scholarly journals Anti-Human CD117 CAR T-Cells Efficiently Eliminate Hematopoietic Stem and CD117-Positive AML Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4063-4063 ◽  
Author(s):  
Renier Myburgh ◽  
Jonathan Kiefer ◽  
Norman F Russkamp ◽  
Alexander Simonis ◽  
Surema Pfister ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Lines ◽  
Surface Expression ◽  
Hematopoietic Stem ◽  
Car T Cells ◽  
Car T

Abstract Introduction: Acute Myeloid Leukemia (AML) is a clonal disease of the hematopoietic system that originates from immature hematopoietic stem and progenitor cells (HSPC). Because some AML-initiating cells are comparatively resistant to conventional cytotoxic agents, disease relapses are common with current treatment approaches. As an alternative, immunological eradication of leukemic cells by adoptively transferred chimeric-antigen receptor T-cells (CAR T-cells) might be considerably more efficient. To date, however, the search for AML-specific surface antigens has remained largely elusive. To circumvent this problem, we propose to target the stem cell antigen c-Kit (CD117) that is expressed by physiological HSPC as wells as by leukemic blasts in >90% of AML patients. For translation into a clinical setting, CAR T cell treatment must then be followed by depletion of CAR T-cells as well subsequent healthy/allogeneic HSC transplantation. Methods: A lentiviral vector was generated which incorporates the CAR (scFv linked to intracellular CD3ζ and 4-1BB signaling domains via stalk and transmembrane regions derived from CD8), followed by a T2A ribosomal skip sequence and RQR8 as selection marker and depletion gene (surface expression of CD34 and CD20 epitopes). The scFv was extracted from a previously published bivalent anti-CD117 antibody (clone 79D) that was derived from an artificial human phage library (Reshetnyak et al., PNAS, 2013). 79D exhibits high binding affinity to an epitope in the membrane-proximal domain of human CD117. Human CD117 was cloned in human CD117 negative HL-60 AML cells and cell lines with stable expression of CD117 at various levels were derived from these. Results: T-cells were isolated from healthy donors or AML patients in complete remission and both healthy donor and AML pateint derived T-cells exhibited sustained growth after activation with recombinant human IL-2 and CD3/CD28 beads. Lentiviral transduction yielded consistently high transduction rates, ranging from 55 - 75% as determined by staining for RQR8 and the scFv. In co-culture assays, CAR T-cells eliminated more than 90% of CD117high leukemia cell lines within 24 hours at effector-to target ratios (E:T) of 4:1 and 1:1 and more than 50% at E:T of 1:4. CAR-mediated cytotoxicity correlated with levels of CD117 surface expression as the elimination of CD117low target cells was less efficient compared to CD117high and CD117intermediate cells. In long-term cytotoxicity assays (45d), only CD117low cells were able to escape CAR-mediated killing. In the setting of primary cells, anti-CD117 CAR T-cells effectively depleted >90% of lin-CD117+CD34+CD38+ and >70% of lin-CD117+CD34+CD38- cells from healthy bone marrow in vitro within 48 hours. Similarly, >70% of patient derived leukemic blasts were eliminated by autologous anti-CD117 CAR T-cells within 48 hours (1:1 ratio of CAR T cells:blasts). In a long-term assay, no outgrowth of leukemic blasts was observed in the presence of autologous CAR T-cells over 3 weeks. To determine effectivity of CAR T-cells in vivo, humanized mice (NSG & MTRG-SKI) were engrafted with umbilical cord blood derived CD34+ cells. A single injection of 2x106 anti-CD117 CAR T-cells resulted in >90% depletion of CD117+ cells in the bone marrow within 6 days. Finally, humanized mice transplanted with bone marrow from AML patients expressing CD117 were treated with patient-derived autologous CAR T-cells. At 6 weeks after injection of CAR T-cells, >98% of hu-CD45 CD117+ cells were depleted in the bone marrow while control human T-cell treated mice showed full-blown CD117 positive AML. Conclusions: We provide proof of concept for the generation of highly-potent CAR T-cells re-directed against CD117 from healthy human donors and AML patients. Anti-CD117 CAR T-cells exhibit high cytotoxic activity against CD117+ cell lines as well as primary healthy HSPC and patient AML cells in vitro and in vivo in murine xenograft models. Strategies for the complete elimination of CAR T-cells (immunologic or small molecule based) are required before translation of this approach to the clinical setting. Disclosures Neri: Philochem AG: Equity Ownership.

