scholarly journals Multiplexed Engineering of CD19CAR T Cells for Post-Transplant Consolidative Immunotherapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1159-1159 ◽  
Author(s):  
Corinne Summers ◽  
Alexandra Grier ◽  
Rebecca Gardner ◽  
Colleen Delaney ◽  
Michael C Jensen
Keyword(s):  
T Cells ◽  
T Cell ◽  
Statistical Significance ◽  
Transduction Efficiency ◽  
P Value ◽  
Drug Resistant ◽  
A Cells ◽  
Car T

Abstract Introduction: Relapse of CD19+ acute lymphoblastic leukemia (ALL) post-hematopoietic cell transplant (HCT) portends a dismal prognosis and opportunities to intensify the anti-tumor potency of HCT are limited by regimen related toxicities. While pre-transplant CD19 chimeric antigen receptor (CAR) T cell therapy can increase the numbers of patients who achieve minimal residual disease negative remissions, there remains an unmet need to further reduce the incidence of early post-HCT relapses through the use of adoptive immunotherapy. However, the use of CAR T cell therapies in this setting is severely limited as patients are on immunosuppressive therapy (IST) to prevent graft versus host disease (GVHD). Here, we report on a multiplexed engineering strategy to generate donor-derived CD19CAR T cells that are resistant to combinations of cyclosporine (CSA)/Tacrolimus (FK506) and/or mycophenolate mofetil (MMF), and devoid of GVHD reactivity for this patient population. Methods and Results: We developed CNA22 or CNA12-P2A-IMPDH2Δ-T2A-Her2tG vectors using the CNA22, CNA12 [Brewin M, et al, Blood 2009] and IMPDH2Δ [Jonnalagadda J, et al, PLoS One 2013] mutants conferring CSA, FK506 and MMF resistance, respectively. Her2tG is a truncated HER2 extracellular protein developed in our lab and used for selection and transduction efficiency determination using Herceptin. The drug resistant lentivirus packaged vector was used to co-transduce human T cells with our CD19CAR vector which includes a 4-1BB co-stimulatory domain and truncated EGFR protein (EGFRt) for cell selection, transduction efficiency and in vivo suicide using Erbitux [Wang, X, et al, Blood 2011]. We coupled dual transduction with mRNA TALEN transfection technology using a TALEN pair targeting the first exon of the TcRα constant region to transfect T cells for efficient knockout of the endogenous T cell receptor (TcR) thus eliminating the risk of causing GVHD. Our multiplexed technologies yield CD3/TcR knockouts rates greater than 80% with successful dual transduction of both vectors demonstrated by EGFRt and Her2tG expression (Figure A). Cells demonstrated MMF resistance in vitro as evidenced by increased Her2tG expression for culture purification following mycophenolic acid (MPA, active metabolite of MMF) exposure. In addition, prolonged MPA drug exposure at multiple drug concentrations (0-10uM) demonstrated improved culture expansion and viability for cultures containing cells transduced to express IMPDH2Δ over a 21 day culture period (Figure B). CD19CAR expressing cells demonstrated CD19 antigen targeted cell lysis as evaluated by chromium release assay. Furthermore, cytokine evaluations of cells following co-culturing with target cells at multiple CSA concentrations (0-1000ng/ml) yielded sustained IL-2, IFNg and TNFa secretion by CNA22 expressing cells following CD19CAR activation (Figure C). In vivo experiments are in progress to assess the functional attributes of these multiplexed engineered CAR T cells in relevant murine model systems. Conclusions: We successfully used multiplexed engineering strategies to generate CD3/TcR- CD19CAR+EGFRt+ CNA22-IMPDH2Δ-HER2tG+ T cells. Cells expressing CNA22 and IMPDH2Δ mutants exhibited activation and expansion in vitro in the presence of immunosuppressive agents CSA and MPA with targeted CD19CAR activity. These studies suggest the ability to generate modified T cells that remain functional in vivo even in the presence of IST and without induction of GVHD, allowing for this approach in the post-transplant setting to prevent/treat disease relapse. Figure A) Cells underwent successful dual transduction followed by mRNA TALEN transfection for development of CD3/TcR- CD19CAR+EGFRt+ CNA22-IMPDH2Δ-HER2tG+ T cells. B) Transduced T cells underwent selection for their respective markers, were stimulated and cultured for 21 days at varying MPA concentrations. Cells transduced to express IMPDH2Δ exhibited improved expansion as compared to CD19CAR and mock control cells. Data was normalized to the cell group's no drug control expansion. C) Following co-culture with K562-CD19 antigen cells with CSA concentrations of 0-1000ng/ml CD19CAR cells transduced with the drug resistant vector secreted significantly increased IFNg and IL-2 levels compared to CD19CAR only cells. NS = not significant; Horizontal bars indicate statistical significance defined as