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Acute myeloid leukemia (AML) is a rapidly progressive malignancy without effective therapies for refractory disease. So far, chimeric antigen receptor (CAR) T cell therapy in AML has not recapitulated the efficacy seen in B cell malignancies. Here we report a pilot study of autologous anti-CD123 CAR T cells in 12 adults with relapsed or refractory AML. CAR T cells targeting CD123 + cells were successfully manufactured in 90.4% of runs. Cytokine release syndrome was observed in 10 of 12 infused individuals (83.3%, 90% confidence interval 0.5-0.97). Three individuals achieved clinical response (25%, 90% confidence interval 0.07-0.53). We found that myeloid-supporting cytokines are secreted during cell therapy and support AML blast survival via kinase signaling, leading to CAR T cell exhaustion. The prosurvival effect of therapy-induced cytokines presents a unique resistance mechanism in AML that is distinct from any observed in B cell malignancies. Our findings suggest that autologous CART manufacturing is feasible in AML, but treatment is associated with high rates of cytokine release syndrome and relatively poor clinical efficacy. Combining CAR T cell therapies with cytokine signaling inhibitors could enhance immunotherapy efficacy in AML and achieve improved outcomes (ClinicalTrials.gov identifier: NCT03766126 )., Competing Interests: Competing interests: D.L.P. declares funding from the National Marrow Donor Program; membership on an entity’s Board of Directors or advisory committees of Kite/Gilead, Janssen, Genentech, DeCart, Sana Biotechnology, Verismo and Novartis; is a current equity holder of the American Society for Transplantation and Cellular Therapy and Verismo; declares honoraria for Incyte; and has patents and royalties in Tmunity and Wiley and Sons Publishing. J.A.F. is a member of the scientific advisory boards of Cartography Bio and Shennon Biotechnologies and has patents, royalties and other intellectual property. M.R.L. is an employee of Hematoloics, Inc. J.L.B. is an employee of Novartis. C.H.J. is an inventor of patents related to CAR therapy products and may be eligible to receive a select portion of royalties paid from Kite to the University of Pennsylvania. C.H.J. is a scientific cofounder and holds equity in Capstan Therapeutics, Dispatch Biotherapeutics and Bluewhale Bio. C.H.J. serves on the board of AC Immune and is a scientific advisor to BlueSphere Bio, Cabaletta, Carisma, Cartography, Cellares, Cellcarta, Celldex, Danaher, Decheng, ImmuneSensor, Kite, Poseida, Verismo, Viracta and WIRB-Copernicus group. S.I.G. has patents related to CAR therapy with royalties paid from Novartis to the University of Pennsylvania. S.I.G. is a scientific cofounder and holds equity in Interius Biotherapeutics and Carisma Therapeutics. S.I.G. is a scientific advisor to Carisma, Cartography, Currus, Interius, Kite, NKILT and Mission Bio. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

We previously showed that chimeric antigen receptor (CAR) T-cell therapy targeting epidermal growth factor receptor variant III (EGFRvIII) produces upregulation of programmed death-ligand 1 (PD-L1) in the tumor microenvironment (TME). Here we conducted a phase 1 trial (NCT03726515) of CAR T-EGFRvIII cells administered concomitantly with the anti-PD1 (aPD1) monoclonal antibody pembrolizumab in patients with newly diagnosed, EGFRvIII + glioblastoma (GBM) (n = 7). The primary outcome was safety, and no dose-limiting toxicity was observed. Secondary outcomes included median progression-free survival (5.2 months; 90% confidence interval (CI), 2.9-6.0 months) and median overall survival (11.8 months; 90% CI, 9.2-14.2 months). In exploratory analyses, comparison of the TME in tumors harvested before versus after CAR + aPD1 administration demonstrated substantial evolution of the infiltrating myeloid and T cells, with more exhausted, regulatory, and interferon (IFN)-stimulated T cells at relapse. Our study suggests that the combination of CAR T cells and PD-1 inhibition in GBM is safe and biologically active but, given the lack of efficacy, also indicates a need to consider alternative strategies., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

