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Despite demonstrating promising outcomes in treating hematologic malignancies, the efficacy of chimeric antigen receptor-modified T (CAR-T) cell therapy remains limited when applied to solid tumors due to tumor immune microenvironment (TIME). Strategies to augment CAR-T cell efficacy against solid tumors have been investigated by ameliorating TIME to a certain extent. In this study, Cabozantinib was utilized in combination with CAR-T cells targeting carbonic anhydrase IX (CAIX) for the treatment of renal cancer. Our findings indicate that combination therapy with CAIX-CAR-T and Cabozantinib demonstrated synergistic efficacy against an orthotopic xenograft tumor model and a subcutaneous tumor model of renal cell carcinoma in mice. Mechanistically, it was observed that CAR-T cells combined with Cabozantinib led to an increase in the infiltration of tumor-infiltrating T cells, while reducing tumor-associated macrophages and M2 polarization. Additionally, Cabozantinib blocked the programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) axis by decreasing the expression of PD-L1 in tumor cells and PD-1 in T cells. Furthermore, Cabozantinib promoted CAR-T cell effector function and reduced T cell exhaustion. This combination therapy represents a novel approach to enhancing CAR-T cell efficacy against solid tumors and holds significant promise for advancing CAR-T cell therapy in clinical settings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Chimeric antigen receptor T (CAR-T) cells show remarkable efficacy for some hematological malignancies. However, CAR targets that are expressed at high level and selective to tumors are scarce. Several strategies have been proposed to tackle the on-target off-tumor toxicity of CAR-T cells that arise from suboptimal selectivity, but these are complicated, with many involving dual gene expression for specificity. In this study, we show that switchable CAR-T cells with a tumor targeting adaptor can mitigate on-target off-tumor toxicity against a low selectivity tumor antigen that cannot be targeted by conventional CAR-T cells, such as CD40. Our system is composed of anti-cotinine murine CAR-T cells and cotinine-labeled anti-CD40 single chain variable fragments (scFv), with which we show selective tumor killing while sparing CD40-expressing normal cells including macrophages in a mouse model of lymphoma. Simple replacement of the tumor-targeting adaptor with a suicidal drug-conjugated tag may further enhance safety by enabling permanent in vivo depletion of the switchable CAR-T cells when necessary. In summary, our switchable CAR system can control CAR-T cell toxicity while maintaining therapeutic efficacy, thereby expanding the range of CAR targets., Competing Interests: Competing interests K.C. and E.Y.C. are founders and shareholders of Ticaros Inc. H.B.P., J.E.L., and G.R.J. are currently employees of Ticaros. K.C., J.C., H.B.P., K.H.K., S.I.K., and G.R.J. are co-inventors on the pending patent for anti-CD40 switchable CAR T cells. The other authors declare no competing interests., (© 2024. The Author(s).)

The redirection of T lymphocytes against tumor-associated or tumor-specific antigens, using bispecific antibodies or chimeric antigen receptors (CAR), has shown therapeutic success against certain hematological malignancies. However, this strategy has not been effective against solid tumors. Here, we describe the development of CAR T cells targeting p95HER2, a tumor-specific antigen found in HER2-amplified solid tumors. These CAR T cells display robust activity against p95HER2-expressing cell lines but demonstrate limited efficacy against patient-derived xenografts. As p95HER2 is invariably detectable on tumor cells that overexpress HER2, but not those that express HER2 at normal levels, we arm p95HER2-specific CAR T cells with affinity-tuned bispecific antibodies against HER2 and CD3 in order to redirect them only to HER2-amplified cells. The combination of p95HER2.CAR T cells and HER2 x CD3 bispecific antibodies lead to a complete regression in three HER2-positive, patient-derived mouse xenografts tumor models. This combination represents a promising strategy to redirect T cells against a subset of HER2-positive tumors., Competing Interests: Competing interests J.A. has received research funds from Roche, Byondis, Menarini and Molecular Partners and consultancy honoraria from Menarini, Mnemo and ARKIN. J.A. is an inventor of patent applications EP22382294, EP20382457, EP16191933.7, EP0930183.5 and P200801652. M.R.A., I.R.R., C.K., E.M., are inventors of patent application EP20382457. M.R.A., S.D., A.G.E., I.R.R., V.N. are inventors of patent application EP22382294. C.K. declares employment, patents and stock ownership with Roche. E.M. declares employment with Roche. E.G. reports: research agreements with Novartis, Roche, Thermo Fisher, AstraZeneca, Taiho, BeiGene, Janssen; consultant/advisor at Roche, Ellipses Pharma, Boehringer Ingelheim, Janssen Global Services, Seattle Genetics, Thermo Fisher, MabDiscovery, Anaveon, F-Star Therapeutics, Hengrui, Sanofi, Incyte; and payment or honoraria for speakers’ bureaus from Merck Sharp & Dohme, Roche, Thermo Fisher, Lilly, Novartis, SeaGen. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Background: CAR-T-cell therapy and bispecific antibody have revolutionized the treatment landscape for multiple myeloma. However, there is currently a lack of studies comparing the efficacy and safety of these two approaches. This meta-analysis assesses the efficacy and safety of B-cell maturation antigen (BCMA)-directed CAR-T-cell therapies and BCMA×CD3 bispecific antibodies as third-line or later interventions for relapsed/refractory multiple myeloma (RRMM)., Methods: We searched PubMed, Embase, Web of Science, and Cochrane databases up to May 31, 2024, identifying 11 eligible studies encompassing 1269 participants. Random-effects models evaluated the primary (complete response (CR) rate) and secondary (overall response rate (ORR)) outcomes, while meta-regression analyses adjusted for relevant covariates., Results: CAR-T-cell therapy achieved significantly higher pooled CR rate (0.54 (95% CI 0.42-0.69) vs bispecific antibodies 0.35 (0.30-0.41), p<0.01) and pooled ORR (0.83 (0.76-0.90) vs 0.65 (0.59-0.71), p<0.01). However, CAR-T therapy had a higher incidence of adverse events, particularly cytokine release syndrome (CRS 0.83 (0.70-0.97) vs bispecific antibodies 0.59 (0.43-0.74), p<0.05). Severe CRS (grade ≥3) occurred at a rate of 0.07 (0.03-0.14) in the CAR-T cell group, contrasting with a negligible rate of 0.01 (0.00-0.02) in the bispecific antibody group (p<0.01). Hematologic adverse events, including neutropenia (grade ≥3; 0.88 (0.81-0.95) vs 0.48 (0.30-0.67), p<0.01) and anemia (grade≥3; 0.55 (0.47-0.62) vs 0.34 (0.28 to 0.40), p<0.01), were also more frequent in the CAR-T-cell group. Furthermore, differences in efficacy were observed among various CAR-T products, with ciltacabtagene autoleucel showing greater efficacy in CR rate (0.77 (0.71-0.84) vs 0.37 (0.32-0.41), p<0.01) and ORR (0.91 (0.83-0.99) vs 0.73 (0.68-0.77), p<0.01) compared with idecabtagene vicleucel., Conclusion: CAR-T-cell therapy demonstrated superior CR rates compared with bispecific antibodies, although with an increase in severe adverse events., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

CD8 + T cell exhaustion hampers control of cancer and chronic infections and limits chimeric antigen receptor (CAR) T cell efficacy. Targeting TET2 in CAR T cells provides therapeutic benefit; however, TET2's role in exhausted T cell (T EX ) development is unclear. In chronic lymphocytic choriomeningitis virus (LCMV) infection, TET2 drove conversion from stem cell-like T EX progenitors toward terminally differentiated and effector (T EFF )-like T EX . TET2 also enforced a terminally differentiated state in the early bifurcation between T EFF and T EX , indicating broad roles for TET2 in acquisition of effector biology. To exploit the therapeutic potential of TET2, we developed clinically actionable TET2- targeted CAR T cells by disrupting TET2 via knock-in of a safety switch alongside CAR knock-in at the TRAC locus. TET2 -targeted CAR T cells exhibited restrained terminal exhaustion in vitro and enhanced antitumor responses in vivo. Thus, TET2 regulates fate transitions in T EX differentiation and can be targeted with a safety mechanism in CAR T cells for improved tumor control.