p-value <0.05. Figure. A) Cells underwent successful dual transduction followed by mRNA TALEN transfection for development of CD3/TcR- CD19CAR+EGFRt+ CNA22-IMPDH2Δ-HER2tG+ T cells. B) Transduced T cells underwent selection for their respective markers, were stimulated and cultured for 21 days at varying MPA concentrations. Cells transduced to express IMPDH2Δ exhibited improved expansion as compared to CD19CAR and mock control cells. Data was normalized to the cell group's no drug control expansion. C) Following co-culture with K562-CD19 antigen cells with CSA concentrations of 0-1000ng/ml CD19CAR cells transduced with the drug resistant vector secreted significantly increased IFNg and IL-2 levels compared to CD19CAR only cells. NS = not significant; Horizontal bars indicate statistical significance defined as p-value <0.05. Disclosures Jensen: Juno Therapeutics, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals 131 Genetic disruption of negative immune regulators to enhance CAR-T efficacy against solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A140-A141
Author(s):  
David Mai ◽  
Omar Johnson ◽  
Carl June
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Solid Tumor ◽  
Transduction Efficiency ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundCAR-T cell therapy has demonstrated remarkable success in hematological malignancies but displays limited efficacy in solid tumors, which comprise most cancer cases. Recent studies suggest that CAR-T cell failure via T cell exhaustion is characterized by decreased surface CAR expression, cytotoxicity, and Th1 cytokine production, leading to reduced antitumor functionality.1 2 3 To address these issues, studies have turned to genetically knocking out or overexpressing targets associated with an exhaustion or effector phenotype, such as PD-1 knockout (KO) and c-Jun overexpression, among other candidates that are typically receptors or transcription factors.4 5 6 However, there are other underexplored factors that mediate various aspects of immune regulation. While genome-wide CRISPR screens may discover such factors, they are unlikely to reveal phenotypes for genes whose function is partially redundant, therefore promising candidates may be missed. Such candidates include post-transcriptional regulators (PTRs) that coordinate immune responses, which are less well-studied in the context of CAR-T cell function. We hypothesized that KO of these PTRs may increase CAR-T cell cytokine activity, phenotype, and persistence, potentially under long-term or exhaustion-inducing conditions, leading to increased tumor control. Ultimately, disruption of negative immune regulators could produce CAR-T cells with enhanced activity and persistence, narrowing the gap between efficacy in hematological and solid tumors.MethodsTo explore whether the disruption of two target PTRs improves solid tumor efficacy, we used CRISPR-Cas9 to genetically delete one or both PTRs in mesothelin-targeting human CAR-T cells and assayed their function in vitro and in vivo in NSG mice.ResultsWe show successful genetic deletion of these genes in post-thymic human T cells and that their disruption does not affect primary expansion (figure 1) or transduction efficiency (figure 2). These KO CAR-T cells display increased expression of co-stimulatory receptors and various cytokines (figure 3). While KO CAR-T cells are functionally similar to WT CAR-T cells in in vitro assays (figure 4), KO CAR-T cells demonstrate superior activity in vivo and can clear large, established tumors compared to WT CAR-T cells at low dose (figure 5).Abstract 131 Figure 1Expansion kinetics of KO CAR-T cellsAbstract 131 Figure 2Transduction efficiency and baseline phenotype of KO CAR-T cellsAbstract 131 Figure 3Costimulatory receptor and cytokine expression of KO CAR-T cellsAbstract 131 Figure 4In vitro cytotoxicity of KO CAR-T cellsAbstract 131 Figure 5In vivo activity of KO CAR-T cellsConclusionsThese results indicate that KO of our target PTRs may improve the potency of CAR-T cells in solid tumors and may have important implications on the development of effective solid-tumor cell therapies.ReferencesJE Wherry and M Kurachi, Molecular and cellular insights into T cell exhaustion, Nature Reviews Immunology 2015;15:486–499.EW Weber, et al. Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science 2021;372:6537.S Kuramitsu et al. Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment. Cell, in revision.BD Choi et al, CRISPR-Cas9 disruption of PD-1 enhances activity of university EGFRvIII CAR T cells in a preclinical model of human glioblastoma. Journal for ImmunoTherapy of Cancer 2019;7:304.RC Lynn et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 2019;576:293–300.LJ Rupp et al. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Scientific Reports 2017;7:737.

