scholarly journals Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR

Blood ◽  
2020 ◽  
Vol 136 (12) ◽  
pp. 1407-1418 ◽  
Author(s):  
Dana Stenger ◽  
Tanja A. Stief ◽  
Theresa Kaeuferle ◽  
Semjon Willier ◽  
Felicitas Rataj ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Third Party ◽  
Leukemia Cell Line ◽  
Effector Memory ◽  
Childhood All ◽  
Car T Cells ◽  
Car T

Abstract Anti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCRβ chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon γ), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched third-party adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.

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 Enhanced CAR-T activity against established tumors by polarizing human T cells to secrete interleukin-9

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Lintao Liu ◽  
Enguang Bi ◽  
Xingzhe Ma ◽  
Wei Xiong ◽  
Jianfei Qian ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
Effector Memory ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Ifn Γ

AbstractCAR-T cell therapy is effective for hematologic malignancies. However, considerable numbers of patients relapse after the treatment, partially due to poor expansion and limited persistence of CAR-T cells in vivo. Here, we demonstrate that human CAR-T cells polarized and expanded under a Th9-culture condition (T9 CAR-T) have an enhanced antitumor activity against established tumors. Compared to IL2-polarized (T1) cells, T9 CAR-T cells secrete IL9 but little IFN-γ, express central memory phenotype and lower levels of exhaustion markers, and display robust proliferative capacity. Consequently, T9 CAR-T cells mediate a greater antitumor activity than T1 CAR-T cells against established hematologic and solid tumors in vivo. After transfer, T9 CAR-T cells migrate effectively to tumors, differentiate to IFN-γ and granzyme-B secreting effector memory T cells but remain as long-lived and hyperproliferative T cells. Our findings are important for the improvement of CAR-T cell-based immunotherapy for human cancers.

scholarly journals Application of Novel T Cell Immunotherapeutics to Drive Antigen-Specific Activation, Expansion, and Differentiation of CD19 Chimeric Antigen Receptor T Cells (CAR T-cells)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-35
Author(s):  
Moriah Rabin ◽  
Mengyan Li ◽  
Scott Garforth ◽  
Jacqueline Marino ◽  
Jian Hua Zheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Effector Memory ◽  
Mhc Molecules ◽  
Car T Cells ◽  
Car T

Background: While chimeric antigen receptor T cells (CAR T-cells) induce dramatic remissions of refractory or recurrent B cell malignancies, the durability of these remissions is frequently limited by subsequent reduction in circulating CAR T-cells and/or by diminution of their effector function. We hypothesized that we could overcome this therapeutic limitation and increase the functional activity and longevity of CAR T-cells by selectively deriving them from virus-specific effector memory T cells. We have developed biologics we termed synTacs (artificial immunological synapse for T-cell activation), which selectively activate and expand antigen-specific CD8+ T cells in vitro and in vivo by recapitulating signals delivered at the immunological synapse. The synTacs consist of dimeric Fc domain scaffolds linking CD28- or 4-1BB-specific ligands to HLA-A2 MHC molecules covalently tethered to virus-derived peptides. Treatment of PBMCs from CMV-exposed donors with synTacs presenting a CMV-derived peptide (pp65-NLVPMVATV) induce vigorous and selective ex vivo and in vivo expansion of highly functional CMV-specific CD8+ T cells, with potent antiviral activity. We used these synTacs to selectively generate CAR T-cells from CMV-specific effector memory CD8+ T cells, which could be further expanded by restimulation with the CMV-specific synTacs. Methods: We treated PBMCs from CMV-exposed donors in media supplemented with either IL-2 or IL-7/12/15 with a synTac containing the CMV-derived pp65 peptide presented by HLA-A2 MHC molecules linked to ligands capable of stimulating CD28- or 4-1BB-dependent costimulatory pathways. PBMCs activated either with anti-CD3/CD28 or the CMV-specific synTacs were transduced with lentivirus expressing an anti-CD19 CAR and a GFP reporter gene. CMV-specific CD8+ T cells were quantified by tetramer staining and CAR T-cells were detected by GFP expression determined by flow cytometric analysis. The functional activity of the CD19 CAR T-cells was determined by a B cell-specific cytotoxic assay. Results: After 7 days, treatment of PBMCs with CMV-specific synTacs rapidly induced robust activation and >50-fold expansion of CMV-specific CD8+ T cells expressing effector memory markers. Treatment of the PBMCs with CMV-specific synTacs selectively activated CMV-specific T cells and enabled them to be specifically transduced with a CD19-specific CAR lentivirus and converted into CD19 CAR T-cells. These CMV-specific CD19 CAR T-cells displayed potent dose-responsive cytotoxic activity targeting purified primary B cells. Furthermore, these CMV-specific CD19 CAR T-cells could be selectively expanded by in vitro treatment with CMV-specific synTacs. Conclusions: SynTacs are versatile immunotherapeutics capable of selective in vitro and in vivo activation and expansion of virus-specific CD8+ T cells with potent antiviral cytotoxic activity. After selective lentiviral transduction and conversion into CD19 CAR T-cells, their co-expression of the CMV-specific T cell receptor enabled them to be potently stimulated and activated by in vitro treatment with CMV synTacs. The modular design of synTacs facilitates efficient coupling of other costimulatory ligands - such as OX40 or GITRL - or cytokines, such as IL-2, IL-7, or IL-15, to enable the selective in vivo delivery of defined costimulatory signals or cytokines to the CAR T-cells expressing CMV-specific TCR. This strategy has the potential to boost the in vivo activity of tumor-specific CAR T-cells after infusion and enable more durable and potent treatment of refractory/recurrent B cell malignancies. Disclosures Almo: Cue Biopharma: Current equity holder in publicly-traded company, Patents & Royalties: Patent number: 62/013,715, Research Funding. Goldstein:Cue Biopharma: Research Funding.

