scholarly journals Common trajectories of highly effective CD19-specific CAR T cells identified by endogenous T cell receptor lineages

2021 ◽  
Author(s):  
Taylor L. Wilson ◽  
Hyunjin Kim ◽  
Ching-Heng Chou ◽  
Deanna Langfitt ◽  
E. Kaitlynn Allen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T ◽  
Post Infusion

AbstractCurrent chimeric antigen receptor-modified (CAR) T cell therapy products are evaluated in bulk, without assessment of the possible heterogeneity in effector potential between cells. Conceivably, only a subset of the pre-infusion product differentiates into optimal effectors. We generated a comprehensive single-cell gene expression and T cell receptor (TCR) sequencing dataset using both pre- and post-infusion CD19-CAR T cells from peripheral blood and bone marrow of pediatric patients with B cell acute lymphoblastic leukemia (B-ALL). We identified potent effector post-infusion cells with identical TCRs to a subset of pre-infusion CAR T cells. Effector precursor CAR T cells exhibited a unique transcriptional profile compared to other pre-infusion cells, and the number of effector precursor cells infused correlated with peak CAR T cell expansion. Additionally, we identified an unexpected cell surface phenotype (TIGIT+, CD62Llo, CD27-), conventionally associated with inhibiting effective T cell responses, that we used to successfully enrich for subsequent effector potential. Collectively, these results demonstrate that highly diverse effector potentials are present among cells in pre-infusion cell products, which can be exploited for diagnostic and therapeutic applications. Furthermore, we provide an integrative experimental and analytical framework for elucidating the biological mechanisms underlying effector development in other CAR T cell therapy products.

scholarly journals A Novel Method for Molecular Enumeration of Circulating Tisa-Cel and Axi-Cel in Lymphoma Patients

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 48-49
Author(s):  
Chiara Monfrini ◽  
Vanessa Aragona ◽  
Martina Magni ◽  
Riccardo Betta ◽  
Cristina Vella ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell Lymphoma ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Large B Cell Lymphoma ◽  
Car T Cell Therapy ◽  
Car T ◽  
Post Infusion