scholarly journals Immunotherapeutic Targeting of TSLPR with Chimeric Antigen Receptor T Cells in AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2789-2789
Author(s):  
Lindsey F Call ◽  
Sommer Castro ◽  
Thao T. Tang ◽  
Cynthia Nourigat-Mckay ◽  
LaKeisha Perkins ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Surface ◽  
Peripheral Blood ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Car T Cells ◽  
Car T

Abstract Adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) has achieved impressive outcomes in the treatment of refractory/relapsed B-ALL, providing potentially curative treatment options for these patients. The use of CAR T in AML, however, is still in its infancy with limitations due to the innate heterogeneity associated with AML and the lack of AML-specific targets for therapeutic development. The CRLF2 gene encodes for thymic stromal lymphopoietin receptor (TSLPR) and has previously been shown to be highly upregulated in a subset of children and adults with B-ALL. Targeting TSLPR with CAR T cells demonstrates potent anti-leukemia activity against TSLPR-positive B-ALL (PMID 26041741). Through Target Pediatric AML (TpAML), we profiled the transcriptome of nearly 3000 children and young adults with AML and identified CRLF2 (TSLPR) to be highly expressed in a subset of AML, including the majority of AML harboring KM2TA (aka MLL) fusions. TSLPR cell surface expression was validated in primary patient samples using flow cytometry, which showed uniform expression of TSLPR on AML blasts. Given that TSLPR is expressed in AML with confirmed cell surface expression, we developed TSLPR-directed CAR T for preclinical evaluation in AML. We generated a TSLPR-directed CAR using the single-chain variable fragment (scFv) derived from an anti-TSLPR binder (clone 3G1, MD Anderson), IgG4 spacer and 41-BB/CD3zeta signaling domains. The in vitro cytotoxicity of TSLPR CAR T cells was evaluated against the REH-1 cell line and primary AML specimens. TSLPR CAR T cells demonstrated anti-leukemia activity against REH-1 as well as against primary AML specimens. To evaluate the in vivo efficacy of TSLPR CAR T cells, we developed a patient-derived xenograft (PDX) model using bone marrow cells from a TSLPR-positive patient. These cells provided a robust model system to evaluate the in vivo activity of TSLPR CAR T cells, as they produced an aggressive leukemia in humanized NSG-SGM3 mice. The PDX generated from these cells died within 2 months of transplant with significant leukemia infiltration into the bone marrow, liver, and spleen. In the in vivo study, the leukemia burden was assessed by flow cytometric analysis of AML cells in the peripheral blood and bone marrow aspirates following treatment with unmodified control or TSLPR CAR T cells given at 10x10 6 T cells per mouse. After CAR T treatment, we detected a significant decrease in leukemia infiltration into the peripheral blood and bone marrow in the CAR T-treated mice compared to mice that received unmodified T cells. In this study, we report that similar to B-ALL, CRLF2 (TSLPR) is overexpressed in a subset of AML, providing a strategy to eliminate AML cells with CAR T cell therapy. We validated the cell surface expression of TSLPR and showed that the expression is uniform across AML specimens. We further demonstrate that CAR T cells targeting TSLPR were effective in eliminating AML cells in vitro and in vivo. Given that TSLPR is highly expressed in the KMT2A-rearranged AML, a subtype that is associated with poor outcomes, TSLPR-directed CAR T cells represent a promising immunotherapy for this high-risk AML subset. Disclosures Pardo: Hematologics, Inc.: Current Employment.

scholarly journals A New Promising Third Generation CAR-CD30 T-Cell Therapy for CD30+ Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2069-2069
Author(s):  
Biagio De Angelis ◽  
Marika Guercio ◽  
Domenico Orlando ◽  
Stefano Di Cecca ◽  
Matilde Sinibaldi ◽  
...  
Keyword(s):  
T Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Short Term ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T

