of CAR T cell therapy has always been under investigation and still in its early stage. An allogeneic CAR was constructed utilizing a CD7-binding domain fused with an endoplasmic reticulum retention signal sequence lysine, aspartic acid, glutamic acid, leucine related sequence antibody (KDEL) that enables intracellular retention of CD7 molecules. With the allogeneic CD7-targeting CAR T therapy, a clinical trial enrolling 20 patients with R/R T-ALL showed a favorable response with a CR rate of 90%. Seven of 18 patients who achieved CR underwent allogeneic hematopoietic cell transplantation (allo-HCT). At a median follow-up of 6.3 months, 15 remained in remission. All patients experienced CRS, including 10% of grade 3 to 4, and there was no grade 3 to 4 graft-versus-host disease (GvHD). Avoiding fratricide is challenging for CAR T cell therapy to treat T cell malignancy. In a trial (NCT05377827), the surface CD7 of CAR T cells was knocked out to prevent fratricide, and the TCR alpha constant (TRAC) was also deleted to avoid GvHD caused by allogeneic CAR T cells. In another trial, an anti-CD7 protein expression blocker which can effectively downregulate surface CD7 expression, is used as another strategy to alleviate fratricide. Recently, naturally selected CD7 CAR T cells were manufactured with fratricide resistance, which was achieved by CAR-mediated CD7 epitope masking or by intracellular sequestration of the CD7 protein. In the first-in-human clinical trial, the naturally selected CD7 CAR T cells were well tolerated and effective against CD7 T cell malignancies.
Several B cell maturation antigen (BCMA)-targeting CAR T therapies have been evaluated in patients with R/R multiple myeloma (MM) in phase I/II clinical trials, and remarkable rates of CR, MRD negativity, and improved survival were demonstrated. Among them are idecabtagene vicleucel (ide-cel and bb2121) and ciltacabtagene autoleucel (cilta-cel, JNJ68284528, or LCAR-B38M), which have been approved recently by the US FDA. Ide-cel, reported in a phase II KarMMa trial, at a median follow-up of 13.3 months, led to the best ORR of 73%, CR or better of 33%, MRD-negative rate of 26% and median PFS of 8.8 months. CARTITUDE-1 trial showed an ORR of 97.9% with strict CR (sCR) 82.5% and the 27-month PFS and OS rates were 54.9% and 70.4%, respectively.[57,58] A 4-year follow-up study of Chinese cohorts, LEGEND-2, in which patients with fewer prior lines of treatment received LCAR-B38M, showed an ORR of 87.8% and a CR rate of 73.0%. The median PFS was 18.0 months and the OS was still not reached. The median DOR was 23.3 months.
Despite high response rates, up to 30% of patients with R/R lymphoma or myeloma did not respond well to CAR T therapy. In recent years, many studies have revealed various intrinsic resistance mechanisms of cancer cells against CAR T cell therapy, providing an important reference for developing new CAR T strategies [Table 1].
Table 1 -
CAR-T clinical trials in hematological malignancies.
||Number of patients
||ORR was 83%, with a CR at 58%; the median PFS was 5.9 months
||ORR was 52% including 40% CR
||ORR was 81.8%
||I and IIa
||54.5% CR; the median PFS was 10 months and 2-year PFS was 41.7%
||91.7% CR; 6-month PFS rate was 81.8%
||39% PR and 33% SD; the median PFS was 6 months
||ORR was 72%, with a CR of 59%; 1-year PFS rate was 36% and 1-year OS rate was 94%
||T-ALL and T-LBL
||95% CR in BM
||ORR was 73%, with a CR of 33%; the median PFS was 8.8 months
||ORR was 97.9%, with a CR of 82.5%; 27-month PFS and OS rates were 54.9% and 70.4%
||ORR was 87.8%, with a CR of 73.0%; the median PFS was 18.0 months
ALL: Acute lymphoblastic leukemia; AML: Acute myelocytic leukemia; B-ALL: B cell acute lymphoblastic leukemia; BCMA: B cell maturation antigen; BM: Bone marrow; B-NHL: B-cell non-Hodgkin's lymphoma; CAR T: Chimeric antigen receptor T-cell therapy; CLL: Chronic lymphocytic leukemia; CLL-1: C-type lectin-like molecule-1; CNS: Central nervous system; CR: Complete remission; CRi: Complete remission with incomplete recovery; DLBCL: Diffuse large B-cell lymphoma; HL: Hodgkin's lymphoma; LBCL: Large B cell lymphoma; MM: Multiple myeloma; NHL: Non-Hodgkin's lymphoma; ORR: Overall response rate; OS: Overall survival; PFS: Progression-free survival; PR: Partial response; SD: Stable disease; T-ALL: T-cell acute lymphoblastic leukemia; T-LBL: T-cell lymphoblastic lymphoma.
Dysfunction of CAR T Cells
Singh et al performed a loss-of-function screening in B-ALL and B-lymphoma cell lines and demonstrated that impaired death receptor signaling contributed to the dysfunction of CAR T cells, which was independently confirmed by Dufva et al. Recently, Yan et al revealed that NOXA (phorbol-12-myristate-13-acetate-induced protein-1 [pmaip-1]), a B-cell lymphoma 2 (BCL-2) family protein, was a pivotal regulator of resistance to CAR T therapy for B-cell malignancies. To overcome the intrinsic resistance, agents that upregulate death receptors, such as inhibitors against cyclooxygenase-2, histone deacetylase, proteasome, and BCL-2, could be used in combination with CAR T therapy.
In addition to the intracellular signaling network, abnormal expression or modification of molecules outside tumor cells also plays a role in the development of resistance. Among them is CD58, a ligand of CD2, and the CD58/CD2 axis is essential for T cell activation. About a quarter of B cell lymphomas had CD58 alteration, which was associated with poor response to CAR T therapy. Using an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas) 9 screen, Yan et al confirmed that loss of CD58 significantly impaired the interaction between tumor and CAR T cells and interfered with the function of CAR T cells. The re-establishment of CD2 activation domain could be genetically engineered to restore the CD58/CD2 signaling in favor of T cell activation. In addition, CD 19 hyperglycosylation, caused by loss of Golgi-resident intra-membrane protease signal peptide peptidase-like 3 in malignant B cells, could directly inhibit CAR T cell function. Since programed cell death-ligand 1 (PD-L1)/L2 is often expressed in some DLBCLs, anti-programed death 1 (PD-1)/PD-L1 therapy can potentially reverse their inhibitory effects on CAR T cells.
Loss of Antigen
About half of the patients would relapse within 6 months after getting CR, and loss of target antigen is a common mechanism.[64-66] For example, the loss of CD19 expression is mostly caused by gene mutation. It was estimated that CD19 loss accounts for 7% to 25% of B-ALL relapses, and ∼30% of DLBCL relapses. The CD19-negative relapse may be due to growth of pre-existing CD19 negative clones, or the secondary mutation or down-regulation of CD19 during CAR T treatment. The mechanisms of antigen-negative tumor clonogenesis vary among different antigens. In contrast to CD19, CD22 is more likely to be silenced by upstream signaling or epigenetic modification, rather than gene mutation.[66,68]
CAR T cells targeting multiple antigens have been developed to overcome antigen-negative relapses, such as administration of cocktail CAR T cells, sequential infusing different CAR T cells, and dual-targeting CAR T cells.[18,69] Among these, tandem CAR, targeting two different antigens simultaneously with one CAR molecule, may be a better option. Because tandem CAR does not require the simultaneous expression of two genes, it exhibits better anti-tumor function by improving the interaction with tumor cells. In 2020, Tong et al tested tandem CART-19/20 cells in R/R B-NHL patients, and a total of 28 patients were treated. Up to 71% patients achieved CR, and two patients achieved PR. In addition, the PFS rate at 12 months was as high as 64%. Later, Shah et al released their results of phase 1 clinical trial of tandem CAR T-19/20 therapy. The ORR (n = 12) was 100% in the high CAR T cells dose group with 11 CR and 1 PR. Tandem CAR targeting CD19/CD22 has also been tested for preventing relapse with CD19-negative disease. According to a report by Dai et al in 2020, six B-ALL patients received tandem CAR T-19/22 treatment and all achieved MRD-CR responses. One patient relapsed with CD19-negative and CD22-low blast cells 5 months after treatment. In 2021, Spiegel et al released their phase I trial results of tandem CAR T-19/22, MRD-CR responses were achieved in 88% of B-ALL patients (n = 17) and 29% of large B cell lymphoma (LBCL) patients (n = 21). CD19 negative/low relapses were observed in five patients with B-ALL and four patients with LBCL, and further study demonstrated that the CAR T-19/22 exhibited lower function to CD22 compared with CD19.