CAR T-cell product quality and stemness (Tstem) are major determinants of in vivo expansion, efficacy, and clinical response. Prolonged ex vivo culturing is known to deplete Tstem, affecting clinical outcome. YTB323, a novel autologous CD19-directed CAR T-cell therapy expressing the same validated CAR as tisagenlecleucel, is manufactured using a next-generation platform in <2 days. Here, we report the preclinical development and preliminary clinical data of YTB323 in adults with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL; NCT03960840). In preclinical mouse models, YTB323 exhibited enhanced in vivo expansion and antitumor activity at lower doses than traditionally manufactured CAR T cells. Clinically, at doses 25-fold lower than tisagenlecleucel, YTB323 showed (i) promising overall safety [cytokine release syndrome (any grade, 35%; grade ≥3, 6%), neurotoxicity (any grade, 25%; grade ≥3, 6%)]; (ii) overall response rates of 75% and 80% for DL1 and DL2, respectively; (iii) comparable CAR T-cell expansion; and (iv) preservation of T-cell phenotype. Current data support the continued development of YTB323 for r/r DLBCL., Significance: Traditional CAR T-cell manufacturing requires extended ex vivo cell culture, reducing naive and stem cell memory T-cell populations and diminishing antitumor activity. YTB323, which expresses the same validated CAR as tisagenlecleucel, can be manufactured in <2 days while retaining T-cell stemness and enhancing clinical activity at a 25-fold lower dose. See related commentary by Wang, p. 1961. This article is featured in Selected Articles from This Issue, p. 1949., (©2023 The Authors; Published by the American Association for Cancer Research.)

Multiple clinical studies have treated mesothelin (MSLN)-positive solid tumors by administering MSLN-directed chimeric antigen receptor (CAR) T cells. Although these products are generally safe, efficacy is limited. Therefore, we generated and characterized a potent, fully human anti-MSLN CAR. In a phase 1 dose-escalation study of patients with solid tumors, we observed two cases of severe pulmonary toxicity following intravenous infusion of this product in the high-dose cohort (1-3 × 10 8 T cells per m 2 ). Both patients demonstrated progressive hypoxemia within 48 h of infusion with clinical and laboratory findings consistent with cytokine release syndrome. One patient ultimately progressed to grade 5 respiratory failure. An autopsy revealed acute lung injury, extensive T cell infiltration, and accumulation of CAR T cells in the lungs. RNA and protein detection techniques confirmed low levels of MSLN expression by benign pulmonary epithelial cells in affected lung and lung samples obtained from other inflammatory or fibrotic conditions, indicating that pulmonary pneumocyte and not pleural expression of mesothelin may lead to dose-limiting toxicity. We suggest patient enrollment criteria and dosing regimens of MSLN-directed therapies consider the possibility of dynamic expression of mesothelin in benign lung with a special concern for patients with underlying inflammatory or fibrotic conditions., Competing Interests: Declaration of interests C.H.J., R.M.Y., and M.M.D. are inventors on patents and/or patent applications licensed to Novartis Institutes of Biomedical Research and receive license revenue from such licenses. J.L.B., B.E., K.M., and L.Z. are holders of stock options and patents with Novartis Institutes for Biomedical Research. R.M.Y. and M.M.D. are inventors on patents and/or patent applications licensed to Tmunity Therapeutics and receive license revenue from such licenses. C.H.J. and A.C. are scientific cofounders of Tmunity Therapeutics. C.H.J. is a scientific cofounder of Capstan Therapeutics and is a member of the scientific advisory boards of AC Immune, Alaunos, BluesphereBio, Cabaletta, Carisma, Cartography, Cellares, Celldex, Decheng, Poseida, Verismo, and WIRB-Copernicus. B.E. and R.J.G. completed work on this study before becoming employees at Miltenyi Biotec and AstraZeneca, respectively. R.J.G. holds or may hold AstraZeneca stock. N.C.S. holds equity in Fate Therapeutics and Pfizer. J.A.F. has received grants and personal fees from Cartography Bio., grants from Tmunity Therapeutics, and personal fees from Retro Bio and Shennon Bio outside the submitted work. Additionally, J.A.F. holds patents related to CAR T cells for cancer that are licensed and associated with royalties. S.F.L. is an inventor on patents in the areas of CAR T and biomarkers at Penn that were assigned to Novartis; received research funding from Novartis, Tmunity, and Cabaletta; and consults for Kite/Gilead. M.M.D. is a consultant for Tmunity Therapeutics and is a Consultant and Member on the Scientific Advisory Board of Cellares Corporation., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

We conducted a phase I clinical trial of anti-BCMA chimeric antigen receptor T cells (CART-BCMA) with or without anti-CD19 CAR T cells (huCART19) in multiple myeloma (MM) patients responding to third- or later-line therapy (phase A, N = 10) or high-risk patients responding to first-line therapy (phase B, N = 20), followed by early lenalidomide or pomalidomide maintenance. We observed no high-grade cytokine release syndrome (CRS) and only one instance of low-grade neurologic toxicity. Among 15 subjects with measurable disease, 10 exhibited partial response (PR) or better; among 26 subjects responding to prior therapy, 9 improved their response category and 4 converted to minimal residual disease (MRD)-negative complete response/stringent complete response. Early maintenance therapy was safe, feasible, and coincided in some patients with CAR T-cell reexpansion and late-onset, durable clinical response. Outcomes with CART-BCMA + huCART19 were similar to CART-BCMA alone. Collectively, our results demonstrate favorable safety, pharmacokinetics, and antimyeloma activity of dual-target CAR T-cell therapy in early lines of MM treatment., Significance: CAR T cells in early lines of MM therapy could be safer and more effective than in the advanced setting, where prior studies have focused. We evaluated the safety, pharmacokinetics, and efficacy of CAR T cells in patients with low disease burden, responding to current therapy, combined with standard maintenance therapy. This article is highlighted in the In This Issue feature, p. 101., (©2022 The Authors; Published by the American Association for Cancer Research.)