Background: Chimeric antigen receptor (CAR) T-cell therapy is an effective treatment in approximately 40% of relapsed/refractory lymphomas. High rates of complete remission (CR) observed in clinical trials, led to fast-track FDA and EMA approval and commercialization of two CD19-directed CAR T-cell products (tisagenlecleucel/Tisa-cel/Kymriah and axicabtagene ciloleucel/Axi-cel/Yescarta) for refractory/relapsed diffuse large B cell lymphoma (DLBCL) and primary mediastinal large B cell lymphoma (PMBCL). In these studies, CAR T-cell engraftment and in vivo expansion have a crucial impact on disease response and toxicity. It is of paramount importance to develop homogeneous standardized approaches for monitoring expansion and persistence of CAR T-cells also in the case of cells not detectable by flow cytometry (FCM) as these data can guide clinical decision making. Aims of this study were: to assess relevant time-points for FCM analysis across the different products to develop a unique quantitative PCR assay to detect both tisa-cel and axi-cel. Methods: We prospectively collected samples from 28 patients (pts) (16 DLBCL, 5 transformed follicular lymphoma and 7 PMBCL) treated with axi-cel (n=15) and tisa-cel (n=13) at the Fondazione IRCCS Istituto Nazionale Tumori between Nov 2019 and July 2020. CAR T-cells were monitored in the peripheral blood (PB) on days 0, 4, 7, 9, 14, 21, 28 and monthly post infusion by FCM. Cells were stained with CD19 CAR Detection Reagent (Miltenyi), Anti-Biotin-PE, CD3-FITC, CD4-VioGreen, CD8-APC-Vio770, CD45-VioBlue, CD14-APC and 7-AAD Staining Solution and analyzed by FCM using the MACSQuant® Analyzer (Miltenyi). To evaluate CAR T-cell persistence, we also generated a unique droplet digital PCR (ddPCR) primer-probe assay to target both tisagen-cel and axi-cel. We obtained a partial cDNA sequence of the two CAR constructs by amplification and direct sequencing of fragments generated with primers designed by educated guess to bind to the anti-CD19 murine single-chain variable fragment (scFV) and to the CD8α or CD28 trans-membrane regions of tisagen-cel and axi-cel vectors respectively. The assay was used to quantify CAR+ cells on genomic DNA extracted from PB cells collected serially after infusion. FCM and molecular data were correlated to outcome. Results: Peak expansion of CAR T-cells by FCM occurred within the first 14 days post-infusion. At the point of maximal expansion, collectively a mean of 17% of circulating CD3+ T cells were CAR+ and CAR+ cells were more abundant among CD3+ cells in pts receiving axi-cel (26% vs 8.5% for tisagen-cel, p<0.05). Patients achieving CR at 1 month from CAR T-cell therapy (n=10) had significantly more CAR+ cells at day 9 than non-CR pts (mean 146 vs 18 CAR+ cells/ul, p<0.05) and their magnitude of expansion in the first 14 days was higher than the one of non-CR pts (mean area under the curve (AUC)= 1110 vs 177; p<0.05). A cut-off value of CAR+ cells at day 9>62/ul was prognostic with sensitivity of 67% and specificity of 76% (Receiver operating characteristic analysis). At subsequent time points the number of CAR T-cells decreased, but continued to be detectable by FCM up to 6 months post infusion although at very low levels (mean 2 CAR+ cell/ul) irrespective of outcome. To evaluate whether these low counts reliably represent CAR T+ cells or are rather signals due to aspecific binding of the antibodies, a ddPCR assay was set up. The assay displayed an excellent separation between positive and negative droplets on both axi-cel and tisagen-cel products, good reproducibility and specificity with max standard deviation of 0.06 and no CAR positive signal in healthy donors. Additionally, the CAR specific signal was detectable up to the 0,01% dilution. FCM and ddPCR data had a good correlation (r=0.93, p<0.001): in accordance with FCM, ddPCR revealed a peak expansion of CAR+ signals at day 9 post infusion, followed by a progressive reduction of the CAR+ signal over time in all pts that becomes undetectable in case of relapse despite the presence of 1-2 cells/ul by FCM that probably represent aspecific findings. Conclusion: The combined use of FCM and the novel molecular assay facilitates the precise enumeration of commercial anti-CD19 CAR T-cells. Higher frequencies of CAR+ T cells at day 9 distinguish responders irrespectively of the product they received in our small prospective series. Updated results will be presented. Disclosures Chiappella: Servier: Honoraria; Roche: Honoraria; Takeda: Honoraria; Iqone: Honoraria; Janssen: Honoraria; Gilead-Kite: Honoraria; Celgene: Honoraria. Corradini:Kite: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Incyte: Consultancy; BMS: Other; Takeda: Consultancy, Honoraria, Other; Servier: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; F. Hoffman-La Roche Ltd: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Other: Travel and accommodations paid by for; KiowaKirin: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Travel and accommodations paid by for; AbbVie: Consultancy, Honoraria, Other: Travel and accommodations paid by for; Gilead: Consultancy, Honoraria, Other: Travel and accommodations paid by for; Daiichi Sankyo: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Other: Travel and accommodations paid by for.

scholarly journals Anti-Mesothelin CAR T cell therapy for malignant mesothelioma

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Laura Castelletti ◽  
Dannel Yeo ◽  
Nico van Zandwijk ◽  
John E. J. Rasko
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Malignant Mesothelioma ◽  
Oncolytic Viruses ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