Different dual-targeting strategies, such as pooled co-infusion of CAR T cells or tandem CAR T cells, have been evaluated in clinical trials enrolling patients with R/R MM. A long-term follow-up of the phase II study demonstrates more durable responses in 62 patients receiving co-infusion of CD19- and BCMA-targeting CAR T cells. At a median follow-up of 21.3 months, the ORR was 92% with CR 60% and the median PFS was 18.3 months. The patients with a CR or better had a DOR up to 45.8 months, suggesting long-term disease control and possible survival plateau in heavily pretreated patients. Compared with co-infusion strategy, CAR T therapy using a single dual-targeting CAR has become more common in clinical trials. GC012F, a BCMA/CD19 dual-targeting CAR T product, was developed on the FasT CAR-T platform enabling 22–36 h manufacturing. A multicenter first-in-human study of GC012F enrolled 28 R/R MM patients, including 89.3% (25/28) high-risk MM, at a median 6.3-month follow-up, the ORR was 89.3% (25/28), and MRD negativity plus sCR was achieved in 75.0% (21/28) of patients. A phase I trial of BCMA/CD38 dual-targeting CAR-T therapy, in which a humanized CAR was constructed with tandem single chain antibody fragment (scFv) targeting both BCMA and CD38, recruited 23 R/R MM patients. At a median follow-up of 9.0 months, 87% (20/23) patients achieved a clinical response with MRD negativity, and the median PFS was 17.2 months. Of note, total and partial resolution of extra-medullary diseases were observed in 56% and 33% of patients, respectively. Other dual-targeting CAR T therapies for MM, such as BCMA/signaling lymphocytic activation molecule family (SLAMF) 7 and BCMA/G protein-coupled receptor, class C, group 5, member D (GPCR5D), are also being tested with pending results.
Sequential therapy with two different CAR T cells delivered superior antitumor efficacy and prevented B-ALL relapse. The first infusion of CAR T cells can reduce tumor burden, to some extent, to prevent the exhaustion of the second infused CAR T cells due to excessive antigen stimulation. As reported by Liu et al, sequential CAR T cell therapy (CD19 and CD22) achieved 18-month event-free survival (EFS) among 67.5% of patients who relapsed after allo-HSCT, 21 of 27 patients received the second CD22 CAR T infusion and were followed up for a median of 19.7 months with 14 cases who remained in CR.
It has been confirmed that the expression of CD19 will be rapidly down-regulated after contact with CAR T cells, which is considered one of the mechanisms by which tumor cells evade CAR T killing. A similar phenomenon also exists in BCMA antigen. Following BCMA CAR T-cell infusions, BCMA expression levels decreased on residual MM cells in 67% of patients. Decreased or lost BCMA expression was found in patients with post-CAR-T relapse.[56,80,81] Biallelic loss or deletion on one allele and a loss of function mutation on the second allele causes the lack of BCMA expression, leading to resistance or relapse following anti-BCMA CAR-T therapy, and demonstrates the possibility of clonal selection of MM cells lacking BCMA.[82,83] To prevent the recurrence of low antigen density tumors, researchers have developed some new strategies to increase the responsiveness of CAR T cells to low antigen density tumor cells. For example, in the clinical practice of CAR T-22 therapy of B-ALL, Sigh et al found that shortening the linker between the heavy and light chains of scFv can effectively improve the function of CAR T-22 cells. Majzner et al demonstrated that the hinge from CD28 can significantly affect the sensitivity of CAR T cells to low-density antigens compared with that from CD8. Liu et al designed a new type of chimeric receptor molecule, named synthetic TCR and antigen receptor (STAR), which can kill low-density antigen tumor cells more effectively with the help of the natural TCR structure. Given the importance of baseline BCMA in conferring responsiveness, pharmacological upregulation of BCMA could sensitize the BCMA-targeting CAR-T therapy. It has been well-characterized that the process of BCMA shedding from cell membrane is catalyzed by γ-secretase, which could be inactivated by γ-secretase inhibitor (GSI). Preclinical studies suggested that combining CAR T cells with a small molecule GSI increases BCMA expression on the surface of myeloma cells. Recently, a phase I first-in-human trial of BCMA targeted CAR T cells in combination with a GSI (JSMD194) was conducted in 18 R/R MM patients who received a median of 10 prior lines of therapy, and 7/18 (39%) had prior BCMA targeted therapy. One patient experienced a dose-limiting toxicity (DLT). 95% of patients experienced CRS, mostly grade 1 to 2 (83%), and 66% of patients experienced immune effector cell-associated neurotoxicity syndrome (ICANS), predominantly grade 1 to 2. GSI treatment increased BCMA surface density by a median of 12-fold. The ORR was 89%, including eight CRs or better. In addition, all-trans retinoic acid (ATRA) was also found to be able to increase BCMA expression through epigenetic modulation and enhance its recognition by BCMA-CAR T cells. Thus, whether ATRA in combination with GSI could improve BCMA CAR-T efficacy needs further evaluation.
CAR T Cell Exhaustion
Failure of CAR T therapy is often caused by the reduction in number of CAR T cells, which is considered one of the intrinsic defects of CAR T cells, also known as CAR T exhaustion. Inflammatory environment, immunosuppressive cells, and metabolic stress promote the dysfunction of CAR T cells. However, the mainstream view believes that “continuous stimulation of antigens” is the main reason for the CAR T cell exhaustion. Although T cell exhaustion is an intrinsic process, we investigators could regulate this process by various means to achieve the purpose of improving CAR T cell persistence in vivo. These strategies include: (1) optimization of CAR structure; (2) improvement of CAR T cell products; (3) modification of the signaling regulatory network of T cells; and (4) combination with other modalities.
Each motif of CAR has a significant impact on its function. As for scFv, the spontaneous aggregation will trigger the self-activating signals (tonic signaling), thereby triggering rapid exhaustion of CAR T cells. The hinge region provides a certain rigidity to the CAR molecule, and transmits mechanical torque to induce the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM), leading to downstream signaling activation; different hinge sequences or adjusting their length significantly affects the long-term antitumor ability of CAR T cells.[85,92-98] The intracellular activation signaling domain with different co-stimulatory motifs, such as the most commonly used CD28 and 4-1BB,[7,99-102] would elicit discrepant downstream activation signals. It has been proved that 4-1BB CAR can better avoid tonic signaling than CD28, and its downstream signaling pathway helps CAR T cells to maintain longevity.[103-105] According to the report of Shah and Fry in 2019, the average duration of CAR T cells carrying 4-1BB in vivo can reach 168 days, while that of CAR T cells using CD28 is only 30 days. However, the overall difference in clinical response is not very significant. In addition, recent studies have shown that appropriately reducing the activation level of CAR molecules, such as mutating the ITAM motifin CD28 or introducing inhibitory CD3-ε, can significantly improve the persistence of CAR T cells.