Purpose: Greater disease burden is a well-established predictor of poorer outcomes following chimeric antigen receptor T-cell (CAR T) therapy. Although bridging therapy (BT) is widely used between leukapheresis and CAR T infusion, limited data have evaluated the impact of BT on CAR T outcomes. In this study, we hypothesized that the quantitative dynamics of radiomic cytoreduction during bridging are prognostic., Experimental Design: Patients with large B-cell lymphoma treated with CD19-CAR T from 2016 to 2022 were included in the study. Metabolic tumor volume (MTV) was determined for all patients on pre-leukapheresis PET and on post-BT/pre-infusion PET in those who received BT. Patients were stratified into "High" and "Low" disease burden using an MTV cutpoint of 65.4cc established by maximally selected log-rank statistic for progression-free survival (PFS)., Results: Of 191 patients treated with CAR T, 144 (75%) received BT. In the BT cohort, 56% had a reduction in MTV post-BT. On multivariate analysis, the MTV trajectory across the bridging period remained significantly associated with PFS (P < 0.001); however, notably, patients with improved MTV (High->Low) had equivalent PFS compared with those with initially and persistently low MTV (Low->Low; HR for High->Low MTV: 2.74; 95% confidence interval, 0.82-9.18). There was a reduction in any grade immune effector cell-associated neurotoxicity syndrome in the High->Low MTV cohort as compared with the High->High MTV cohort (13% vs. 41%; P = 0.05)., Conclusions: This is the first study to use radiomics to quantify disease burden pre- and post-BT in a large real-world large B-cell lymphoma cohort. We demonstrate that effective BT can enable initially high-disease burden patients to achieve post-CAR T outcomes comparable with low-disease burden patients., (©2024 American Association for Cancer Research.)

Abstract: Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of patients with relapsed or refractory hematologic malignancies, but it comes with unique toxicities, notably cytokine release syndrome and ICANS (immune effector cell-associated neurotoxicity syndrome). As experience with CAR T-cell therapy grows, distinct and infrequent neurologic complications are becoming increasingly evident. Recently, reports of acute myelopathy after the administration of CAR T-cell therapies have been accumulating. Despite the establishment of consensus guidelines for managing ICANS, there remains limited guidance on the appropriate investigations and treatments for this rare complication. In this manuscript, we delve into the clinical features, pathophysiology, and strategies for the optimal management of acute myelitis after CAR T-cell therapy and draw insights from reported cases in the literature., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Background: Immunotherapy, such as chimeric antigen receptor T (CAR-T) cells targeting CD33 or CD123, has been well developed over the past decade for the treatment of acute myeloid leukemia (AML). However, the inability to sustain tumor-free survival and the possibility of relapse due to antigen loss have raised concerns. A dual targeting of CD33 and CD123 is needed for better outcomes., Methods: Based on our previously constructed CD33 and CD123 monovalent CAR-T, Loop33 × 123 and Loop123 × 33 CAR-T were constructed with molecular cloning techniques. All CAR-T cells were generated by lentivirus transduction of T cells from healthy donors. Phenotype detection was evaluated on day 7 concerning activation, exhaustion, and subtype proportions. Coculture killing assays were conducted using various AML cell lines and primary AML cells. Degranulation and cytokine secretion levels were detected by flow cytometry. Cell-derived xenograft models were established using wild-type Molm 13 cell lines, or a mixture of Molm 13-KO33 and Molm 13-KO123 cells as an ideal model of immune escape. By monitoring body weight and survival of tumor-bearing mice, Loop33 × 123 and Loop123 × 33 CAR-T cells were further assessed for their efficacy in vivo., Results: In vitro study, our results demonstrated that Loop33 × 123 CAR-T cells could efficiently eliminate AML cell lines and primary AML cells with elevated degranulation and cytokine secretion levels. Compared with our previously constructed monovalent CD33 or CD123 CAR-T cells, Loop33 × 123 CAR-T cells showed superior advantages in an immune escape model. In vivo studies further confirmed that Loop33 × 123 CAR-T cells could effectively prolong the survival of mice without significant toxicity. However, Loop123 × 33 CAR-T cells failed to show the same effects. Furthermore, Loop33 × 123 CAR-T cells efficiently circumvented potential immune escape, a challenge where monovalent CAR-T cells failed., Conclusions: Loop33 × 123 CAR-T targeting CD33 and CD123 could efficiently eliminate AML cells and prolong survival of tumor-bearing mice, while addressing the issue of immune escape., Competing Interests: Declarations Conflict of interest The authors declare no competing interests., (© 2024. The Author(s).)

Chimeric antigen receptor (CAR) cell therapy has achieved groundbreaking success in treating hematological malignancies. However, its application to solid tumors remains challenging due to complex manufacturing processes, limited in vivo persistence, and transient therapeutic effects. In vivo CAR-immune cells induced by gene delivery systems loaded with CAR genes and gene-editing tools have shown efficiency for anti-tumor immunotherapy. In situ programming of autologous immune cells avoids the safety concerns of allogeneic immune cells, and the manufacture of gene delivery systems could be standardized. Therefore, the in vivo editing and in situ generation of CAR-immune cells might potentially overcome the abovementioned limitations of current CAR cell therapy. This review mainly focuses on CAR structures, gene-editing tools, and gene delivery techniques applied in anti-tumor immunotherapy to help design and develop in situ CAR-immune cell therapy. The recent applications of in vivo CAR-immune cell therapy in both hematologic malignancies and solid tumors are investigated. To sum up, the in vivo editing and in situ generation of CAR therapy holds promise for offering a practical, cost-effective, efficient, safe, and widely applicable approach to the next-generation anti-tumor immunotherapy., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Several reports have indicated that chimeric antigen receptor (CAR) T-cell therapy following autologous hematopoietic stem cell transplantation (ASCT) is a promising strategy for refractory/relapsed (r/r) central nervous system lymphoma (CNSL), but the number of reported cases is limited., Methods: The cohort in this retrospective study consisted of 38 patients with r/r CNSL who received CAR T-cell therapy following ASCT at our center between January 2019 and April 2024. Group comparisons of continuous variables were tested using the unpaired Student's t-test or the Mann-Whitney U-test, while categorical variables were analyzed using Fisher's exact test. The Kaplan-Meier method was employed to estimate survival curves, and group comparisons were performed using the log-rank test., Results: The cohort comprised 38 patients with r/r CNSL, all of whom had active CNS involvement. After therapy, the best overall response rate (ORR) of all patients was 78.9%. Subgroup analysis found that a lower ORR was observed in patients with lactate dehydrogenase levels above the upper limit of normal (60.0% vs. 91.3%, P = 0.039). With a median follow-up of 37.5 months, the estimated 1-year overall survival (OS) and progression-free survival (PFS) rates were 72.8% and 57.4%, respectively. The risk factors associated with PFS was no response to current therapy (adjusted hazard ratio: 22.87, P < 0.001). The incidence rates of severe cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome were both 13.2%. Among the 25 patients with secondary CNSL (SCNSL), the best ORRs were 91.7% for those with CNS lesions only and 61.5% for those with CNS and systemic lesions (P = 0.160), while the estimated 1-year PFS rates were 83.3% and 38.5%, respectively (P = 0.030)., Conclusions: CAR T-cell therapy following ASCT shows promising efficacy for r/r CNSL patients. Besides, SCNSL patients with CNS and systemic lesions have inferior treatment efficacy compared to those with CNS lesions only., Competing Interests: Declarations Conflict of interest The authors declare no competing interests. Ethical approval This study was approved by the Ethics Committee of the Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (No: TJ-IRB20160314). Consent to participate The clinical trial was conducted in accordance with the principles of the Declaration of Helsinki, and informed consent was obtained from all eligible patients. Consent for publication All authors have agreed the submission., (© 2024. The Author(s).)

