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Lung cancer is the leading cause of cancer-related death worldwide, with poor outcomes even for those diagnosed at early stages. Current standard-of-care for most non-small cell lung cancer (NSCLC) patients involves an array of chemotherapy, radiotherapy, immunotherapy, targeted therapy, and surgical resection depending on the stage and location of the cancer. While patient outcomes have certainly improved, advances in highly personalized care remain limited. However, there is growing excitement around harnessing the power of tumor-infiltrating lymphocytes (TILs) through the use of adoptive cell transfer (ACT) therapy. These TILs are naturally occurring, may already recognize tumor-specific antigens, and can have direct anti-cancer effect. In this review, we highlight comparisons of various ACTs, including a brief TIL history, show current advances and successes of TIL therapy in NSCLC, discuss the potential roles for epigenetics in T cell expansion, and highlight challenges and future directions of the field to combat NSCLC in a personalized manner., 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 B.V. All rights reserved.)

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.)

Self-renewal programs in leukemia stem cells (LSCs) predict poor prognosis in patients with acute myeloid leukemia (AML). We identify CD4 + T cell-derived interleukin (IL)-21 as an important negative regulator of self-renewal of LSCs. IL-21/IL-21R signaling favors asymmetric cell division and differentiation in LSCs through the activation of p38-MAPK signaling, resulting in reduced LSC numbers and significantly prolonged survival in murine AML models. In human AML, serum IL-21 at diagnosis is identified as an independent positive prognostic biomarker for outcome and correlates with improved survival and higher complete remission rates in patients that underwent high-dose chemotherapy. IL-21 treatment inhibits primary LSC function and enhances the effect of cytarabine and CD70 CAR T cell treatment on LSCs in vitro. Low-dose IL-21 treatment prolongs the survival of AML mice in syngeneic and xenograft experiments. Therefore, promoting IL-21/IL-21R signaling on LSCs may be an approach to reduce stemness and increase differentiation in AML., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.)

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.)

Background: Adoptive T-cell therapy has demonstrated clinical activity in B-cell malignancies, offering hope for its application to a broad spectrum of cancers. However, a significant portion of patients with solid tumors experience primary or secondary resistance to this treatment modality. Target antigen loss resulting either from non-uniform antigen expression or defects in antigen processing and presentation machinery is one well-characterized resistance mechanism. Constitutively expressed membrane-anchored interleukin-12 (caIL-12) has demonstrated enhanced antitumor activity and low systemic exposure in multiple preclinical adoptive T-cell treatment models with homogeneous tumor antigen expression. In this study, we assess the therapeutic impact of caIL-12 on target antigen-negative variants in syngeneic mouse models., Methods: Target antigen-positive tumors were generated by transducing B16F10 melanoma cells (B16) or Lewis Lung Carcinoma cells (LLC) with a construct expressing the OVA antigen, SIINFEKL, tagged to ubiquitin (B16-U-OVA, LLC-U-OVA), while B16 or LLC tumors served as antigen-negative variants. C57BL/6J mice were subcutaneously injected with heterogeneous tumors composed of 80% B16-U-OVA and 20% B16. Bilateral tumors were established by injecting the left flank with B16-U-OVA or LLC-U-OVA tumors and the right flank injected with B16 or LLC tumors. The tumor-bearing mice then underwent 5.5 Gy total body irradiation, followed by adoptive transfer of OT-I TCR-T cells engineered with or without caIL-12., Results: TCR-T cells (OT-I) delivered caIL-12 to the B16-U-OVA tumor sites and induced robust tumor control and survival benefits in mice bearing a heterogeneous tumor with OVA-negative variants. caIL-12 exerted its effect on OVA-negative B16 variants primarily by priming and activating endogenous antitumor CD8 T cells via antigen spreading. In addition, antigen spreading induced by OT-I-caIL-12 resulted in controlling OVA-negative tumors implanted at distant sites. This therapeutic effect required antigen-specific TCR-T cells and caIL-12 to colocalize at the tumor site, along with endogenous CD8 T cells capable of recognizing shared tumor antigens., Conclusion: Expression of caIL-12 by tumor-targeting T cells demonstrated therapeutic effect against target-antigen-negative tumor variants, primarily through the induction of antigen spreading. These findings highlight the potential of caIL-12 to address challenges of antigen escape and tumor heterogeneity that may limit the efficacy of T-cell therapy against solid tumors., Competing Interests: Competing interests: LZ is an inventor on NIH patent of inducible IL-12. CSH owns patents and royalties from multiple inventions in the field of immunotherapy and cellular therapy at the NIH and at Rutgers Cancer Institute, serves as a consultant or advisory board for Capstan Therapeutics, Neogene Therapeutics, Vir Biotechnology, sponsored research from Neogene Therapeutics, Iovance Biotherapeutics, equity and company officer for Scarlet TCR. The remaining authors declare no competing financial 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.)