Defined Central Memory and Stem Memory T Cell Phenotype of CD4 and CD8 CAR T Cells for the Treatment of CD19+ Acute Lymphoblastic Leukemia in an Automated Closed System

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4558-4558 ◽  
Author(s):  
Franziska Blaeschke ◽  
Theresa Kaeuferle ◽  
Judith Feucht ◽  
Dana Weber ◽  
Ramin Lotfi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Automated System ◽  
Cell Phenotype ◽  
Target Cells ◽  
Cell Numbers ◽  
Car T Cells ◽  
Car T

Abstract Relapsed and refractory B-precursor acute lymphoblastic leukemia (B-ALL) remains a major therapeutic problem. Chimeric antigen receptor (CAR) modified T cells targeting CD19 are promising treatment options for these patients with the potential to induce hematological remission in adult and pediatric patients with refractory B-ALL. Despite the promising data, some patients do not respond to T-cell treatment. Until now it is not possible to fully understand and predict critical factors for response or non-response, but proliferation and persistence of CAR T cells in vivo is an essential precondition for treatment efficacy. Central memory T cells (Tcm) and stem cell-like memory T cells (Tscm) are known to be the best candidates for a sustained in vivo expansion after T-cell therapy with small cell doses. Therefore, we set up a protocol for the generation of anti-CD19 CAR T cells in a closed system that is compliant to current GMP regulations. Starting samples were mononuclear cells from pediatric ALL patients at diagnosis and under chemotherapy using up to 100cc peripheral blood. After separation for CD4+/CD8+ cells, T cells were activated with anti-CD3/CD28 beads. The lentiviral vector encoded the anti-CD19 single-chain variable fragment, 4-1BB (CD137) co-stimulation and T cell receptor (TCR) zeta chain. The whole process including separation of T cells, activation, transduction and cultivation was performed in a closed and fully automated system. Despite a broad variety in cellular composition including high blast counts, low cell numbers and a rather exhausted phenotype in the starting fraction, a robust T-cell composition was achieved at day five after activation with a mean of 63% CD4+ and 37% CD8+ T cells and a transduction rate of up to 38 %. The vast majority of CAR T cells were of a Tcm (47%) and Tscm (44%) phenotype leading to a strong proliferative potential of more than 100-fold expansion. In addition, a reduced sensitivity to inhibitory signals was documented (programmed cell death protein 1 (PD-1) expression ≤10%). CAR T cells showed effective cytotoxic functionality when co-cultured with CD19+ target cells with only little background of the un-transduced control. At an effector to target ratio of 5:1 up to 80 % of the CD19+ target cells were killed. In addition, a significant release of Interferon gamma (IFN-ɣ), Tumor necrosis factor (TNF-α) and Interleukin-2 (IL-2) was detected upon recognition of the target cell lines, confirming a strong and target-specific Th1 response. In conclusion, generation of CAR T cells from small pediatric blood samples was feasible in a closed GMP-compatible fully automated system. Despite variety of cell numbers, cellular composition and T cell phenotype in the starting sample, a uniform T cell product of Tcm and Tscm could be produced with a balanced CD4 / CD8 ratio leading to high expansion potential and functionality of the T cell graft. Disclosures Blaeschke: Miltenyi Biotec GmbH: Other: Miltenyi Biotec GmbH provided reagents free of charge.. Kaiser:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment.