To address immune escape, multi-specific CAR-T-cell strategies use natural ligands that specifically bind multiple receptors on malignant cells. In this context, we propose a split CAR design comprising a universal receptor expressed on T cells and ligand-based switch molecules, which preserves the natural trimeric structure of ligands like APRIL and BAFF. Following optimization of the hinges and switch labeling sites, the split-design CAR-T cells ensure the native conformation of ligands, facilitating the optimal formation of immune synapses between target cancer cells and CAR-T cells. Our CAR-T-cell strategy demonstrates antitumor activities against various B-cell malignancy models in female mice, potentially preventing immune escape following conventional CAR-T-cell therapies in the case of antigen loss or switching. This ligand-based split CAR design introduces an idea for optimizing CAR recognition, enhancing efficacy and potentially improving safety in clinical translation, and may be broadly applicable to cellular therapies based on natural receptors or ligands., Competing Interests: Competing interests The Chinese patent “A method to prepare natural ligand-mediated switchable gene-modified immune cells against multiple targets” with the application number CN202010073487.1 is related to the present work. The applicant for the patent is Peking University Shenzhen Graduate School. The authors declare that they have no competing interests., (© 2024. The Author(s).)

Background: The failure of conventional therapies and the propensity for recurrence and metastasis make triple-negative breast cancer (TNBC) a formidable challenge with grim prognoses and diminished survival rates. Immunotherapy, including immune checkpoint blockade and chimeric antigen receptor (CAR)-T cell therapy, presents innovative and potentially more effective strategies for addressing TNBC. Within this context, the inducible costimulator (ICOS), a member of the CTLA4/CD28 family, plays a crucial role in regulating immune responses and T-cell differentiation by binding to its ligand ICOSL. However, the impact of the ICOS/ICOSL axis on cancer varies., Methods: In this study, immunohistochemistry was conducted to examine the expression level of ICOSL in TNBC tumor tissues. We developed ICOS-enhanced B7H3-CAR-T cells (ICOS-B7H3-CAR) using the third-generation CAR-T cell technology, which featured magnified ICOS expression and targeted the B7H3 antigen. Xenograft and metastasis models of TNBC were conducted to examine the cytotoxicity and durability of CAR-T cells in tumors. Overexpression and CRISPR/Cas9-mediated knockout (KO) techniques were employed to regulate the expression of ICOSL on TNBC cell lines., Results: Notably, we observed elevated ICOSL expression in TNBC tumor tissues, which correlated with poor survival prognosis in patients with TNBC. Compared with conventional B7H3-CAR-T cells, ICOS-B7H3-CAR-T cells significantly inhibited the tumor growth of TNBC cells both in vitro and in vivo, accompanied by increased secretion of cytokines such as interferon gamma and tumor necrosis factor alpha. Furthermore, the in vivo experiments illustrated that ICOS-B7H3-CAR-T cells exhibited prolonged antitumor activity and could effectively eradicate metastases in a TNBC metastasis model, consequently extending survival. Importantly, manipulating the expression of ICOSL on TNBC cells through overexpression or KO significantly influenced the function of ICOS-B7H3-CAR-T cells. This suggests that the level of ICOSL expression on TNBC cells is critical for enhancing the potent antitumor effects of ICOS-B7H3-CAR-T cells., Conclusion: Overall, our study highlights the potential clinical application of ICOS as a promising strategy for combating TNBC recurrence and metastasis., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

A significant challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is its immunosuppressive microenvironment, which is densely populated by protumoral glioma-associated microglia and macrophages (GAMs). Myeloid immune checkpoint therapy targeting the CD47-signal regulatory protein alpha (SIRPα) axis induces GAM phagocytic function, but CD47 blockade monotherapy is associated with toxicity and low bioavailability in solid tumors. In this work, we engineer a CAR T cell against epidermal growth factor receptor variant III (EGFRvIII), constitutively secreting a signal regulatory protein gamma-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eradicate orthotopic EGFRvIII-mosaic GBM in vivo, promoting GAM-mediated tumor cell phagocytosis. In a subcutaneous CD19 + lymphoma mouse model, anti-CD19-SGRP CAR T cell therapy is superior to conventional anti-CD19 CAR T. Thus, combination of CAR and SGRP eliminates bystander tumor cells in a manner that could overcome main mechanisms of CAR T cell therapy resistance, including immune suppression and antigen escape., (© 2024. The Author(s).)

Research has demonstrated the high mortality and morbidity associated with B-Acute lymphoblastic lymphoma (B-ALL). Researchers have developed several therapeutic approaches to combat the disorder. Recently, researchers developed chimeric antigen receptors (CARs)-T cells, which recognize antigens independently of major histocompatibility complexes (MHCs) and activate at a higher level with additional persistence. However, relapsing B-ALL has been reported in several cases. This review article was aimed to collecting recent information regarding the mechanisms used by B-ALL-related lymphocytes to escape from CAR-T cells and the plausible resolution projects., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Acute myeloid leukemia (AML) is a malignant cancer characterized by abnormal differentiation of hematopoietic stem and progenitor cells (HSPCs). While chimeric antigen receptor T (CAR-T) cell immunotherapies target AML cells, they often induce severe on-target/off-tumor toxicity by attacking normal cells expressing the same antigen. Here, we used base editors (BEs) and a prime editor (PE) to modify the epitope of CD123 on HSPCs, protecting healthy cells from CAR-T-induced cytotoxicity while maintaining their normal function. Although BE effectively edits epitopes, complex bystander products are a concern. To enhance precision, we optimized prime editing, increasing the editing efficiency from 5.9% to 78.9% in HSPCs. Epitope-modified cells were resistant to CAR-T lysis while retaining normal differentiation and function. Furthermore, BE- or PE-edited HSPCs infused into humanized mice endowed myeloid lineages with selective resistance to CAR-T immunotherapy, demonstrating a proof-of-concept strategy for treating relapsed AML., Competing Interests: Declaration of interests Y.Z., G.-H.C., and R.-J.J. filed a PCT patent related to this work., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment of hematologic malignancies, yet it carries significant risks, including acute kidney injury (AKI). In this study, we investigated the risk factors and clinical impact of AKI in patients undergoing CAR-T cell therapy. This retrospective study involved hematologic patients treated with CAR-T therapy. Clinical and laboratory data were collected, and clinical outcomes were monitored during follow-up after CAR-T infusion. AKI was defined according to KDIGO criteria. The outcome measures included early mortality, overall survival (OS), and disease-free survival (DFS). Among the 48 patients analyzed, 14 (29%) developed AKI, with a mean onset of 6 days after CAR-T infusion. The risk of AKI was associated with baseline performance status (OR 8.65, IC95% 6.2-12, p = 0.032) and the development of severe cytokine release syndrome post-therapy (OR 16.4 95%CI 1.9-138.5, p = 0.01). Patients with AKI more frequently required intensive care. Furthermore, severe AKI was independently associated with worse clinical outcomes, including reduced OS and DFS (HR 18.2, 95%CI 2.6-27.3, p = 0.003). Additionally, patients who developed AKI post-CAR-T therapy were more likely to progress to chronic kidney disease during follow-up. In conclusion, frail patients undergoing CAR-T therapy are at an increased risk of developing AKI, which can significantly affect both short- and long-term outcomes. Preventive strategies and early recognition of AKI are essential in these patients., (© 2024. The Author(s).)

Chimeric antigen receptor T cell (CAR-T) therapy has emerged as a revolutionary approach in the treatment of malignancies. Despite its remarkable successes, this field continues to grapple with challenges such as scalability, safety concerns, limited therapeutic effect, in vivo persistence, and the need for precise control over CAR expression. In the post-pandemic era of COVID-19 vaccine immunization, the application of messenger RNA (mRNA) encapsulated within lipid nanoparticles (LNPs) has recently garnered significant attention as a potential solution to address these challenges. This review delves into the dynamic landscape of mRNA-LNP technology and its potential implications for CAR-engineered immune cell-based immunotherapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Under compassionate use, chimeric antigen receptor (CAR) T cells have elicited durable remissions in patients with refractory idiopathic inflammatory myopathies (IIMs). Here, we report on the safety, efficacy, and correlative data of the first subject with the immune-mediated necrotizing myopathy (IMNM) subtype of IIM who received a fully human, 4-1BBz anti-CD19-CAR T cell therapy (CABA-201) in the RESET-Myositis phase I/II trial (NCT06154252). CABA-201 was well-tolerated following infusion. Notably, no evidence of cytokine release syndrome or immune effector cell-associated neurotoxicity syndrome was observed. Creatine kinase levels decreased, and muscular strength improved post-infusion. Peripheral B cells were depleted rapidly following infusion, and the subject achieved peripheral B cell aplasia by day 15 post-infusion. Peripheral B cells returned at 2 months post-infusion and were almost entirely transitional. Autoantibodies to SRP-9, SRP-72, SRP-54, and Ro-52, decreased relative to baseline, whereas antibodies associated with pathogens and vaccinations remained stable. The infusion product consisted of predominantly CD4 + effector memory T cells and exhibited in vitro cytolytic activity. Post-infusion, CABA-201 expansion peaked at day 15 and was preceded by a serum IFN-γ peak on day 8 with peaks in serum IL-12p40 and IP-10 on day 15. These data detail the safety, efficacy, and pharmacodynamics of CABA-201 in the first IMNM subject., Competing Interests: Declaration of interests J.V., D.N., J.S., M.W., Z.V., A.E., J.W., J.C., Q.L., T.F., C.S., F.H.-N., D.T., C.M., C.L., D.C., and S.B. are employees of Cabaletta Bio., (Copyright © 2024 Cabaletta Bio. Published by Elsevier Inc. All rights reserved.)