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: Chimeric antigen receptor T (CAR-T) cell therapy has been shown remarkable efficacy in the treatment of hematological malignancies in recent years. However, a considerable proportion of patients would experience tumor recurrence and deterioration. Insufficient CAR-T cell persistence is the major reason for relapse. Multiple strategies to enhance the long-term antitumor effects of CAR-T cells have been explored and developed. In this study, we focused on tyrosine kinase inhibitors (TKIs), which have emerged immunomodulatory potential besides direct tumoricidal effects., Methods: Here, we screened 50 approved TKIs drugs and identified that afatinib (AFA) markedly enhanced the expressing of CD62L and inhibited reactive oxygen species level in T cells. And the underlying mechanisms of AFA medicating T cells were explored by detecting signal transduction, and metabolism pattern. Furthermore, we co-cultured AFA with CAR-T cells during the preparation stage and multianalyses of differentiation characteristics, metabolic profiling, and RNA sequencing revealed that AFA induce comprehensive metabolism remodeling and fate reprogramming. Based on it, we finally identified the antitumor efficacy of AFA-pretreatment CAR-T compared with negative-control CAR-T., Results: We identified that AFA blocked the T-cell receptor (TCR) and phosphoinositide 3-kinase-protein kinase B-mechanistic target of rapamycin signaling pathways, induced metabolic reprogramming and modulated T-cell differentiation. When combined with CAR-T cells, AFA inhibited the exhaustion and enhanced the persistence and cytotoxicity. Our results revealed that the pretreatment of AFA enables to boost CAR-T cells with strong antitumor cytotoxicity in leukemia mouse model., Conclusions: Our study systematically demonstrated that AFA pretreatment effectively enhanced CAR-T cells antitumor performance, which presents a novel optimization strategy for potent and durable CAR-T cell therapy., Competing Interests: Competing interests: None declared., (© 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.)

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.)

Background: Despite remarkable achievements in applying chimeric antigen receptor (CAR)-T cells to treat hematological malignancies, they remain much less effective against solid tumors, facing several challenges affecting their clinical use. We previously showed that multichain DNAX-activating protein (DAP) CAR structures could enhance the safety and efficacy of CAR-T cells when used against solid tumors. In particular, mesothelin (MSLN)-targeted CAR-T cell therapy has therapeutic potential in MSLN-positive solid tumors, including ovarian cancer and mesothelioma., Methods: In vitro cell killing assays and xenograft model were utilized to determine the anti-tumor efficacy of MSLN targeting DAP-CAR-T cells and other CAR-T cells. ELISA and flow cytometry analysis were used to assess the cytokine secretion capacity and proliferation ability. Eight patients with MSLN expression were enrolled to evaluate the safety and efficacy of MSLN-DAP CAR-T cell therapy. Single-cell sequencing was performed to explore the dynamics of immune cells in patients during treatment and to identify the transcriptomic signatures associated with efficacy and toxicity., Results: We found that multichain DAP-CAR formed by combining a natural killer cell immunoglobulin-like receptor truncator and DAP12 exhibited better cytotoxicity and tumor-killing capacity than other natural killer cell-activated receptors associated with DAP12, DAP10, or CD3Z. The safety and efficacy of MSLN-DAP CAR-T cell therapy in patients with ovarian cancer and mesothelioma were evaluated in a single-arm, open-label clinical trial (ChiCTR2100046544); two patients achieved partial response, while four patients had a stable disease status. Furthermore, single-cell sequencing analysis indicated that KT032 CAR-T cell infusion could recruit more immune cells and temporarily remodel the TME., Conclusions: Our study highlights the safety and therapeutic efficacy of multiple-chain DAP-CAR-T cell therapy targeting MSLN to treat patients with ovarian cancer and mesothelioma., Trial Registration: ChiCTR.org.cn, ChiCTR2100046544 . May 21, 2021., Competing Interests: Declarations Ethics approval and consent to participate This study was approved by the Institution Review Board of The First Affiliated Hospital of Nanjing Medical University (2021-SR-084). This study conformed to the principles of the Helsinki Declaration. Healthy donors or patients consented to participate in the study. All participants provided written informed consent prior to participation. The animal experiment in this study was approved by the Institutional Ethics Committee of Nanjing Medical University (IACUC2103036). Consent for publication All participants provided written informed consent for publication. Competing interests Authors EX-W, C-W, and JZ-Z were employed by Nanjing CART Medical Technology Co., Ltd. The other authors declare that they have no competing interests., (© 2024. The Author(s).)