AbstractMalignant mesothelioma (MM) is a treatment-resistant tumor originating in the mesothelial lining of the pleura or the abdominal cavity with very limited treatment options. More effective therapeutic approaches are urgently needed to improve the poor prognosis of MM patients. Chimeric Antigen Receptor (CAR) T cell therapy has emerged as a novel potential treatment for this incurable solid tumor. The tumor-associated antigen mesothelin (MSLN) is an attractive target for cell therapy in MM, as this antigen is expressed at high levels in the diseased pleura or peritoneum in the majority of MM patients and not (or very modestly) present in healthy tissues. Clinical trials using anti-MSLN CAR T cells in MM have shown that this potential therapeutic is relatively safe. However, efficacy remains modest, likely due to the MM tumor microenvironment (TME), which creates strong immunosuppressive conditions and thus reduces anti-MSLN CAR T cell tumor infiltration, efficacy and persistence. Various approaches to overcome these challenges are reviewed here. They include local (intratumoral) delivery of anti-MSLN CAR T cells, improved CAR design and co-stimulation, and measures to avoid T cell exhaustion. Combination therapies with checkpoint inhibitors as well as oncolytic viruses are also discussed. Preclinical studies have confirmed that increased efficacy of anti-MSLN CAR T cells is within reach and offer hope that this form of cellular immunotherapy may soon improve the prognosis of MM patients.

scholarly journals Metabolic and Mitochondrial Functioning in Chimeric Antigen Receptor (CAR)—T Cells

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1229
Author(s):  
Ali Hosseini Rad S. M. ◽  
Joshua Colin Halpin ◽  
Mojtaba Mollaei ◽  
Samuel W. J. Smith Bell ◽  
Nattiya Hirankarn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Chimeric Antigen Receptor ◽  
Metabolic State ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized adoptive cell therapy with impressive therapeutic outcomes of >80% complete remission (CR) rates in some haematological malignancies. Despite this, CAR T cell therapy for the treatment of solid tumours has invariably been unsuccessful in the clinic. Immunosuppressive factors and metabolic stresses in the tumour microenvironment (TME) result in the dysfunction and exhaustion of CAR T cells. A growing body of evidence demonstrates the importance of the mitochondrial and metabolic state of CAR T cells prior to infusion into patients. The different T cell subtypes utilise distinct metabolic pathways to fulfil their energy demands associated with their function. The reprogramming of CAR T cell metabolism is a viable approach to manufacture CAR T cells with superior antitumour functions and increased longevity, whilst also facilitating their adaptation to the nutrient restricted TME. This review discusses the mitochondrial and metabolic state of T cells, and describes the potential of the latest metabolic interventions to maximise CAR T cell efficacy for solid tumours.

IMMU-45. COMBINATION OF EGFRVIII CAR T-CELL THERAPY AND PARACRINE GAM MODULATION FOR THE TREATMENT OF GBM

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi102-vi103
Author(s):  
Tomás A Martins ◽  
Marie-Françoise Ritz ◽  
Tala Shekarian ◽  
Philip Schmassmann ◽  
Deniz Kaymak ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Differential Expression Analysis ◽  
Dependent Manner ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Abstract The GBM immune tumor microenvironment mainly consists of protumoral glioma-associated microglia and macrophages (GAMs). We have previously shown that blockade of CD47, a ‘don't eat me’-signal overexpressed by GBM cells, rescued GAMs' phagocytic function in mice. However, monotherapy with CD47 blockade has been ineffective in treating human solid tumors to date. Thus, we propose a combinatorial approach of local CAR T cell therapy with paracrine GAM modulation for a synergistic elimination of GBM. We generated humanized EGFRvIII CAR T-cells by lentiviral transduction of healthy donor human T-cells and engineered them to constitutively release a soluble SIRPγ-related protein (SGRP) with high affinity towards CD47. Tumor viability and CAR T-cell proliferation were assessed by timelapse imaging analysis in co-cultures with endogenous EGFRvIII-expressing BS153 cells. Tumor-induced CAR T-cell activation and degranulation were confirmed by flow cytometry. CAR T-cell secretomes were analyzed by liquid chromatography-mass spectrometry. Immunocompromised mice were orthotopically implanted with EGFRvIII+ BS153 cells and treated intratumorally with a single CAR T-cell injection. EGFRvIII and EGFRvIII-SGRP CAR T-cells killed tumor cells in a dose-dependent manner (72h-timepoint; complete cytotoxicity at effector-target ratio 1:1) compared to CD19 controls. CAR T-cells proliferated and specifically co-expressed CD25 and CD107a in the presence of tumor antigen (24h-timepoint; EGFRvIII: 59.3±3.00%, EGFRvIII-SGRP: 52.6±1.42%, CD19: 0.1±0.07%). Differential expression analysis of CAR T-cell secretomes identified SGRP from EGFRvIII-SGRP CAR T-cell supernatants (-Log10qValue/Log2fold-change= 3.84/6.15). Consistent with studies of systemic EGFRvIII CAR T-cell therapy, our data suggest that intratumoral EGFRvIII CAR T-cells were insufficient to eliminate BS153 tumors with homogeneous EGFRvIII expression in mice (Overall survival; EGFRvIII-treated: 20%, CD19-treated: 0%, n= 5 per group). Our current work focuses on the functional characterization of SGRP binding, SGRP-mediated phagocytosis, and on the development of a translational preclinical model of heterogeneous EGFRvIII expression to investigate an additive effect of CAR T-cell therapy and GAM modulation.

