Search

NKG2D ligands (NKG2DLs) are broadly expressed in cancer. To target these, we describe an adaptor chimeric antigen receptor (CAR) termed NKG2D/Dap10-12. Herein, T cells are engineered to co-express NKG2D with a fusion protein that comprises Dap10 joined to a Dap12 endodomain. NKG2D/Dap10-12 T cells elicit compelling efficacy, eradicating or controlling NKG2DL-expressing tumors in several established xenograft models. Importantly, durable responses, long-term survival, and rejection of tumor re-challenge are reproducibly achieved. Efficacy is markedly superior to a clinical stage CAR analog, comprising an NKG2D-CD3ζ fusion. Structure-function analysis using an extended CAR panel demonstrates that potency is dependent on membrane proximity of signaling units, high NKG2D cell surface expression, adaptor structure, provision of exogenous Dap10, and inclusion of one rather than three immune tyrosine activation motifs per signaling unit. Potent therapeutic impact of NKG2D/Dap10-12 T cells is also underpinned by enhanced oxidative phosphorylation, reduced senescence, and transcriptomic re-programming for increased ribosomal biogenesis., Competing Interests: Declaration of interests J.M. is CSO, scientific founder, and shareholder of Leucid Bio. M.G., F.K., C.M.H., R.M., C.B., C.T., and D.M.D. are employees of Leucid Bio while P.D. is a former Leucid Bio employee. D.L.-Y. is undertaking a PhD studentship funded by Leucid Bio and has acted as a consultant for Leucid Bio. J.M., D.M.D., D.L.-Y., and F.K. are co-inventors on patent filings in relation to adaptor CAR technology., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Glioblastoma is the most common lethal primary brain tumor, urging evaluation of new treatment options. Chimeric antigen receptor (CAR)-T cells targeting B7 homolog 3 (B7-H3) are promising because of the overexpression of B7-H3 on glioblastoma cells but not on healthy brain tissue. Nanobody-based (nano)CARs are gaining increasing attention as promising alternatives to classical single-chain variable fragment-based (scFv)CARs, because of their single-domain nature and low immunogenicity. Still, B7-H3 nanoCAR-T cells have not been extensively studied in glioblastoma., Methods: B7-H3 nanoCAR- and scFvCAR-T cells were developed and evaluated in human glioblastoma models. NanoCAR-T cells targeting an irrelevant antigen served as control. T cell activation, cytokine secretion and killing capacity were evaluated in vitro using ELISA, live cell imaging and flow cytometry. Antigen-specific killing was assessed by generating B7-H3 knock-out cells using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-genome editing. The tumor tracing capacity of the B7-H3 nanobody was first evaluated in vivo using nuclear imaging. Then, the therapeutic potential of the nanoCAR-T cells was evaluated in a xenograft glioblastoma model., Results: We showed that B7-H3 nanoCAR-T cells were most efficient in lysing B7-H3 pos glioblastoma cells in vitro. Lack of glioblastoma killing by control nanoCAR-T cells and lack of B7-H3 neg glioblastoma killing by B7-H3 nanoCAR-T cells showed antigen-specificity. We showed in vivo tumor targeting capacity of the B7-H3 nanobody-used for the nanoCAR design-in nuclear imaging experiments. Evaluation of the nanoCAR-T cells in vivo showed tumor control in mice treated with B7-H3 nanoCAR-T cells in contrast to progressive disease in mice treated with control nanoCAR-T cells. However, we observed limiting toxicity in mice treated with B7-H3 nanoCAR-T cells and showed that the B7-H3 nanoCAR-T cells are activated even by low levels of mouse B7-H3 expression., Conclusions: B7-H3 nanoCAR-T cells showed promise for glioblastoma therapy following in vitro characterization, but limiting in vivo toxicity was observed. Off-tumor recognition of healthy mouse tissue by the cross-reactive B7-H3 nanoCAR-T cells was identified as a potential cause for this toxicity, warranting caution when using highly sensitive nanoCAR-T cells, recognizing the low-level expression of B7-H3 on healthy tissue., 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.)