T cells with memory phenotype have better in vivo expansion potential. Therefore, optimizing the preparation process of CAR T cells, such as shortening the culture time, replacing interleukin (IL)-2 with IL-7, IL-15, and/or IL-21 cytokines,[110,111] and priming CAR T cells with epigenetic drugs such as decitabine, can improve the proportion of memory phenotype cells in CAR T products and the long-term anti-tumor potential. In addition, recent studies have shown that CD4-positive T cells are decisive for maintaining the long-term efficacy of CAR T products; therefore, increasing the proportion of CD4-positive cells is also one of the strategies to overcome CAR T cell exhaustion. Wang et al purified CD8+ central memory T cells to generate CS1-targeting CAR T cells, in which cytotoxicity, cytokine production, and maintenance of memory phenotype were enhanced by the addition of lenalidomide during T cell expansion. Furthermore, lenalidomide improved CS1 (CD319, SLAM-7)-CAR T cell persistence and anti-myeloma response in vivo. Similarly, lenalidomide enhanced BCMA-CAR T cell functions in a dose-dependent manner and potentiated its anti-myeloma activity in vivo. The CAR T cell persistence was increased due to delayed functional exhaustion.
With the CRISPR/Cas9 screening system, many genes affecting the persistence of T cells have been identified in recent years. In 2019, Lynn et al showed that CAR T cells engineered to overexpress c-JUN, one of the canonical activator protein-1 (AP-1) factors, presented enhanced expansion potential, less terminal differentiation and improved anti-tumor activity. In 2021, Patrick Hogan's team screened T cells and found that basic leucine zipper activating transcription factor-like transcription factor can cooperate with interferon regulatory factor 4 to prevent CAR T cell exhaustion.[117,118] Pinzing et al suggested that knockout of de novo DNA methyltransferase 3 alpha gene improve the fitness of CAR T cells and enhance their anti-tumor ability through epigenetic regulation. Good et al demonstrated that CAR T exhaustion was accompanied by up-regulated expression of inhibitor of differentiation 3 and SRY-related HMG-box 4 (SOX4), and silencing these two genes enhances CAR T cells’ anti-tumor function even after long-term exposure to tumor cells. In addition, in the follow-up analysis of a patient, Carl June's team found that disruption of tet methylcytosine dioxygenase-2 endowed CAR T cells with prominent in vivo continuous expansion ability.
In addition to improving CAR T itself, combining other interventions can also improve the long-term anti-tumor function of CAR T cells. For example, chemotherapy preconditioning is critical for in vivo proliferation of CAR T cells. In 2010, the Rosenberg's team first reported the clinical scheme of CAR T-19 therapy after preconditioning. In 2011, Brentjens et al reported that eight patients with refractory chronic lymphocytic leukemia were treated with CAR T-19. Among the five patients who received preconditioning, one achieved PR, two achieved SD, and one patient with relapsed ALL obtained CR. At the same time, three patients who did not receive preconditioning had no clinical benefit. These small-scale clinical data preliminarily suggested the necessity of combined chemotherapy preconditioning. Today, preconditioning has become an essential step in the CAR T treatment process. Combining PD-1 antibodies is also one of the means enhancing the efficacy of CAR T therapy,[124,125] which has been shown to specifically expand T cells in the exhausted precursor state, thereby rescuing CAR T exhaustion and improving the anti-tumor function.
Universal or Off-the-shelf CAR T Therapy
The universal or off-the-shelf CAR T cells are scaled-up allogeneic CAR T cells manufactured by using third-party healthy T cells derived from various sources, such as human leukocyte antigen-compatible peripheral mononuclear cells, cord blood, and induced pluripotent stem cells. Individual allogeneic CAR T cells are generated using donor lymphocytes in the setting of allo-HCT. Unlike autologous CAR T cells, universal CAR T (UCAR T) cells are readily available for advanced patients without delay, can reduce the production cost through scalable manufacturing, and have potential to enhance clinical efficacies due to superior T cell fitness. However, UCAR T cells may cause life-threatening GvHD and are susceptible to graft rejection by the host immune system, resulting in limited persistence. Gene editing technologies, such as transcription activator-like effector nucleases (TALEN) and CRISPR/Cas9, have been introduced in allogeneic CAR T cells to disrupt the endogenous TCR subunit and beta-2-microglobulin to reduce the risk of GvHD and graft rejection, respectively.[128,129] The gene-edited UCAR T cell therapies are being evaluated in many clinical trials. In phase I pediatric (PALL) and adult (CALM) trials, UCAR T-19, an “off-the-shelf” CAR T product in which genes encoding TCR α and β chains and CD52 are simultaneously disrupted by TALEN, showed impressive clinical responses with minimal GvHD and acceptable persistence in children and adult patients with high-risk B-cell ALL. The application of UCAR T cells will provide opportunities for increased treatment access and combination with other immunotherapeutic options.
Role of Transplantation
Consolidation of CAR T cell therapy with allo-HSCT is potential strategy to prevent recurrence. The high response rate of CAR T cell therapy provides patients with valuable transplant opportunities. One study obtained 5-year EFS following allo-HSCT in 61.9% of patients receiving CAR-T cell therapy. A parallel comparison of outcomes in R/R B-ALL patients who achieved remission from either CAR T cell therapy or chemotherapy and who subsequently underwent allo-HSCT is conducted and the 4-year leukemia-free survival and OS were all similar. On the other hand, allo-HSCT may prolong EFS and relapse-free survival when patients have high (≥5%) pre-infusion bone marrow MRD assessed by flow cytometry (BM-FCM-MRD)or poor prognostic markers (P < 0.05). Although there is currently no consensus on the timing of transplantation after CAR-T cell therapy, a study showed later utilization of allo-HCT after CAR-T cell therapy is associated with higher mortality.
Autologous stem cell transplantation (ASCT) remains as frontline treatment for transplant-eligible MM patients. There are several rationales for the combination of CAR T-cell therapy with ASCT. A low tumor burden at the time CAR T-cell infusion prevents T-cell exhaustion and translates into improved activity; besides, post-ASCT T cell reconstitution provides a window for immune-based therapies, and might improve safety by reducing cytokine productions. CTL019, a CD19-CAR T cell product, has been shown to improve PFS and DOR following second ASCT in a subset of MM patients who progressed early after first ASCT, possibly by modulating secondary immune responses against myeloma-propagating cells. A single-arm exploratory clinical trial was conducted to evaluate the safety and efficacy of sequential anti-CD19 and anti-BCMA CAR-T cell infusion, followed by lenalidomide maintenance after ASCT. The treatments were tolerable among 10 high-risk newly diagnosed MM patients, and the ORR was 100%, including sCR 90% and CR 10%. At a median follow-up of 42 months, 70% of patients achieved MRD negativity for >2 years, with PFS and OS not reached. Thus, these data provided clinical evidence that CAR T therapy following ASCT is safe and feasible for both newly diagnosed multiple myeloma and R/R MM patients. An important open question for future studies is whether CAR T-cell therapy can replace ASCT.
Quality Control of CAR T Cell Manufacturing
Leukapheresis is an essential step to produce sufficient and effective CAR T cells. To ensure good quality and expansion potential of the collected cells, appropriate washout period, T cell yield, and reduction of non-T cells should be considered. In addition, high platelet count, circulating malignant cells, patient's age and granulocyte colony-stimulating factors can also adversely impact on leukapheresis. A shift of CAR T manufacturing platform toward automated and integrated closed system will substantially reduce the production cost. Despite general consensus on critical parameters of cell products, there is no specific technical requirements or standards for CAR T cell products. We suggest that preparing CAR T cells require whole-process quality control,[138,139] which includes: (1) control of starting materials; (2) in-process control and testing; (3) release testing; (4) validation of the production process; and (5) stability study. Because of the potential risks of insertion mutation and viral replication, the quality control of vectors for gene modification should follow the gene therapy-related guidance from regulatory agencies of the government [Figure 2].
The deep and durable responses have been achieved in a fraction of patients who fail to respond to standard of care, highlighting the great potential of CAR T therapy to transform the current treatment paradigm for hematologic cancers. Allogeneic transplantation is necessary for most of adult ALL patients who achieve CR after CAR T therapy to reduce relapse and maintain long-term remission. Increasing studies in B-NHL, such as Scholar-1, Scholar-5, ZUMA-7, Belinda, and Transform, suggest that CAR T therapy produces better survival data than ASCT; however, whether CAR T-cell therapy can replace ASCT remains an important open question for future studies. As CAR T therapy continues to evolve, there are still challenges and bottlenecks. More efforts are needed to elucidate the underlying mechanisms of CAR T cell resistance, which will aid in developing strategies of CAR design optimization, improving CAR-T cell fitness and persistence, and counteracting immunosuppressive TME. It is also critical to identify novel targets to mitigate antigen escape after CAR T therapy. Different combination approaches involving CAR-T therapy, such as dual-targeting CAR T, proceeding with transplantation, immunomodulatory drugs and immune checkpoint blockade, warrant further studies in either real-world setting or clinical trials. We expect that accumulating advances in the field of CAR T technology will improve clinical efficacy and safety, broaden the application of CAR-T therapy, and bring greater benefits to patients with hematological malignancies.