scholarly journals 221 CRISPR screen identifies loss of IFNγR signaling and downstream adhesion as a resistance mechanism to CAR T-cell cytotoxicity in solid but not liquid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A234-A234
Author(s):  
Rebecca Larson ◽  
Michael Kann ◽  
Stefanie Bailey ◽  
Nicholas Haradhvala ◽  
Kai Stewart ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Solid Tumors ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundChimeric Antigen Receptor (CAR) therapy has had a transformative impact on the treatment of hematologic malignancies1–6 but success in solid tumors remains elusive. We hypothesized solid tumors have cell-intrinsic resistance mechanisms to CAR T-cell cytotoxicity.MethodsTo systematically identify resistance pathways, we conducted a genome-wide CRISPR knockout screen in glioblastoma cells, a disease where CAR T-cells have had limited efficacy.7 8 We utilized the glioblastoma cell line U87 and targeted endogenously expressed EGFR with CAR T-cells generated from 6 normal donors for the screen. We validated findings in vitro and in vivo across a variety of human tumors and CAR T-cell antigens.ResultsLoss of genes in the interferon gamma receptor (IFNγR) signaling pathway (IFNγR1, JAK1, JAK2) rendered U87 cells resistant to CAR T-cell killing in vitro. IFNγR1 knockout tumors also showed resistance to CAR T cell treatment in vivo in a second glioblastoma line U251 in an orthotopic model. This phenomenon was irrespective of CAR target as we also observed resistance with IL13Ralpha2 CAR T-cells. In addition, resistance to CAR T-cell cytotoxicity through loss of IFNγR1 applied more broadly to solid tumors as pancreatic cell lines targeted with either Mesothelin or EGFR CAR T-cells also showed resistance. However, loss of IFNγR signaling did not impact sensitivity of liquid tumor lines (leukemia, lymphoma or multiple myeloma) to CAR T-cells in vitro or in an orthotopic model of leukemia treated with CD19 CAR. We isolated the effects of decreased cytotoxicity of IFNγR1 knockout glioblastoma tumors to be cancer-cell intrinsic because CAR T-cells had no observable differences in proliferation, activation (CD69 and LFA-1), or degranulation (CD107a) when exposed to wildtype versus knockout tumors. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell adhesion pathways compared to wildtype glioblastoma cells after exposure to CAR T-cells. We found that loss of IFNγR1 reduced CAR T-cell binding avidity to glioblastoma.ConclusionsThe critical role of IFNγR signaling for susceptibility of solid tumors to CAR T-cells is surprising given that CAR T-cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumors, IFNγR signaling was required for sufficient adhesion of CAR T-cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumors differ in their interactions with CAR T-cells and suggests that enhancing T-cell/tumor interactions may yield improved responses in solid tumors.AcknowledgementsRCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.ReferencesMaude SL, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448.Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377:2531–2544.Locke FL, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. The Lancet Oncology 2019;20:31–42.Schuster SJ, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017;377:2545–2554.Wang M, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2020;382:1331–1342.Cohen AD, et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 2019;129:2210–2221.Bagley SJ, et al. CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges. Neuro-oncology 2018;20:1429–1438.Choi BD, et al. Engineering chimeric antigen receptor T cells to treat glioblastoma. J Target Ther Cancer 2017;6:22–25.Ethics ApprovalAll human samples were obtained with informed consent and following institutional guidelines under protocols approved by the Institutional Review Boards (IRBs) at the Massachusetts General Hospital (2016P001219). Animal work was performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) (2015N000218 and 2020N000114).