The impact of phenotypic, clonal, and functional heterogeneity of chimeric antigen receptor (CAR)-T cells on clinical outcome remains understudied. Here, we integrate clonal kinetics with transcriptomic heterogeneity resolved by single-cell omics to interrogate cellular dynamics of non-transduced (CAR neg ) and transduced (CAR pos ) T cells, in the infusion product (IP) and at the CAR-T cell expansion peak in five B cell acute lymphoblastic leukemia (B-ALL) patients treated with CD19CAR-T cells (varni-cel). We identify significant differences in cellular dynamics in response to therapy. CAR pos T cells at IP of complete response patients exhibit a significantly higher CD4:CD8 ratio, validated in a larger cohort B-ALL patients (n = 47). Conversely, at the expansion peak, there is a clonal expansion of CD8 + effector memory and cytotoxic T cells. Cytotoxic CAR pos γδ-T cells expansion correlates with treatment efficacy validated in a cohort of B-ALL (n = 18) and diffuse large B cell lymphoma (DLBCL) patients (n = 58). Our data provide insights into the complexity of T cell responses following CAR-T cell therapy and suggest drivers of immunotherapy response., Competing Interests: Declaration of interests P.M. is a founder of the spin-off OneChain ImmunoTx, which has no connection with the present research. V.O.-M. reports honoraria and/or consulting fees from BMS/Celgene, Novartis, Gilead/Kite, Miltenyi Biomedicine, Pfizer, and Janssen. A.M.G.-S. reports honoraria and/or consulting fees from Roche, BMS/Celgene, Kyowa Kirin, Novartis, Gilead/Kite, Incyte, Lilly, ADC Therapeutics America, Miltenyi, Ideogen, AbbVie, and Sobi., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lambert-Eaton myasthenic syndrome (LEMS) is an autoantibody-mediated disease of the neuromuscular junction characterized by muscular weakness. Autoantibodies to presynaptic P/Q-type voltage-gated calcium channels (VGCCs) induce defective neuromuscular function. In severe cases, current immunosuppressive and immunomodulatory treatment strategies are often insufficient. First reports show beneficial effects of anti-CD19 chimeric antigen receptor (CAR)-T cell therapy in patients with autoantibody-mediated myasthenia gravis. We report a patient with isolated idiopathic LEMS treated with autologous anti-CD19-CAR-T cells. In this patient, CAR-T infusion leads to expansion of predominantly CD4 + CAR-T cells with a terminally differentiated effector memory cells re-expressing CD45RA (TEMRA)-like phenotype indicating cytotoxic capabilities and subsequent B cell depletion. VGCC antibody titers decrease, resulting in a clinical improvement of LEMS symptoms, e.g., 8-fold increase in walking distance. The patient does not show relevant side effects except for cytokine release syndrome grade 2 and intermittent neutropenia suggesting that anti-CD19 CAR-T cell therapy may be a treatment option in patients with LEMS., Competing Interests: Declaration of interests D.B. is an employee and shareholder of Kyverna Therapeutics with pending patent applications., (Copyright © 2024 The Author(s). Published by 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.

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).)

Hepatocellular carcinoma (HCC) ranks among the most prevalent cancers worldwide, highlighting the urgent need for improved diagnostic and therapeutic methodologies. The standard treatment regimen generally involves surgical intervention followed by systemic therapies; however, the median survival rates for patients remain unsatisfactory. Chimeric antigen receptor (CAR) T-cell therapy has emerged as a pivotal advancement in cancer treatment. Both clinical and preclinical studies emphasize the notable efficacy of CAR T cells in targeting HCC. Various molecules, such as GPC3, c-Met, and NKG2D, show significant promise as potential immunotherapeutic targets in liver cancer. Despite this, employing CAR T cells to treat solid tumors like HCC poses considerable challenges within the discipline. Numerous innovations have significant potential to enhance the efficacy of CAR T-cell therapy for HCC, including improvements in T cell trafficking, strategies to counteract the immunosuppressive tumor microenvironment, and enhanced safety protocols. Ongoing efforts to discover therapeutic targets for CAR T cells highlight the need for the development of more practical manufacturing strategies for CAR-modified cells. This review synthesizes recent findings and clinical advancements in the use of CAR T-cell therapies for HCC treatment. We elucidate the therapeutic benefits of CAR T cells in HCC and identify the primary barriers to their broader application. Our analysis aims to provide a comprehensive overview of the current status and future prospects of CAR T-cell immunotherapy for HCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhou, Wei, Xu, Wu, Dou, Li, Zhang and Zhang.)

