Predictive value of pre-infusion tumor growth rate for the occurrence and severity of CRS and ICANS in lymphoma under CAR T-cell therapy.

Chimeric antigen receptor T-cell therapy (CART) can be administered outpatient yet requires management of potential side effects such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). The pre-infusion tumor burden is associated with CRS, yet there is no data on the relevance of pre-infusion tumor growth rate (TGR). Our objective was to investigate TGR for the occurrence and severity of CRS and ICANS. Consecutive patients with available pre-baseline and baseline (BL) imaging before CART were included. TGR was determined as both absolute (abs) and percentage change (%) of Lugano criteria-based tumor burden in relation to days between exams. CRS and ICANS were graded according to ASTCT consensus criteria. Clinical metadata was collected including the international prognostic index (IPI), patient age, ECOG performance status, and LDH. Sixty-two patients were included (median age: 62 years, 40% female). The median pre-BL TGR [abs] and pre-BL TGR [%] was 7.5 mm2/d and 30.9%/d. Pre-BL TGR [abs] and pre-BL TGR [%] displayed a very weak positive correlation with the grade of CRS (r[abs] = 0.14 and r[%] = 0.13) and no correlation with ICANS (r[abs] = - 0.06 and r[%] = - 0.07). There was a weak positive correlation between grade of CRS and grade of ICANS (r = 0.35; p = 0.005) whereas there was no significant correlation of CRS or ICANS to any other of the examined parameters. The pre-infusion TGR before CART was weakly associated with the occurrence of CRS, but not the severity, whereas there were no significant differences in the prediction of ICANS. There was no added information when compared to pre-infusion tumor burden alone. Outpatient planning and toxicity management should not be influenced by the pre-infusion TGR.

Ocular adverse events following CAR-T cell therapy: A pharmacovigilance study and systematic review.

The rise of immuno-oncology, including the use of chimeric antigen receptor T-cell (CAR-T) therapy is bringing in a new wave of cancer treatments, particularly in hematologic malignancies. However, data on their adverse events, particularly of the eye, is under-reported. To assess the ocular adverse events associated with the six FDA-approved CAR-T cell therapies, a disproportionality analysis utilizing the FAERS database was conducted from the first quarter of 2017 to the third quarter of 2023, as well as a systematic review of case reports of ocular events following CAR-T cell therapy up to December 20, 2023. A total of 53 ocular adverse events were identified from the FDAs FAERS database. The adverse events most frequently observed were mydriasis and xerophthalmia with tisagenlecleucel (Kymriah). The systematic review resulted in 8 case reports encompassing 19 patients which included a total of 27 events. This study demonstrates the importance of anticipation of potential ocular adverse events by ophthalmologists and oncologists as they can greatly contribute to morbidity in patients with cancer.

[CAR T cells as drugs for novel therapies (advanced therapy medicinal products)]. / CAR-T-Zellen als Arzneimittel für neuartige Therapien (Advanced Therapy Medicinal Products).

BACKGROUND: Two commercial chimeric antigen receptor (CAR) T cell products, axicabtagene-ciloleucel (Yescarta®) and tisagenlecleucel (Kymriah®), are registered for the treatment of B cell neoplasia, for which an increased supply of CAR T cell products is required. PROBLEM: The production of patient-specific CAR T cells as advanced therapy medicinal products (ATMPs) poses considerable challenges with respect to logistics, regulation, and manufacturing. METHOD: Review of the CAR T cell manufacturing process and the regulatory network, the current challenges, and future development capabilities of CAR T cells for adoptive immunotherapy. RESULTS: CAR T cells are manufactured under individualized, laborious, good manufacturing practice-conforming processes in decentralized or in specialized centers. Starting from the patient's leukapheresis product, T cells are genetically engineered ex vivo with a CAR, amplified, and after extensive quality control re-applied to the patient. Most CAR T cell products are manufactured in a manual or semi-automated process; fully automated, supervised, and closed systems are increasingly applied to meet the need for a growing number of CAR T cell products. In this setting, research aims at providing allogeneic CAR T cell products or non-T cells such as natural killer cells for broad applications. CONCLUSION: The significance of CAR T cells in adoptive immunotherapy is continuously growing. As individualized cell products, manufacturing requires highly efficient processes under the control of harmonized protocols and regulations so as to ensure the quality of the ATMP in view of increasing demand and to develop new fields in therapy.