scholarly journals 124 Functionalizing CAR T cells for selective proliferation and dual-targeting using the meditope technology

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A133-A133
Author(s):  
Cheng-Fu Kuo ◽  
Yi-Chiu Kuo ◽  
Miso Park ◽  
Zhen Tong ◽  
Brenda Aguilar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundMeditope is a small cyclic peptide that was identified to bind to cetuximab within the Fab region. The meditope binding site can be grafted onto any Fab framework, creating a platform to uniquely and specifically target monoclonal antibodies. Here we demonstrate that the meditope binding site can be grafted onto chimeric antigen receptors (CARs) and utilized to regulate and extend CAR T cell function. We demonstrate that the platform can be used to overcome key barriers to CAR T cell therapy, including T cell exhaustion and antigen escape.MethodsMeditope-enabled CARs (meCARs) were generated by amino acid substitutions to create binding sites for meditope peptide (meP) within the Fab tumor targeting domain of the CAR. meCAR expression was validated by anti-Fc FITC or meP-Alexa 647 probes. In vitro and in vivo assays were performed and compared to standard scFv CAR T cells. For meCAR T cell proliferation and dual-targeting assays, the meditope peptide (meP) was conjugated to recombinant human IL15 fused to the CD215 sushi domain (meP-IL15:sushi) and anti-CD20 monoclonal antibody rituximab (meP-rituximab).ResultsWe generated meCAR T cells targeting HER2, CD19 and HER1/3 and demonstrate the selective specific binding of the meditope peptide along with potent meCAR T cell effector function. We next demonstrated the utility of a meP-IL15:sushi for enhancing meCAR T cell proliferation in vitro and in vivo. Proliferation and persistence of meCAR T cells was dose dependent, establishing the ability to regulate CAR T cell expansion using the meditope platform. We also demonstrate the ability to redirect meCAR T cells tumor killing using meP-antibody adaptors. As proof-of-concept, meHER2-CAR T cells were redirected to target CD20+ Raji tumors, establishing the potential of the meditope platform to alter the CAR specificity and overcome tumor heterogeneity.ConclusionsOur studies show the utility of the meCAR platform for overcoming key challenges for CAR T cell therapy by specifically regulating CAR T cell functionality. Specifically, the meP-IL15:sushi enhanced meCAR T cell persistence and proliferation following adoptive transfer in vivo and protects against T cell exhaustion. Further, meP-ritiuximab can redirect meCAR T cells to target CD20-tumors, showing the versatility of this platform to address the tumor antigen escape variants. Future studies are focused on conferring additional ‘add-on’ functionalities to meCAR T cells to potentiate the therapeutic effectiveness of CAR T cell therapy.

scholarly journals Feasibility, and Efficacy of Donor-Derived cd19-Targeted Car t-Cell Therapy in Refractory/Relapsed(r/r)b-Cell Acute Lymphoblastic Leukemia (b-all) Patients

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-6
Author(s):  
Xian Zhang ◽  
Junfang Yang ◽  
Wenqian Li ◽  
Gailing Zhang ◽  
Yunchao Su ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T ◽  
Grade 3