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).)

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.)

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).)

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.)

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.

Background Aims: Chimeric antigen receptor T (CAR-T) cells targeting single antigens show limited activity against solid tumors due to poor T cell persistence, low efficiency infiltration, and exhaustion together with heterogeneous tumor-associated antigen (TAA) expression. This is also true in high-risk neuroblastoma (HRNB), a lethal pediatric extracranial malignancy. To overcome these obstacles, a combinational strategy using GD2-specific and GPC2-specific CAR-T cells was developed to improve immunotherapeutic efficacy., Methods: We individually developed GD2-specific and GPC2-specific CARs containing a selective domain (sCAR) which was a peptide of 10 amino acids derived from human nuclear autoantigen La/SS-B. These constructs allowed us to generate two different HRNB antigen-specific CAR-T cells with enhanced biological activity through stimulating sCAR-engrafted T cells via a selective domain-specific monoclonal antibody (SmAb). Binding affinity and stimulation of GD2- and GPC2-specific sCARs by SmAb were measured, and transient and persistent anti-tumor cytotoxicity of GD2sCAR-T and GPC2sCAR-T cells were quantified in neuroblastoma cell lines expressing different TAA levels. The anti-tumor pharmaceutical effects and cellular mechanisms mediated by single or combinational sCAR-T cells were evaluated in vitro and in vivo., Results: GD2- and GPC2-specific sCARs had antigen-specific binding affinity similar to their parental counterparts and were recognized by SmAb. SmAb-mediated stimulation selectively activated sCAR-T proliferation and increased central memory T cells in the final products. SmAb-stimulated sCAR-T cells had enhanced transient cytolytic activity, and combination therapy extended long-term anti-tumor activity in vitro through TNF-α and IL-15 release. Stimulated sCAR-T cells overcame heterogeneous antigen expression in HRNB, and the multi-TAA-targeting strategy was especially efficacious in vivo, inducing apoptosis through the caspase-3/PARP pathway and inhibiting the release of several tumor-promoting cytokines., Conclusions: These data suggest that combined targeting of multiple TAAs is a promising strategy to overcome heterogenous antigen expression in solid tumors and extend CAR-T cell persistence for HRNB immunotherapy., Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

The success of chimeric antigen receptor (CAR) T cell therapy in treating several hematopoietic malignancies has been difficult to replicate in solid tumors, in part because of T cell exhaustion and eventually dysfunction. To counter T cell dysfunction in the tumor microenvironment, we metabolically armored CAR T cells by engineering them to secrete interleukin-10 (IL-10). We show that IL-10 CAR T cells preserve intact mitochondrial structure and function in the tumor microenvironment and increase oxidative phosphorylation in a mitochondrial pyruvate carrier-dependent manner. IL-10 secretion promoted proliferation and effector function of CAR T cells, leading to complete regression of established solid tumors and metastatic cancers across several cancer types in syngeneic and xenograft mouse models, including colon cancer, breast cancer, melanoma and pancreatic cancer. IL-10 CAR T cells also induced stem cell-like memory responses in lymphoid organs that imparted durable protection against tumor rechallenge. Our results establish a generalizable approach to counter CAR T cell dysfunction through metabolic armoring, leading to solid tumor eradication and long-lasting immune protection., Competing Interests: Competing interests Y.G., L.T. and Y.Z. are inventors on patents related to the technology described in this paper. L.T. and Y.G. are cofounders, share-holders and advisors for Leman Biotech. The interests of L.T. were reviewed and managed by EPFL. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