[CAR T-cell therapy for malignant B-cell lymphoma : A new treatment paradigm]. / CAR-T-Zellen zur Behandlung von malignen B-Zell-Lymphomen : Ein neues Therapieprinzip.

Following the first demonstration of efficacy of anti-CD19-directed chimeric antigen receptor (CAR) T cells in a patient with relapsed chronic lymphocytic leukemia (CLL) in 2011, pivotal studies for this innovative therapy were initially conducted in multiple relapsed or refractory (r/r) childhood and young adult acute B­cell leukemia and in aggressive adult B­cell lymphoma. The studies demonstrated efficacy even in chemotherapy-refractory disease, resulting in the first approval of autologous and genetically engineered T cells for the treatment of r/r B­cell acute lymphoblastic leukemia (B-ALL) in the US for the product tisagenlecleucel (Kymriah®, Novartis) back in 2018. Approval for the treatment of r/r aggressive B­cell lymphoma followed shortly thereafter for tisagenlecleucel and axicabtagene ciloleucel (Yescarta, Kite/Gilead). This review focuses on the treatment of aggressive B­cell lymphoma and other CD19 positive B­cell lymphomas by summarizing the study results of clinically tested CAR T cells, discussing possible resistance mechanisms, and providing an outlook on ongoing studies with new target antigens for the treatment of B­cell lymphomas.

[CAR T-cell therapy for multiple myeloma]. / CAR-T-Zell-Therapie beim multiplen Myelom.

Chimeric antigen receptor (CAR) T-cell treatment is a novel immunotherapy utilizing the patient's own immune system as a "weapon against tumor cells". In patients with multiple myeloma (MM), CAR T-cell therapy has been investigated in clinical trials. The current data on B­cell maturation antigen (BCMA)-targeted CAR T-cells have shown impressive efficacy, and official approval is expected shortly. However, the majority of patients relapse after CAR T-cell therapy. Moreover, the treatment can cause severe adverse events such as cytokine release syndrome and neurotoxicity with lethal outcome. The cost-benefit ratio of this treatment also needs to be optimized. Despite these limitations, CAR T-cell therapy represents an attractive option for patients with MM and has the potential to be incorporated into the standard of care.

Stealth transgenes enable CAR-T cells to evade host immune responses.

BACKGROUND: Adoptive cell therapy, such as chimeric antigen receptor (CAR)-T cell therapy, has improved patient outcomes for hematological malignancies. Currently, four of the six FDA-approved CAR-T cell products use the FMC63-based αCD19 single-chain variable fragment, derived from a murine monoclonal antibody, as the extracellular binding domain. Clinical studies demonstrate that patients develop humoral and cellular immune responses to the non-self CAR components of autologous CAR-T cells or donor-specific antigens of allogeneic CAR-T cells, which is thought to potentially limit CAR-T cell persistence and the success of repeated dosing. METHODS: In this study, we implemented a one-shot approach to prevent rejection of engineered T cells by simultaneously reducing antigen presentation and the surface expression of both Classes of the major histocompatibility complex (MHC) via expression of the viral inhibitors of transporter associated with antigen processing (TAPi) in combination with a transgene coding for shRNA targeting class II MHC transactivator (CIITA). The optimal combination was screened in vitro by flow cytometric analysis and mixed lymphocyte reaction assays and was validated in vivo in mouse models of leukemia and lymphoma. Functionality was assessed in an autologous setting using patient samples and in an allogeneic setting using an allogeneic mouse model. RESULTS: The combination of the Epstein-Barr virus TAPi and an shRNA targeting CIITA was efficient and effective at reducing cell surface MHC classes I and II in αCD19 'stealth' CAR-T cells while retaining in vitro and in vivo antitumor functionality. Mixed lymphocyte reaction assays and IFNγ ELISpot assays performed with T cells from patients previously treated with autologous αCD19 CAR-T cells confirm that CAR T cells expressing the stealth transgenes evade allogeneic and autologous anti-CAR responses, which was further validated in vivo. Importantly, we noted anti-CAR-T cell responses in patients who had received multiple CAR-T cell infusions, and this response was reduced on in vitro restimulation with autologous CARs containing the stealth transgenes. CONCLUSIONS: Together, these data suggest that the proposed stealth transgenes may reduce the immunogenicity of autologous and allogeneic cellular therapeutics. Moreover, patient data indicate that repeated doses of autologous FMC63-based αCD19 CAR-T cells significantly increased the anti-CAR T cell responses in these patients.