This study was funded by the National Natural Science Foundation of China (Nos. 82150108, 31991171, and 81830002 to Weidong Han and 32070951 to Yajing Zhang); the National Natural Science Foundation of China (No. 81572920) and the Natural Science Foundation of Zhejiang Province of China (No. LY21H080005) to Yang Xu; the National Natural Science Foundation of China (Nos. 81830006 and 82170219) and the Science Technology Department of Zhejiang Province (No. 2021C03117) to Wenbing Qian; and the National Natural Science Foundation of China (No. 81830004), Translational Research Grant of National Center for Clinical Medical Research of Hematology (No. 2020ZKZC04), the Ministry of Science and Technology of China (No. 2021YFA1100800), and the Shanghai Municipal Health Commission (No. 2020CXJQ02) to Aibin Liang.
Conflicts of interest
1. Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci U S A 1989;86:10024–10028. doi: 10.1073/pnas.86.24.10024.
2. Garrido F, Aptsiauri N. Cancer immune escape: MHC expression in primary tumours versus metastases. Immunology 2019;158:255–266. doi: 10.1111/imm.13114.
3. Fangazio M, Ladewig E, Gomez K, Garcia-Ibanez L, Kumar R, Teruya-Feldstein J, et al. Genetic mechanisms of HLA-I loss and immune escape in diffuse large B cell lymphoma. Proc Natl Acad Sci U S A 2021;118:e2104504118. doi: 10.1073/pnas.2104504118.
4. Kershaw MH, Westwood JA, Parker LL, Wang G, Eshhar Z, Mavroukakis SA, et al. A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer. Clin Cancer Res 2006;12:6106–6115. doi: 10.1158/1078-0432.CCR-06-1183.
5. Lamers CH, Sleijfer S, Vulto AG, Kruit WH, Kliffen M, Debets R, et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J Clin Oncol 2006;24:e20–e22. doi: 10.1200/JCO.2006.05.9964.
6. Savoldo B, Ramos CA, Liu E, Mims MP, Keating MJ, Carrum G, et al. CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients. J Clin Invest 2011;121:1822–1826. doi: 10.1172/JCI46110.
7. Cappell KM, Kochenderfer JN. A comparison of chimeric antigen receptors containing CD28 versus 4-1BB costimulatory domains. Nat Rev Clin Oncol 2021;18:715–727. doi: 10.1038/s41571-021-00530-z.
8. Kowolik CM, Topp MS, Gonzalez S, Pfeiffer T, Olivares S, Gonzalez N, et al. CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. Cancer Res 2006;66:10995–11004. doi: 10.1158/0008-5472.CAN-06-0160.
9. Long AH, Haso WM, Shern JF, Wanhainen KM, Murgai M, Ingaramo M, et al. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med 2015;21:581–590. doi: 10.1038/nm.3838.
10. Louis CU, Savoldo B, Dotti G, Pule M, Yvon E, Myers GD, et al. Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma. Blood 2011;118:6050–6056. doi: 10.1182/blood-2011-05-354449.
11. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med 2011;365:725–733. doi: 10.1056/NEJMoa1103849.
12. Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 2013;368:1509–1518. doi: 10.1056/NEJMoa1215134.
13. Tong C, Zhang Y, Liu Y, Ji X, Zhang W, Guo Y, et al. Optimized tandem CD19/CD20 CAR-engineered T cells in refractory/relapsed B cell lymphoma. Blood 2020;136:1632–1644. doi: 10.1182/blood.2020005278.
14. Schuster SJ, Bishop MR, Tam CS, Waller EK, Borchmann P, McGuirk JP, et al. Tisagenlecleucel in adult relapsed or refractory diffuse large B-cell lymphoma. N Engl J Med 2019;380:45–56. doi: 10.1056/NEJMoa1804980.
15. Zhao WH, Liu J, Wang BY, Chen YX, Cao XM, Yang Y, et al. A phase 1, open-label study of LCAR-B38M, a chimeric antigen receptor T cell therapy directed against B cell maturation antigen, in patients with relapsed or refractory multiple myeloma. J Hematol Oncol 2018;11:141. doi: 10.1186/s13045-018-0681-6.
16. Nie Y, Lu W, Chen D, Tu H, Guo Z, Zhou X, et al. Mechanisms underlying CD19-positive ALL relapse after anti-CD19 CAR T cell therapy and associated strategies. Biomarker Res 2020;8:18. doi: 10.1186/s40364-020-00197-1.
17. Wang L. Clinical determinants of relapse following CAR-T therapy for hematologic malignancies: coupling active strategies to overcome therapeutic limitations. Curr Res Transl Med 2022;70:103320. doi: 10.1016/j.retram.2021.103320.
18. Sterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J 2021;11:69. doi: 10.1038/s41408-021-00459-7.
19. Minton K. Overcoming CAR T cell exhaustion. Nat Rev Immunol 2020;20:72–73. doi: 10.1038/s41577-019-0265-x.
20. Delgoffe GM, Xu C, Mackall CL, Green MR, Gottschalk S, Speiser DE, et al. The role of exhaustion in CAR T cell therapy. Cancer Cell 2021;39:885–888. doi: 10.1016/j.ccell.2021.06.012.
21. Kasakovski D, Xu L, Li Y. T cell senescence and CAR-T cell exhaustion in hematological malignancies. J Hematol Oncol 2018;11:91. doi: 10.1186/s13045-018-0629-x.
22. Singh N, Lee YG, Shestova O, Ravikumar P, Hayer KE, Hong SJ, et al. Impaired death receptor signaling in leukemia causes antigen-independent resistance
by inducing CAR T-cell dysfunction. Cancer Discov 2020;10:552–567. doi: 10.1158/2159-8290.CD-19-0813.
23. Peng W, Chen JQ, Liu C, Malu S, Creasy C, Tetzlaff MT, et al. Loss of PTEN promotes resistance
to T cell-mediated immunotherapy. Cancer Discov 2016;6:202–216. doi: 10.1158/2159-8290.CD-15-0283.
24. Jain MD, Zhao H, Wang X, Atkins R, Menges M, Reid K, et al. Tumor interferon signaling and suppressive myeloid cells are associated with CAR T-cell failure in large B-cell lymphoma. Blood 2021;137:2621–2633. doi: 10.1182/blood.2020007445.
25. Yan X, Chen D, Ma X, Wang Y, Guo Y, Wei J, et al. CD58 loss in tumor cells confers functional impairment of CAR T cells. Blood Adv 2022;6:5844–5856. doi: 10.1182/bloodadvances.2022007891.
26. Majzner RG, Frank MJ, Mount C, Tousley A, Kurtz DM, Sworder B, et al. CD58 aberrations limit durable responses to CD19 CAR in large B cell lymphoma patients treated with axicabtagene ciloleucel but can be overcome through novel CAR engineering. Blood 2020;136:53–54. doi: 10.1182/blood-2020-139605.
27. Wang K, Wei G, Liu D. CD19: a biomarker for B cell development, lymphoma diagnosis and therapy. Exp Hematol Oncol 2012;1:36. doi: 10.1186/2162-3619-1-36.
28. Blüml S, McKeever K, Ettinger R, Smolen J, Herbst R. B-cell targeted therapeutics in clinical development. Arthritis Res Ther 2013;15:S4. doi: 10.1186/ar3906.
29. Locke FL, Ghobadi A, Jacobson CA, Miklos DB, Lekakis LJ, Oluwole OO, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1-2 trial. Lancet Oncol 2019;20:31–42. doi: 10.1016/S1470-2045(18)30864-7.