With recent advances in tumor immunotherapy, chimeric antigen receptor T (CAR-T) cell therapy has achieved unprecedented success in several hematologic tumors, significantly improving patient prognosis. However, in solid tumors, the efficacy of CAR-T cell therapy is limited because of high antigen uncertainty and the extremely restrictive tumor microenvironment (TME). This challenge has led to the exploration of new targets, among which fibroblast activation protein (FAP) has gained attention for its relatively stable and specific expression in the TME of various solid tumors, making it a potential new target for CAR-T cell therapy. This study comprehensively analyzed the biological characteristics of FAP and discussed its potential application in CAR-T cell therapy, including the theoretical basis, and preclinical and clinical research progress of targeting FAP with CAR-T cell therapy for solid tumor treatment. The challenges and future optimization directions of this treatment strategy were also explored, providing new perspectives and strategies for CAR-T cell therapy in solid tumors., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background Aims: Hu8F4 is a T-cell receptor-like antibody with high affinity for the leukemia-associated antigen PR1/HLA-A2 epitope. Adapted into a chimeric antigen receptor (CAR) format, Hu8F4-CAR is composed of the Hu8F4 single-chain variable fragment, the human IgG1 CH2CH3 extracellular spacer domain, a human CD28 costimulatory domain and the human CD3ζ signaling domain. We have demonstrated high efficacy of Hu8F4-CAR-T cells against PR1/HLA-A2-expressing cell lines and leukemic blasts from patients with acute myeloid leukemia in vitro. Previous studies have shown that modification of the Fc domains of IgG4 CH2CH3 spacer regions can eliminate activation-induced cell death and off-target killing mediated by mouse Fc gamma receptor-expressing cells., Methods: We generated Hu8F4-CAR(PQ) with mutated Fc receptor binding sites on the CH2 domain of Hu8F4-CAR to prevent unwanted interactions with Fc gamma receptor-expressing cells in vivo., Results: The primary human T cells transduced with Hu8F4-CAR(PQ) can specifically lyse HLA-A2 + PR1-expressing leukemia cell lines in vitro. Furthermore, both adult donor-derived and cord blood-derived Hu8F4-CAR(PQ)-T cells are active and can eliminate U937 leukemia cells in NSG mice., Conclusions: Herein, we demonstrate that modification of the IgG1-based spacer can eliminate Fc receptor binding-induced adverse effects and Hu8F4-CAR(PQ)-T cells can kill leukemia in vivo., Competing Interests: Declaration of competing interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Adoptive immunotherapy with T cells, genetically modified to express a tumor-reactive chimeric antigen receptor (CAR), is an innovative and rapidly developing life-saving treatment for cancer patients without other therapeutic opportunities. CAR-T cell therapy has proven effective only in hematological malignancies. However, although by now only a few clinical trials had promising outcomes, we predict that CAR-T therapy will eventually become an established treatment for several solid tumors. Oncolytic viruses (OVs) can selectively replicate in and kill cancer cells without harming healthy cells. They can stimulate an immune response against the tumor, because OVs potentially stimulate adaptive immunity and innate components of the host immune system. Using CAR-T cells along with oncolytic viruses may enhance the efficacy of CAR-T cell therapy in destroying solid tumors by increasing the tumor penetrance of T cells and reducing the immune suppression by the tumor microenvironment. This review describes recent advances in the design of oncolytic viruses and CAR-T cells while providing an overview of the potential combination of oncolytic virotherapy with CAR-T cells for solid cancers. In this review, we will focus on the host-virus interaction in the tumor microenvironment to reverse local immunosuppression and to develop CAR-T cell effector function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ponterio, Haas and De Maria.)

The ability to remotely control the activity of chimeric antigen receptors (CARs) with small molecules can improve the safety and efficacy of gene-modified T cells. Split ON- or OFF-switch CARs involve the dissociation of tumor-antigen binding from T cell activation (i.e., CD3ζ) on the receptor (R-) and signaling (S-) chains, respectively, that either associate or are disrupted in the presence of a small molecule. Here, we have developed an inducible (i)ON-CAR comprising the anti-apoptotic B cell lymphoma protein 2 protein in the ectodomain of both chains which associate in the presence of venetoclax. We showed that inducible ON (iON)-CAR T cells respond to target tumors cells in the presence of venetoclax or the BH3 mimetic navitoclax in a dose-dependent manner, while there is no impact of the drugs on equivalent second generation-CAR T cells. Within 48 h of venetoclax withdrawal, iON-CAR T cells lose the ability to respond to target tumor cells in vitro as evaluated by Interferon-gamma (IFNγ) production, and they are reliant upon the presence of venetoclax for in vivo activity. Finally, by fusing a degron sequence to the endodomain of the iON-CAR S-chain we generated an all-in-one ON/OFF-switch CAR, the iONØ-CAR, down-regulated by lenalidomide within 4 to 6 for functionally inactive T cells (no IFNγ production) within 24 h. We propose that our remote-control CAR designs can reduce toxicity in the clinic. Moreover, the periodic rest of iON and iONØ-CAR T cells may alleviate exhaustion and hence augment persistence and long-term tumor control in patients., Competing Interests: Competing interests statement:Provisional intellectual property filing has been filed for iON and iONØ CAR designs with inventors including G.M.P.G.A., B.E.C., SS., and M.I.