PD-1 downregulation enhances CAR-T cell antitumor efficiency by preserving a cell memory phenotype and reducing exhaustion.

BACKGROUND: Despite the encouraging outcome of chimeric antigen receptor T cell (CAR-T) targeting B cell maturation antigen (BCMA) in managing relapsed or refractory multiple myeloma (RRMM) patients, the therapeutic side effects and dysfunctions of CAR-T cells have limited the efficacy and clinical application of this promising approach. METHODS: In this study, we incorporated a short hairpin RNA cassette targeting PD-1 into a BCMA-CAR with an OX-40 costimulatory domain. The transduced PD-1KD BCMA CAR-T cells were evaluated for surface CAR expression, T-cell proliferation, cytotoxicity, cytokine production, and subsets when they were exposed to a single or repetitive antigen stimulation. Safety and efficacy were initially observed in a phase I clinical trial for RRMM patients. RESULTS: Compared with parental BCMA CAR-T cells, PD-1KD BCMA CAR-T cell therapy showed reduced T-cell exhaustion and increased percentage of memory T cells in vitro. Better antitumor activity in vivo was also observed in PD-1KD BCMA CAR-T group. In the phase I clinical trial of the CAR-T cell therapy for seven RRMM patients, safety and efficacy were initially observed in all seven patients, including four patients (4/7, 57.1%) with at least one extramedullary site and four patients (4/7, 57.1%) with high-risk cytogenetics. The overall response rate was 85.7% (6/7). Four patients had a stringent complete response (sCR), one patient had a CR, one patient had a partial response, and one patient had stable disease. Safety profile was also observed in these patients, with an incidence of manageable mild to moderate cytokine release syndrome and without the occurrence of neurological toxicity. CONCLUSIONS: Our study demonstrates a design concept of CAR-T cells independent of antigen specificity and provides an alternative approach for improving the efficacy of CAR-T cell therapy.

Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16.

BACKGROUND: Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies in the Western world. Contributing factors include a high frequency of late-stage diagnosis, the development of chemoresistance, and the evasion of host immune responses. Currently, debulking surgery and platinum-based chemotherapy are the treatment cornerstones, although recurrence is common. As the clinical efficacy of immune checkpoint blockade is low, new immunotherapeutic strategies are needed. Chimeric antigen receptor (CAR) T cell therapy empowers patients' own T cells to fight and eradicate cancer, and has been tested against various targets in OC. A promising candidate is the MUC16 ectodomain. This ectodomain remains on the cell surface after cleavage of cancer antigen 125 (CA125), the domain distal from the membrane, which is currently used as a serum biomarker for OC. CA125 itself has not been tested as a possible CAR target. In this study, we examined the suitability of the CA125 as a target for CAR T cell therapy. METHODS: We tested a series of antibodies raised against the CA125 extracellular repeat domain of MUC16 and adapted them to the CAR format. Comparisons between these candidates, and against an existing CAR targeting the MUC16 ectodomain, identified K101 as having high potency and specificity. The K101CAR was subjected to further biochemical and functional tests, including examination of the effect of soluble CA125 on its activity. Finally, we used cell lines and advanced orthotopic patient-derived xenograft (PDX) models to validate, in vivo, the efficiency of our K101CAR construct. RESULTS: We observed a high efficacy of K101CAR T cells against cell lines and patient-derived tumors, in vitro and in vivo. We also demonstrated that K101CAR functionality was not impaired by the soluble antigen. Finally, in direct comparisons, K101CAR, which targets the CA125 extracellular repeat domains, was shown to have similar efficacy to the previously validated 4H11CAR, which targets the MUC16 ectodomain. CONCLUSIONS: Our in vitro and in vivo results, including PDX studies, demonstrate that the CA125 domain of MUC16 represents an excellent target for treating MUC16-positive malignancies.

Development of a compact bidirectional promoter-driven dual chimeric antigen receptor (CAR) construct targeting CD19 and CD20 in the Sleeping Beauty (SB) transposon system.