30. Schuster SJ, Bishop MR, Tam C, Borchmann P, Jaeger U, Waller EK, et al. Sustained disease control for adult patients with relapsed or refractory diffuse large B-cell lymphoma: an updated analysis of Juliet, a global pivotal phase 2 trial of tisagenlecleucel. Blood 2018;132:1684. doi: 10.1182/blood-2018-99-115252.
31. Westin JR, Tam CS, Borchmann P, Jaeger U, McGuirk JP, Holte H, et al. Correlative analyses of patient and clinical characteristics associated with efficacy in tisagenlecleucel-treated relapsed/refractory diffuse large B-cell lymphoma patients in the Juliet trial. Blood 2019;134:4103. doi: 10.1182/blood-2019-129107.
32. Abramson JS, Palomba ML, Gordon LI, Lunning MA, Wang M, Arnason J, et al. Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study. Lancet 2020;396:839–852. doi: 10.1016/S0140-6736(20)31366-0.
33. Zhang WY, Wang Y, Guo YL, Dai HR, Yang QM, Zhang YJ, et al. Treatment of CD20-directed chimeric antigen receptor-modified T cells in patients with relapsed or refractory B-cell non-hodgkin lymphoma: an early phase IIa trial report. Signal Transduct Target Ther 2016;1:16002. doi: 10.1038/sigtrans.2016.2.
34. Zhang WY, Liu Y, Wang Y, Wang CM, Yang QM, Zhu HL, et al. Long-term safety and efficacy of CART-20 cells in patients with refractory or relapsed B-cell non-hodgkin lymphoma: 5-years follow-up results of the phase I and IIa trials. Signal Transduct Target Ther 2017;2:17054. doi: 10.1038/sigtrans.2017.54.
35. Tan Y, Cai H, Li C, Deng B, Song W, Ling Z, et al. A novel full-human CD22-CAR T cell therapy with potent activity against CD22low
B-ALL. Blood Cancer J 2021;11:71. doi: 10.1038/s41408-021-00465-9.
36. Pan J, Niu Q, Deng B, Liu S, Wu T, Gao Z, et al. CD22 CAR T-cell therapy in refractory or relapsed B acute lymphoblastic leukemia. Leukemia 2019;33:2854–2866. doi: 10.1038/s41375-019-0488-7.
37. Zhu H, Deng H, Mu J, Lyu C, Jiang Y, Deng Q. Anti-CD22 CAR-T cell therapy as a salvage treatment in B cell malignancies refractory or relapsed after anti-CD19 CAR-T therapy. Onco Targets Ther 2021;14:4023–4037. doi: 10.2147/OTT.S312904.
38. Baird JH, Frank MJ, Craig J, Patel S, Spiegel JY, Sahaf B, et al. CD22-directed CAR T-cell therapy induces complete remissions in CD19-directed CAR-refractory large B-cell lymphoma. Blood 2021;137:2321–2325. doi: 10.1182/blood.2020009432.
39. Anagnostou T, Riaz IB, Hashmi SK, Murad MH, Kenderian SS. Anti-CD19 chimeric antigen receptor T-cell therapy in acute lymphocytic leukaemia: a systematic review and meta-analysis. Lancet Haematol 2020;7:e816–e826. doi: 10.1016/S2352-3026(20)30277-5.
40. Tan Y, Pan J, Deng B, Ling Z, Song W, Xu J, et al. Toxicity and effectiveness of CD19 CAR T therapy in children with high-burden central nervous system refractory B-ALL. Cancer Immunol Immunother 2021;70:1979–1993. doi: 10.1007/s00262-020-02829-9.
41. Baroni ML, Sanchez Martinez D, Gutierrez Aguera F, Roca Ho H, Castella M, Zanetti SR, et al. 41BB-based and CD28-based CD123-redirected T-cells ablate human normal hematopoiesis in vivo. J Immunother Cancer 2020;8:e000845. doi: 10.1136/jitc-2020-000845.
42. Nguyen DH, Ball ED, Varki A. Myeloid precursors and acute myeloid leukemia cells express multiple CD33-related Siglecs. Exp Hematol 2006;34:728–735. doi: 10.1016/j.exphem.2006.03.003.
43. Ritchie DS, Neeson PJ, Khot A, Peinert S, Tai T, Tainton K, et al. Persistence and efficacy of second generation CAR T cell against the LeY antigen in acute myeloid leukemia. Mol Ther 2013;21:2122–2129. doi: 10.1038/mt.2013.154.
44. Wang QS, Wang Y, Lv HY, Han QW, Fan H, Guo B, et al. Treatment of CD33-directed chimeric antigen receptor-modified T cells in one patient with relapsed and refractory acute myeloid leukemia. Mol Ther 2015;23:184–191. doi: 10.1038/mt.2014.164.
45. Fiorenza S, Turtle CJ. CAR-T cell therapy for acute myeloid leukemia: preclinical rationale, current clinical progress, and barriers to success. BioDrugs 2021;35:281–302. doi: 10.1007/s40259-021-00477-8.
46. Zhang H, Wang P, Li Z, He Y, Gan W, Jiang H. Anti-CLL1 chimeric antigen receptor T-cell therapy in children with relapsed/refractory acute myeloid leukemia. Clin Cancer Res 2021;27:3549–3555. doi: 10.1158/1078-0432.CCR-20-4543.
47. Ma H, Padmanabhan IS, Parmar S, Gong Y. Targeting CLL-1 for acute myeloid leukemia therapy. J Hematol Oncol 2019;12:41. doi: 10.1186/s13045-019-0726-5.
48. Jin X, Zhang M, Sun R, Lyu H, Xiao X, Zhang X, et al. First-in-human phase I study of CLL-1 CAR-T cells in adults with relapsed/refractory acute myeloid leukemia. J Hematol Oncol 2022;15:88. doi: 10.1186/s13045-022-01308-1.
49. Jetani H, Navarro-Bailón A, Maucher M, Frenz S, Verbruggen C, Yeguas A, et al. Siglec-6 is a novel target for CAR T-cell therapy in acute myeloid leukemia. Blood 2021;138:1830–1842. doi: 10.1182/blood.2020009192.
50. Wang CM, Wu ZQ, Wang Y, Guo YL, Dai HR, Wang XH, et al. Autologous T cells expressing CD30 chimeric antigen receptors for relapsed or refractory hodgkin lymphoma: an open-label phase I trial. Clin Cancer Res 2017;23:1156–1166. doi: 10.1158/1078-0432.CCR-16-1365.
51. Ramos CA, Grover NS, Beaven AW, Lulla PD, Wu MF, Ivanova A, et al. Anti-CD30 CAR-T cell therapy in relapsed and refractory hodgkin lymphoma. J Clin Oncol 2020;38:3794–3804. doi: 10.1200/JCO.20.01342.
52. Grover NSIA, Moore DT, Et AL. CD30-directed CAR-T cells co-expressing CCR4 in relapsed/refractory hodgkin lymphoma and CD30+ cutaneous T cell lymphoma. Transplant Cell Ther 2022;28 (3 Suppl):S54–S55. doi: 10.1016/S2666-6367(22)00225-1.
53. Pan J, Tan Y, Wang G, Deng B, Ling Z, Song W, et al. Donor-derived CD7 chimeric antigen receptor T cells for T-cell acute lymphoblastic leukemia: first-in-human, phase I trial. J Clin Oncol 2021;39:3340–3351. doi: 10.1200/JCO.21.00389.
54. Lu P, Liu Y, Yang J, Zhang X, Yang X, Wang H, et al. Naturally selected CD7 CAR-T therapy without genetic manipulations for T-ALL/LBL: first-in-human phase 1 clinical trial. Blood 2022;140:321–334. doi: 10.1182/blood.2021014498.
55. Sharma P, Kanapuru B, George B, Lin X, Xu Z, Bryan WW, et al. FDA approval summary: idecabtagene vicleucel for relapsed or refractory multiple myeloma. Clin Cancer Res 2022;28:1759–1764. doi: 10.1158/1078-0432.CCR-21-3803.