Immune cell therapy based on chimeric antigen receptor (CAR) technology platform has been greatly developed. The types of CAR immune cell therapy have expanded from T cells to innate immune cells such as NK cells and macrophages, and the diseases treated have expanded from hematological malignancies to non-tumor fields such as infectious diseases and autoimmune diseases. Among them, CAR-T and CAR-NK therapy have observed examples of rapid remission in approved clinical trials, but the efficacy is unstable and plagued by tumor resistance. Trogocytosis is a special phenomenon of intercellular molecular transfer that is common in the immune system and is achieved by recipient cells through acquisition and internalization of donor cell-derived molecules and mediates immune effects. Recently, a novel short-term drug resistance mechanism based on trogocytosis has been proposed, and the bidirectional molecular exchange between CAR immune cells and tumor cells triggered by trogocytosis partially explains the long-term relapse phenomenon after treatment with CAR immune cells. In this review, we summarize the research progress of trogocytosis in CAR immunotherapy, discuss the influencing factors of trogocytosis and its direct and indirect interference with CAR immune cells and emphasize that the interference of trogocytosis can further release the potential of CAR immune cell therapy., (© 2024. The Author(s).)

Few studies evaluated patient-reported outcomes (PROs) for patients with hematologic malignancies receiving with Chimeric Antigen Receptor T (CAR-T) cell therapy. We performed a systematic review and meta-analysis to evaluate the benefits of CAR-T cell therapies focused on PROs. A systematic literature searched from PubMed, Cochrane, and the Web of Science from inception to September 2023. Study selection and data extraction were conducted independently by two reviewers based on pre-specified criteria. The COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) Risk of Bias checklist was used to evaluate the methodological quality of the included studies. The random-effects model was employed to calculate the combined effect and 95% Confidence intervals. This study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline and the protocol was registered with PROSPERO (CRD42024586706). We identified 20,110 studies. Of those, 15 studies with 16 different PRO measures (PROMs) were included in the meta-analysis. CAR-T cell therapy improved PROs in the six domains of general health status, pain, fatigue, depression, social function, and cognitive function: from the general health status (SMD: 0.57, 95% CI: 0.34 to 0.81) to cognitive function (SMD: 0.25, 95% CI: 0.14 to 0.37). The current meta-analysis shows that CAR-T cell therapy produces clinically meaningful differences in PROs. These results suggest that the professional perspective and patient values and preferences should be weighed equally when considering CAR-T cell therapy for hematologic malignancies., (© 2024. The Author(s).)

Introduction: TGFβ is a major immunoinhibitory factor present in the microenvironment of solid tumors. Various cancer types acquire the ability to overexpress TGFβ to escape immune response. Specifically, TGFβ dampens cytotoxic T cell activity, and its presence has been correlated with tumor invasion and poor prognosis., Methods: In this study, we developed two approaches to counteract the effects of TGFβ and provide a functional advantage to genetically engineered T cells in the immunoinhibitory tumor milieu. We designed a TGFβRI-based co-stimulatory switch receptor (CSRI), comprising the TGFβ receptor I extracellular binding domain and a 4-1BB co-stimulatory signaling moiety. Additionally, we tested the efficacy of a TGFβ-binding scFv trap produced by T cells., Results: We demonstrated that both approaches enhanced tumor-specific T cell cytokine secretion, upregulated activation markers, and reduced inhibition markers upon co-culture with melanoma targets. Furthermore, CSRI and the anti-TGFβ trap exhibited improved anti-tumor function in vivo ., Conclusion: Overall, we show that targeting the TGFβ pathway can enhance cellular immunotherapy., Competing Interests: TM and CC are inventors on a submitted US provisional patent application 63/683,259. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Matikhina and Cohen.)