Backgrounds As CAR T-cell therapy is a highly personalized therapy, process of generating autologous CAR-T cells for each patient is complex and can still be problematic, particularly for heavily pre-treated patients and patients with significant leukemia burden. Here, we analyzed the feasibility and efficacy in 37 patients with refractory/relapsed (R/R) B-ALL who received CAR T-cells derived from related donors. Patients and Methods From April 2017 to May 2020, 37 R/R B-ALL patients with a median age of 19 years (3-61 years), were treated with second-generation CD19 CAR-T cells derived from donors. The data was aggregated from three clinical trials (www.clinicaltrials.gov NCT03173417; NCT02546739; and www.chictr.org.cn ChiCTR-ONC-17012829). Of the 37 patients, 28 were relapsed following allogenic hematopoietic stem cell transplant (allo-HSCT) and whose lymphocytes were collected from their transplant donors (3 HLA matched sibling and 25 haploidentical). For the remaining 9 patients without prior transplant, the lymphocytes were collected from HLA identical sibling donors (n=5) or haploidentical donors (n=4) because CAR-T cells manufacture from patient samples either failed (n=5) or blasts in peripheral blood were too high (>40%) to collect quality T-cells. The median CAR-T cell dose infused was 3×105/kg (1-30×105/kg). Results For the 28 patients who relapsed after prior allo-HSCT, 27 (96.4%) achieved CR within 30 days post CAR T-cell infusion, of which 25 (89.3%) were minimal residual disease (MRD) negative. Within one month following CAR T-cell therapy, graft-versus-host disease (GVHD) occurred in 3 patients including 1 with rash and 2 with diarrhea. A total of 19 of the 28 (67.9%) patients had cytokine release syndrome (CRS), including two patients (7.1%) with Grade 3-4 CRS. Four patients had CAR T-cell related neurotoxicity including 3 with Grade 3-4 events. With a medium follow up of 103 days (1-669days), the median overall survival (OS) was 169 days (1-668 days), and the median leukemia-free survival (LFS) was 158 days (1-438 days). After CAR T-cell therapy, 15 patients bridged into a second allo-HSCT and one of 15 patients (6.7%) relapsed following transplant, and two died from infection. There were 11 patients that did not receive a second transplantation, of which three patients (27.3%) relapsed, and four parents died (one due to relapse, one from arrhythmia and two from GVHD/infection). Two patients were lost to follow-up. The remaining nine patients had no prior transplantation. At the time of T-cell collection, the median bone marrow blasts were 90% (range: 18.5%-98.5%), and the median peripheral blood blasts were 10% (range: 0-70%). CR rate within 30 days post CAR-T was 44.4% (4/9 cases). Six patients developed CRS, including four with Grade 3 CRS. Only one patient had Grade 3 neurotoxicity. No GVHD occurred following CAR T-cell therapy. Among the nine patients, five were treated with CAR T-cells derived from HLA-identical sibling donors and three of those five patients achieved CR. One patient who achieved a CR died from disseminated intravascular coagulation (DIC) on day 16. Two patients who achieved a CR bridged into allo-HSCT, including one patient who relapsed and died. One of two patients who did not response to CAR T-cell therapy died from leukemia. Four of the nine patients were treated with CAR T-cells derived from haploidentical related donors. One of the four cases achieved a CR but died from infection on day 90. The other three patients who had no response to CAR T-cell therapy died from disease progression within 3 months (7-90 days). Altogether, seven of the nine patients died with a median time of 19 days (7-505 days). Conclusions We find that manufacturing CD19+ CAR-T cells derived from donors is feasible. For patients who relapse following allo-HSCT, the transplant donor derived CAR-T cells are safe and effective with a CR rate as high as 96.4%. If a patient did not have GVHD prior to CAR T-cell therapy, the incidence of GVHD following CAR T-cell was low. Among patients without a history of transplantation, an inability to collect autologous lymphocytes signaled that the patient's condition had already reached a very advanced stage. However, CAR T-cells derived from HLA identical siblings can still be considered in our experience, no GVHD occurred in these patients. But the efficacy of CAR T-cells from haploidentical donors was very poor. Disclosures No relevant conflicts of interest to declare.

scholarly journals Allogeneic Hematopoietic Cell Transplantation Is Critical to Maintain Remissions after CD19-CAR T-Cell Therapy for Pediatric ALL: A Single Center Experience

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 39-40
Author(s):  
Aimee C Talleur ◽  
Renee M. Madden ◽  
Amr Qudeimat ◽  
Ewelina Mamcarz ◽  
Akshay Sharma ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Allogeneic Hct ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