BACKGROUND: A bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy. METHODS: We developed a second-generation CAR directing CD19 and CD20 antigens, incorporating them in a head-to-head orientation from a bidirectional promoter using a single Sleeping Beauty transposon system. The efficacy of bidirectional promoter-driven dual CD19 and CD20 CAR T cells was determined in vitro against cell lines expressing either, or both, CD19 and CD20 antigens. In vivo antitumor activity was tested in Raji lymphoma-bearing immunodeficient NOD-scid IL2Rgammanull (NSG) mice. RESULTS: Of all tested promoters, the bidirectional EF-1α promoter optimally expressed transcripts from both sense (CD19-CAR) and antisense (GFP.CD20-CAR) directions. Superior cytotoxicity, cytokine production and antigen-specific activation were observed in vitro in the bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells. In contrast, a unidirectional construct driven by the EF-1α promoter, but using self-cleaving peptide-linked CD19 and CD20 CARs, showed inferior expression and in vitro function. Treatment of mice bearing advanced Raji lymphomas with bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells effectively controlled tumor growth and extended the survival of mice compared with group treated with single antigen targeted CAR T cells. CONCLUSION: The use of bidirectional promoters in a single vector offers advantages of size and robust CAR expression with the potential to expand use in other forms of gene therapies like CAR T cells.

Liquid biopsy approach to monitor the efficacy and response to CAR-T cell therapy.

BACKGROUND: Chimeric antigen receptor (CAR)-T cells are approved for use in the treatment of hematological malignancies. Axicabtagene ciloleucel (YESCARTA) and brexucabtagene autoleucel (TECARTUS) genetically modified autologous T cells expressing an anti-CD19 scFv based on the FMC63 clone have shown impressive response rates for the treatment of CD19+B cell malignancies, but there remain challenges in monitoring long-term persistence as well as the functional characterization of low-level persisting CAR-T cells in patients. Furthermore, due to CD19-negative driven relapse, having the capability to monitor patients with simultaneous detection of the B cell malignancy and persisting CAR-T cells in patient peripheral blood is important for ensuring timely treatment optionality and understanding relapse. METHODS: This study demonstrates the development and technical validation of a comprehensive liquid biopsy, high-definition single cell assay (HDSCA)-HemeCAR for (1) KTE-X19 CAR-T cell identification and analysis and (2) simultaneously monitoring the CD19-epitope landscape on neoplastic B cells in cryopreserved or fresh peripheral blood. Proprietary anti-CD19 CAR reagents, healthy donor transduced CAR-T cells, and patient samples consisting of malignant B cell fractions from manufacturing were used for assay development. RESULTS: The CAR-T assay showed an approximate limit of detection at 1 cell in 3 million with a sensitivity of 91%. Genomic analysis was additionally used to confirm the presence of the CAR transgene. This study additionally reports the successful completion of two B cell assays with multiple CD19 variants (FMC63 and LE-CD19) and a unique fourth channel biomarker (CD20 or CD22). In patient samples, we observed that CD19 isoforms were highly heterogeneous both intrapatient and interpatient. CONCLUSIONS: With the simultaneous detection of the CAR-T cells and the B cell malignancy in patient peripheral blood, the HDSCA-HemeCAR workflow may be considered for risk monitoring and patient management.

Metastatic gastric cancer target lesion complete response with Claudin18.2-CAR T cells.

Treatment of hematologic malignancies with patient-derived anti-CD19 chimeric antigen receptor (CAR) T-cells has demonstrated long-term remissions for patients with otherwise treatment-refractory advanced leukemia and lymphoma. Conversely, CAR T-cell treatment of solid tumors, including advanced gastric cancer (GC), has proven more challenging due to on-target off-tumor toxicities, poor tumor T-cell infiltration, inefficient CAR T-cell expansion, immunosuppressive tumor microenvironments, and demanding preconditioning regimens. We report the exceptional results of autologous Claudin18.2-targeted CAR T cells (CT041) in a patient with metastatic GC, who had progressed on four lines of combined systemic chemotherapy and immunotherapy. After two CT041 infusions, the patient had target lesion complete response and sustained an 8-month overall partial response with only minimal ascites. Moreover, tumor-informed circulating tumor DNA (ctDNA) reductions coincided with rapid CAR T-cell expansion and radiologic response. No severe toxicities occurred, and the patient's quality of life significantly improved. This experience supports targeting Claudin18.2-positive GC with CAR T-cell therapy and helps to validate ctDNA as a biomarker in CAR T-cell therapy. Clinical Insight: Claudin18.2-targeted CAR T cells can safely provide complete objective and ctDNA response in salvage metastatic GC.