Cytotoxic T lymphocytes (CTLs) play a crucial role in cancer rejection. However, CTLs encounter dysfunction and exhaustion in the immunosuppressive tumor microenvironment (TME). Although the reactive oxygen species (ROS)-rich TME attenuates CTL function, the underlying molecular mechanism remains poorly understood. The nuclear factor erythroid 2-related 2 (Nrf2) is the ROS-responsible factor implicated in increasing susceptibility to cancer progression. Therefore, we examined how Nrf2 is involved in anti-tumor responses of CD8 + T and chimeric antigen receptor (CAR) T cells in the ROS-rich TME. Here, we demonstrated that tumor growth in Nrf2 -/- mice was significantly controlled and was reversed by T cell depletion and further confirmed that Nrf2 deficiency in T cells promotes anti-tumor responses using an adoptive transfer model of antigen-specific CD8 + T cells. Nrf2-deficient CTLs are resistant to ROS, and their effector functions are sustained in the TME. Furthermore, Nrf2 knockdown in human CAR-T cells enhanced the survival and function of intratumoral CAR-T cells in a solid tumor xenograft model and effectively controlled tumor growth. ROS-sensing Nrf2 inhibits the anti-tumor T cell responses, indicating that Nrf2 may be a potential target for T cell immunotherapy strategies against solid tumors., Competing Interests: Declaration of interests C.H. received funding from NeoImmuneTech, Inc. D.C., S.-K.I., and B.H.L. are currently employed by NeoImmuneTech, Inc., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Adoptive cell therapy using chimeric antigen receptor (CAR) T cells has proven to be lifesaving for many cancer patients. However, its therapeutic efficacy has been limited in solid tumors. One key factor for this is cancer-associated fibroblasts (CAFs) that modulate the tumor microenvironment (TME) to inhibit T cell infiltration and induce "T cell dysfunction." Additionally, the sparsity of tumor-specific antigens (TSA) and expression of CAR-directed tumor-associated antigens (TAA) on normal tissues often results in "on-target off-tumor" cytotoxicity, raising safety concerns. Using TALEN-mediated gene editing, we present here an innovative CAR T cell engineering strategy to overcome these challenges. Our allogeneic "Smart CAR T cells" are designed to express a constitutive CAR, targeting FAP + CAFs in solid tumors. Additionally, a second CAR targeting a TAA such as mesothelin is specifically integrated at a TCR signaling-inducible locus like PDCD1. FAPCAR-mediated CAF targeting induces expression of the mesothelin CAR, establishing an IF/THEN-gated circuit sensitive to dual antigen sensing. Using this approach, we observe enhanced anti-tumor cytotoxicity, while limiting "on-target off-tumor" toxicity. Our study thus demonstrates TALEN-mediated gene editing capabilities for design of allogeneic IF/THEN-gated dual CAR T cells that efficiently target immunotherapy-recalcitrant solid tumors while mitigating potential safety risks, encouraging clinical development of this strategy., Competing Interests: Declaration of interests S.Dharani, H.C., A.J., J.V., P.D., L.P., and S.Das are current employees and equity holders at Cellectis. TALEN is a Cellectis patented technology. J.P.F. is a former Cellectis employee and is a current employee of Bristol Myers Squibb., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chimeric antigen receptor (CAR)-T cell therapy demonstrates substantial efficacy in various hematological malignancies. However, its application in solid tumors is still limited. Clinical studies report suboptimal outcomes such as reduced cytotoxicity of CAR-T cells and tumor evasion, underscoring the need to address the challenges of sliding cytotoxicity in CAR-T cells. Despite improvements from fourth and next-generation CAR-T cells, new challenges include systemic toxicity from continuously secreted proteins, low productivity, and elevated costs. Recent research targets genetic modifications to boost killing potential, metabolic interventions to hinder tumor progression, and diverse combination strategies to enhance CAR-T cell therapy. Efforts to reduce the duration and cost of CAR-T cell therapy include developing allogenic and in-vivo approaches, promising significant future advancements. Concurrently, innovative technologies and platforms enhance the potential of CAR-T cell therapy to overcome limitations in treating solid tumors. This review explores strategies to optimize CAR-T cell therapies for solid tumors, focusing on enhancing cytotoxicity and overcoming application restrictions. We summarize recent advances in T cell subset selection, CAR-T structural modifications, infiltration enhancement, genetic and metabolic interventions, production optimization, and the integration of novel technologies, presenting therapeutic approaches that could improve CAR-T cell therapy's efficacy and applicability in solid tumors., (© 2024. The Author(s).)

Surgical resection is a primary treatment option for patients with triple-negative breast cancer (TNBC), but it is associated with a high rate of postoperative local and metastatic relapse. Although chimeric antigen receptor-engineered NK (CAR-NK) cell therapy can specifically recognize and eradicate tumor cells, its therapeutic potency toward TNBCs is markedly suppressed by the hostile tumor microenvironment, which restricts the infiltration, survival, and effector functions of CAR-NK cells inside tumor masses. In this study, HER1-overexpressing TNBC-targeted CAR-NK (HER1-CAR-NK) cells were genetically engineered with catalase to endow them with tolerance toward the high levels of oxidative stress and hypoxia inside TNBC tumors through the catalytic decomposition of hydrogen peroxide, which is a principle reactive oxygen species inside tumors, into O2. We refer to these cells as HER1-CAR-CAT-NK cells. Upon intratumoral fixation with an injectable alginate hydrogel, HER1-CAR-CAT-NK cells enabled sustained tumor hypoxia attenuation and exhibited markedly enhanced persistence and effector functions inside TNBC tumors. As a result, locoregional HER1-CAR-CAT-NK cell therapy not only inhibited the growth of local primary residual tumors but also elicited systemic antitumor activity to suppress the growth of distant tumors. This study highlights that genetic engineering of HER1-CAR-NK cells with catalase is a promising strategy to suppress the postoperative local and distant relapse of TNBC tumors., (©2024 American Association for Cancer Research.)

The use of chimeric antigen receptor (CAR)-T cells has enhanced the range of available therapeutic modalities in the context of cancer treatment. CAR-T cells have demonstrated considerable efficacy in the targeted eradication of blood cancer cells, thereby stimulating substantial interest in the advancement of such therapeutic approaches. However, the efficacy of CAR-T cells against solid tumor cells has been limited due to the presence of various obstacles. Solid tumors exhibit antigenic diversity and an immunosuppressive microenvironment, which presents a challenge for immune cells attempting to penetrate the tumor. CAR-T cells also demonstrate decreased proliferative activity and cytotoxicity. Furthermore, concerns exist regarding tumor antigen loss and therapy-associated toxicity. Currently, scientists are working to enhance the structure of the CAR and improve the survival and efficiency of CAR-T cells in recognizing tumor antigens in solid tumors. Chemotherapy drugs are frequently employed in the treatment of malignant neoplasms and can also be used prior to cell therapy to enhance CAR-T cell engraftment. Recent studies have demonstrated that chemotherapy drugs can mitigate the suppressive impact of TME, eliminate the physical barrier by destroying the tumor stroma, and facilitate greater penetration of immune cells and CAR-T cells into the tumor. This, in turn, increases their survival, persistence, and cytotoxicity, as well as affects the metabolism of immune cells inside the tumor. However, the effectiveness of the combined approach against solid tumors depends on several factors, including the type of tumor, dosage, population of CAR-T cells, and individual characteristics of the body. This review examines the principal obstacles to the utilization of CAR-T cells against solid tumors, proposes solutions to these issues, and assesses the potential advantages of a combined approach to radiation exposure, which has the potential to enhance the sensitivity of the tumor to other agents., (© 2024. The Author(s).)