In chronic lymphocytic leukemia (CLL) patients who achieve a complete remission (CR) to anti-CD19 chimeric antigen receptor T cells (CART-19), remissions are remarkably durable. Preclinical data suggesting synergy between CART-19 and the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib prompted us to conduct a prospective single-center phase 2 trial in which we added autologous anti-CD19 humanized binding domain T cells (huCART-19) to ibrutinib in patients with CLL not in CR despite ≥6 months of ibrutinib. The primary endpoints were safety, feasibility, and achievement of a CR within 3 months. Of 20 enrolled patients, 19 received huCART-19. The median follow-up for all infused patients was 41 months (range, 0.25-58 months). Eighteen patients developed cytokine release syndrome (CRS; grade 1-2 in 15 of 18 subjects), and 5 developed neurotoxicity (grade 1-2 in 4 patients, grade 4 in 1 patient). While the 3-month CR rate among International Working Group on CLL (iwCLL)-evaluable patients was 44% (90% confidence interval [CI], 23-67%), at 12 months, 72% of patients tested had no measurable residual disease (MRD). The estimated overall and progression-free survival at 48 months were 84% and 70%, respectively. Of 15 patients with undetectable MRD at 3 or 6 months, 13 remain in ongoing CR at the last follow-up. In patients with CLL not achieving a CR despite ≥6 months of ibrutinib, adding huCART-19 mediated a high rate of deep and durable remissions. ClinicalTrials.gov number, NCT02640209., (© 2022 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.)