98 ATA3271: an armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A109-A109
Author(s):  
Jiangyue Liu ◽  
Xianhui Chen ◽  
Jason Karlen ◽  
Alfonso Brito ◽  
Tiffany Jheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Phase 3 ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T

BackgroundMesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein with high expression levels in an array of malignancies including mesothelioma, ovaria, non-small cell lung cancer, and pancreatic cancers and is an attractive target antigen for immune-based therapies. Early clinical evaluation of autologous MSLN-targeted chimeric antigen receptor (CAR)-T cell therapies for malignant pleural mesothelioma has shown promising acceptable safety1 and have recently evolved with incorporation of next-generation CAR co-stimulatory domains and armoring with intrinsic checkpoint inhibition via expression of a PD-1 dominant negative receptor (PD1DNR).2 Despite the promise that MSLN CAR-T therapies hold, manufacturing and commercial challenges using an autologous approach may prove difficult for widespread application. EBV T cells represent a unique, non-gene edited approach toward an off-the-shelf, allogeneic T cell platform. EBV-specific T cells are currently being evaluated in phase 3 trials [NCT03394365] and, to-date, have demonstrated a favorable safety profile including limited risks for GvHD and cytokine release syndrome.3 4 Clinical proof-of-principle studies for CAR transduced allogeneic EBV T cell therapies have also been associated with acceptable safety and durable response in association with CD19 targeting.5 Here we describe the first preclinical evaluation of ATA3271, a next-generation allogeneic CAR EBV T cell therapy targeting MSLN and incorporating PD1DNR, designed for the treatment of solid tumor indications.MethodsWe generated allogeneic MSLN CAR+ EBV T cells (ATA3271) using retroviral transduction of EBV T cells. ATA3271 includes a novel 1XX CAR signaling domain, previously associated with improved signaling and decreased CAR-mediated exhaustion. It is also armored with PD1DNR to provide intrinsic checkpoint blockade and is designed to retain functional persistence.ResultsIn this study, we characterized ATA3271 both in vitro and in vivo. ATA3271 show stable and proportional CAR and PD1DNR expression. Functional studies show potent antitumor activity of ATA3271 against MSLN-expressing cell lines, including PD-L1-high expressors. In an orthotopic mouse model of pleural mesothelioma, ATA3271 demonstrates potent antitumor activity and significant survival benefit (100% survival exceeding 50 days vs. 25 day median for control), without evident toxicities. ATA3271 maintains persistence and retains central memory phenotype in vivo through end-of-study. Additionally, ATA3271 retains endogenous EBV TCR function and reduced allotoxicity in the context of HLA mismatched targets. ConclusionsOverall, ATA3271 shows potent anti-tumor activity without evidence of allotoxicity, both in vitro and in vivo, suggesting that allogeneic MSLN-CAR-engineered EBV T cells are a promising approach for the treatment of MSLN-positive cancers and warrant further clinical investigation.ReferencesAdusumilli PS, Zauderer MG, Rusch VW, et al. Abstract CT036: A phase I clinical trial of malignant pleural disease treated with regionally delivered autologous mesothelin-targeted CAR T cells: Safety and efficacy. Cancer Research 2019;79:CT036-CT036.Kiesgen S, Linot C, Quach HT, et al. Abstract LB-378: Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial. Cancer Research 2020;80:LB-378-LB-378.Prockop S, Doubrovina E, Suser S, et al. Off-the-shelf EBV-specific T cell immunotherapy for rituximab-refractory EBV-associated lymphoma following transplantation. J Clin Invest 2020;130:733–747.Prockop S, Hiremath M, Ye W, et al. A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disease (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy. Blood 2019; 134: 5326–5326.Curran KJ, Sauter CS, Kernan NA, et al. Durable remission following ‘Off-the-Shelf’ chimeric antigen receptor (CAR) T-Cells in patients with relapse/refractory (R/R) B-Cell malignancies. Biology of Blood and Marrow Transplantation 2020;26:S89.