Intraperitoneal cellular immunotherapy with CAR-redirected lymphocytes is an intriguing approach to target peritoneal carcinomatosis (PC) from ovarian cancer (OC), which is currently evaluated in clinical trials. PC displays a composite structure with floating tumor cells within ascites and solid-like masses invading the peritoneum. Therefore, a comprehensive experimental model is crucial to optimize CAR-cell therapies in such a peculiar environment. Here, we explored the activity of cytokine-induced killer lymphocytes (CIK), redirected by CAR against mesothelin (MSLN-CAR.CIK), within reductionistic 3D models resembling the structural complexity of both liquid and solid components of PC. MSLN-CAR.CIK effectively killed and were functionally efficient against OC targets. In a "floating-like" 3D context with floating OC spheroids, both tumor localization and killing by MSLN-CAR.CIK were significantly boosted by fluid flow. In a "solid-like" context, MSLN-CAR.CIK were recruited through the extracellular matrix on embedded tumor aggregates, with variable kinetics depending on the effector-target distance. Furthermore, MSLN-CAR.CIK penetrated the inner levels of OC spheroids exerting effective tumor killing. Our findings provide currently unknown therapeutically relevant information on intraperitoneal approaches with CAR.CIK, supporting further developments and improvements for clinical studies in the context of locoregional cell therapy approaches for patients with PC from OC., (© 2024. The Author(s).)

Patients with advanced gastric and colorectal cancers have limited treatment options. Since mesothelin is highly expressed in these tumour types, we evaluated the therapeutic benefits of anti-mesothelin hYP218 CAR T cells alone, and in combination with anti-PD1 antibody, pembrolizumab. GEPIA analysis was performed using human gastric (n = 408) and colon cancer tumours (n = 275) in TCGA database, to evaluate mRNA expression of mesothelin, compared to normal tissues. Mesothelin expression in gastric and colorectal cancer cell-lines (n = 5) was analysed using flow cytometry. In vitro efficacy by hYP218 CAR T cells was tested by cytotoxicity and cytokine release assays. In vivo anti-tumour efficacy of hYP218 CAR T cells alone, and in combination with pembrolizumab, was evaluated in NSG mice bearing human gastric (HGC27) and colorectal (SW48) tumour xenografts. Additionally, hYP218 CAR-T cell persistence, activation and exhaustion marker-expression were studied. Mesothelin expression was significantly higher in gastric and colon cancer biopsies compared to normal tissues (p < .005). Mesothelin expression in gastric and colon cancer cell lines ranged from 10 000 to 70 000 molecules per cell. hYP218 CAR T cells demonstrated strong cytotoxic activity at low effector to target ratio, ranging from 0.24 to 1.0. In NSG mouse-models, hYP218 CAR T cells demonstrated anti-tumour efficacy and persisted in the tumour microenvironment in a functional state at day 40 posttreatment with expression of activation markers CD39 and CD69, increased production of IFN-γ and TNF-α and ability to kill tumour cells in vitro when isolated from tumours. There was increased PD1 expression. In combination with pembrolizumab, hYP218 CAR T cells led to slower tumour growth in NSG mice bearing large but not small HGC27 tumours. Anti-tumour efficacy of hYP218 CAR T cells is due to increased accumulation of activated CAR T cells in the tumour and combination with pembrolizumab resulted in improvement in anti-tumour activity of large established tumours. HIGHLIGHTS: Mesothelin expression is significantly higher in gastric and colorectal cancers than normal tissues. hYP218 CAR T cells demonstrate strong anti-tumour activity against mesothelin-positive gastric and colorectal carcinomas. Activated hYP218 CAR T cells persist in the tumour microenvironment and retain their cytotoxic activity. Addition of pembrolizumab in larger tumours enhance CAR T cell efficacy., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

OX40L-CAR-T regs show promise for treating autoimmunity and transplantation rejection.

Cellular immunotherapy has emerged as one of the most potent approaches to treating cancer patients. Adoptive transfer of chimeric antigen receptor (CAR) T cells as well as the use of haploidentical natural killer (NK) cells can induce remission in patients with lymphoma and leukemia. Although the use of CAR T cells has been established, this approach is currently limited for wider use by the risk of severe adverse events, including cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. Moreover, the risk of triggering graft vs host reactions in settings of allogeneic T cell infusion limits the use to autologous CAR T cells if advanced CRISPR engineering is not applied. In contrast, NK cell-based cancer immunotherapy has emerged as a safe approach even in allogeneic settings. However, efficient transduction of primary blood NK cells with vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentivirus commonly used for T cell modification remains challenging. This article presents a detailed method that significantly enhances the transduction efficiency of NK cells by utilizing a short-term culture in cytokine-supplemented medium. It also encompasses the preparation of high-titer and high-quality lentiviral particles for optimal NK cell transduction. Overall, this protocol details the step-by-step culture of NK cells in cytokine-supplemented medium, their transduction with VSV-G lentiviral vectors, and subsequent expansion for functional assays. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of NK cells from human peripheral blood mononuclear cells (PBMCs) Basic Protocol 2: NK cell expansion and transduction with lentivirus for generating CAR-NK cells Support Protocol 1: Plasmid amplification Support Protocol 2: Lentivirus preparation Support Protocol 3: Lentivirus titration., (© 2024 Wiley Periodicals LLC.)

The prognosis of children with acute myeloid leukemia (AML) has improved incrementally over the last decades. However, at relapse, overall survival (OS) ∼40% to 50% and is even lower for patients with chemorefractory disease. Effective and less-toxic therapies are urgently needed for these children. In the last years, immune-directed therapies such as chimeric antigen receptor (CAR)-T cells were introduced, which showed outstanding clinical activity against B-cell malignancies. CART therapies are being developed for AML on the basis of the results obtained for other hematologic malignancies. The biggest challenge of CART therapy for AML is to identify a specific target antigen, since antigens expressed in AML cells are usually shared with healthy hematopoietic stem cells. An overview of prospects of CART in pediatric AML, focused on the common antigens targeted by CART in AML that have been tested or are currently under investigation, is provided in this manuscript., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: Acute myeloid leukemia (AML) is a complex and heterogeneous disease characterized by an aggressive clinical course and limited efficacious treatment options in the relapsed/refractory (R/R) setting. Chimeric antigen receptor (CAR)-modified T (CAR-T) cell immunotherapy is an investigational treatment strategy for R/R AML that has shown some promise. However, obstacles to successful CAR-T cell immunotherapy for AML remain., Areas Covered: In analyses of clinical trials of CAR-T cell therapy for R/R AML, complete responses without measurable residual disease have been reported, but the durability of those responses remains unclear. Significant barriers to successful CAR-T cell therapy in AML include the scarcity of suitable tumor-target antigens (TTA), inherent T cell functional deficits, and the immunoinhibitory and hostile tumor microenvironment (TME). This review will focus on these barriers to successful CAR-T cell therapy in AML, and discuss scientific advancements and evolving strategies to overcome them., Expert Opinion: Achieving durable remissions in R/R AML will likely require a multifaceted approach that integrates advancements in TTA selection, enhancement of the intrinsic quality of CAR-T cells, and development of strategies to overcome inhibitory mechanisms in the AML TME.