Armored TGFßRIIDN ROR1-CAR T cells reject solid tumors and resist suppression by constitutively-expressed and treatment-induced TGFß1.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy target receptor tyrosine kinase-like orphan receptor 1 (ROR1) is broadly expressed in hematologic and solid tumors, however clinically-characterized ROR1-CAR T cells with single chain variable fragment (scFv)-R12 targeting domain failed to induce durable remissions, in part due to the immunosuppressive tumor microenvironment (TME). Herein, we describe the development of an improved ROR1-CAR with a novel, fully human scFv9 targeting domain, and augmented with TGFßRIIDN armor protective against a major TME factor, transforming growth factor beta (TGFß). METHODS: CAR T cells were generated by lentiviral transduction of enriched CD4+ and CD8+ T cells, and the novel scFv9-based ROR1-CAR-1 was compared with the clinically-characterized ROR1-R12-scFv-based CAR-2 in vitro and in vivo. RESULTS: CAR-1 T cells exhibited greater CAR surface density than CAR-2 when normalized for %CAR+, and produced more interferon (IFN)-γ tumor necrosis factor (TNF)-α and interleukin (IL)-2 in response to hematologic (Jeko-1, RPMI-8226) and solid (OVCAR-3, Capan-2, NCI-H226) tumor cell lines in vitro. In vivo, CAR-1 and CAR-2 both cleared hematologic Jeko-1 lymphoma xenografts, however only CAR-1 fully rejected ovarian solid OVCAR-3 tumors, concordantly with greater expansion of CD8+ and CD4+CAR T cells, and enrichment for central and effector memory phenotype. When equipped with TGFß-protective armor TGFßRIIDN, CAR-1 T cells resisted TGFß-mediated pSmad2/3 phosphorylation, as compared with CAR-1 alone. When co-cultured with ROR-1+ AsPC-1 pancreatic cancer line in the presence of TGFß1, armored CAR-1 demonstrated improved recovery of killing function, IFN-γ, TNF-α and IL-2 secretion. In mouse AsPC-1 pancreatic tumor xenografts overexpressing TGFß1, armored CAR-1, in contrast to CAR-1 alone, achieved complete tumor remissions, and yielded accelerated expansion of CAR+ T cells, diminished circulating active TGFß1, and no apparent toxicity or weight loss. Unexpectedly, in AsPC-1 xenografts without TGFß overexpression, TGFß1 production was specifically induced by ROR-1-CAR T cells interaction with ROR-1 positive tumor cells, and the TGFßRIIDN armor conferred accelerated tumor clearance. CONCLUSIONS: The novel fully human TGFßRIIDN-armored ROR1-CAR-1 T cells are highly potent against ROR1-positive tumors, and withstand the inhibitory effects of TGFß in solid TME. Moreover, TGFß1 induction represents a novel, CAR-induced checkpoint in the solid TME, which can be circumvented by co-expressing the TGßRIIDN armor on T cells.

CAR-mediated targeting of NK cells overcomes tumor immune escape caused by ICAM-1 downregulation.

BACKGROUND: The antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric antigen receptors (CARs). Despite antibody or CAR targeting, some tumors remain resistant towards NK cell attack. While the importance of ICAM-1/LFA-1 interaction for natural cytotoxicity of NK cells is known, its impact on ADCC induced by the ErbB2 (HER2)-specific antibody trastuzumab and ErbB2-CAR-mediated NK cell cytotoxicity against breast cancer cells has not been investigated. METHODS: Here we used NK-92 cells expressing high-affinity Fc receptor FcγRIIIa in combination with trastuzumab or ErbB2-CAR engineered NK-92 cells (NK-92/5.28.z) as well as primary human NK cells combined with trastuzumab or modified with the ErbB2-CAR and tested cytotoxicity against cancer cells varying in ICAM-1 expression or alternatively blocked LFA-1 on NK cells. Furthermore, we specifically stimulated Fc receptor, CAR and/or LFA-1 to study their crosstalk at the immunological synapse and their contribution to degranulation and intracellular signaling in antibody-targeted or CAR-targeted NK cells. RESULTS: Blockade of LFA-1 or absence of ICAM-1 significantly reduced cell killing and cytokine release during trastuzumab-mediated ADCC against ErbB2-positive breast cancer cells, but not so in CAR-targeted NK cells. Pretreatment with 5-aza-2'-deoxycytidine induced ICAM-1 upregulation and reversed NK cell resistance in ADCC. Trastuzumab alone did not sufficiently activate NK cells and required additional LFA-1 co-stimulation, while activation of the ErbB2-CAR in CAR-NK cells induced efficient degranulation independent of LFA-1. Total internal reflection fluorescence single molecule imaging revealed that CAR-NK cells formed an irregular immunological synapse with tumor cells that excluded ICAM-1, while trastuzumab formed typical peripheral supramolecular activation cluster (pSMAC) structures. Mechanistically, the absence of ICAM-1 did not affect cell-cell adhesion during ADCC, but rather resulted in decreased signaling via Pyk2 and ERK1/2, which was intrinsically provided by CAR-mediated targeting. Furthermore, while stimulation of the inhibitory NK cell checkpoint molecule NKG2A markedly reduced FcγRIIIa/LFA-1-mediated degranulation, retargeting by CAR was only marginally affected. CONCLUSIONS: Downregulation of ICAM-1 on breast cancer cells is a critical escape mechanism from trastuzumab-triggered ADCC. In contrast, CAR-NK cells are able to overcome cancer cell resistance caused by ICAM-1 reduction, highlighting the potential of CAR-NK cells in cancer immunotherapy.