Prognosis of a significant proportion of patients with chemotherapy-refractory or multiply-relapsed CD30+ Non-Hodgkin's Lymphoma (NHL) or Hodgkin lymphoma (HL) still remain poor. Targeting CD30 with monoclonal antibodies in HL and anaplastic large cell lymphoma was shown to induce remarkable clinical activity; however, occurrence of adverse events (mainly neuropathy) may result into treatment discontinuation in many patients. Immunotherapeutic approaches targeting CD30 by chimeric antigen receptor (CAR) has been demonstrated to be of value in two independent clinical trials, although clinical benefit was sub-optimal. We designed a new CAR construct characterized by an anti-CD30 single-chain variable-fragment cassette (AC10), linked to CD3ζ by the signaling domains of two costimulatory molecules, namely either CD28.4-1BB or CD28.OX40. The inducible Caspase-9 (iCasp9) safety switch was included in both constructs with the goal of promptly controlling undue toxicity. As a selectable marker, we added in frame the CD34 antigen. The in vitro anti-tumor efficacy was evaluated by using either the NHL cell line: Karpas299, or the HL cell lines: L428, in both short-term cytotoxic assay (51Cr release assays) and long-term co-cultures for 6 days. Supernatant from co-culture experiments was analyzed by Elisa. We assessed the antitumor effect of CAR.CD30 T cells in a in vivo NSG mouse model engrafted i.v. with lymphoma FF-luciferase cell lines Karpas299 or L428, and monitored tumor growth by IVIS Imaging system. For tumor re-challenging, mice of the NHL model surviving until day +140, were i.v. infused with 0.2x106 Karpas299 cells, and subsequently followed for additional 110 days. Persistence of CAR.CD30 T cells was evaluated, together with a deep characterization of memory profile of T cells. Independently from the costimulatory domains CD28.OX40 or CD28.4-1BB, the generated retroviral vectors showed similar transduction efficiency of T cells (86.5±5.1% and 79.3±5.3%, respectively). Nevertheless, CD28.OX40 costimulatory domains was associated with more stable expression of the CAR over time, during extensive in vitro culture (84.72±5.30% vs 63.98±11.51% CD28.4-1BB CAR T cells at 30 days after transduction; p=0.002). For both CAR constructs, we did not observe any significant difference in the suicide gene iCasp9 activity, both in vitro and in vivo. In short-term cytotoxic assay, both CAR.CD30 T cells significantly and specifically lysed CD30+ NHL and HL tumor cell lines. In long-term co-culture, CD28.OX40 showed a superior anti-lymphoma in vitro activity as compared to CD28.41BB T cells, when challenged at very high tumor/effector ratio (8:1) (for Karpas 299; p=0.03). Moreover, the antigen stimulation was associated to higher levels of Th1 cytokine production, with CD28.OX40 T cells secreting a significantly higher amount of IFNγ, IL2 and TNFα as compared to CD28.41BB T cells (p= 0.040; p=0.008; p=0.02; respectively). Bioluminescence in HL (L428) tumor-bearing mice, treated with NT T cells, rapidly increased up to 5 log in less than 50 days and mice either died or were sacrificed due to morbidity. The best outcome was observed in mice treated with CD28.OX40, as three out of five mice were still alive at the experimental end-point of day+165, as compared with mice treated with CD28.4-1BB (60% vs 0%, p=0.0021). In NHL (Karpas 299) mouse models, CD28.OX40 had an extensive anti-tumor control superior to that of CD28.41BB T cells, leading to a significant reduction of tumor bioluminescence at day 45 (3.32x10 vs 2.29x10, p=0.04). The median survival of mice treated with NT and CD28.4-1BB CAR T cells was 45.5 and 58 days respectively, but undetermined for mice treated with CD28.OX40 CAR T cells (p=0.0002). After 140 days, cured mice were re-challenged with Karpas 299; mice were followed for other 100 days. Bioluminescence analysis showed rapid progression of the tumor in the control mice cohort, as well as in CD28.4-1BB treated mice. In contrast, in CD28.OX40 treated mice, at day+240 days, 4 out of 6 mice were tumor-free, resulting into a statistically significant survival benefit (p=0.0014). Only in mice treated with 28.OX40 T cells, we observed a long-lasting persistence of circulating CAR-T cells up to day +221. In summary, we have developed a novel CAR.CD30 construct displaying features that make it a particularly suitable candidate for a clinical trial in patients suffering from CD30+ tumors. Disclosures Merli: Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy. Locatelli:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BluebirdBio: Consultancy; Miltenyi: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