Glycosaminoglycans are often deprioritized as targets for synthetic immunotherapy due to the complexity of glyco-epitopes and limited options for obtaining specific subtype binding. Solid tumors express proteoglycans that are modified with oncofetal chondroitin sulfate (CS), a modification normally restricted to the placenta. Here, we report the design and functionality of transient chimeric antigen receptor (CAR) T cells with selectivity to oncofetal CS. Following expression in T cells, the CAR could be "armed" with recombinant VAR2CSA lectins (rVAR2) to target tumor cells expressing oncofetal CS. While unarmed CAR T cells remained inactive in the presence of target cells, VAR2-armed CAR T cells displayed robust activation and the ability to eliminate diverse tumor cell types in vitro. Cytotoxicity of the CAR T cells was proportional to the concentration of rVAR2 available to the CAR, offering a potential molecular handle to finetune CAR T cell activity. In vivo, armed CAR T cells rapidly targeted bladder tumors and increased the survival of tumor-bearing mice. Thus, our work indicates that cancer-restricted glycosaminoglycans may be exploited as potential targets for CAR T cell therapy., Competing Interests: Disclosure and competing interests statement MD as the corresponding author certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (e.g., employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: MD, AS, and PHS are co-founders of, and shareholders in, VAR2 Pharmaceuticals. NAN and TG are consultants for VAR2 Pharmaceuticals. VAR2 Pharmaceuticals is a biotechnology company that specializes in the therapeutic development of the VAR2CSA technology (www.var2pharma.com). The remaining authors declare no competing interests., (© 2024. The Author(s).)

Background: Macrophages engineered with chimeric antigen receptors (CAR) are suitable for immunotherapy based on their immunomodulatory activity and ability to infiltrate solid tumours. However, the production and application of genetically edited, highly effective, and mass-produced CAR-modified macrophages (CAR-Ms) are challenging., Methods: Here, we used homology-independent targeted insertion (HITI) for site-directed CAR integration into the safe-harbour region of human pluripotent stem cells (hPSCs). This approach, together with a simple differentiation protocol, produced stable and highly effective CAR-Ms without heterogeneity., Findings: These engineered cells phagocytosed cancer cells, leading to significant inhibition of cancer-cell proliferation in vitro and in vivo. Furthermore, the engineered CARs, which incorporated a combination of CD3ζ and Megf10 (referred to as FRP5 Mζ ), markedly enhanced the antitumour effect of CAR-Ms by promoting M1, but not M2, polarisation. FRP5 Mζ promoted M1 polarisation via nuclear factor kappa B (NF-κB), ERK, and STAT1 signalling, and concurrently inhibited STAT3 signalling even under M2 conditions. These features of CAR-Ms modulated the tumour microenvironment by activating inflammatory signalling, inducing M1 polarisation of bystander non-CAR macrophages, and enhancing the infiltration of T cells in cancer spheroids., Interpretation: Our findings suggest that CAR-Ms have promise as immunotherapeutics. In conclusion, the guided insertion of CAR containing CD3ζ and Megf10 domains is an effective strategy for the immunotherapy of solid tumours., Funding: This work was supported by KRIBB Research Initiative Program Grant (KGM4562431, KGM5282423) and a Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korean government (Ministry of Science and ICT,Ministry of Health and Welfare) (22A0304L1-01)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The potential of macrophage-mediated phagocytosis as a cancer treatment is promising. Blocking the CD47-SIRPα interaction with a CD47-specific antibody significantly enhances macrophage phagocytosis. However, concerns regarding their toxicity to nontumor cells remain substantial. Here, we engineered chimeric antigen receptor macrophages (CAR-Ms) by fusing a humanized single-chain variable fragment with FcγRIIa and integrating short hairpin RNA to silence SIRPα, thereby disrupting the CD47-SIRPα signaling pathway. These modified CAR-shSIRPα-M cells exhibited an M1-like phenotype, superior phagocytic function, substantial cytotoxic effects on HER2-positive tumor cells, and the ability to eliminate patient-derived organoids. In vivo, CAR-M cells significantly inhibited tumor growth and prolonged survival in tumor-bearing mice. Notably, CAR-shSIRPα-M cells enhanced cytotoxic T-cell infiltration into tumors, thereby enhancing the antitumor response in both the humanized immune system mouse model and immunocompetent mice. Mechanistically, SIRPα inhibition activated inflammatory pathways and the cGAS-STING signaling cascade in CAR-M cells, leading to increased production of proinflammatory cytokines, reactive oxygen species, and nitric oxide, thereby enhancing their antitumor effects. These findings underscore the potential of SIRPα inhibition as a novel strategy to increase the antitumor efficacy of CAR-M cells in cancer immunotherapy, particularly against solid tumors., (© 2024. The Author(s).)

Measurable residual disease (MRD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an independent risk factor for relapse in patients with acute lymphoblastic leukemia (ALL). This study aimed to assess the efficacy, safety, and immune reconstitution of chimeric antigen receptor T-cell (CAR-T) therapy in patients with molecular relapse after allo-HSCT. Eleven patients with molecular relapse of B-cell-ALL who underwent CAR-T therapy after allo-HSCT were enrolled. The rate of MRD negativity after a month of CAR-T infusion was 81.8%. Patients who bridged to second-HSCT after CAR-T therapy ( n  = 3) showed a trend of higher 3-year leukemia-free survival and 3-year overall survival than those who did not ( n  = 8; 100% vs. 75.0%; 95% CI, 45.0-104.9%; p  = 0.370). No treatment-related mortalities were observed. Among patients who did not bridge to second-HSCT and remained in complete remission until the last follow-up ( n  = 6), five of them had not recovered normal immunoglobulin concentrations with a median follow-up of 43 months. CAR-T therapy may be a safe and effective treatment strategy to improve survival after allo-HSCT; however, the problem of prolonged hypogammaglobulinemia in patients who do not bridge to second-HSCT is worth noting.

Glioblastoma multiforme (GBM) is the most common CNS cancer, it has dismal survival rates despite several effective mediators: intensified cytotoxic therapy, chimeric antigen receptor (CAR)-T cell therapy, viral therapy, adoptive cell therapy, immune checkpoint blockade therapy, radiation therapy and vaccine therapy. This review examines the basic concepts underlying immune targeting and examines products such as checkpoint blockade drugs, CAR-T cells, oncolytic viruses, combinatory multimodal immunotherapy and cancer vaccines. New approaches to overcoming current constraints and challenges in GBM therapy are discussed, based on recent studies into these tactics, findings from ongoing clinical trials, as well as previous trial results., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: For decades cancer remained the center of attention in the scientific community as its survival rates are low. Researchers from all around the world wanted to know the core of the problem as to what initiates cancer in a patient and helps with its progression. Many postulations came to light, but Cancer Stem Cells (CSC) was the most appealing and convincing., Main Body: In this review, we shed light on a potential solution to the problem by reviewing CAR-T cells (Chimeric antigen receptor T cells). These specialized T cells are designed to detect specific antigens on cancer cells. We analyse the steps of their formation from the collection of T cells from the patient's bloodstream and modifying it to exhibit specific CAR structures on their surfaces, to reinjecting them back and evaluating their efficacy. We thoroughly investigate the structure of the CAR design with improvements across different generations. The focus extends to the unique properties of CSCs as in how targeting specific markers on them can enhance the precision of cancer therapy., Conclusion: Despite the successes, the review discusses the existing limitations and toxicities associated with CAR-derived therapies, highlighting the ongoing need for research and refinement. Looking ahead, we explore proposed strategies aimed at optimizing CAR-T cell therapy to mitigate adverse effects for improved cancer treatments., Competing Interests: Declarations Ethics Approval and Consent to Participate Not applicable. Consent of Publication Not applicable. Competing Interests The authors declare that there is no competing interest., (© 2024. The Author(s).)