CD19-CAR T-cell therapy has shown remarkable efficacy in pediatric patients with relapsed and/or refractory B-cell acute lymphoblastic leukemia (r/r ALL). Despite high short-term remission rates, many responses are not durable and the best management of patients who achieve a complete response (CR) post-CAR T-cell therapy remains controversial. In particular, it is unclear if these patients should be observed or proceed to consolidative allogeneic hematopoietic cell transplantation (HCT). To address this question, we reviewed the clinical course of all patients (n=22) who received either an investigational CAR T-cell product (Phase I study: SJCAR19 [NCT03573700]; n=12) or tisagenlecleucel (n=10) at our institution. The investigational CD19-CAR T cells were generated by a standard cGMP-compliant procedure using a lentiviral vector encoding a 2nd generation CD19-CAR with a FMC63-based CD19 binding domain, CD8a stalk and transmembrane domain, and 41BB.ζ signaling domain. Patients received therapy between 8/2018 and 3/2020. All products met manufacturing release specifications. Within the entire cohort, median age at time of infusion was 12.3 years old (range: 1.8-23.5) and median pre-infusion marrow burden using flow-cytometry minimal residual disease (MRD) testing was 6.8% (range: 0.003-100%; 1 patient detectable by next-generation sequencing [NGS] only). All patients received lymphodepleting chemotherapy (fludarabine, 25mg/m2 daily x3, and cyclophosphamide, 900mg/m2 daily x1), followed by a single infusion of CAR T-cells. Phase I product dosing included 1x106 CAR+ T-cells/kg (n=6) or 3x106 CAR+ T-cells/kg (n=6). Therapy was well tolerated, with a low incidence of cytokine release syndrome (any grade: n=10; Grade 3-4: n=4) and neurotoxicity (any grade: n=8; Grade 3-4: n=3). At 4-weeks post-infusion, 15/22 (68.2%) patients achieved a CR in the marrow, of which 13 were MRDneg (MRDneg defined as no detectable leukemia by flow-cytometry, RT-PCR and/or NGS, when available). Among the 2 MRDpos patients, 1 (detectable by NGS only) relapsed 50 days after CAR T-cell infusion and 1 died secondary to invasive fungal infection 35 days after infusion. Within the MRDneg cohort, 6/13 patients proceeded to allogeneic HCT while in MRDneg/CR (time to HCT, range: 1.8-2.9 months post-CAR T-cell infusion). All 6 HCT recipients remain in remission with a median length of follow-up post-HCT of 238.5 days (range 19-441). In contrast, only 1 (14.3%) patient out of 7 MRDneg/CR patients who did not receive allogeneic HCT, remains in remission with a follow up of greater 1 year post-CAR T-cell infusion (HCT vs. no HCT: p<0.01). The remaining 6 patients developed recurrent detectable leukemia within 2 to 9 months post-CAR T-cell infusion (1 patient detectable by NGS only). Notably, recurring leukemia remained CD19+ in 4 of 5 evaluable patients. All 4 patients with CD19+ relapse received a 2nd CAR T-cell infusion (one in combination with pembrolizumab) and 2 achieved MRDneg/CR. There were no significant differences in outcome between SJCAR19 study participants and patients who received tisagenlecleucel. With a median follow up of one year, the 12 month event free survival (EFS) of all 22 patients is 25% (median EFS: 3.5 months) and the 12 month overall survival (OS) 70% (median OS not yet reached). In conclusion, infusion of investigational and FDA-approved autologous CD19-CAR T cells induced high CR rates in pediatric patients with r/r ALL. However, our current experience shows that sustained remission without consolidative allogeneic HCT is not seen in most patients. Our single center experience highlights not only the need to explore maintenance therapies other than HCT for MRDneg/CR patients, but also the need to improve the in vivo persistence of currently available CD19-CAR T-cell products. Disclosures Sharma: Spotlight Therapeutics: Consultancy; Magenta Therapeutics: Other: Research Collaboration; CRISPR Therapeutics, Vertex Pharmaceuticals, Novartis: Other: Clinical Trial PI. Velasquez:St. Jude: Patents & Royalties; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees. Gottschalk:Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; TESSA Therapeutics: Other: research collaboration; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy.

scholarly journals CAR T-cell therapy of solid tumors: promising approaches to modulating antitumor activity of CAR T cells