The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers 1-7 . However, little is known about the long-term potential and clonal stability of the infused cells. Here we studied long-lasting CD19-redirected chimeric antigen receptor (CAR) T cells in two patients with chronic lymphocytic leukaemia 1-4 who achieved a complete remission in 2010. CAR T cells remained detectable more than ten years after infusion, with sustained remission in both patients. Notably, a highly activated CD4 + population emerged in both patients, dominating the CAR T cell population at the later time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by a small number of clones. Single-cell profiling demonstrated that these long-persisting CD4 + CAR T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation. In addition, longitudinal profiling revealed a population of gamma delta CAR T cells that prominently expanded in one patient concomitant with CD8 + CAR T cells during the initial response phase. Our identification and characterization of these unexpected CAR T cell populations provide novel insight into the CAR T cell characteristics associated with anti-cancer response and long-term remission in leukaemia., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Purpose: CD19-targeted chimeric antigen receptor (CAR)-modified T cells demonstrate unprecedented responses in B-cell acute lymphoblastic leukemia (B-ALL); however, relapse remains a substantial challenge. Short CAR T-cell persistence contributes to this risk; therefore, strategies to improve persistence are needed., Methods: We conducted a pilot clinical trial of a humanized CD19 CAR T-cell product (huCART19) in children and young adults with relapsed or refractory B-ALL (n = 72) or B-lymphoblastic lymphoma (n = 2), treated in two cohorts: with (retreatment, n = 33) or without (CAR-naive, n = 41) prior CAR exposure. Patients were monitored for toxicity, response, and persistence of huCART19., Results: Seventy-four patients 1-29 years of age received huCART19. Cytokine release syndrome developed in 62 (84%) patients and was grade 4 in five (6.8%). Neurologic toxicities were reported in 29 (39%), three (4%) grade 3 or 4, and fully resolved in all cases. The overall response rate at 1 month after infusion was 98% (100% in B-ALL) in the CAR-naive cohort and 64% in the retreatment cohort. At 6 months, the probability of losing huCART19 persistence was 27% (95% CI, 14 to 41) for CAR-naive and 48% (95% CI, 30 to 64) for retreatment patients, whereas the incidence of B-cell recovery was 15% (95% CI, 6 to 28) and 58% (95% CI, 33 to 77), respectively. Relapse-free survival at 12 and 24 months, respectively, was 84% (95% CI, 72 to 97) and 74% (95% CI, 60 to 90) in CAR-naive and 74% (95% CI, 56 to 97) and 58% (95% CI, 37 to 90) in retreatment cohorts., Conclusion: HuCART19 achieved durable remissions with long-term persistence in children and young adults with relapsed or refractory B-ALL, including after failure of prior CAR T-cell therapy., Competing Interests: David M. BarrettTravel, Accommodations, Expenses: Therakos David T. TeacheyConsulting or Advisory Role: SobiResearch Funding: Novartis, Beam Therapeutics, NeoImmuneTech Colleen CallahanConsulting or Advisory Role: NovartisSpeakers' Bureau: Peerview Susan R. RheingoldEmployment: OptiNoseStock and Other Ownership Interests: OptiNoseConsulting or Advisory Role: PfizerResearch Funding: Pfizer Richard AplencExpert Testimony: Vorys Pamela A. ShawPatents, Royalties, Other Intellectual Property: I am part of a patent owned by UPenn and currently licensed to Novartis for an algorithm that predicts severe cytokine release syndrome for the CART 19 therapy. I receive 10% of the licensing fees Edward PequignotPatents, Royalties, Other Intellectual Property: As part of Penn's role in the FDA-approval of CAR-T therapy, and for my part as an employee of Penn involved in their research in CAR-T, I have received royalties of approximately $300 in US dollars over the past 2 years Jennifer L. BrogdonEmployment: Novartis Institutes for BioMedical ResearchStock and Other Ownership Interests: Novartis Institutes for BioMedical Research Donald L. SiegelResearch Funding: Tmunity Therapeutics IncPatents, Royalties, Other Intellectual Property: A patent, owned by the Trustees of the University of Pennsylvania, on which I am listed as an inventor is licensed to Alexion Pharmaceuticals. I receive royalties as stipulated in the University of Pennsylvania Faculty HandbookExpert Testimony: Regeneron Megan M. DavisLeadership: Cellares CorporationConsulting or Advisory Role: Tmunity Therapeutics IncResearch Funding: Tmunity Therapeutics IncPatents, Royalties, Other Intellectual Property: Novartis Institutes for Biomedical Research—royalties and milestones for patents/knowhow, Tmunity Therapeutics—royalties andmilestones from patents/knowhow Simon F. LaceyConsulting or Advisory Role: Gilead SciencesResearch Funding: Tmunity Therapeutics Inc, Cabaletta Bio, Novartis Institutes for BioMedical ResearchPatents, Royalties, Other Intellectual Property: Patents and IP related to CTL019 (Kymriah) assigned by the University of Pennsylvania to Novartis Elizabeth O. HexnerConsulting or Advisory Role: Blueprint Medicines, ABIM Subspecialty BoardResearch Funding: Blueprint Medicines, Tmunity Therapeutics Inc Gerald B. WertheimEmployment: Johnson & JohnsonStock and Other Ownership Interests: Johnson & Johnson Bruce L. LevineStock and Other Ownership Interests: Tmunity Therapeutics IncHonoraria: Novartis, TerumoConsulting or Advisory Role: Avectas, Ori Biotech, Vycellix, Immuneel Therapeutics, In8bio, Patheon/ThermoFisher Viral Vector ServicesResearch Funding: Tmunity Therapeutics IncPatents, Royalties, Other Intellectual Property: Intellectual property and patents in the field of cell and gene therapyTravel, Accommodations, Expenses: Avectas, Terumo Carl H. JuneLeadership: AC ImmuneStock and Other Ownership Interests: Celldex, Tmunity Therapeutics Inc, Cabaletta Bio, Carisma Therapeutics, DeCART Therapeutics, Bluesphere Bio, Cellares, ZIOPHARM Oncology, Decheng Capital, Posieda Therapeutics, VerismaHonoraria: PfizerConsulting or Advisory Role: Celldex, Viracta Therapeutics, Cabaletta Bio, Carisma Therapeutics, Kiadis Pharma, WIRB-Copernicus Group, Janssen OncologyResearch Funding: Novartis, Tmunity Therapeutics IncPatents, Royalties, Other Intellectual Property: IP licensed to Novartis; Royalties paid to University of Pennsylvania, Office of Naval Research; IP and patent royalties, IP licensed to Tmunity Stephan A. GruppConsulting or Advisory Role: Novartis, Jazz Pharmaceuticals, Janssen, Cellular Biomedicine Group, TCR2 Therapeutics, Humanigen, Roche, Adaptimmune, Alimera Sciences, Cabaletta Bio, CRISPR Therapeutics/VertexResearch Funding: Novartis, Kite/Gilead, Servier, Jazz Pharmaceuticals, VertexPatents, Royalties, Other Intellectual Property: UPenn Toxicity management patentExpert Testimony: Juno Therapeutics Shannon L. MaudeConsulting or Advisory Role: Novartis, Wugen IncResearch Funding: NovartisTravel, Accommodations, Expenses: Novartis, Kite, a Gilead company, Wugen IncNo other potential conflicts of interest were reported.