scholarly journals The Decrease of Anti-CD3 Antibody Concentration Improved the Cytotoxicity of Chimeric Antigen Receptor (CAR) T Cells in the Treatment of Chronic Lymphoblastic Leukemia (CLL)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4792-4792
Author(s):  
Sanmei Wang ◽  
Yilian Yang ◽  
Yu Zhu ◽  
Lei Fan ◽  
Michael Schmitt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Antibody Concentration ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell Therapy ◽  
Car T

Abstract Purpose: Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated impressive responses in refractory and relapsed acute lymphoblastic leukemia (ALL) and non-hodgkin's lymphoma (NHL), however, the outcome among chronic lymphoblastic leukemia (CLL) seems to be inferior compared to other lymphoblastic malignancies, indicating that efficacy of CAR-T cell therapy may be attributed to inherent T cell defects that are characteristic of CLL which impaired their proliferative capacity and sustained persistence in vivo. Thereby, infusion of less-differentiated T cells which have the capacity to persist and engraft long-term in vivo may enhance the anti-tumor activity. Materials and methods: On day 0, cryopreserved PBMCs from healthy donors (HDs) and CLL patients were thawed and seeded on anti-CD3 antibody (0.1μg/ml vs 1μg/ml) in combination with anti-CD28 antibody (1μg/ml) coated 24-well plates. On day 3, activated T cells (ATCs) supplied with retroviral supernatant of the third-generation RV-SFG.CD19.CD28.4-1BBzeta vector were transferred into 24-well plates previously coated with retronectin. Transduction efficiencies and phenotypes of CAR-T cells were evaluated on days 7, 10 and 14 after transduction using flow cytometry analysis. On a functional level, chromium 51 (Cr-51) release assay and intracellular staining (ICS) analysis were performed to explore the altered cytotoxic capability of CAR-T cells. Results: We observed that the decrease of anti-CD3 antibody concentration (0.1μg/ml) showed no influence on viability, expansion, transduction efficiency of CAR-T cells generated from HDs or CLL patients compared to standard anti-CD3 antibody concentration (1μg/ml). Moreover, the decrease of anti-CD3 antibody (0.1μg/ml)-mediated T cell activation resulted in an enrichment of less-differentiated naïve (CD45RA +CCR7 +) and central memory (CD45RA -CCR7 +)-like T cells both among CD4 + and CD8 + CAR-T cells. Additionally, cytokines-production (TNF-α, IFN-γ) were dramatically increased evaluated with ICS analysis from HDs and CLL patients in two different concentrations (0.1μg/ml vs. 1μg/ml) . Notably, CAR-T cells derived from HDs displayed decreased cytotoxic capability while CLL patients-derived CAR-T cells demonstrated increased cytotoxicity with lower anti-CD3 antibody concentration (0.1μg/ml) in the assessment of Cr-51 release assay, indicating that the proliferative capacity and sustained persistence of CAR-T cells derived from CLL patients were obtained in vivo. Conclusion: Anti-CD3 antibody-mediated activation of T cells altered anti-tumor efficiency of CAR-T cells before the transduction of ACTs with virus vectors. Consequently, when exploring the strategies to improve the efficacy of CAR-T cells, especially among CLL patients with inherent T cell defects, improvement of the functionality of T cells has to be taken into account. Figure 1 Figure 1. Disclosures Schmitt: TolerogenixX: Current holder of individual stocks in a privately-held company; Apogenix: Research Funding; Hexal: Other: Travel grants, Research Funding; Kite Gilead: Other: Travel grants; MSD: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Travel grants, Research Funding; Bluebird Bio: Other: Travel grants.