We report the case of long-term persisting rheumatoid arthritis (RA), treated with CD20-CD19 CAR-T when it became associated with diffuse large B cell lymphoma (DLBCL), resulting in a sustained drug-free remission of the preceding RA, as well as of the subsequent DLBCL that formed the indication of the CAR-T therapy using zamtocabtagene autoleucel, with a 1-year follow-up. According to our best knowledge, this is the first published clinical case report of long-term persisting RA treated with CAR-T cell therapy., Competing Interests: Competing interests: DS, PT, PvH, GZ, SH, TO and SM are employees of Miltenyi Biomedicine GmbH. GN received speaker honoraria from Astra Zeneca, AbbVie, GSK, Lilly, Pfizer, MSD, Roche, Miltenyi, SOBI and Swixx., (© 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.)

Enhancing the efficacy of CD19 CAR-T cell therapy can significantly improve patient outcomes by reducing relapse rates in CD19 + B cell malignancies. Exogenous or transgenic cytokines are often used to boost the expansion and durability of CAR-T cells but pose risks of severe toxicities. A promising approach to address these limitations is to immobilize cytokines on the surface of CAR-T cells using transmembrane (TM) anchor domains. Given IL-7 can enhance T-cell proliferation and antitumor activity, our study developed membrane-bound IL-7 constructs using different TM anchor domains (CD8, CD28 and B7-1). We primarily found that the CD8 TM provided superior anchoring for IL-7 compared to CD28 and B7-1. Moreover, the IL-7 construct with a CD8 TM (IL7/CD8) enhanced naïve T cell proliferation and effector functions, and improved the in vitro and in vivo antitumor activity of CD19 CAR-T cells. Importantly, although IL7/CD8 could promote T-cell proliferation, it did not sustain long-term autonomous expansion, which could ensure the safety of CD19 CAR-T cells expressing IL7/CD8 in clinical applications. Collectively, the IL7/CD8 construct represents a promising strategy for enhancing the therapeutic potential of CD19 CAR-T cell therapy., (© 2024. The Author(s).)

Background: Gastrointestinal (GI) cancers represent a significant health burden worldwide. Their incidence continues to increase, and their management remains a clinical challenge. Chimeric antigen receptor (CAR) natural killer (NK) cells have emerged as a promising alternative to CAR-T cells for immunotherapy of GI cancers. Notably, CAR-NK cells offer several advantages, including reduced risk of graft-versus-host disease, lower cytokine release syndrome, and the ability to target cancer cells through both CAR-dependent and natural cytotoxic mechanisms., Main Body: This review comprehensively discusses the development and applications of CAR-NK cells in the treatment of GI cancers. We explored various sources of NK cells, CAR design strategies, and the current state of CAR-NK cell therapy for GI cancers, highlighting recent preclinical and clinical trials. Additionally, we addressed existing challenges and propose potential strategies to enhance the efficacy and safety of CAR-NK cell therapy., Conclusions: Our findings highlight the potential of CAR-NK cells to revolutionize GI cancer treatment and pave the way for future clinical applications., (© 2024. The Author(s).)

Chimeric antigen receptor T-cell (CAR-T) therapy has ushered in a new era for the treatment of multiple myeloma (MM). Numerous clinical studies, especially those involving B-cell maturation antigen (BCMA)-directed CAR-T, have shown remarkable efficacy in patients with relapsed or refractory multiple myeloma (R/R MM). However, a considerable number of patients still experience disease recurrence or progression after BCMA CAR-T treatment, which is attributed to various factors, including antigen escape, CAR-T manufacturing factors, T cell exhaustion, inhibitory effects of tumor microenvironment and impact of prior treatments. The scarcity of effective treatment options following post-CAR-T disease recurrence, coupled with the lack of well-established salvage regimens, leaves patients who do relapse facing a bleak prognosis. In recent years, some academic institutions have achieved certain results in salvage treatments of patients with relapse after BCMA CAR-T treatment through secondary infusion of BCMA CAR-T, changing to non-BCMA-directed CAR-T, double-target CAR-T, bispecific antibodies or other novel therapies. This review summarizes the mechanisms of resistance or relapse after BCMA CAR-T administration and the available data on current salvage treatments, hoping to provide ideas for optimizing clinical salvage therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Fu, Liu, Gao, Lin and He.)