56. Munshi NC, Anderson LD Jr, Shah N, Madduri D, Berdeja J, Lonial S, et al. Idecabtagene vicleucel in relapsed and refractory multiple myeloma. N Engl J Med 2021;384:705–716. doi: 10.1056/NEJMoa2024850.
57. Berdeja JG, Madduri D, Usmani SZ, Jakubowiak A, Agha M, Cohen AD, et al. Ciltacabtagene autoleucel, a B-cell maturation antigen-directed chimeric antigen receptor T-cell therapy in patients with relapsed or refractory multiple myeloma (CARTITUDE-1): a phase 1b/2 open-label study. Lancet 2021;398:314–324. doi: 10.1016/S0140-6736(21)00933-8.
58. Martin T, Usmani SZ, Berdeja JG, Agha M, Cohen AD, Hari P, et al. Ciltacabtagene autoleucel, an anti-B-cell maturation antigen chimeric antigen receptor T-cell therapy, for relapsed/refractory multiple myeloma: CARTITUDE-1 2-year follow-up. J Clin Oncol 2022;JCO2200842. doi: 10.1200/JCO.22.00842.
59. Zhao WH, Wang BY, Chen LJ, Fu WJ, Xu J, Liu J, et al. Four-year follow-up of LCAR-B38M in relapsed or refractory multiple myeloma: a phase 1, single-arm, open-label, multicenter study in China (LEGEND-2). J Hematol Oncol 2022;15:86. doi: 10.1186/s13045-022-01301-8.
60. Zhang H, Zhao P, Huang H. Engineering better chimeric antigen receptor T cells. Exp Hematol Oncol 2020;9:34. doi: 10.1186/s40164-020-00190-2.
61. Dufva O, Koski J, Maliniemi P, Ianevski A, Klievink J, Leitner J, et al. Integrated drug profiling and CRISPR screening identify essential pathways for CAR T-cell cytotoxicity. Blood 2020;135:597–609. doi: 10.1182/blood.2019002121.
62. Yan X, Chen D, Wang Y, Guo Y, Tong C, Wei J, et al. Identification of NOXA as a pivotal regulator of resistance
to CAR T-cell therapy in B-cell malignancies. Signal Transduct Target Ther 2022;7:98. doi: 10.1038/s41392-022-00915-1.
63. Heard A, Landmann JH, Hansen AR, Papadopolou A, Hsu YS, Selli ME, et al. Antigen glycosylation regulates efficacy of CAR T cells targeting CD19. Nat Commun 2022;13:3367. doi: 10.1038/s41467-022-31035-7.
64. Wei J, Han X, Bo J, Han W. Target selection for CAR-T therapy. J Hematol Oncol 2019;12:62. doi: 10.1186/s13045-019-0758-x.
65. Orlando EJ, Han X, Tribouley C, Wood PA, Leary RJ, Riester M, et al. Genetic mechanisms of target antigen loss in CAR19 therapy of acute lymphoblastic leukemia. Nat Med 2018;24:1504–1506. doi: 10.1038/s41591-018-0146-z.
66. Fry TJ, Shah NN, Orentas RJ, Stetler-Stevenson M, Yuan CM, Ramakrishna S, et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nat Med 2018;24:20–28. doi: 10.1038/nm.4441.
67. Rabilloud T, Potier D, Pankaew S, Nozais M, Loosveld M, Payet-Bornet D. Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy. Nat Commun 2021;12:865. doi: 10.1038/s41467-021-21168-6.
68. Yang X, Yu Q, Xu H, Zhou J. Upregulation of CD22 by chidamide promotes CAR T cells functionality. Sci Rep 2021;11:20637. doi: 10.1038/s41598-021-00227-4.
69. Zhang X, Zhu L, Zhang H, Chen S, Xiao Y. CAR-T cell therapy in hematological malignancies: Current opportunities and challenges. Front Immunol 2022;13:927153. doi: 10.3389/fimmu.2022.927153.
70. Shah NN, Johnson BD, Schneider D, Zhu F, Szabo A, Keever-Taylor CA, et al. Bispecific anti-CD20, anti-CD19 CAR T cells for relapsed B cell malignancies: a phase 1 dose escalation and expansion trial. Nat Med 2020;26:1569–1575. doi: 10.1038/s41591-020-1081-3.
71. Dai H, Wu Z, Jia H, Tong C, Guo Y, Ti D, et al. Bispecific CAR-T cells targeting both CD19 and CD22 for therapy of adults with relapsed or refractory B cell acute lymphoblastic leukemia. J Hematol Oncol 2020;13:30. doi: 10.1186/s13045-020-00856-8.
72. Spiegel JY, Patel S, Muffly L, Hossain NM, Oak J, Baird JH, et al. CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell malignancies: a phase 1 trial. Nat Med 2021;27:1419–1431. doi: 10.1038/s41591-021-01436-0.
73. Wang Y, Cao J, Gu W, Shi M, Lan J, Yan Z, et al. Long-term follow-up of combination of B-cell maturation antigen and CD19 chimeric antigen receptor T cells in multiple myeloma. J Clin Oncol 2022;40:2246–2256. doi: 10.1200/JCO.21.01676.
74. Du J, Jiang H, Dong B, Gao L, Liu L, Ge J, et al. Updated results of a multicenter first-in-human study of BCMA/CD19 dual-targeting fast CAR-T GC012F for patients with relapsed/refractory multiple myeloma (RRMM). J Clin Oncol 2022;40 (16_suppl):8005. doi: 10.1200/JCO.2022.40.16_suppl.8005.
75. Mei H, Li C, Jiang H, Zhao X, Huang Z, Jin D, et al. A bispecific CAR-T cell therapy targeting BCMA and CD38 in relapsed or refractory multiple myeloma. J Hematol Oncol 2021;14:161. doi: 10.1186/s13045-021-01170-7.
76. Zah E, Nam E, Bhuvan V, Tran U, Ji BY, Gosliner SB, et al. Systematically optimized BCMA/CS1 bispecific CAR-T cells robustly control heterogeneous multiple myeloma. Nat Commun 2020;11:2283. doi: 10.1038/s41467-020-16160-5.
77. Fernández de Larrea C, Staehr M, Lopez AV, Ng KY, Chen Y, Godfrey WD, et al. Defining an optimal dual-targeted CAR T-cell therapy approach simultaneously targeting BCMA and GPRC5D to prevent BCMA escape-driven relapse in multiple myeloma. Blood Cancer Discov 2020;1:146–154. doi: 10.1158/2643-3230.BCD-20-0020.
78. Liu S, Deng B, Yin Z, Lin Y, An L, Liu D, et al. Combination of CD19 and CD22 CAR-T cell therapy in relapsed B-cell acute lymphoblastic leukemia after allogeneic transplantation. Am J Hematol 2021;96:671–679. doi: 10.1002/ajh.26160.
79. Blanco B, Ramírez-Fernández Á, Bueno C, Argemí-Muntadas L, Fuentes P, Aguilar-Sopeña Ó, et al. Overcoming CAR-mediated CD19 downmodulation and leukemia relapse with T lymphocytes secreting anti-CD19 T-cell engagers. Cancer Immunol Res 2022;10:498–511. doi: 10.1158/2326-6066.CIR-21-0853.
80. Cohen AD, Garfall AL, Stadtmauer EA, Melenhorst JJ, Lacey SF, Lancaster E, et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 2019;129:2210–2221. doi: 10.1172/JCI126397.
81. Brudno JN, Maric I, Hartman SD, Rose JJ, Wang M, Lam N, et al. T cells genetically modified to express an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of poor-prognosis relapsed multiple myeloma. J Clin Oncol 2018;36:2267–2280. doi: 10.1200/JCO.2018.77.8084.
82. Da Vià MC, Dietrich O, Truger M, Arampatzi P, Duell J, Heidemeier A, et al. Homozygous BCMA gene deletion in response to anti-BCMA CAR T cells in a patient with multiple myeloma. Nat Med 2021;27:616–619. doi: 10.1038/s41591-021-01245-5.