GRP78 Is Expressed on the Cell Surface of Pediatric AML Blasts and Can be Targeted with GRP78-CAR T Cells without Toxicity to Hematopoietic Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-12 ◽  
Author(s):  
Nikhil Hebbar ◽  
Rebecca Epperly ◽  
Abishek Vaidya ◽  
Sujuan Huang ◽  
Cheng Cheng ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Cell Lines ◽  
Target Cells ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T ◽  
Pediatric Aml

Finding the ideal immunotherapy target for AML has proven challenging and is limited by overlapping expression of antigens on hematopoietic progenitor cells (HPCs) and AML blasts. Intracellular Glucose-regulated-protein 78 (GRP78) is a key UPR regulator, which normally resides in the endoplasmic reticulum (ER). GRP78 is overexpressed and translocated to the cell surface in a broad range of solid tumors and hematological malignancies in response to elevated ER stress, making it an attractive target for immune-based therapies with T cells expressing chimeric antigen receptors (CARs). The goal of this project was to determine the expression of GRP78 on pediatric AML samples, generate GRP78-CAR T cells, and evaluate their effector function against AML blasts in vitro and in vivo. To demonstrate overexpression of GRP78 in AML, we performed gene expression analysis by RNAseq on a cohort of cord blood CD34+ cell samples (N=5) and 74 primary AML samples. Primary AML samples included RUNX1-RUNX1T1 (N=7), CBFB-MYH11(N=17), KMT2A rearrangement (N=28) and NUP98 (N=22). Analysis showed increased GRP78 expression in AML samples, especially in KMT2A- and NUP98-rearranged AML. To demonstrate surface expression of GRP78, we performed flow cytometry of AML (Kg1a, MOLLM13, THP-1, MV4-11) cell lines as well as 11 primary AML samples and 5 PDX samples; non transduced (NT) T cells served as control. All AML samples, including cell lines, primary AML blasts, and PDX samples, showed increased expression of GRP78 on their cell surface in comparison to NT T cells We then designed a retroviral vector encoding a GRP78-CAR using a GRP78-specific peptide as an antigen recognition domain, and generated GRP78-CAR T cells by retroviral transduction of primary human T cells. Median transduction efficiency was 82% (± 5-8%, N=6), and immunophenotypic analysis showed a predominance of naïve and terminal effector memory subsets on day 7 after transduction (N=5). To determine the antigen specificity of GRP78-CAR T cells, we performed coculture assays in vitro with cell surface GRP78+ (AML cell lines: MOLM13, MV-4-11, and THP-1 and 3 AML PDX samples) or cell surface GRP78- (NT T cells) targets. T cells expressing CARs specific for HER2-, CD19-, or a non-functional GRP78 (DGRP78)-CAR served as negative controls. GRP78-CAR T cells secreted significant amounts of IFNg and IL-2 only in the presence of GRP78+ target cells (N=3, p<0.005); while control CAR T cells did not. GRP78-CAR T cells only killed GRP78+ target cells in standard cytotoxicity assays confirming specificity. To test the effects of GRP78-CAR T cells on normal bone marrow derived HPCs, we performed standard colony forming unit (CFU) assays post exposure to GRP78-CAR or NT T cells (effector to target (E:T) ratio 1:1 and 5:1) and determined the number of BFU-E, CFU-E, CFU-GM, and CFU-GEMM. No significant differences between GRP78-CAR and NT T cells were observed except for CFU-Es at an E:T ratio of 5:1 that was not confirmed for BFU-Es. Finally, we evaluated the antitumor activity of GRP78-CAR T cells in an in vivo xenograft AML model (MOLM13). Tumor growth was monitored by serial bioluminescence imaging. A single intravenous dose of GRP78-CAR T cells induced tumor regression, which resulted in a significant (p<0.001) survival advantage in comparison to mice that had received control CAR T cells. In conclusion, GRP78 is expressed on the cell surface of AML. GRP78-CAR T cells have potent anti-AML activity in vitro and in vivo and do not target normal HPCs. Thus, our cell therapy approach warrants further active exploration and has the potential to improve outcomes for patients with AML. Disclosures Hebbar: St. Jude: Patents & Royalties. Epperly:St. Jude: Patents & Royalties. Vaidya:St. Jude: Patents & Royalties. Gottschalk:TESSA Therapeutics: Other: research collaboration; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties. Velasquez:St. Jude: Patents & Royalties; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees.