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.)

Background: Neoadjuvant therapy with trastuzumab, pertuzumab and paclitaxel (THP) has significantly improved the prognosis of patients with human epidermal growth factor receptor 2 (HER2) + breast cancer (BC). However, there remains a subset of non-responsive patients. Thus, this study sought to identify key regulators of THP neoadjuvant therapy resistance and potential targets to sensitize sensitivity., Methods: The Cancer Genome Atlas database, Gene Expression Omnibus and membrane protein database were used to identify the key regulator of THP neoadjuvant resistance. The biological functions and mechanisms of delta-like 4 proteins (DLL4) in THP therapy resistance were investigated in vitro and in vivo using the bioinformatic analysis, multiplex immunofluorescence, flow cytometry, sphere formation assays and chromatin immunoprecipitation, etc. Furthermore, DLL4-targeted chimeric antigen receptor (CAR)-T cells were established to sensitize THP therapy., Results: DLL4 was identified as a key target in THP neoadjuvant therapy resistance for HER2 + BC. Mechanistically, DLL4 + tumor cells exhibited enhanced stemness and resistance to the THP neoadjuvant chemotherapy. Additionally, soluble DLL4 can split away from tumor cells and diffuse into the stroma, where it can activate the Notch signaling pathway in neutrophils, inducing the formation and release of neutrophil extracellular traps (NETs) by regulating the transcription of MPO, PDIA4 and ELANE. This led to the exclusion of lymphocyte infiltration, thereby enhancing therapy resistance. What is more, we designed a DLL4-targeted CAR-T to eliminate DLL4 + tumor cells and reverse the resistant status., Conclusions: Our study revealed novel functions of DLL4 in cell stemness and immune infiltration, including NET formation and T cell exclusion, which collectively contributed to THP neoadjuvant therapy resistance in HER2 + BC. Furthermore, we provided a CAR-T-based therapy to sensitize the THP neoadjuvant therapy., (© 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.)

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: Adoptive cell therapies (ACT) exhibit excellent efficacy in hematological malignancy. However, its application in solid tumors still has many challenges partly due to the tumor immune microenvironment. Cryo-thermal therapy (CTT) can induce an acute inflammatory response and remold the immune environment, providing an appropriate environment for the activation of adaptive immunity. However, it remains unclear whether CTT can enhance the efficacy of ACT., Methods: A bilateral B16F10 tumor-bearing mouse model was used to assess whether CTT could enhance the efficacy of ACT. The right large tumor was subjected to CTT, and the left small tumor was collected for flow cytometry, RNA-seq, immunohistochemistry and TCR Vβ sequencing. Finally, bilateral B16F10 tumor-bearing mice and 4T1 tumor-bearing mice were used to assess the efficacy after CTT combined with ACT., Results: CTT dramatically reshaped the immune microenvironment in distal tumors to an acute inflammatory state by promoting innate cell infiltration, increasing cytokine production by macrophages and DCs. The remodeling of the tumor immune microenvironment further enhanced the antitumor efficiency of ACT by increasing the proliferation of T cells, promoting activation of the effector functions of T cells and boosting the expansion of TCR clones., Conclusions: Our results suggest that CTT can significantly reshape the tumor immunosuppressive microenvironment and convert "cold tumors" into "hot tumors," thereby enhancing ACT-induced immune responses and maximizing the therapeutic effect of ACT., Competing Interests: Declarations Conflict of interest The authors declare no competing interests. Ethics approval All animal experiments were approved by the Animal Welfare Committee of Shanghai Jiao Tong University, and experimental methods were performed in accordance with the guidelines of Shanghai Jiao Tong University Animal Care (approved by Shanghai Jiao Tong University Scientific Ethics Committee, Registration No. 2020017)., (© 2024. The Author(s).)

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).)