scholarly journals A Feasibility and Safety Study of CD19 and CD22 Chimeric Antigen Receptors-Modified T Cell Cocktail for Therapy of B Cell Acute Lymphoblastic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 277-277 ◽  
Author(s):  
Junfang Yang ◽  
Jianqiang Li ◽  
Xian Zhang ◽  
Fanyong LV ◽  
Xiaoling Guo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Median Number ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Post Infusion

Abstract Introduction: Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has recently demonstrated high success but also shown limitations regarding their toxicity and development of CD19negative variants. Here we reported results from a phase I study designed to determine the safety of the CD19 CAR-T and CD22 CAR-T cocktail and the feasibility of making enough quantities to treat patients with CD19+CD22+ relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). Patients and Methods: From July 2017 to July 2018, a total of 15 patients with CD19+CD22+ relapsed/refractory B-ALL were treated, including 5 children and 10 adults (Table 1). All patients received fludarabine 30mg/m2/d´3d and cyclophosphamide 250mg/m2/d´2d before infusion of CAR-T cells, followed by a cocktail CAR-T cell infusion with a median number of 2 (0.9-5)´105 CD19 CAR+ T cells/kg and a median number of 0.5 (0.4-12)´105 CD22 CAR+ T cells/kg. The lentiviral backbone containing constructs of CD19 CAR and CD22 CAR are shown in Figure 1. CD19 CAR includes a truncated EGFR sequence which can be used to identify and select CAR+ cells. CD22 CAR includes a single chain variable fragment (ScFv) sequence derived from a monoclonal antibody against human PD-L1 which attempts to reduce the exhaustion of CAR-T cells by blocking the PD-1/PD-L1. Real-time quantitative PCR using primers with specificity for the ScFv of CD19 CAR and ScFv of CD22 CAR can detect the in vivo CAR-T persistence for either CAR. Sequential transduction was performed 2 days after activation of sorted T cells stimulated with CD3 and CD28 antibodies. Percentages of CD19 CAR+ and CD22 CAR+ T cells were determined by flow cytometry through staining with an antibody against EGFR and a fusion protein of CD22-Fc, respectively, and expression of anti-PDL1 ScFv in CD22 CAR-T cells were demonstrated by flow cytometry through intracellular staining with a PD-L1-Fc fusion protein. The primary end points of this study were to evaluate feasibility and toxicity, and the secondary end points included disease response and persistent CAR-T engraftment of infused CAR-T cell. Results: The median observation period was 133 days (24-392 days). The median percentage of pre-treatment bone marrow CD19+CD22+ blasts was 21.5%(0.11-74.1%). On day 20-30 after CAR-T infusion, 15/15 (100%) cases achieved complete remission (CR) or incomplete CR(CRi), 14/15 (93.3%) cases had negative minimal residual disease (MRD). Patient P098 had residual (0.58%) CD19+CD22+ BM blasts at day 30 post-infusion and thereafter achieved negative MRD after re-infusion with CD19 CAR-T cells. 11/17 patients were bridged into allo-HSCT and have remained in remission state with a median follow up of 133 (97-214) days. 2/5 patients without bridging allo-HSCT relapsed on day 240 and day 105 post-infusion, respectively. Notably, both patients (100%) relapsed with CD19+CD22+ leukemia cells. Despite achievement of a very high CR rate, a very low treatment-related toxicity was observed in this trial. Only 1 patients experienced grade 3 cytokine release syndrome (CRS) and another patient (6.7%) developed grade 3 central nervous system (CNS) toxicity; all other patients were CRS grade<2 and CNS grade 0. On days -1, 1, 4, 7, 10, 14, 21 and 28 after infusion, peripheral blood (PB) was drawn and the level of infused CD19 and CD22 CAR-T cells were analyzed by either qPCR or flow cytometry assay. Results demonstrated obvious in vivo proliferation of both CD19 and CD22 CAR-T cells. The median peak level was 3.5 (0.47-79.1)´104 copy number/mg PB genomic DNA for CD19 CAR-T, and 0.9 (0.08-80.8)´104 copy number/mg PB genomic DNA for CD22 CAR-T. The median day to reach the peak value was day 10 for both CARs, ranging mostly from day 7 to day 14. Conclusion: This study demonstrates technical feasibility, high efficacy and low toxicities of CD19 and CD22 CAR-T cocktail in treating patients with CD19+CD22+ relapsed/refractory B-ALL. Both patients relapsed with CD19+ leukemia suggests this cocktail treatment may reduce the risk of CD19 negative relapse. Low toxicities may relate with small number of infused CAR-T, but involvement of anti-PDL1 ScFv which is co-expressed with CD22 CAR construct cannot be excluded. Therefore, related mechanisms are currently being investigated in the lab. Disclosures No relevant conflicts of interest to declare.