scholarly journals Nanoparticle T-cell engagers as a modular platform for cancer immunotherapy

Leukemia ◽  
2021 ◽  
Author(s):  
Kinan Alhallak ◽  
Jennifer Sun ◽  
Katherine Wasden ◽  
Nicole Guenthner ◽  
Julie O’Neal ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Escape ◽  
Multiple Cancer ◽  
Cancer Antigen ◽  
Cancer Antigens ◽  
Car T ◽  
Modular Platform

AbstractT-cell-based immunotherapy, such as CAR-T cells and bispecific T-cell engagers (BiTEs), has shown promising clinical outcomes in many cancers; however, these therapies have significant limitations, such as poor pharmacokinetics and the ability to target only one antigen on the cancer cells. In multiclonal diseases, these therapies confer the development of antigen-less clones, causing tumor escape and relapse. In this study, we developed nanoparticle-based bispecific T-cell engagers (nanoBiTEs), which are liposomes decorated with anti-CD3 monoclonal antibodies (mAbs) targeting T cells, and mAbs targeting the cancer antigen. We also developed a nanoparticle that targets multiple cancer antigens by conjugating multiple mAbs against multiple cancer antigens for T-cell engagement (nanoMuTEs). NanoBiTEs and nanoMuTEs have a long half-life of about 60 h, which enables once-a-week administration instead of continuous infusion, while maintaining efficacy in vitro and in vivo. NanoMuTEs targeting multiple cancer antigens showed greater efficacy in myeloma cells in vitro and in vivo, compared to nanoBiTEs targeting only one cancer antigen. Unlike nanoBiTEs, treatment with nanoMuTEs did not cause downregulation (or loss) of a single antigen, and prevented the development of antigen-less tumor escape. Our nanoparticle-based immuno-engaging technology provides a solution for the major limitations of current immunotherapy technologies.

scholarly journals Base-edited CAR T cells for combinational therapy against T cell malignancies

Leukemia ◽  
2021 ◽  
Author(s):  
Christos Georgiadis ◽  
Jane Rasaiyaah ◽  
Soragia Athina Gkazi ◽  
Roland Preece ◽  
Aniekan Etuk ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Specific Binding ◽  
Base Editing ◽  
Car T Cells ◽  
Dna And Rna ◽  
Car T ◽  
T Cell Malignancies

AbstractTargeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by ‘T v T’ fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in ‘self-enrichment’ yielding populations 99.6% TCR−/CD3−/CD7−. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic ‘off-target’ activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