Background: Adoptive immunotherapy using natural killer (NK) cells has attracted considerable interest in numerous clinical trials targeting both hematological and solid tumors. Traditionally, NK cells are primarily derived from either peripheral blood (PB) or umbilical cord blood (UCB). However, these methods can lead to variability and heterogeneity within the NK cell population. In contrast, induced pluripotent stem cell (iPSC)-derived NK (iNK) cells provide a more controlled and uniform cellular population, suitable for large-scale clinical applications. This makes iNK cells a promising option for developing "off-the-shelf" immunotherapeutic products. Nevertheless, current NK cell differentiation protocols, which rely on embryoid body (EB) cultures, are labor-intensive and susceptible to unwanted heterogeneity during differentiation. Here, we developed a more efficient approach for generating iNK cells by employing a monolayer and feeder-free differentiation protocol, alongside optimized culture media., Methods: The iNK cells were generated using a two-step in vitro monolayer feeder-free system following NK cell development. To evaluate their maturity, phenotypic analysis was performed using flow cytometry, comparing with PB-NK cells and the NK-92 cell line. Additionally, single-cell RNA sequencing was performed to examine their transcriptomic profiles. The cytotoxic activity of the iNK cells was evaluated by co-culturing with cholangiocarcinoma (CCA) and breast cancer (BCA) cell lines in both monolayer (2D) and tumor spheroid (3D) co-culture systems., Results: We successfully differentiated iPSCs into mesoderm (ME), hematopoietic stem/progenitor cells (HSPCs), and NK cells. The resulting iNK cells exhibited typical NK cell markers such as CD45, CD56, and CD16, and expressed key functional proteins, including both activating and inhibitory receptors. Single-cell RNA sequencing confirmed that the transcriptomic profile of our iNK cells closely resembles that of PB-NK cells. Importantly, our iNK cells demonstrated strong cytotoxic abilities against various CCA and BCA cell lines, surpassing the NK-92 cell line in both monolayer cultures and tumor spheroid cultures., Conclusion: This study highlights the potential of iPSCs as an effective alternative cell source for generating NK cells. Using a two-step in vitro monolayer feeder-free system, we successfully generated iNK cells that not only expressed key NK cell markers and their receptors but also displayed a transcriptomic profile closely resembling PB-NK cells. Furthermore, iNK cells exhibited cytotoxicity against CCA and BCA cell lines comparable to that of PB-NK cells. This approach could pave the way for off-the-shelf NK cell products, potentially enhancing the effectiveness of adoptive NK cell therapy., Competing Interests: Declarations Ethics approval and consent to participate The use of peripheral blood samples in this study was approved by the Siriraj Institutional Review Board, Thailand (Si 320/2021) on May 5, 2021, in accordance with the Helsinki Declaration of 1975. This research was part of the project titled” Generation of “off-the-shelf” iPSC-derived natural killer (NK) cells as a prototype for cell-based therapy”. Each patient was given an information sheet explaining the study and provided written informed consent. Consent for publication All authors have contributed to, read, and approved the manuscript for submission. Competing interests The authors declare that they have 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).)

Chimeric antigen receptor T-cell (CAR-T) immunotherapy has achieved remarkable success in treating chemotherapy-refractory hematological malignancies. However, its efficacy in solid and brain tumors remains limited due to challenges such as insufficient target antigens, poor T-cell adaptability, inefficient tumor site trafficking, and the immunosuppressive tumor microenvironment. To address these challenges, Shaw and colleagues proposed an innovative strategy targeting cancer-specific exons (CSEs) in pediatric solid and brain tumors. Using RNA sequencing data from 16 tumor types, the study identified 157 highly tumor-specific targets, including both known and novel proteins. The researchers validated several targets, including FN1 and COL11A1, demonstrating their therapeutic potential in in vitro and in vivo models. The study's approach of integrating exon-level analysis with a broad search for extracellular matrix proteins offers a new frontier for CAR-T therapy, providing valuable insights for improving immunotherapy in pediatric solid tumors. Although promising, the study also highlights the need for further evaluation of tumor recurrence and CAR-T cell exhaustion. The identification of novel pan-tumor targets may revolutionize CAR-T therapy design and expand its application in pediatric cancer treatment., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication All authors have read and agreed to the published version of the manuscript. Competing interests The authors declare that they have no competing interests., (© 2024. The Author(s).)