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 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).

Combinatorial Antigen Targeting Strategy for Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23
Author(s):  
Pinar Ataca Atilla ◽  
Mary K McKenna ◽  
Norihiro Watanabe ◽  
Maksim Mamonkin ◽  
Malcolm K. Brenner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Tumor Control ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Introduction: Efforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia associated antigen with chimeric antigen receptor T (CAR T) cells have had limited success. We determined whether combinatorial expression of chimeric antigen receptors directed to two different AML associated antigens would augment tumor eradication and prevent relapse in targets with heterogeneous expression of myeloid antigens. Methods: We generated CD123 and CD33 targeting CARs; each containing a 4-1BBz or CD28z endodomain. We analyzed the anti-tumor activity of T cells expressing each CAR alone or in co-transduction with a CLL-1 CAR with CD28z endodomain and CD8 hinge previously optimized for use in our open CAR-T cell trial for AML (NCT04219163). We analyzed CAR-T cell phenotype, expansion and transduction efficacy by flow cytometry and assessed function by in vitro and in vivo activity against AML cell lines expressing high, intermediate or low levels of the target antigens (Molm 13= CD123 high, CD33 high, CLL-1 intermediate, KG1a= CD123 low, CD33 low, CLL-1 low and HL60= CD123 low, CD33 intermediate, CLL-1 intermediate/high) For in vivo studies we used NOD.SCID IL-2Rg-/-3/GM/SF (NSGS) mice with established leukemia, determining antitumor activity by bioluminescence imaging. Results: We obtained high levels of gene transfer and expression with both single (CD33.4-1BBʓ, CD123.4-1BBʓ, CD33.CD28ʓ, CD123.CD28ʓ, CLL-1 CAR) and double transduction CD33/CD123.4-1BBʓ or CD33/CD123.CD28ʓ) although single-transductants had marginally higher total CAR expression of 70%-80% versus 60-70% after co-transduction. Constructs containing CD28 co-stimulatory domain exhibited rapid expansion with elevated peak levels compared to 41BB co-stim domain irrespective of the CAR specificity. (p&lt;0.001) (Fig 1a). In 72h co-culture assays, we found consistently improved anti-tumor activity by CAR Ts expressing CLL-1 in combination either with CD33 or with CD123 compared to T cells expressing CLL-1 CAR alone. The benefit of dual expression was most evident when the target cell line expressed low levels of one or both target antigens (e.g. KG1a) (Fig 1b) (P&lt;0.001). No antigen escape was detected in residual tumor. Mechanistically, dual expression was associated with higher pCD3ʓ levels compared to single CAR T cells on exposure to any given tumor (Fig 1c). Increased pCD3ʓ levels were in turn associated with augmented CAR-T degranulation (assessed by CD107a expression) in both CD4 and CD8 T cell populations and with increased TNFα and IFNɣ production (p&lt;0.001 Fig 1d). In vivo, combinatorial targeting with CD123/CD33.CD28ʓ and CLL-1 CAR T cells improved tumor control and animal survival in lines (KG1a, MOLM13 and HL60) expressing diverse levels of the target antigens (Fig 2). Conclusion: Combinatorial targeting of T cells with CD33 or CD123.CD28z CARs and CLL-1-CAR improves CAR T cell activation associated with superior recruitment/phosphorylation of CD3ʓ, producing enhanced effector function and tumor control. The events that lead to increased pCD3ʓ after antigen engagement in the dual transduced cells may in part be due to an overall increase in CAR expression but may also reflect superior CAR recruitment after antigen engagement. We are now comparing the formation, structure, and stability of immune synapses in single and dual targeting CARs for AML. Disclosures Brenner: Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Maker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Memmgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Atilla:Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: founder; Marker Therapeuticsa: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Memgen: Membership on an entity's Board of Directors or advisory committees; KUUR: Membership on an entity's Board of Directors or advisory committees.

Sign in / Sign up

Export Citation Format

Share Document