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 Checkpoint Blockade in Combination with CD33 Chimeric Antigen Receptor T Cell Therapy and Hypomethylating Agent Against Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1383-1383 ◽  
Author(s):  
Tongyuan Xue ◽  
Marissa Del Real ◽  
Emanuela Marcucci ◽  
Candida Toribio ◽  
Sonia Maryam Setayesh ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Myeloid Leukemia ◽  
T Cell Therapy ◽  
Programmed Death ◽  
Car T ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. The cure rate for primary AML patients is only 35% and decreases with age. Novel and effective immunotherapies for patients with relapsed and/or refractory (r/r) AML remain an urgent unmet need. CD33 is an attractive immunotherapeutic target for myeloid malignancies given its expression on more than 85% of AML patient samples. We therefore set out to design and test CD33 chimeric antigen receptor (CD33CAR) T cells preclinically as a single agent and in combinational therapy. To assess antileukemic responses of CD33CAR T cells in vitro and in vivo, we enriched CD4/CD8 T cells from peripheral blood mononuclear cells (PBMCs) and genetically modified them to express a second-generation CD33CAR. CD33CAR T cells exhibited potent antigen dependent CD107a degranulation, IFN-γ production and killing activities against AML cells in vitro. Using a NOD-SCID-IL2Rgnull (NSG) xenograft model engrafted with MOLM-14-ffluc, a CD33 expressing AML cell line transduced with lentivirus carrying firefly luciferase (ffluc) and enhanced green fluorescent protein (eGFP), 3 million CD33CAR or mock T cells were introduced intravenously. CD33 CAR T cell-treated group displayed 98.2% leukemic regression 4 days post CAR T infusion, and 99.6% reduction on day 31. Bioluminescent imaging (BLI) and Kaplan-Meier analysis demonstrated that CD33CAR T cells significantly decreased leukemic burden and prolonged overall survival compared to mock T cells in vivo. Decitabine, a DNA hypomethylating agent (HMA), is a main therapeutic agent for treating AML. We observed HMA treatment led to increased CD33 expression on MOLM-14 cells in vitro. We hypothesized that decitabine can potentiate CD33CAR T cell-mediated AML killing by increasing CD33 expression. MOLM-14 cells were treated with either decitabine alone, CD33CAR T cells alone, or sequential treatment using various concentrations of decitabine or DMSO followed by CD33CAR or mock T cells in an E:T ratio of 1:100. We determined the target specific killing activities in each group using flow cytometric based analysis 48 and 96 hours later. The decitabine followed by CD33CAR T cells treatment reproducibly resulted in the most robust antileukemic activity with 80.6% MOLM-14 cells killed. In comparison, CD33CAR T cells or decitabine monotherapy resulted in 11.5% and 50.9% killing, respectively. In vivo testing of the combinational effects of decitabine and CD33CAR T cells are underway and will be updated at the meeting. Finally, checkpoint blockade targeting programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) has shown survival benefits, particularly in combination with HMA, for patients with r/r AML (Daver et al. 2019). We observed elevated PD-L1 expression on residual AML blasts that survived the treatment with decitabine in combination with CD33CAR T cells. Therefore, we hypothesized that blockade of PD-1/PD-L1 interaction might further improve the antileukemic effect of CD33CAR T cells against AML cells post antigen induction by decitabine. MOLM-14 cells were treated with decitabine for 2 days and CD33CAR T cells were added in an E:T ratio of 1:75. Anti-PD-1 or IgG4 antibody was added to the culture at various concentrations. The most robust CD33 specific killing was seen in the culture with anti-PD-1 antibody added. Further characterization are underway and will be presented. Taken together, our preclinical findings have demonstrated the potency of the CD33CAR T cell therapy and ways to optimize its efficacy. Our results support clinical translation of CD33CAR T cells for patients with AML. Disclosures Budde: F. Hoffmann-La Roche Ltd: Consultancy.

scholarly journals Targeting glycosylation of PD-1 to enhance CAR-T cell cytotoxicity