The recent development of successful CAR (chimeric antigen receptor) T cell therapies has been accompanied by a need to better control potentially fatal toxicities that can arise from adverse immune reactions. Here we present a ligand-controlled CAR system, based on the IKZF3 ZF2 β-hairpin IMiD-inducible degron, which allows for the reversible control of expression levels of type I membrane proteins, including CARs. Testing this system in an established mouse xenotransplantation model for acute lymphoblastic leukemia, we validate the ability of the CAR19-degron to target and kill CD19-positive cells displaying complete control/clearance of the tumor. We also demonstrate that the activity of CAR19-degron can be regulated in vivo when dosing a US Food and Drug Administration-approved drug, lenalidomide., Competing Interests: Declaration of interests Seth Carbonneau, James E. Bradner, Marc Hild, and Carla P. Guimaraes are co-inventors on a patent application (PCT/US2018/056472) submitted by Novartis AG., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19 - disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.

Chimeric antigen receptor (CAR)-T cell therapies reprogram T cells to engage and eliminate cancer cells. Patients' T cells are transduced in vitro using lentiviral or retroviral vectors containing a CAR transgene. Following infusion, CAR-T cells expand in vivo and may persist in the peripheral blood and bone marrow for years. Therefore, monitoring in vivo copies of the CAR transgene requires highly sensitive, validated analytical methods. Herein, we describe the validation of a qPCR assay to detect tisagenlecleucel transgene in patient samples. The limit of detection and lower limit of quantitation were 3.1 and 10 copies/200 ng genomic DNA, respectively, equivalent to ∼50 copies/μg genomic DNA and in alignment with US Food and Drug Administration guidance on bioanalytical method validation. The assay allowed quantitation of the tisagenlecleucel transgene over a wide dynamic range with a high degree of linearity, that is, 10 1- 10 6 copies/200 ng genomic DNA (R 2 ≥ 0.9988). Coefficients of variation of measured transgene copies ranged from 0.2% to 12.8%. A droplet digital PCR assay was performed as a method of validation and showed a strong correlation with the qPCR assay (R 2  = 0.9980, p < 0.0001). This qPCR assay is being utilized to monitor tisagenlecleucel expansion and persistence in clinical trials., Competing Interests: All authors are employees of Novartis Pharmaceuticals, Novartis Institutes for BioMedical Research, or Navigate BioPharma Services, a subsidiary of Novartis Pharmaceuticals., (© 2020 Navigate BioPharma Services, Inc.)

Background: Chimeric antigen receptor (CAR) T cells are a promising therapy for hematologic malignancies. B-cell maturation antigen (BCMA) is a rational target in multiple myeloma (MM)., Methods: We conducted a phase I study of autologous T cells lentivirally-transduced with a fully-human, BCMA-specific CAR containing CD3ζ and 4-1BB signaling domains (CART-BCMA), in subjects with relapsed/refractory MM. Twenty-five subjects were treated in 3 cohorts: 1) 1-5 x 108 CART-BCMA cells alone; 2) Cyclophosphamide (Cy) 1.5 g/m2 + 1-5 x 107 CART-BCMA cells; and 3) Cy 1.5 g/m2 + 1-5 x 108 CART-BCMA cells. No pre-specified BCMA expression level was required., Results: CART-BCMA cells were manufactured and expanded in all subjects. Toxicities included cytokine release syndrome and neurotoxicity, which were grade 3-4 in 8 (32%) and 3 (12%) subjects, respectively, and reversible. One subject died at day 24 from candidemia and progressive myeloma, following treatment for severe CRS and encephalopathy. Responses (based on treated subjects) were seen in 4/9 (44%) in cohort 1, 1/5 (20%) in cohort 2, and 7/11 (64%) in cohort 3, including 5 partial, 5 very good partial, and 2 complete responses, 3 of which were ongoing at 11, 14, and 32 months. Decreased BCMA expression on residual MM cells was noted in responders; expression increased at progression in most. Responses and CART-BCMA expansion were associated with CD4:CD8 T cell ratio and frequency of CD45RO-CD27+CD8+ T cells in the pre-manufacturing leukapheresis product., Conclusion: CART-BCMA infusions with or without lymphodepleting chemotherapy are clinically active in heavily-pretreated MM patients., Trial Registration: NCT02546167., Funding: University of Pennsylvania-Novartis Alliance and NIH.

We identified genetic mutations in CD19 and loss of heterozygosity at the time of CD19 - relapse to chimeric antigen receptor (CAR) therapy. The mutations are present in the vast majority of resistant tumor cells and are predicted to lead to a truncated protein with a nonfunctional or absent transmembrane domain and consequently to a loss of surface antigen. This irreversible loss of CD19 advocates for an alternative targeting or combination CAR approach.

Tolerance to self-antigens prevents the elimination of cancer by the immune system 1,2 . We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27 + CD45RO - CD8 + T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27 + PD-1 - CD8 + CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies.