Abstract: Hematologic toxicity is a common side effect of chimeric antigen receptor T-cell (CAR-T) therapies, being particularly severe among patients with relapsed or refractory multiple myeloma (MM). In this study, we characterized 48 patients treated with B-cell maturation antigen (BCMA) CAR-T cells to understand kinetics of cytopenia, identify predictive factors, and determine potential mechanisms underlying these toxicities. We observed that overall incidence of cytopenia was 95.7%, and grade >3 thrombocytopenia and neutropenia, 1 month after infusion, was observed in 57% and 53% of the patients, respectively, being still present after 1 year in 4 and 3 patients, respectively. Baseline cytopenia and high peak inflammatory markers were highly correlated with cytopenia that persisted up to 3 months. To determine potential mechanisms underlying cytopenias, we evaluated the paracrine effect of BCMA CAR-T cells on hematopoietic stem and progenitor cell (HSPC) differentiation using an ex vivo myeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-T cells (spCAR) halted HSPC differentiation, promoting more immature phenotypes, which could be prevented with a combination of interferon γ, tumor necrosis factor α/β, transforming growth factor β, interleukin-6 (IL-6) and IL-17 inhibitors. Single-cell RNA sequencing demonstrated upregulation of transcription factors associated with early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1, CEBPA) and a decrease in the activity of key regulons involved in neutrophil and monocytic maturation (ID2 and MAFB). These results suggest that CAR-T activation induces HSPC maturation arrest through paracrine effects and provides potential treatments to mitigate the severity of this toxicity., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

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.)

Background and Purpose: In patients with metastatic melanoma who respond to anti-PD-1 therapy, the proliferation of intra-tumour CD8+ T cells is directly correlated with the clinical response, making tumour-infiltrating lymphocytes (TILs) a treatment of interest in combination with a PD-1 inhibitor, which is the undisputed gold standard in the management of metastatic melanoma. The aim of this trial was, therefore, to evaluate the safety and efficacy of sequential combination therapy consisting of nivolumab (a PD-1 inhibitor) and TILs adoptive T cells in patients with metastatic melanoma., Materials and Methods: We performed an exploratory, prospective, single-centre, open-label, non-randomised, uncontrolled phase I/II study. We enrolled 10 previously untreated patients with advanced melanoma. The treatment regimen was neoadjuvant anti-PD-1 therapy followed by 2 injections of TILs and a second sequence of anti-PD-1 therapy., Results and Interpretation: Among the four patients who received the autologous TILs + nivolumab combination, three (75%) achieved an objective response (two achieved a partial response [PR] at the end of the study, two achieved a complete response [CR]), and one achieved a CR at the end of the study. Among these three patients, one had a PR, and two had stable disease (SD) after the nivolumab course and before any TILs administration, reinforcing the importance of the tumour response after TILs injection. These responses were persistent, ranging from 9 months to 3.4 years.

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.)

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 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).)

Background: One of the significant challenges for cell therapies, such as chimeric antigen receptor (CAR)-T cell therapy, is the poor infiltration of immune cells into tumor tissues. CAR-monocytes/macrophages (CAR-M) are promising therapies because of their enrichment in the tumor microenvironment. Thus, we constructed a novel CAR-M to facilitate the infiltration of T cells and other immune cells., Methods: The suicide gene inducible caspase-9 ( iCasp9 ) and anti-erb-b2 receptor tyrosine kinase 2 (HER2) CAR elements were transfected into THP1 (an immortalized human monocyte cell line) by lentivirus. The suicide efficiency and specific anti-tumor efficacy were assessed using flow cytometry, inCucyte, and tumor-bearing BALB/c-nude mouse models. The activation of related signaling pathways in CAR-THP1 activation was explored by transcriptome sequencing. Finally, the synergistic therapeutic efficacy of CAR-THP1 combined with RAK cell treatment was demonstrated in tumor-bearing NOD.CB17-Prkdc scid Il2rg tm1 /Bcgen mouse models., Results: We developed a novel CAR-THP1, which incorporated iCasp9, CD3ζ, and CD147 intracellular segments, based on the first-generation HER2-CAR backbone. By constructing and comparing a series of CARs with different permutations, CAR-CD3ζ-CD147-iCasp9-THP1 was selected as the optimal combination. CAR-CD3ζ-CD147-iCasp9-THP1 initiated suicide quickly and efficiently under the control of iCasp9 gene, which enabled us to achieve controlled proliferation of CAR-THP1. CAR-THP1 also exhibited robust specific anti-tumor efficacy independently of T cells in vitro and in vivo . Through transcriptional sequencing, we found that CAR-THP1 tended to differentiate into the M1 phenotype and bridged innate and adaptive immunity. A combination of CAR-THP1 and Retronectin actived killer cells (RAKs) showed better therapeutic efficiency, as the metalloproteinases (MMPs) secreted by CAR-THP1 facilitated the degradation of the dense tumor matrix. This further assisted intratumoral infiltration of T cells and augmented the anti-tumor immune response., Conclusion: CAR-THP1 might be effective against HER2-positive tumor cells and has great potential for combination therapy with other immune cells., (Copyright © 2024 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.)