BACKGROUND AND OBJECTIVE: Currently, there is no available method for the prediction of first-in-human (FIH) dose for chimeric antigen receptor-T (CAR-T) cells. The objective of this work was to predict the FIH dose of CAR-T cells from different doses given to mice., Methods: In this study, six scaling methods were evaluated for the prediction of FIH dose for CAR-T cells. The methods were body weight-based fixed exponents such as 1.0 and 0.75, human equivalent dose (HED) using exponents 0.33, two modified HED methods such as using total animal dose (in place of per kg basis) and body surface area in place of body weight using total animal dose with exponent 0.33 and a physiological factor derived from physiological parameters. The FIH doses of six CAR-T cells were predicted in this study. The predicted human doses were compared with the recommended human dose by the US-FDA for four CAR-T cell products, and the literature data were used for the remaining two CAR-T cells., Results: The results indicated that the two modified HED methods and physiological factor are the best and reliable methods for the prediction of FIH dose for CAR-T cells., Conclusions: The proposed methods are simple and accurate in their predictive power and can be used on a spreadsheet., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The human-derived NK-92 cell-based CAR-NK therapy exhibits inconsistency with overall suboptimal efficacy and rapid in vivo clearance of CAR-NK92 cells in cancer patients. Analysis indicates that although pre-existing IgM in healthy individuals (n = 10) strongly recognizes both NK-92 and CAR-NK92 cells, IgG and IgE do not. However, only a subset of cancer patients (3/8) exhibit strong IgM recognition of these cells, with some (2/8) showing pre-existing IgG recognition. These results suggest a natural immunoreactivity between NK-92 and CAR-NK92 cells and pre-existing human Abs. Furthermore, the therapy's immunogenicity is evidenced by enhanced IgG and IgM recognition postinfusion of CAR-NK92 cells. We also confirmed that healthy plasma's cytotoxicity toward these cells is reduced by complement inhibitors, suggesting that Abs may facilitate the rapid clearance of CAR-NK92 cells through complement-dependent cytotoxicity. Given that NK-92 cells lack known receptors for IgG and IgM, identifying and modifying the recognition targets for these Abs on NK-92 and CAR-NK92 cells may improve clinical outcomes. Moreover, we discovered that the 72nd amino acid of the NKG2D receptor on NK-92 cells is alanine. Previous studies have demonstrated polymorphism at the 72nd amino acid of the NKG2D on human NK cells, with NKG2D72Thr exhibiting a superior activation effect on NK cells compared with NKG2D72Ala. We confirmed this conclusion also applies to NK-92 cells by in vitro cytotoxicity experiments. Therefore, reducing the immunoreactivity and immunogenicity of CAR-NK92 and directly switching NK-92 bearing NKG2D72Ala to NKG2D72Thr represent pressing challenges in realizing NK-92 cells as qualified universal off-the-shelf cellular therapeutics., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Chimeric antigen receptor T-cell (CAR T-cell) therapies have emerged as a promising treatment modality for several malignancies, particularly haematological malignancies, by inducing robust antitumour responses. However, CAR T-cell therapies are associated with a spectrum of adverse events, including neurological complications. We here provide a review of neurological adverse events observed in patients undergoing CAR T-cell therapy, focusing on their incidence, clinical manifestations, underlying mechanisms and potential management strategies., (Copyright © 2024. Published by Elsevier Masson SAS.)

Chimeric antigen receptor (CAR) T-cell therapy has achieved significant success in achieving durable and potentially curative responses in patients with hematological malignancies. CARs are tailored fusion proteins that direct T cells to a specific antigen on tumor cells thereby eliciting a targeted immune response. The approval of several CD19-targeted CAR T-cell therapies has resulted in a notable surge in clinical trials involving CAR T cell therapies for hematological malignancies. Despite advancements in understanding response mechanisms, resistance patterns, and adverse events associated with CAR T-cell therapy, the translation of these insights into robust clinical efficacy has shown modest outcomes in both clinical trials and real-world scenarios. Therefore, the assessment of CAR T-cell functionality through rigorous preclinical studies plays a pivotal role in refining therapeutic strategies for clinical applications. This review provides an overview of the various in vitro and animal models used to assess the functionality of CAR T-cells. We discuss the findings from preclinical research involving approved CAR T-cell products, along with the implications derived from recent preclinical studies aiming to optimize the functionality of CAR T-cells. The review underscores the importance of robust preclinical evaluations and the need for models that accurately replicate human disease to bridge the gap between preclinical success and clinical efficacy., Competing Interests: Declaration of competing interest Dr. Melenhorst has patents related to Biomarkers and manufacturing of chimeric antigen receptor-engineered T cells. The remaining authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Natural killer (NK) cells play a vital role in innate immunity and show great promise in cancer immunotherapy. Traditional sources of NK cells, such as the peripheral blood, are limited by availability and donor variability. In addition, in vitro expansion can lead to functional exhaustion and gene editing challenges. This study aimed to harness induced pluripotent stem cell (iPSC) technology to provide a consistent and scalable source of NK cells, overcoming the limitations of traditional sources and enhancing the potential for cancer immunotherapy applications. We developed human placental-derived iPSC lines using reprogramming techniques. Subsequently, an optimized two-step differentiation protocol was introduced to generate high-purity NK cells. Initially, iPSCs were differentiated into hematopoietic-like stem cells using spin-free embryoid bodies (EBs). Subsequently, the EBs were transferred to ultra-low attachment plates to induce NK cell differentiation. iPSC-derived NK (iNK) cells expressed common NK cell markers (NKp46, NKp30, NKp44, CD16 and eomesodermin) at both RNA and protein levels. iNK cells demonstrated significant resilience to cryopreservation and exhibited enhanced cytotoxicity. The incorporation of a chimeric antigen receptor (CAR) construct further augmented their cytotoxic potential. This study exemplifies the feasibility of generating iNK cells with high purity and enhanced functional capabilities, their improved resilience to cryopreservation and the potential to have augmented cytotoxicity through CAR expression. Our findings offer a promising pathway for the development of potential cellular immunotherapies, highlighting the critical role of iPSC technology in overcoming challenges associated with traditional NK cell sources., (© 2024 The Author(s). Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of the Australian and New Zealand Society for Immunology, Inc.)

Autologous chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of lymphoid malignancies, leading to the approval of CD19-CAR T cells for B-cell lymphomas and acute leukaemia, and more recently, B-cell maturation antigen-CAR T cells for multiple myeloma. The long-term follow-up of patients treated in the early clinical trials demonstrates the possibility for long-term remission, suggesting a cure. This is associated with a low incidence of significant long-term side effects and a rapid improvement in the quality of life for responders. In contrast, other types of immunotherapies require prolonged treatments or carry the risk of long-term side effects impairing the quality of life. Despite impressive results, some patients still experience treatment failure or ultimately relapse, underscoring the imperative to improve CAR T-cell therapies and gain a better understanding of their determinants of efficacy to maximize positive outcomes. While the next-generation of CAR T cells will undoubtingly be more potent, there are already opportunities for optimization when utilizing the currently available CAR T cells. This review article aims to summarize the current evidence from clinical, translational and fundamental research, providing clinicians with insights to enhance their understanding and use of CAR T cells., (© 2024 The Author(s). British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

This narrative review examines the promising potential of integrating artificial intelligence (AI) with CRISPR-Cas9 genome editing to advance CAR T-cell therapy. AI algorithms offer unparalleled precision in identifying genetic targets, essential for enhancing the therapeutic efficacy of CAR T-cell treatments. This precision is critical for eliminating negative regulatory elements that undermine therapy effectiveness. Additionally, AI streamlines the manufacturing process, significantly reducing costs and increasing accessibility, thereby encouraging further research and development investment. A key benefit of AI integration is improved safety; by predicting and minimizing off-target effects, AI enhances the specificity of CRISPR-Cas9 edits, contributing to safer CAR T-cell therapy. This advancement is crucial for patient safety and broader clinical adoption. The convergence of AI and CRISPR-Cas9 has transformative potential, poised to revolutionize personalized immunotherapy. These innovations could expand the application of CAR T-cell therapy beyond hematologic malignancies to various solid tumors and other non-hematologic conditions, heralding a new era in cancer treatment that substantially improves patient outcomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Adoptive cell therapy (ACT) enhances the patient's own immune cells' ability to identify and eliminate cancer cells. Several immune cell types are currently being applied in autologous ACT, including T cells, natural killer (NK) cells, and macrophages. The cells' inherent antitumor capacity can be used, or they can be targeted toward tumor-associated antigen through expression of a chimeric antigen receptor (CAR). Although CAR-based ACT has achieved great results in hematologic malignancies, the accessibility of ACT is limited by the autologous nature of the therapy. Induced pluripotent stem cells (iPSCs) hold the potential to address this challenge, because they can provide an unlimited source for the in vitro generation of immune cells. Various immune subsets have been generated from iPSC for application in ACT, including several T-cell subsets (αβT cells, mucosal-associated invariant T cells, invariant NKT [iNKT] cells, and γδT cells), as well as NK cells, macrophages, and neutrophils. iPSC-derived αβT, NK, and iNKT cells are currently being tested in phase I clinical trials. The ability to perform (multiplexed) gene editing at the iPSC level and subsequent differentiation into effector populations not only expands the arsenal of ACT but allows for development of ACT utilizing cell types which cannot be efficiently obtained from peripheral blood or engineered and expanded in vitro., Competing Interests: Conflicts of Interest Disclosure MPD and SJCvdS are members of the Michel Sadelain laboratory, which receives research funds from Fate Therapeutics. This conflict has been disclosed to the Memorial Sloan Kettering Cancer Center who manages this conflict according to institutional policy., (Copyright © 2024 International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Third-generation chimeric antigen receptor T cells (CARTs) for relapsed or refractory (r/r) chronic lymphocytic leukemia (CLL) may improve efficacy compared to second-generation CARTs due to their enhanced CAR design. We performed the first phase 1/2 investigator-initiated trial evaluating escalating doses of third-generation CARTs (HD-CAR-1) targeting CD19 in patients with r/r CLL and B-cell lymphoma. CLL eligibility criteria were failure to two therapy lines including at least one pathway inhibitor and/or allogeneic hematopoietic cell transplantation. Nine heavily pretreated patients received HD-CAR-1 at dose levels ranging from 1 × 10 6 to 200 × 10 6 CART/m 2 . In-house HD-CAR-1 manufacturing was successful for all patients. While neurotoxicity was absent, one case of grade 3 cytokine release syndrome was observed. By day 90, six patients (67%) attained a CR, five of these (83%) with undetectable MRD. With a median follow-up of 27 months, 2-year PFS and OS were 30% and 69%, respectively. HD-CAR-1 products of responders contained significantly more CD4 + T cells compared to non-responders. In non-responders, a strong enrichment of effector memory-like CD8 + T cells with high expression of CD39 and/or CD197 was observed. HD-CAR-1 demonstrated encouraging efficacy and exceptionally low treatment-specific toxicity, presenting new treatment options for patients with r/r CLL. Trial registration: #NCT03676504., (© 2024. The Author(s).)