Background: Patients with diffuse large B-cell lymphoma or follicular lymphoma that is refractory to or that relapses after immunochemotherapy and transplantation have a poor prognosis. High response rates have been reported with the use of T cells modified by chimeric antigen receptor (CAR) that target CD19 in B-cell cancers, although data regarding B-cell lymphomas are limited., Methods: We used autologous T cells that express a CD19-directed CAR (CTL019) to treat patients with diffuse large B-cell lymphoma or follicular lymphoma that had relapsed or was refractory to previous treatments. Patients were monitored for response to treatment, toxic effects, the expansion and persistence of CTL019 cells in vivo, and immune recovery., Results: A total of 28 adult patients with lymphoma received CTL019 cells, and 18 of 28 had a response (64%; 95% confidence interval [CI], 44 to 81). Complete remission occurred in 6 of 14 patients with diffuse large B-cell lymphoma (43%; 95% CI, 18 to 71) and 10 of 14 patients with follicular lymphoma (71%; 95% CI, 42 to 92). CTL019 cells proliferated in vivo and were detectable in the blood and bone marrow of patients who had a response and patients who did not have a response. Sustained remissions were achieved, and at a median follow-up of 28.6 months, 86% of patients with diffuse large B-cell lymphoma who had a response (95% CI, 33 to 98) and 89% of patients with follicular lymphoma who had a response (95% CI, 43 to 98) had maintained the response. Severe cytokine-release syndrome occurred in 5 patients (18%). Serious encephalopathy occurred in 3 patients (11%); 2 cases were self-limiting and 1 case was fatal. All patients in complete remission by 6 months remained in remission at 7.7 to 37.9 months (median, 29.3 months) after induction, with a sustained reappearance of B cells in 8 of 16 patients and with improvement in levels of IgG in 4 of 10 patients and of IgM in 6 of 10 patients at 6 months or later and in levels of IgA in 3 of 10 patients at 18 months or later., Conclusions: CTL019 cells can be effective in the treatment of relapsed or refractory diffuse large B-cell lymphoma and follicular lymphoma. High rates of durable remission were observed, with recovery of B cells and immunoglobulins in some patients. Transient encephalopathy developed in approximately one in three patients and severe cytokine-release syndrome developed in one in five patients. (Funded by Novartis and others; ClinicalTrials.gov number, NCT02030834 .).

We conducted a first-in-human study of intravenous delivery of a single dose of autologous T cells redirected to the epidermal growth factor receptor variant III (EGFRvIII) mutation by a chimeric antigen receptor (CAR). We report our findings on the first 10 recurrent glioblastoma (GBM) patients treated. We found that manufacturing and infusion of CAR-modified T cell (CART)-EGFRvIII cells are feasible and safe, without evidence of off-tumor toxicity or cytokine release syndrome. One patient has had residual stable disease for over 18 months of follow-up. All patients demonstrated detectable transient expansion of CART-EGFRvIII cells in peripheral blood. Seven patients had post-CART-EGFRvIII surgical intervention, which allowed for tissue-specific analysis of CART-EGFRvIII trafficking to the tumor, phenotyping of tumor-infiltrating T cells and the tumor microenvironment in situ, and analysis of post-therapy EGFRvIII target antigen expression. Imaging findings after CART immunotherapy were complex to interpret, further reinforcing the need for pathologic sampling in infused patients. We found trafficking of CART-EGFRvIII cells to regions of active GBM, with antigen decrease in five of these seven patients. In situ evaluation of the tumor environment demonstrated increased and robust expression of inhibitory molecules and infiltration by regulatory T cells after CART-EGFRvIII infusion, compared to pre-CART-EGFRvIII infusion tumor specimens. Our initial experience with CAR T cells in recurrent GBM suggests that although intravenous infusion results in on-target activity in the brain, overcoming the adaptive changes in the local tumor microenvironment and addressing the antigen heterogeneity may improve the efficacy of EGFRvIII-directed strategies in GBM., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Over the past decade, our understanding of cancer immunotherapy has evolved from assessing peripheral responses in the blood to monitoring changes in the tumour microenvironment. Both preclinical and clinical experience has taught us that modulation of the tumour microenvironment has significant implications to generating robust antitumour immunity. Clinical benefit has been well documented to correlate with a tumour microenvironment that contains a dense infiltration of CD8 + CD45RO + T effectors and a high ratio of CD8 + T cells to FoxP3 + regulatory T cells (Tregs). In preclinical tumour models, modulation of the Glucocorticoid induced TNF receptor (GITR)/GITR ligand (GITRL) axis suggests this pathway may provide the desired biological outcome of inhibiting Treg function while activating CD8 + T effector cells. This review will focus on the scientific rationale and considerations for the therapeutic targeting of GITR for cancer immunotherapy and will discuss possible combination strategies to enhance clinical benefit., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376)., (Copyright © 2015, American Association for the Advancement of Science.)