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.)

Purpose of Review: While anti-CD19 CAR T-cell therapy represents a major advance in systemic diffuse large B-cell lymphomas, central nervous system (CNS) lymphomas have been excluded from pivotal trials because of the fear of neurotoxicity. The purpose of this review was to assess the efficacy and tolerance of CAR T-cells in CNS lymphomas based on recently published studies., Recent Findings: All the studies on CAR T-cell therapy for both primary and secondary CNS lymphomas reported high response rates (complete response rates ranging from 32 to 67%) in highly pre-treated patients. One-year PFS reached 40 to 60% in several studies. Neurotoxicity occurred in 36 to 68% of patients, including grade 3-4 neurotoxicity in 4.5 to 29% of patients. CAR T-cell therapy appears to be a very promising treatment in CNS lymphomas, with efficacy results close to those observed in systemic lymphomas. The toxicity profile, notably regarding neurotoxicity, is reassuring and should not prevent the development of CAR T-cells in the disease., Competing Interests: Declarations. Competing Interests: Caroline Houillier declares no conflict of interest. Sylvain Choquet has received consultant fees from Atara, Pierre Fabre, Astra Zeneca, Novartis and Takeda; has received honoraria for presentations from Pierre Fabre, Takeda, Novartis, Gilead, Janssen; has received support for attending meetings from Pierre Fabre, Janssen. Human and Animal Rights: All reported studies with human subjects performed by the authors have been previously published and complied with all applicable ethical standards (including the Helsinki declaration and its amendments, institutional/national research committee standards, and international/national/institutional guidelines)., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

T-cell-mediated therapeutic strategies are the most potent effectors of cancer immunotherapy. However, an essential barrier to this therapy in solid tumors is disrupting the anti-cancer immune response, cancer-immunity cycle, T-cell priming, trafficking and T-cell cytotoxic capacity. Thus, reinforcing the anti-cancer immune response is needed to improve the effectiveness of T-cell-mediated therapy. Tumor-associated protease ADAM10, endothelial cells (ECs) and cytotoxic CD8 + T cells engage in complex communication via adhesion, transmigration and chemotactic mechanisms to facilitate an anti-cancer immune response. The precise impact of ADAM10 on the intricate mechanisms underlying these interactions remains unclear. This paper broadly explores how ADAM10, through different routes, influences the efficacy of T-cell-mediated therapy. ADAM10 cleaves CD8 + T-cell-targeting genes and impacts their expression and specificity. In addition, ADAM10 mediates the interactions of adhesion molecules with T cells and influences CD8 + T-cell activity and trafficking. Thus, understanding the role of ADAM10 in these events may lead to innovative strategies for advancing T-cell-mediated therapies., (© 2024 the Australian and New Zealand Society for Immunology, Inc.)

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).)

Intratumorally injection of CAR-T cells to treat melanoma can reduce the incidence of systemic side effects and ensure an adequate concentration of CAR-T cells. However, few targeting ligands and hostile tumor microenvironment (TME) inhibit the CAR-T cells' survival and infiltration. An in situ hyaluronic acid‑manganese-capsaicin complex nanogel (HA-Mn-CAP Gel) was designed by forming complex nanogel based on the main skeleton material of hyaluronic acid (HA). It targeted CD44 and TRPV 1 receptors through HA and CAP, concentrated and released Mn at melanoma, subsequently relieving the hypoxia in the tumor and increasing the expression of CAR-T cells' specific ligands. Cell experiments showed that HA-Mn-CAP Gel significantly enhanced the expression of representative NKG 2 D ligands (MICA/B and ULBP 2/5/6 ) >2.7 times on A375 melanoma cells. Sequential administration of HA-Mn-CAP Gel and NKG 2 D/CAR-T increased the tumor inhibition rate about 1.5 times that of the NKG 2 D/CAR-T group in melanoma-bearing NSG mice. The results of immunohistochemistry and immunofluorescence assays showed that the combined HA-Mn-CAP Gel and NKG 2 D/CAR-T significantly increased the proliferation and infiltration of T cells, especially for CD8 + cells. Therefore, the new promising strategy of increasing the target ligand expression and adjusting TME before administering CAR-T cells is suitable for treating solid tumors., 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 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).)