Clinical evidence supports the notion that T cell exhaustion and terminal differentiation pose challenges to the persistence and effectiveness of chimeric antigen receptor-T (CAR-T) cells. MEK1/2 inhibitors (MEKIs), widely used in cancer treatment due to their ability to inhibit aberrant MAPK signaling, have shown potential synergistic effects when combined with immunotherapy. However, the impact and mechanisms of MEKIs on CAR-T cells remain uncertain and controversial. To address this, we conducted a comprehensive investigation to determine whether MEKIs enhance or impair the efficacy of CAR-T cells. Our findings revealed that MEKIs attenuated CAR-T cell exhaustion and terminal differentiation induced by tonic signaling and antigen stimulation, thereby improving CAR-T cell efficacy against hematological and solid tumors. Remarkably, these effects were independent of the specific scFvs and costimulatory domains utilized in CARs. Mechanistically, analysis of bulk and single-cell transcriptional profiles demonstrates that the effect of MEK inhibition was related to diminish anabolic metabolism and downregulation of c-Fos and JunB. Additionally, the overexpression of c-Fos or JunB in CAR-T cells counteracted the effects of MEK inhibition. Furthermore, our Cut-and-Tag assay revealed that MEK inhibition downregulated the JunB-driven gene profiles associated with exhaustion, differentiation, anergy, glycolysis, and apoptosis. In summary, our research unveil the critical role of the MAPK-c-Fos-JunB axis in driving CAR-T cell exhaustion and terminal differentiation. These mechanistic insights significantly broaden the potential application of MEKIs to enhance the effectiveness of CAR-T therapy., (© 2024. The Author(s).)

Objective: To investigate whether modification with IL-21 and CCL19 enhances killing and tumor-infiltrating efficiency of NKP30 CAR-T cells in lung cancer., Methods: The modified IL-21-CCL19 NKP30 CAR-T cells expressing IL-21 and CCL19 fusion gene was constructed based on NKP30 CAR-T cells and stimulated with CD3CD28 antibodies and IL-2. The immunophenotype and migration of the cells in the presence of IL-21 were investigated using flow cytometry and migration experiments. Lactate dehydrogenase (LDH) release and sphere formation assays were used to assess the killing and infiltration capabilities of CAR-T cells, and the secretion levels of IFN-γ, IL-21 and CCL19 were determined with enzyme-linked immunospot assay (ELISPOT) and ELISA. A zebrafish model bearing HCG-27 cell xenograft was established by microinjection of the tumor cells into the yolk sac followed 24 h later by injection of the immune cells at the same site, and the fluorescence signals were captured using a fluorescent microscopy., Results: The NKP30 ligand B7H6, which was almost undetectable in normal tissues and blood cells, was highly expressed (over 90%) in lung cancer cells. Compared with NKP30 CAR-T cells and conventional T cells, IL-21-CCL19 NKP30 CAR-T cells exhibited stronger proliferative and migration capabilities with the formation of central memory T cells. The reduced expressions of CTLA4 and PD1 in the constructed cells resulted in enhanced killing efficiency against lung cancer cells accompanied by significantly increased production of IFN-γ, IL-21 and CCL19. In the zebrafish models, CAR-T cells exhibited stronger cytotoxicity and proliferative abilities than typical T cells, but these differences were not statistically significant between the two CAR-T cells., Conclusion: Modification of NKP30 CAR-T cells with IL-21 and CCL19 facilitates their access into solid tumors for more effective tumor cell killing while producing a large number of memory T cells.