TCRab Deficient CAR T-Cells Targeting CD123: An Allogeneic Approach of Adoptive Immunotherapy for the Treatment of Acute Myeloid Leukemia (AML)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2555-2555 ◽  
Author(s):  
Roman Galetto ◽  
Céline Lebuhotel ◽  
Agnès Gouble ◽  
Nuria Mencia-Trinchant ◽  
Cruz M Nicole ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Surface Expression ◽  
Car T Cells ◽  
Healthy Donors ◽  
Car T ◽  
Acute Myeloid

Abstract The remissions achieved using autologous T-cells expressing chimeric antigen receptors (CARs) in patients with advanced B cell leukemia and lymphomas have encouraged the use of CAR technology to treat different types of cancers by targeting distinct tumor-specific antigens. Since the current autologous approach utilizes CAR T-cells manufactured on a "per patient" basis, we propose an alternative approach based on the use of a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic "off-the-shelf" CAR T-cell-based frozen products. In the present work we have adapted this allogeneic platform to the production of T-cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed on tumor cells from the majority of patients with Acute Myeloid Leukemia (AML). Multiple antigen recognition domains were screened in the context of different CAR architectures to identify candidates displaying activity against cells expressing variable levels of the CD123 antigen. The three lead candidates were tested in an orthotopic human AML cell line xenograft mouse model. From the three candidates that displayed comparable activity in vitro, we found two candidates capable of eradicating tumor cells in vivo with high efficiency. Subsequently, Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology was used to inactivate the TCRα constant (TRAC) gene, eliminating the potential for engineered T-cells to mediate Graft versus Host Disease (GvHD). Editing of the TRAC gene can be achieved at high frequencies, and allows efficient amplification of TCR-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. In addition, we show that TCR-deficient T-cells display equivalent in vitro and in vivo activity to non-edited T-cells expressing the same CAR. We have performed an initial evaluation of the expression of CD123 in AML patients and found an average cell surface expression of CD123 was of 67% in leukemic blasts (95% CI 48-82), 71% in CD34+CD38+ cells (95% CI 56-86), and 64% in CD34+CD38- (95% CI 41-87). Importantly, we have found that CD123 surface expression persists in CD34+CD38-CD90- cells after therapy in at least 20% of patients in remission (n=25), thus emphasizing the relevance of the target. Currently, the sensitivity of primary AML cells to CAR T-cells is being tested. Finally, we will also present our large scale manufacturing process of allogeneic CD123 specific T-cells from healthy donors, showing the feasibility for this off-the-shelf T-cell product that could be available for administration to a large number of AML patients. Disclosures Galetto: Cellectis SA: Employment. Lebuhotel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Smith:Cellectis: Employment, Patents & Royalties.