Clinically relevant orthotopic pancreatic cancer models for adoptive T cell transfer therapy.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor. Prognosis is poor and survival is low in patients diagnosed with this disease, with a survival rate of ~12% at 5 years. Immunotherapy, including adoptive T cell transfer therapy, has not impacted the outcomes in patients with PDAC, due in part to the hostile tumor microenvironment (TME) which limits T cell trafficking and persistence. We posit that murine models serve as useful tools to study the fate of T cell therapy. Currently, genetically engineered mouse models (GEMMs) for PDAC are considered a "gold-standard" as they recapitulate many aspects of human disease. However, these models have limitations, including marked tumor variability across individual mice and the cost of colony maintenance. METHODS: Using flow cytometry and immunohistochemistry, we characterized the immunological features and trafficking patterns of adoptively transferred T cells in orthotopic PDAC (C57BL/6) models using two mouse cell lines, KPC-Luc and MT-5, isolated from C57BL/6 KPC-GEMM (KrasLSL-G12D/+p53-/- and KrasLSL-G12D/+p53LSL-R172H/+, respectively). RESULTS: The MT-5 orthotopic model best recapitulates the cellular and stromal features of the TME in the PDAC GEMM. In contrast, far more host immune cells infiltrate the KPC-Luc tumors, which have less stroma, although CD4+ and CD8+ T cells were similarly detected in the MT-5 tumors compared with KPC-GEMM in mice. Interestingly, we found that chimeric antigen receptor (CAR) T cells redirected to recognize mesothelin on these tumors that signal via CD3ζ and 41BB (Meso-41BBζ-CAR T cells) infiltrated the tumors of mice bearing stroma-devoid KPC-Luc orthotopic tumors, but not MT-5 tumors. CONCLUSIONS: Our data establish for the first time a reproducible and realistic clinical system useful for modeling stroma-rich and stroma-devoid PDAC tumors. These models shall serve an indepth study of how to overcome barriers that limit antitumor activity of adoptively transferred T cells.

PD-1 checkpoint inhibition enhances the antilymphoma activity of CD19-CAR-iNKT cells that retain their ability to prevent alloreactivity.

BACKGROUND: Relapse and graft-versus-host disease (GVHD) are the main causes of death after allogeneic hematopoietic cell transplantation (HCT). Preclinical murine models and clinical data suggest that invariant natural killer T (iNKT) cells prevent acute and chronic GVHD. In addition, iNKT cells are crucial for efficient immune responses against malignancies and contribute to reduced relapse rates after transplantation. Chimeric antigen receptors (CAR) redirect effector cells to cell surface antigens and enhance killing of target cells. With this study, we aimed to combine enhanced cytotoxicity of CD19-CAR-iNKT cells against lymphoma cells with their tolerogenic properties. METHODS: iNKT cells were isolated from peripheral blood mononuclear cells and transduced with an anti-CD19-CAR retrovirus. After in vitro expansion, the functionality of CD19-CAR-iNKT cells was assessed by flow cytometry, image stream analysis and multiplex analysis in single-stimulation or repeated-stimulation assays. Moreover, the immunoregulatory properties of CD19-CAR-iNKT cells were analyzed in apoptosis assays and in mixed lymphocyte reactions. The effect of checkpoint inhibition through nivolumab was analyzed in these settings. RESULTS: In this study, we could show that the cytotoxicity of CD19-CAR-iNKT cells was mediated either through engagement of their CAR or their invariant T-cell receptor, which may circumvent loss of response through antigen escape. However, encounter of CD19-CAR-iNKT cells with their target induced a phenotype of exhaustion. Consequently, checkpoint inhibition increased cytokine release, cytotoxicity and survival of CD19-CAR-iNKT cells. Additionally, they showed robust suppression of alloreactive immune responses. CONCLUSION: In this work, we demonstrate that CAR-iNKT cells are a powerful cytotherapeutic option to prevent or treat relapse while potentially reducing the risk of GVHD after allogeneic HCT.