83. Samur MK, Fulciniti M, Aktas Samur A, Bazarbachi AH, Tai YT, Prabhala R, et al. Biallelic loss of BCMA as a resistance
mechanism to CAR T cell therapy in a patient with multiple myeloma. Nat Commun 2021;12:868. doi: 10.1038/s41467-021-21177-5.
84. Singh N, Frey NV, Engels B, Barrett DM, Shestova O, Ravikumar P, et al. Antigen-independent activation enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells. Nat Med 2021;27:842–850. doi: 10.1038/s41591-021-01326-5.
85. Majzner RG, Rietberg SP, Sotillo E, Dong R, Vachharajani VT, Labanieh L, et al. Tuning the antigen density requirement for CAR T-cell activity. Cancer Discov 2020;10:702–723. doi: 10.1158/2159-8290.CD-19-0945.
86. Liu Y, Liu G, Wang J, Zheng ZY, Jia L, Rui W, et al. Chimeric STAR receptors using TCR machinery mediate robust responses against solid tumors. Sci Transl Med 2021;13:eabb5191. doi: 10.1126/scitranslmed.abb5191.
87. Laurent SA, Hoffmann FS, Kuhn PH, Cheng Q, Chu Y, Schmidt-Supprian M, et al. γ-Secretase directly sheds the survival receptor BCMA from plasma cells. Nat Commun 2015;6:7333. doi: 10.1038/ncomms8333.
88. Pont MJ, Hill T, Cole GO, Abbott JJ, Kelliher J, Salter AI, et al. γ-Secretase inhibition increases efficacy of BCMA-specific chimeric antigen receptor T cells in multiple myeloma. Blood 2019;134:1585–1597. doi: 10.1182/blood.2019000050.
89. Cowan AJ, Pont M, Sather BD, Turtle CJ, Till BG, Libby E, et al. Safety and efficacy of fully human BCMA CAR T cells in combination with a gamma secretase inhibitor to increase BCMA surface expression in patients with relapsed or refractory multiple myeloma. Blood 2021;138 (Suppl 1):551. doi: 10.1182/blood-2021-154170.
90. Garcia-Guerrero E, Rodríguez-Lobato LG, Danhof S, Sierro-Martínez B, Goetz R, Sauer M, et al. ATRA augments BCMA expression on myeloma cells and enhances recognition by BCMA-CAR T-cells. Blood 2020;136:13–14. doi: 10.1182/blood-2020-142572.
91. Gumber D, Wang LD. Improving CAR-T immunotherapy: overcoming the challenges of T cell exhaustion. EBioMedicine 2022;77:103941. doi: 10.1016/j.ebiom.2022.103941.
92. Qin L, Lai Y, Zhao R, Wei X, Weng J, Lai P, et al. Incorporation of a hinge domain improves the expansion of chimeric antigen receptor T cells. J Hematol Oncol 2017;10:68. doi: 10.1186/s13045-017-0437-8.
93. Leick MB, Silva H, Scarfò I, Larson R, Choi BD, Bouffard AA, et al. Non-cleavable hinge enhances avidity and expansion of CAR-T cells for acute myeloid leukemia. Cancer Cell 2022;40:494–508. e5. doi: 10.1016/j.ccell.2022.04.001.
94. McComb S, Nguyen T, Shepherd A, Henry KA, Bloemberg D, Marcil A, et al. Programmable attenuation of antigenic sensitivity for a nanobody-based EGFR chimeric antigen receptor through hinge domain truncation. Front Immunol 2022;13:864868. doi: 10.3389/fimmu.2022.864868.
95. Zhang A, Sun Y, Du J, Dong Y, Pang H, Ma L, et al. Reducing hinge flexibility of CAR-T cells prolongs survival in vivo with low cytokines release. Front Immunol 2021;12:724211. doi: 10.3389/fimmu.2021.724211.
96. Bister A, Ibach T, Haist C, Smorra D, Roellecke K, Wagenmann M, et al. A novel CD34-derived hinge for rapid and efficient detection and enrichment of CAR T cells. Mol Ther Oncolytics 2021;23:534–546. doi: 10.1016/j.omto.2021.11.003.
97. Schäfer D, Henze J, Pfeifer R, Schleicher A, Brauner J, Mockel-Tenbrinck N, et al. A novel Siglec-4 derived spacer improves the functionality of CAR T cells against membrane-proximal epitopes. Front Immunol 2020;11:1704. doi: 10.3389/fimmu.2020.01704.
98. Liu L, Sommermeyer D, Cabanov A, Kosasih P, Hill T, Riddell SR, et al. Inclusion of strep-tag II in design of antigen receptors for T-cell immunotherapy. Nat Biotechnol 2016;34:430–434. doi: 10.1038/nbt.3461.
99. Wu L, Wei Q, Brzostek J, Gascoigne NRJ. Signaling from T cell receptors (TCRs) and chimeric antigen receptors (CARs) on T cells. Cell Mol Immunol 2020;17:600–612. doi: 10.1038/s41423-020-0470-3.
100. Zhao X, Yang J, Zhang X, Lu XA, Xiong M, Zhang J, et al. Efficacy and safety of CD28- or 4-1BB-based CD19 CAR-T cells in B cell acute lymphoblastic leukemia. Mol Ther Oncolytics 2020;18:272–281. doi: 10.1016/j.omto.2020.06.016.
101. Roselli E, Boucher JC, Li G, Kotani H, Spitler K, Reid K, et al. 4-1BB and optimized CD28 co-stimulation enhances function of human mono-specific and bi-specific third-generation CAR T cells. J Immunother Cancer 2021;9:e003354. doi: 10.1136/jitc-2021-003354.
102. Salter AI, Ivey RG, Kennedy JJ, Voillet V, Rajan A, Alderman EJ, et al. Phosphoproteomic analysis of chimeric antigen receptor signaling reveals kinetic and quantitative differences that affect cell function. Sci Signal 2018;11:eaat6753. doi: 10.1126/scisignal.aat6753.
103. Milone MC, Fish JD, Carpenito C, Carroll RG, Binder GK, Teachey D, et al. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol Ther 2009;17:1453–1464. doi: 10.1038/mt.2009.83.
104. Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 2018;131:49–57. doi: 10.1182/blood-2017-06-741041.
105. Philipson BI, O’Connor RS, May MJ, June CH, Albelda SM, Milone MC. 4-1BB costimulation promotes CAR T cell survival through noncanonical NF-κB signaling. Sci Signal 2020;13:eaay8248. doi: 10.1126/scisignal.aay8248.
106. Shah NN, Fry TJ. Mechanisms of resistance
to CAR T cell therapy. Nat Rev Clin Oncol 2019;16:372–385. doi: 10.1038/s41571-019-0184-6.
107. Boucher JC, Li G, Kotani H, Cabral ML, Morrissey D, Lee SB, et al. CD28 costimulatory domain-targeted mutations enhance chimeric antigen receptor T-cell function. Cancer Immunol Res 2021;9:62–74. doi: 10.1158/2326-6066.CIR-20-0253.
108. Wu W, Zhou Q, Masubuchi T, Shi X, Li H, Xu X, et al. Multiple signaling roles of CD3ε and its application in CAR-T cell therapy. Cell 2020;182:855–871. e23. doi: 10.1016/j.cell.2020.07.018.
109. Ghassemi S, Nunez-Cruz S, O’Connor RS, Fraietta JA, Patel PR, Scholler J, et al. Reducing ex vivo culture improves the antileukemic activity of chimeric antigen receptor (CAR) T cells. Cancer Immunol Res 2018;6:1100–1109. doi: 10.1158/2326-6066.CIR-17-0405.
110. Markley JC, Sadelain M. IL-7 and IL-21 are superior to IL-2 and IL-15 in promoting human T cell-mediated rejection of systemic lymphoma in immunodeficient mice. Blood 2010;115:3508–3519. doi: 10.1182/blood-2009-09-241398.