Antibodies targeting checkpoint inhibitors or co-stimulatory receptors on T cells have shown significant antitumor efficacy in preclinical and clinical studies. In mouse tumor models, engagement of activating Fcγ receptor (FcγR)-expressing immune cells was recently shown to be required for the tumoricidal activity of antibodies recognizing the tumor necrosis factor superfamily receptor (TNFR) GITR (CD357) and CTLA-4 (CD152). In particular, activating FcγRs facilitated the selective elimination of intratumoral T-cell populations. However, it remains unclear whether FcγRs contribute to the antitumor efficacy of other immunomodulatory antibodies. Here, we explored the mechanism of antitumor activity mediated by an agonistic antibody (clone OX86) to the co-stimulatory TNFR OX40 (CD134). OX40 was highly expressed by intratumoral T cells, particularly those of the FoxP3(+) regulatory T-cell (Treg) lineage. OX86 administration resulted in the depletion of intratumoral regulatory T cells in an activating FcγR-dependent manner, which correlated with tumor regression. Together with previous data from our group and others, these findings support a mechanism whereby antibodies targeting antigens highly expressed by intratumoral T cells can mediate their elimination by FcγR-expressing immune cells, and facilitate subsequent antitumor immunity.

Fc γ receptor (FcγR) coengagement can facilitate antibody-mediated receptor activation in target cells. In particular, agonistic antibodies that target tumor necrosis factor receptor (TNFR) family members have shown dependence on expression of the inhibitory FcγR, FcγRIIB. It remains unclear if engagement of FcγRIIB also extends to the activities of antibodies targeting immunoregulatory TNFRs expressed by T cells. We have explored the requirement for activating and inhibitory FcγRs for the antitumor effects of antibodies targeting the TNFR glucocorticoid-induced TNFR-related protein (GITR; TNFRSF18; CD357) expressed on activated and regulatory T cells (T reg cells). We found that although FcγRIIB was dispensable for the in vivo efficacy of anti-GITR antibodies, in contrast, activating FcγRs were essential. Surprisingly, the dependence on activating FcγRs extended to an antibody targeting the non-TNFR receptor CTLA-4 (CD152) that acts as a negative regulator of T cell immunity. We define a common mechanism that correlated with tumor efficacy, whereby antibodies that coengaged activating FcγRs expressed by tumor-associated leukocytes facilitated the selective elimination of intratumoral T cell populations, particularly T reg cells. These findings may have broad implications for antibody engineering efforts aimed at enhancing the therapeutic activity of immunomodulatory antibodies.

Histone deacetylases (HDACs) play a critical role in regulating gene expression and key biological processes. However, how HDACs are involved in innate immunity is little understood. Here, in this first systematic investigation of the role of HDACs in immunity, we show that HDAC inhibition by a small-molecule HDAC inhibitor (HDACi), LAQ824, alters Toll-like receptor 4 (TLR4)-dependent activation and function of macrophages and dendritic cells (DCs). Surprisingly, pan-HDAC inhibition modulates only a limited set of genes involved in distinct arms of immune responses. Specifically, it inhibited DC-controlled T helper 1 (Th1) effector but not Th2 effector cell activation and migration. It also inhibited macrophage- and DC-mediated monocyte but not neutrophil chemotaxis. These unexpected findings demonstrate the high specificity of HDAC inhibition in modulating innate and adaptive immune responses, and highlight the potential for HDACi to alter the Th1 and Th2 balance in therapeutic settings.

Previous studies have found that class II-restricted T cells from CD4-deficient mice reconstituted with a tail-less CD4 transgene have a specific defect in the development of Th2 effector cells; however, the reason for this defect was not clear. Following stimulation with a high potency peptide and exogenous IL-4, CD4-dependent signaling is required for optimal generation of a Th2 effector population. However, initial IL-4 and GATA-3 transcription is appropriately induced, suggesting that the initial stages of Th2 development are intact and independent of CD4 after priming with a strong agonist peptide. In addition to the defect in Th2 development, CD4 mutant T cells are also relatively resistant to activation-induced cell death (AICD). Furthermore, inhibition of AICD in wild-type T cells causes a defect in Th2 development similar to that seen in the CD4 mutant T cells. These data support the hypothesis that CD4-dependent signaling pathways regulate a distinct checkpoint in the expansion and commitment phase of Th2 development, which is related to dysregulation of AICD.

T cell activation is associated with the partitioning of TCRs and other signaling proteins, forming an immunological synapse. This study demonstrates a novel function for the CD4 coreceptor in regulating molecular clustering at the immunological synapse site. We show using transgenic mouse and retroviral reconstitution studies that CD4 is required for TCR/protein kinase C (PKC) theta clustering. Specifically, we demonstrate that CD4 palmitoylation sequences are required for TCR/PKCtheta raft association and subsequent clustering, indicating a particular role for raft-associated CD4 molecules in regulating immune synapse organization. Although raft association of CD4 is necessary, it is not sufficient to mediate clustering, as cytoplasmic tail deletion mutants are able to localize to rafts, but are unable to mediate TCR/PKCtheta clustering, indicating an additional requirement for CD4 signaling. These studies suggest that CD4 coreceptor function is regulated not only through its known signaling function, but also by posttranslational lipid modifications which regulate localization of CD4 in lipid rafts.