CD47 expression is critical for CAR T-cell survival in vivo.

BACKGROUND: CD47 is an attractive immunotherapeutic target because it is highly expressed on multiple solid tumors. However, CD47 is also expressed on T cells. Limited studies have evaluated CD47-chimeric antigen receptor (CAR) T cells, and the role of CD47 in CAR T-cell function remains largely unknown. METHODS: Here, we describe the development of CD47-CAR T cells derived from a high affinity signal regulatory protein α variant CV1, which binds CD47. CV1-CAR T cells were generated from human peripheral blood mononuclear cells and evaluated in vitro and in vivo. The role of CD47 in CAR T-cell function was examined by knocking out CD47 in T cells followed by downstream functional analyses. RESULTS: While CV1-CAR T cells are specific and exhibit potent activity in vitro they lacked antitumor activity in xenograft models. Mechanistic studies revealed CV1-CAR T cells downregulate CD47 to overcome fratricide, but CD47 loss resulted in their failure to expand and persist in vivo. This effect was not limited to CV1-CAR T cells, since CD47 knockout CAR T cells targeting another solid tumor antigen exhibited the same in vivo fate. Further, CD47 knockout T cells were sensitive to macrophage-mediated phagocytosis. CONCLUSIONS: These findings highlight that CD47 expression is critical for CAR T-cell survival in vivo and is a 'sine qua non' for successful adoptive T-cell therapy.

Tuned activation of MSLN-CAR T cells induces superior antitumor responses in ovarian cancer models.

BACKGROUND: Limited persistence of functional CAR T cells in the immunosuppressive solid tumor microenvironment remains a major hurdle in the successful translation of CAR T cell therapy to treat solid tumors. Fine-tuning of CAR T cell activation by mutating CD3ζ chain immunoreceptor tyrosine-based activation motifs (ITAMs) in CD19-CAR T cells (containing the CD28 costimulatory domain) has proven to extend functional CAR T cell persistence in preclinical models of B cell malignancies. METHODS: In this study, two conventional second-generation MSLN-CAR T cell constructs encoding for either a CD28 co-stimulatory (M28z) or 4-1BB costimulatory (MBBz) domain and a novel mesothelin (MSLN)-directed CAR T cell construct encoding for the CD28 costimulatory domain and CD3ζ chain containing a single ITAM (M1xx) were evaluated using in vitro and in vivo preclinical models of ovarian cancer. Two ovarian cancer cell lines and two orthotopic models of ovarian cancer in NSG mice were used: SKOV-3 cells inoculated through microsurgery in the ovary and to mimic a disseminated model of advanced ovarian cancer, OVCAR-4 cells injected intraperitoneally. MSLN-CAR T cell treatment efficacy was evaluated by survival analysis and the characterization and quantification of the different MSLN-CAR T cells were performed by flow cytometry, quantitative PCR and gene expression analysis. RESULTS: M1xx CAR T cells elicited superior antitumor potency and persistence, as compared with the conventional second generation M28z and MBBz CAR T cells. Ex vivo M28z and MBBz CAR T cells displayed a more exhausted phenotype than M1xx CAR T cells as determined by co-expression of PD-1, LAG-3 and TIM-3. Furthermore, M1xx CAR T cells showed superior ex vivo IFNy, TNF and GzB production and were characterized by a self-renewal gene signature. CONCLUSIONS: Altogether, our study demonstrates the enhanced therapeutic potential of MSLN-CAR T cells expressing a mutated CD3ζ chain containing a single ITAM for the treatment of ovarian cancer. CAR T cells armored with calibrated activation potential may improve the clinical responses in solid tumors.