111. Alizadeh D, Wong RA, Yang X, Wang D, Pecoraro JR, Kuo CF, et al. IL15 enhances CAR-T cell antitumor activity by reducing mTORC1 activity and preserving their stem cell memory phenotype. Cancer Immunol Res 2019;7:759–772. doi: 10.1158/2326-6066.CIR-18-0466.
112. Wang Y, Tong C, Dai H, Wu Z, Han X, Guo Y, et al. Low-dose decitabine priming endows CAR T cells with enhanced and persistent antitumour potential via epigenetic reprogramming. Nat Commun 2021;12:409. doi: 10.1038/s41467-020-20696-x.
113. Melenhorst JJ, Chen GM, Wang M, Porter DL, Chen C, Collins MA, et al. Decade-long leukaemia remissions with persistence of CD4+
CAR T cells. Nature 2022;602:503–509. doi: 10.1038/s41586-021-04390-6.
114. Wang X, Walter M, Urak R, Weng L, Huynh C, Lim L, et al. Lenalidomide enhances the function of CS1 chimeric antigen receptor - redirected T cells against multiple myeloma. Clin Cancer Res 2018;24:106–119. doi: 10.1158/1078-0432.CCR-17-0344.
115. Works M, Soni N, Hauskins C, Sierra C, Baturevych A, Jones JC, et al. Anti-B-cell maturation antigen chimeric antigen receptor T cell function against multiple myeloma is enhanced in the presence of lenalidomide. Mol Cancer Ther 2019;18:2246–2257. doi: 10.1158/1535-7163.MCT-18-1146.
116. Lynn RC, Weber EW, Sotillo E, Gennert D, Xu P, Good Z, et al. C-Jun overexpression in CAR T cells induces exhaustion resistance
. Nature 2019;576:293–300. doi: 10.1038/s41586-019-1805-z.
117. Seo H, González-Avalos E, Zhang W, Ramchandani P, Yang C, Lio CJ, et al. BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells. Nat Immunol 2021;22:983–995. doi: 10.1038/s41590-021-00964-8.
118. Wei J, Long L, Zheng W, Dhungana Y, Lim SA, Guy C, et al. Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy. Nature 2019;576:471–476. doi: 10.1038/s41586-019-1821-z.
119. Prinzing B, Zebley CC, Petersen CT, Fan Y, Anido AA, Yi Z, et al. Deleting DNMT3A in CAR T cells prevents exhaustion and enhances antitumor activity. Sci Transl Med 2021;13:eabh0272. doi: 10.1126/scitranslmed.abh0272.
120. Good CR, Aznar MA, Kuramitsu S, Samareh P, Agarwal S, Donahue G, et al. An NK-like CAR T cell transition in CAR T cell dysfunction. Cell 2021;184:6081–6100. e26. doi: 10.1016/j.cell.2021.11.016.
121. Fraietta JA, Nobles CL, Sammons MA, Lundh S, Carty SA, Reich TJ, et al. Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells. Nature 2018;558:307–312. doi: 10.1038/s41586-018-0178-z.
122. Kochenderfer JN, Wilson WH, Janik JE, Dudley ME, Stetler-Stevenson M, Feldman SA, et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood 2010;116:4099–4102. doi: 10.1182/blood.
123. Brentjens RJ, Rivière I, Park JH, Davila ML, Wang X, Stefanski J, et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood 2011;118:4817–4828. doi: 10.1182/blood-2011-04-348540.
124. Augmenting CAR T cells with PD-1 blockade. Cancer Discov 2019;9:158. doi: 10.1158/2159-8290.CD-NB2018-165.
125. Chong EA, Melenhorst JJ, Lacey SF, Ambrose DE, Gonzalez V, Levine BL, et al. PD-1 blockade modulates chimeric antigen receptor (CAR)-modified T cells: refueling the CAR. Blood 2017;129:1039–1041. doi: 10.1182/blood-2016-09-738245.
126. Yost KE, Satpathy AT, Wells DK, Qi Y, Wang C, Kageyama R, et al. Clonal replacement of tumor-specific T cells following PD-1 blockade. Nat Med 2019;25:1251–1259. doi: 10.1038/s41591-019-0522-3.
127. Muthuvel M, Srinivasan H, Louis L, Martin S. Engineering off-the-shelf universal CAR T cells: a silver lining in the cloud. Cytokine 2022;156:155920. doi: 10.1016/j.cyto.2022.155920.
128. Depil S, Duchateau P, Grupp SA, Mufti G, Poirot L. ‘Off-the-shelf’ allogeneic CAR T cells: development and challenges. Nat Rev Drug Discov 2020;19:185–199. doi: 10.1038/s41573-019-0051-2.
129. Young RM, Engel NW, Uslu U, Wellhausen N, June CH. Next-generation CAR T-cell therapies. Cancer Discov 2022;12:1625–1633. doi: 10.1158/2159-8290.CD-21-1683.
130. Benjamin R, Graham C, Yallop D, Jozwik A, Mirci-Danicar OC, Lucchini G, et al. Genome-edited, donor-derived allogeneic anti-CD19 chimeric antigen receptor T cells in paediatric and adult B-cell acute lymphoblastic leukaemia: results of two phase 1 studies. Lancet 2020;396:1885–1894. doi: 10.1016/S0140-6736(20)32334-5.
131. Shah NN, Lee DW, Yates B, Yuan CM, Shalabi H, Martin S, et al. Long-term follow-up of CD19-CAR T-cell therapy in children and young adults with B-ALL. J Clin Oncol 2021;39:1650–1659. doi: 10.1200/JCO.20.02262.
132. Zhao YL, Liu DY, Sun RJ, Zhang JP, Zhou JR, Wei ZJ, et al. Integrating CAR T-Cell therapy and transplantation: Comparisons of safety and long-term efficacy of allogeneic hematopoietic stem cell transplantation after CAR T-cell or chemotherapy-based complete remission in B-cell acute lymphoblastic leukemia. Front Immunol 2021;12:605766. doi: 10.3389/fimmu.2021.605766.
133. Jiang H, Li C, Yin P, Guo T, Liu L, Xia L, et al. Anti-CD19 chimeric antigen receptor-modified T-cell therapy bridging to allogeneic hematopoietic stem cell transplantation for relapsed/refractory B-cell acute lymphoblastic leukemia: an open-label pragmatic clinical trial. Am J Hematol 2019;94:1113–1122. doi: 10.1002/ajh.25582.
134. Shadman M, Gauthier J, Hay KA, Voutsinas JM, Milano F, Li A, et al. Safety of allogeneic hematopoietic cell transplant in adults after CD19-targeted CAR T-cell therapy. Blood Adv 2019;3:3062–3069. doi: 10.1182/bloodadvances.2019000593.
135. Garfall AL, Stadtmauer EA, Hwang WT, Lacey SF, Melenhorst JJ, Krevvata M, et al. Anti-CD19 CAR T cells with high-dose melphalan and autologous stem cell transplantation for refractory multiple myeloma. JCI Insight 2019;4:e127684. doi: 10.1172/jci.insight.127684.
136. Shi X, Yan L, Shang J, Kang L, Yan Z, Jin S, et al. Anti-CD19 and anti-BCMA CAR T cell therapy followed by lenalidomide maintenance after autologous stem-cell transplantation for high-risk newly diagnosed multiple myeloma. Am J Hematol 2022;97:537–547. doi: 10.1002/ajh.26486.
137. Qayed M, McGuirk JP, Myers GD, Parameswaran V, Waller EK, Holman P, et al. Leukapheresis guidance and best practices for optimal chimeric antigen receptor T-cell manufacturing. Cytotherapy 2022;24:869–878. doi: 10.1016/j.jcyt.2022.05.003.
138. Vormittag P, Gunn R, Ghorashian S, Veraitch FS. A guide to manufacturing CAR T cell therapies. Curr Opin Biotechnol 2018;53:164–181. doi: 10.1016/j.copbio.2018.01.025.
139. Li Y, Huo Y, Yu L, Wang J. Quality control and nonclinical research on CAR-T cell products: general principles and key issues. Eng 2019;5:122–131. doi: 10.1016/j.eng.2018.12.003.