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiaojuan Shi ◽  
Daiqun Zhang ◽  
Feng Li ◽  
Zhen Zhang ◽  
Shumin Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Inhibitory Effects ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

AbstractAsparagine-linked (N-linked) glycosylation is ubiquitous and can stabilize immune inhibitory PD-1 protein. Reducing N-linked glycosylation of PD-1 may decrease PD-1 expression and relieve its inhibitory effects on CAR-T cells. Considering that the codon of Asparagine is aac or aat, we wondered if the adenine base editor (ABE), which induces a·t to g·c conversion at specific site, could be used to reduce PD-1 suppression by changing the glycosylated residue in CAR-T cells. Our results showed ABE editing altered the coding sequence of N74 residue of PDCD1 and downregulated PD-1 expression in CAR-T cells. Further analysis showed ABE-edited CAR-T cells had enhanced cytotoxic functions in vitro and in vivo. Our study suggested that the single base editors can be used to augment CAR-T cell therapy.

scholarly journals Fratricide-resistant CD1a-specific CAR T cells for the treatment of cortical T-cell acute lymphoblastic leukemia

Blood ◽  
2019 ◽  
Vol 133 (21) ◽  
pp. 2291-2304 ◽  
Author(s):  
Diego Sánchez-Martínez ◽  
Matteo L. Baroni ◽  
Francisco Gutierrez-Agüera ◽  
Heleia Roca-Ho ◽  
Oscar Blanch-Lombarte ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Antileukemic Activity ◽  
Car T Cells ◽  
Car T ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) has a dismal outcome, and no effective targeted immunotherapies for T-ALL exist. The extension of chimeric antigen receptor (CAR) T cells (CARTs) to T-ALL remains challenging because the shared expression of target antigens between CARTs and T-ALL blasts leads to CART fratricide. CD1a is exclusively expressed in cortical T-ALL (coT-ALL), a major subset of T-ALL, and retained at relapse. This article reports that the expression of CD1a is mainly restricted to developing cortical thymocytes, and neither CD34+ progenitors nor T cells express CD1a during ontogeny, confining the risk of on-target/off-tumor toxicity. We thus developed and preclinically validated a CD1a-specific CAR with robust and specific cytotoxicity in vitro and antileukemic activity in vivo in xenograft models of coT-ALL, using both cell lines and coT-ALL patient–derived primary blasts. CD1a-CARTs are fratricide resistant, persist long term in vivo (retaining antileukemic activity in re-challenge experiments), and respond to viral antigens. Our data support the therapeutic and safe use of fratricide-resistant CD1a-CARTs for relapsed/refractory coT-ALL.

scholarly journals Piggybac Transposon Mediated CD19 CAR-T Cells Derived from CD45RA-Positive PBMCs Possess Potent and Sustained Antileukemic Function

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2807-2807
Author(s):  
Masaya Suematsu ◽  
Shigeki Yagyu ◽  
Nobuyoshi Nagao ◽  
Susumu Kubota ◽  
Yuto Shimizu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Research Funding ◽  
Stress Test ◽  
Transduction Efficiency ◽  
Antigen Stimulation ◽  
Car T Cells ◽  
Car T