Patient-reported outcomes in CD30-directed CAR-T cells against relapsed/refractory CD30+ lymphomas.

Chimeric antigen receptor (CAR)-T cells targeting CD30 have demonstrated high response rates with durable remissions observed in a subset of patients with relapsed/refractory CD30+ hematologic malignancies, particularly classical Hodgkin lymphoma. This therapy has low rates of toxicity including cytokine release syndrome with no neurotoxicity observed in our phase 2 study. We collected patient-reported outcomes (PROs) on patients treated with CD30 directed CAR-T cells to evaluate the impact of this therapy on their symptom experience. We collected PROs including PROMIS (Patient-Reported Outcomes Measurement Information System) Global Health and Physical Function questionnaires and selected symptom questions from the NCI PRO-CTCAE in patients enrolled on our clinical trial of CD30-directed CAR-T cells at procurement, at time of CAR-T cell infusion, and at various time points post treatment. We compared PROMIS scores and overall symptom burden between pre-procurement, time of infusion, and at 4 weeks post infusion. At least one PRO measurement during the study period was found in 23 out of the 28 enrolled patients. Patient overall symptom burden, global health and mental health, and physical function were at or above baseline levels at 4 weeks post CAR-T cell infusion. In addition, PROMIS scores for patients who participated in the clinical trial were similar to the average healthy population. CD30 CAR-T cell therapy has a favorable toxicity profile with patient physical function and symptom burden recovering to at least their baseline pretreatment health by 1 month post infusion. Trial registration number: NCT02690545.

Valproic acid increases CAR T cell cytotoxicity against acute myeloid leukemia.

The treatment of B cell malignancies has dramatically changed with the introduction of immunotherapy, especially chimeric antigen receptor T (CAR-T) cell therapy. However, only limited efficacy is observed in acute myeloid leukaemia (AML). In the study, We detected CD123 and CLL-1 expression on leukaemia cells from Relapsed/Refractory AML (R/R AML) patients. Then, we constructed anti-CD123 CAR and CLL-1 CAR with different co-stimulation domains (CD28 or 4-1BB) and detected their anti-AML effects. To increase the efficacy of CAR-T cell therapy, we tested different strategies, including application of combined checkpoint inhibitors and histone deacetylase inhibitors (HDACi) in vivo and in vitro We found CD123 and CLL-1 were highly expressed on AML cells. The proportions of T cell subsets and NK cells involved in anti-tumour or anti-inflammation processes in AML patients significantly decreased when compared with healthy donors. Both CD123 CAR and CLL-1 CAR displayed specific anti-AML effects in vitro To improve the lysis effects of CAR-T cells, we combined CAR-T cell therapy with different agents. PD-1/PD-L1 antibodies only slightly improved the potency of CAR-T cell therapy (CD123 CAR-T 60.92% ± 2.9087% vs. 65.43% ± 2.1893%, 60.92% ± 2.9087% vs. 67.43% ± 3.4973%; 37.37% ± 3.908% vs. 41.89% ± 5.1568%, 37.37% ± 3.908% vs. 42.84% ± 4.2635%). However, one HDACi (valproic acid [VPA]) significantly improved CAR-T cell potency against AML cells (CLL-1 CAR-T 34.97% ± 0.3051% vs. 88.167% ± 1.5327%, p < 0.0001; CD123 CAR-T 26.87% ± 2.7010% vs. 82.56% ± 3.086%, p < 0.0001 in MV411; CLL-1 CAR-T 78.77% ± 1.2061% vs. 93.743% ± 1.2333%, p < 0.0001; CD123 CAR-T 64.10% ± 1.5130% vs. 94.427% ± 0.142%, p = 0.0001 in THP-1). Combination therapy prolonged the overall survival of mice when compared with single CD123 CAR-T cell therapy (median survival: 180 days vs. unfollowed). A possible mechanism is that activated CD8+T cells upregulate natural-killer group 2 member D (NKG2D), and VPA upregulates NKG2D ligand expression in AML cells, contributing to NKG2D-mediated cytotoxicity of CAR-T cells against tumour cells. In conclusion, CD123 and CLL-1 are promising targets for AML CAR-T cell therapy. A combination of VPA pre-treatment and CAR-T against AML exhibits synergic effects.