An ideal vaccine delivery system would elicit persistent protection following a single administration, preferably by a noninvasive route, and be safe even in the face of immunosuppression, either inherited or acquired, of the recipient. We have exploited the unique life cycle of the autonomous parvoviruses to develop a nonproliferating vaccine platform that appears to both induce priming and continually boost a protective immune response following a single inoculation. A crippled parvovirus vector was constructed, based on a chimera between minute virus of mice (MVM) and LuIII, which expresses Borrelia burgdorferi outer surface protein A (OspA) instead of its coat protein. The vector was packaged into an MVM lymphotropic capsid and inoculated into naive C3H/HeNcr mice. Vaccination with a single vector dose, either intravenously or intranasally, elicited high-titer anti-OspA-specific antibody that provided protection from live spirochete challenge and was sustained over the lifetime of the animal. Both humoral and cell-mediated Th(1) immunity was induced, as shown by anti-OspA immunoglobulin G2a antibody and preferential gamma interferon production by OspA-specific CD4(+) T cells.

Small GTPase Ras is capable of mediating activation in T lymphocytes by using Raf kinase-dependent signaling pathway. Other effectors of Ras exist, however, suggesting that targets of Ras alternative to Raf may also contribute to T cell functions. Here we demonstrate that Ras(V12G37) mutant that fails to bind Raf, potently increases intracellular calcium concentration and cytokine production in primary antigen-stimulated T cells. From three known effectors which retain the ability to interact with Ras(V12G37), overexpression of phospholipase C epsilon but not that of RIN1 or Ral guanine nucleotide exchange factors enhanced cytokine and nuclear factor-activated T cell reporter T cell responses. Hence T cell activation can be critically regulated by the Ras effector pathway independent from Raf that can be mimicked by phospholipase C epsilon.

Although extracellular signal-regulated kinase (Erk) activation influences IL-4 production in various experimental systems, its role during Th differentiation is unclear. In this study, we show that Erk plays a critical role in IL-4 expression during TCR-induced Th differentiation of naive CD4(+) T cells. Stimulation of CD4(+) T cells with a high affinity peptide resulted in sustained Erk activation and Th1 differentiation. However, reduction of Erk activity led to a dramatic increase in IL-4 production and Th2 generation. Analysis of RNA and nuclear proteins of CD4(+) T cells 48 h after stimulation revealed that this was due to early IL-4 expression. Interestingly, transient Erk activation resulted in altered AP-1 DNA binding activity and the induction of an AP-1 complex that was devoid of Fos protein and consisted of Jun-Jun dimers. These data show that in the presence of a strong TCR signal, IL-4 expression can be induced in naive CD4(+) T cells by altering the strength of Erk activation. In addition, these data suggest that TCR-induced Erk activation is involved in the regulation of IL-4 expression by altering the composition of the AP-1 complex and its subsequent DNA binding activity.

Antigen exposure via airway epithelia is often associated with a failure to prime or with the preferential priming of Th2 cells. We previously reported that the intranasal delivery of a Th1-inducing antigen promoted Th2-dominated responses, rather than the expected Th1 responses. Thus, we proposed that when pulmonary T cell priming is induced, the lung microenvironment might intrinsically favor the generation of Th2 types of responses. To establish a potential mechanism for such preferential priming, we examined the initial interactions between antigens and resident antigen-presenting cells (APCs) within the lung. We show that intranasally delivered antigens are preferentially taken up and can be presented to antigen-specific T cells by a resident population of CD11c(bright) APCs. Most of these antigen-loaded APCs remained within lung tissues, and migration into secondary lymphoid organs was not crucial for T cell priming to occur within the pulmonary tract. Furthermore, these pulmonary APCs demonstrated a marked expression of IL-6 and IL-10 within hours of antigen uptake, suggesting that resident tissue APCs have the capacity to promote Th2 T cell differentiation in situ.

The potency of TCR signaling can regulate the differentiation of naive CD4(+) T cells into Th1 and Th2 subsets. In this work we demonstrate that TCR signaling by low-affinity, but not high-affinity, peptide ligands selectively induces IL-4 transcription within 48 h of priming naive CD4(+) T cells. This early IL-4 transcription is STAT6 independent and occurs before an increase in GATA-3. Furthermore, the strength of the TCR signal differentially affects the balance of NFATp and NFATc DNA binding activity, thereby regulating IL-4 transcription. Low-potency TCR signals result in high levels of nuclear NFATc and low levels of NFATp, which are permissive for IL-4 transcription. These data provide a model for how the strength of TCR signaling can influence the generation of Th1 and Th2 cells.