Objective: To construct c-Met CAR-T cells secreting PD-1 antibodies to reduce immune inhibitory effect of tumor cells and enhance the efficacy of CAR-T cell therapy against pancreatic cancer., Methods: Kaplan-Meier Plotter, GEPIA, and Timer 2.0 bioinformatics databases were used to analyze c-Met expression in pancreatic cancer and its correlation with survival and immune infiltration status. In clinical samples of pancreatic cancer and pancreatic cancer Aspc-1 cells, c-Met and PD-L1 expressions were detected using immunohistochemistry or flow cytometry. Using gene editing technology, PD-1 secretory antibodies and HIS tags were linked to second-generation c-Met CAR molecules to construct PD-1/c-Met CAR plasmids, which were then packaged into lentiviruses for infection of activated T cells. The positive rate and cell subset distribution of CAR-T cells were analyzed with flow cytometry, and secretory PD-1 antibodies in cell supernatants were detected using Western blotting. The target cell killing efficiency and proliferative activity of the modified CAR-T cells were evaluated after activation, and cytokine secretion was analyzed using ELISA., Results: The expression of c-Met was significantly higher in pancreatic cancer than in normal tissues, and its expression level was negatively correlated with the patients' survival and positively correlated with immune cell infiltration. The clinical samples of pancreatic cancer tissues expressed significantly higher levels of c-Met and PD-L1 than the adjacent tissues, and 90.7% and 57.7% of Aspc-1 cells were positive for c-Met and PD-L1, respectively. The constructed PD-1/c-Met CAR-T cells were capable of secreting PD-1 antibodies and showed a significantly higher killing efficiency against tumor cells than c-Met CAR-T cells at an effector-to-target ratio of 20: 1, with also a higher proliferative activity after target cell stimulation and higher levels of IL-2 and TNF-α secretin., Conclusion: PD-1/c-Met CAR-T cells have higher killing efficiency against pancreatic cancer cells with also higher proliferative activity than c-Met CAR-T cells.

Multiple sclerosis (MS) is an autoimmune disease affecting the central nervous system (CNS) with the immune system attacking myelin sheaths leading to neuronal death. While several disease-modifying therapies are available to treat MS, these therapies are not universally effective and do not stop disease progression. More personalized long-term treatment options that target specific aspects of the disease, such as reducing relapse frequency, delaying disability accumulation, and addressing symptoms that impact daily functioning, as well as therapies that can promote neuroprotection and repair are needed. Chimeric Antigen Receptor (CAR) Tcell therapies have revolutionized cancer treatment by intravenously (IV) administering a defined dose of T cells with high specificity provided by the CAR. An autologous CAR T cell therapy using suppressive regulatory T cells (Tregs) inducing long-lasting tolerance would be the ideal treatment for patients. Hence, we expanded the application of CAR-T cells by introducing a CAR into Tregs to treat MS patients. We developed a myelin oligodendrocyte glycoprotein (MOG)-specific CAR Treg cell therapy for patients with MS. MOG is expressed on the outer membrane of the myelin sheath, the insulating layer the forms around nerves, making it an ideal target for CAR Treg therapy. Our lead candidate is a 2nd generation CAR, composed of an anti-MOG scFv screened from a large human library. In vitro, we demonstrated CAR-dependent functionality and showed efficacy in vivo using a passive EAE mouse model. Additionally, the MOG-CAR Tregs have very low tonic signaling with a desirable signal-to-noise ratio resulting in a highly potent CAR. In summary our data suggest that MOG-CAR Tregs are a promising MS treatment option with the potential to induce long-lasting tolerance in patients., (© 2024. The Author(s).)

Background: Adoptive cell cancer therapies aim to re-engineer a patient's immune cells to mount an anti-cancer response. Chimeric antigen receptor T and natural killer cells have been engineered and proved successful in treating some cancers; however, the genetic methods for engineering are laborious, expensive, and inefficient and can cause severe toxicities when they over-proliferate., Results: We examined whether the cell-killing capacity of activated T and NK cells could be targeted to cancer cells by anchoring antibodies to their cell surface. Using metabolic glycoengineering to introduce azide moieties to the cellular surface, we covalently attached a dibenzocyclooctyne-modified antibody using the strain-promoted alkyne azide cycloaddition reaction, creating antibody-conjugated T and NK cells. We targeted the immune cells to tumors possessing the xenoantigen, N-glycolyl neuraminic acid GM3 ganglioside, using the 14F7hT antibody. These activated T and NK cells are "armed" with tumour-homing capabilities that specifically lyses antigen-positive cancer cells without off-target toxicities. Moreover, when exposed to target cells, 14F7hT-conjugated T cells that are not preactivated exhibit increased perforin, granzyme, CD69, and CD25 expression and specific cell killing., Conclusions: This research shows the potential for a non-genetic method for redirecting cytotoxic immune cells as a feasible and effective approach for tumor-targeted cell immunotherapy., (© 2024. The Author(s).)