2019 ◽  
pp. 5-12 ◽  
Author(s):  
Ya.Yu. Kiseleva ◽  
A.M. Shishkin ◽  
A.V. Ivanov ◽  
T.M. Kulinich ◽  
V.K. Bozhenko
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Solid Tumors ◽  
Functional Activity ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Adoptive immunotherapy that makes use of genetically modified autologous T cells carrying a chimeric antigen receptor (CAR) with desired specificity is a promising approach to the treatment of advanced or relapsed solid tumors. However, there are a number of challenges facing the CAR T-cell therapy, including the ability of the tumor to silence the expression of target antigens in response to the selective pressure exerted by therapy and the dampening of the functional activity of CAR T cells by the immunosuppressive tumor microenvironment. This review discusses the existing gene-engineering approaches to the modification of CAR T-cell design for 1) creating universal “switchable” synthetic receptors capable of attacking a variety of target antigens; 2) enhancing the functional activity of CAR T cells in the immunosuppressive microenvironment of the tumor by silencing the expression of inhibiting receptors or by stimulating production of cytokines.

The efficacy and safety of anti-CLL1 based CAR-T cells in children with relapsed or refractory acute myeloid leukemia: A multicenter interim analysis.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 10000-10000
Author(s):  
Hui Zhang ◽  
Chaoke Bu ◽  
Zhiyong Peng ◽  
min luo ◽  
Chunfu Li
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Myeloid Leukemia ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T ◽  
Refractory Acute Myeloid Leukemia

10000 Background: Chimeric antigen receptor (CAR)-T cell therapy has demonstrated remarkable success in treating a variety of blood cancers, such as CD19 CAR-T for B-cell malignancies and BCMA CAR-T for myeloid myeloma (MM). However, similar achievement has yet to be replicated in patients with relapsed and refractory acute myeloid leukemia (R/R AML), primarily due to the AML heterogeneity, making it difficult to find an ideal CAR-T target. Previous efforts have targeted single CD33, CD123, LeY, NKG2D, or CD70 receptors, but the overall response rate is very disappointed. To address these challenges, we aim to find an effective target for AML without the need for the hematopoietic stem cells transplant (HSCT). In our study, CLL1 is chosen as a promising target as it is not expressed on normal HSCs, but highly expressed on AML blasts cells and leukemia stem cells (LSCs). Here we report the interim analysis from a Phase I clinical trial using anti-CLL1 based CAR-T cells to treat children with R/R AML. The primary and secondary objectives were to evaluate the safety and anti-AML responses, respectively, with long-term prognosis within those patients who did not receive allogeneic HSCT (allo-HSCT) as an additional objective. Methods: We have generated a 2nd generation of CLL1 CAR-T, the extracellular scFv was derived from a murine CLL1 monoclonal antibody, which was generated by hybridoma technology. Autologous CAR-T cells were manufactured in a cGMP facility. Between Oct 2019 and Jan 2021, 11 pediatric R/R AML patients were infused. CLL1 or CLL1-CD33 dual CAR-T cells were given by a dose at 0.3-1x106/kg with a single dose after lymphodepleting conditioning with cyclophosphamide/fludarabine(Cy/Flu). Results: Of the 11 patients infused, Grade 3-4 hematologic adverse events were observed before and during CAR-T cell infusion, and no dose-limiting toxicities were observed. Meanwhile, grade 1-3 cytokine release syndrome was observed but without any lethal events. All the adverse effects were resolved after guideline-directed intervention. Anti-CLL1 CAR-T cells efficiently expanded in vivo, the median expansion peaking time was at Day 8. For these 11 R/R-AML patients, 10 patients completely responded to anti-CLL1 based CAR-T cell therapy, with CLL1 positive AML blast eliminated within one month. Among the responded 10 patients, 5 patients reached CR/MRD-, 3 patients reached CR/MRD+, 1 patient reached PR and 1 patient showed SD, with only CLL1 negative AML cells. Conclusions: Our study demonstrated that 10/11 patients responded to CLL1 CAR-T cell therapy within one month. For patients showing MRD+ with CLL1 negative AML blast, chemotherapy like Azacitidine, and combined with HSCT may help those patients to reach complete response. These initial results suggested that anti-CLL1 base CAR-T cells can be a well-tolerated and candidate option for treating children with R/R-AML. Clinical trial information: ChiCTR1900027684.

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