Abstract Background: The quality of chimeric antigen receptor (CAR)-T cell products, including the expression of memory and exhaustion markers, has been shown to influence their long-term functionality. We previously demonstrated that piggyBac (PB) transposon-mediated CD19 CAR-T cells exhibit memory-rich phenotype that is characterized by a high proportion of CD45RA+/CCR7+ T cell fraction. To further investigate the favorable phenotype of PB-CD19 CAR-T cells, we generated PB-CD19 CAR-T cells from CD45RA+ and CD45RA− peripheral blood mononuclear cells (PBMCs) (RA+ CAR and RA− CAR, respectively), and compared their phenotype and antitumor function. Methods: CD45RA+ and CD45RA− PBMCs were isolated by magnetic selection from whole PBMCs, then the CD19-CAR transgene was transduced into these cells using the PB transposon system, as described previously. Transduction efficiency of CD19 CAR transgene was determined 24 hours by flow cytometry after transduction. The phenotype of CD19 CAR-T was evaluated by flow cytometry on day 14. High throughput RNA sequencing was performed to see the T cell activation/exhaustion profile upon antigen stimulation. Sequential killing assays were performed by adding fresh tumor cells into CAR-T cells co-cultured with tumor cells every three days by restoring an effector target ratio of 1:1. To see the durable antitumor efficacy in vivo, we performed in vivo stress test, in which CAR T-cells dosage was lowered to the functional limits, so that these CAR-T cells should be maintained and expanded in vivo, to achieve the antitumor efficacy. We injected 5 x 10 5 of firefly luciferase-labeled CD19+ tumor cells (REH) into NSG mice via tail vein, then these mice were treated with 1 x 10 5 of CD19 RA+ CAR-T, RA− CAR-T, or control CAR-T cells, respectively, at day 6 after the tumor injection. Results: RA+ CAR T cells demonstrated better transient transduction efficiency 24 h after transduction (RA+ CAR-T: 77.5 ± 9.8% vs RA− CAR-T: 39.7 ± 3.8%), and superior expansion capacity after 14 days of culture than RA− CAR-T cells (RA+ CAR-T: 32.5 ± 9.3-fold vs RA− CAR-T: 11.1 ± 5.4-fold). RA+ CAR-T cells exhibited dominant CD8 expression (RA+ CAR-T: 84.0 ± 3.4% vs RA− CAR-T: 34.1 ± 10.6%), less expression of exhaustion marker PD-1 (RA+ CAR-T: 3.1 ± 2.5% vs RA− CAR-T: 19.2 ± 6.4%) and T cell senescence marker CD57 (RA+ CAR-T: 6.8 ± 3.6% vs RA− CAR-T: 20.2 ± 6.9%), and enrichment of naïve/stem cell memory fraction (CAR+/CD45RA+CCR7+ fraction; RA+ CAR-T: 71.9 ± 9.7% vs RA− CAR-T: 8.0 ± 5.3%), which were associated with longevity of CAR-T cells. Transcriptome analysis revealed that RA+ CAR-T cells exhibited the enrichment of naïve/memory phenotype and less expression of canonical exhaustion markers, and these exhaustion profiles even maintained after the antigen stimulation. RA+ CAR-T cells demonstrated sustained killing activity even after multiple tumor rechallenges in vitro, without inducing exhaustion marker expression of PD-1. Although antigen stimulation could increase CAR expression, leading to tonic CAR signaling and exhaustion, in our study, the expression of CAR molecule on the cell surface following antigen stimulation in RA+ CAR was controlled at a relatively lower level that in RA− CAR-T cells. RA+ CAR-T cells achieved prolonged tumor control with expansion of CAR-T cells than RA− CAR-T cells in in vivo stress test (Fig.1A-C). On day15, bone marrow studies in RA+ CAR group exhibited abundant human CD3 positive T cells with less expression of PD-1, and relatively smaller amount of REH cells than RA− CAR group (Fig.1D). Furthermore, in two of long-lived mice in RA+ CAR group, human CD3 positive T cells were expanded even day 50 after treatment as confirmed by sequential bone marrow studies (Fig.1E), which indicated the antigen-induced proliferation and long-term functionality of RA+ CAR-T cells in vivo. Conclusion: Our results suggest that PB-mediated RA+ CAR-T cells exhibit memory-rich phenotype and superior antitumor function, thereby indicating the usefulness of CD45RA+ PBMC as a starting material of PB-CAR-T cells. Figure 1 Figure 1. Disclosures Yagyu: AGC Inc.: Research Funding. Nagao: AGC Inc.: Current Employment. Kubota: AGC Inc.: Current Employment. Shimizu: AGC Inc.: Current Employment. Nakazawa: AGC Inc.: Research Funding; Toshiba Corporation: Research Funding.

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