Recent years have witnessed the success of αβ T cells engineered to express chimeric antigen receptors (CARs) in treating haematological cancers. CARs combine the tumour antigen binding capability of antibodies with the signalling functions of the T-cell receptor (TCR) ζ chain and co-stimulatory receptors. Despite the success, αβ CAR T cells face limitations. Possible solutions would be the use of γδ T cells and new chimeric receptors, such as TCR fusion constructs (TRuCs). Notably, γδ CAR T cells are gaining traction in pre-clinical and clinical studies, demonstrating a promising safety profile in several pilot studies. This review delves into the current understanding of γδ CAR and TCR fusion construct T cells, exploring the opportunities and challenges they present for cancer treatment., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells has shown promising results in early-phase clinical studies. However, advancing CAR-NK cell therapeutic efficacy is imperative. In this study, we investigated the impact of a fourth-generation CD19-targeted CAR (CAR.19) coexpressing IL-27 on NK-92 cells. We observed a significant improvement in NK-92 cell proliferation and cytotoxicity activity against B-cell cancer cell lines, both in vitro and in a xenograft mouse B-cell lymphoma model. Our systematic transcriptome analysis of the activated NK-92 CAR variants further supports the potential of IL-27 in fourth-generation CARs to overcome limitations of NK cell-based targeted tumor therapies by providing essential growth and activation signals. Integrating IL-27 into CAR-NK cells emerges as a promising strategy to enhance their therapeutic potential and elicit robust responses against cancer cells. These findings contribute substantially to the mounting evidence supporting the potential of fourth-generation CAR engineering in advancing NK cell-based immunotherapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

CD19-directed chimeric antigen receptor (CAR) T-cell therapy has had a dramatic impact on the natural history and survival of patients with high-risk B-cell non-Hodgkin lymphoma. Accompanying this success has been the development of new fields of medicine and investigation into toxicity risks and mitigation therapies, mechanisms of resistance and the development of novel and next generation products and strategies in order to address relapse, and issues related to global access and health care economics. This article is a survey of each of these areas as it pertains to the rapidly evolving field of CAR T-cell therapy, written by an International community of lymphoma experts, who also happen to be women., (© 2023 John Wiley & Sons Ltd.)

Chimeric antigen receptor-natural killer (CAR-NK) cell therapy is emerging as a promising cancer treatment, with notable safety and source diversity benefits over CAR-T cells. This study focused on optimizing CAR constructs for NK cells to maximize their therapeutic potential. We designed seven CD19 CAR constructs and expressed them in NK cells using a retroviral system, assessing their tumour-killing efficacy and persistence. Results showed all constructs enhanced tumour-killing and prolonged survival in tumour-bearing mice. In particular, CAR1 (CD8 TMD-CD3ζ SD)-NK cells showed superior efficacy in treating tumour-bearing animals and exhibited enhanced persistence when combined with OX40 co-stimulatory domain. Of note, CAR1-NK cells were most effective at lower effector-to-target ratios, while CAR4 (CD8 TMD-OX40 CD- FcεRIγ SD) compromised NK cell expansion ability. Superior survival rates were noted in mice treated with CAR1-, CAR2 (CD8 TMD- FcεRIγ SD)-, CAR3 (CD8 TMD-OX40 CD- CD3ζ SD)- and CAR4-NK cells over those treated with CAR5 (CD28 TMD- FcεRIγ SD)-, CAR6 (CD8 TMD-4-1BB CD-CD3ζ 1-ITAM SD)- and CAR7 (CD8 TMD-OX40 CD-CD3ζ 1-ITAM SD)-NK cells, with CAR5-NK cells showing the weakest anti-tumour activity. Increased expression of exhaustion markers, especially in CAR7-NK cells, suggests that combining CAR-NK cells with immune checkpoint inhibitors might improve anti-tumour outcomes. These findings provide crucial insights for developing CAR-NK cell products for clinical applications., (© 2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

The activity of anti-CD19 chimerci antigen receptor (CAR) T-cell therapy in chronic lymphocytic leukemia (CLL) with Richter's transformation (RT) to aggressive large B-cell lymphoma (LBCL) is largely unknown. In a multicenter retrospective study, we report the safety and efficacy of CAR T-cell therapy in patients with RT (N=30) compared to patients with aggressive B-cell lymphoma (N=283) and patients with transformed indolent non-Hodgkin lymphoma (iNHL) (N=141) between April 2016 and January 2023. Two-thirds of patients received prior therapy for CLL before RT and 89% of them received B-cell receptor and B-cell lymphoma 2 inhibitors. Toxicities of CAR T-cell therapy in RT were similar to other lymphomas, with no fatalities related to cytokine release syndrome or immune effector-cell associated neurotoxicity synderome. The 100-day overall response rate and complete response rates in patients with RT were 57% and 47%, respectively. With a median follow-up of 19 months, the median overall survival (OS) was 9.9 months in patients with RT compared to 18 months in de novo LBCL and not reached in patients with transformed iNHL. The OS at 12 months was 45% in patients with RT compared with 62% and 75% in patients with de novo LBCL and transformed iNHL, respectively. In a multivariate analysis, worse OS was associated with RT histology, elevated lactate dehydrogenase, and more prior lines of therapy. CAR T-cell therapy can salvage a proportion of patients with CLL and RT exposed to prior targeted agents; however, efficacy in RT is inferior compared to de novo LBCL and transformed iNHL.

Chimeric antigen receptor (CAR) T cells are in standard clinical use to treat relapsed or refractory hematologic malignancies, such as non-Hodgkin lymphoma, multiple myeloma and acute lymphoblastic leukemia. Owing to the rapidly progressing field of CAR T-cell therapy and the lack of generally accepted treatment guidelines, we hypothesized significant differences between European centers in prevention, diagnosis and management of short- and long-term complications. To capture the current CAR T-cell management among European Society for Blood and Marrow Transplantation (EBMT) centers and to determine the medical need and specific areas for future clinical research the EBMT Transplant Complications Working Party performed a survey among 227 EBMT CAR T-cell centers. We received complete servey answers from 106 centers (47%) addressing questions in the areas of product selection, CAR T-cell logistics, management of cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome as well as management in later phases including prolonged cytopenias. We identified common patterns in complication management, but also significant variety in clinical management of the centers in important aspects. Our results demonstrate a high medical need for treatment harmonization and future clinical research in the following areas: treatment of steroid-refractory and very severe cytokine release syndrome/neurotoxicity, treatment of cytopenia, early discharge and outpatient management, as well as immunoglobulin substitution.