scholarly journals Clinical Development of a Non-Gene-Edited Allogeneic Bcma-Targeting CAR T-Cell Product in Relapsed or Refractory Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 27-28
Author(s):  
A. Samer Al-Homsi ◽  
Sebastien Anguille ◽  
Jason Brayer ◽  
Dries Deeren ◽  
Nathalie Meuleman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Gene Editing ◽  
Cell Product ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Background Autologous CAR T-cell therapy targeting the B-cell maturation antigen (BCMA) has shown impressive objective response rates in patients with advanced multiple myeloma (MM). Clinical grade manufacturing of autologous CAR T-cells has limitations including vein-to-vein delivery time delay and potentially sub-optimal immunological capability of T-cells isolated from patients with advanced disease. Allogeneic CAR T-cell products, whereby cells from healthy third-party donors are used to generate an "off-the-shelf" CAR T-cell product, have the potential to overcome some of these issues. To circumvent the primary potential risk of graft-versus-host disease (GvHD) associated with the use of allogeneic T-cells, abrogation of the T-cell receptor (TCR) expression in the CAR T-cells, via gene editing, is being actively pursued. To avoid the potential safety risks and manufacturing challenges associated with gene editing, the allogeneic CYAD-211 CAR T-cell product exploits short hairpin RNA (shRNA) interference technology to down-regulate TCR expression thus avoiding the risk of life-threatening GvHD. Aim The aim is to generate a BCMA-specific allogeneic CAR T-cell product using a non-gene editing approach and study its activity both in vitro and in vivo. CYAD-211 combines a BCMA-specific CAR with a single optimized shRNA targeting the TCR CD3ζ subunit. Downregulation of CD3ζ impairs the TCR expression on the surface of the donor T-cells, preventing their reactivity with the normal host tissue cells and potential GvHD induction. Maintaining all the elements required for the therapy within a single vector (all-in-one vector) provides some significant manufacturing advantages, as a solitary selection step will isolate cells expressing all the desired traits. Results CYAD-211 cells produce high amounts of interferon-gamma (IFN-γ) during in vitro co-cultures with various BCMA-expressing MM cell lines (i.e., RPMI-8226, OPM-2, U266, and KMS-11). Cytotoxicity experiments confirmed that CYAD-211 efficiently kills MM cell lines in a BCMA-specific manner. The anti-tumor efficacy of CYAD-211 was further confirmed in vivo, in xenograft MM models using the RPMI-8226 and KMS-11 cell lines. Preclinical data also showed no demonstrable evidence of GvHD when CYAD-211 was infused in NSG mice confirming efficient inhibition of TCR-induced activation. Following FDA acceptance of the IND application, IMMUNICY-1, a first-in-human, open-label dose-escalation phase I clinical study evaluating the safety and clinical activity of CYAD-211 for the treatment of relapsed or refractory MM patients to at least two prior MM treatment regimens, is scheduled to begin recruitment. IMMUNICY-1 will evaluate three dose-levels of CYAD-211 (3x107, 1x108 and 3x108 cells/infusion) administered as a single infusion after a non-myeloablative conditioning (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days) according to a classical Fibonacci 3+3 design. Description of the study design and preliminary safety and clinical data from the first cohort will be presented at ASH 2020. Conclusion CYAD-211 is the first generation of non-gene edited allogeneic CAR T-cell product based on shRNA technology. The IMMUNICY-1 clinical study seeks to provide proof of principle that single shRNA-mediated knockdown can generate fully functional allogeneic CAR T-cells in humans without GvHD-inducing potential. We anticipate that subsequent generations of this technology will incorporate multiple shRNA hairpins within a single vector system. This will enable the production of allogeneic CAR T-cells in which multiple genes of interest are modulated simultaneously thereby providing a platform approach that can underpin the future of this therapeutic modality. Figure 1 Disclosures Al-Homsi: Celyad: Membership on an entity's Board of Directors or advisory committees. Brayer:Janssen: Consultancy; Bristol-Myers Squibb, WindMIL Therapeutics: Research Funding; Bristol-Myers Squibb, Janssen, Amgen: Speakers Bureau. Nishihori:Novartis: Other: Research support to institution; Karyopharm: Other: Research support to institution. Sotiropoulou:Celyad Oncology: Current Employment. Twyffels:Celyad Oncology: Current Employment. Bolsee:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

scholarly journals CD19-Directed Fast CART Therapy for Relapsed/Refractory Acute Lymphoblastic Leukemia: From Bench to Bedside

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1340-1340 ◽  
Author(s):  
Cheng Zhang ◽  
Jiaping He ◽  
Li Liu ◽  
Jishi Wang ◽  
Sanbin Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Lymphoblastic Leukemia ◽  
Preclinical Study ◽  
Car T Cells ◽  
Car T ◽  
Manufacturing Time ◽  
Grade Iii

Background CAR-T targeting CD19 has been a success in treating B-cell acute lymphoblastic leukemia (B-ALL). However, relapse rate is high and the long term survival in pateints is not satisfactory, which is partly due to the limited expansion and persistence of the conventionally-manufactured CAR-T cells. In addition, long manufacturing time and high cost of CAR-T product further limit the wider applications of CAR-T therapy. To solve these issues, we have developed a new manufacturing platform, FasT CAR-T, which shorten the manufacturing time to one day as compared to the conventional CAR-T manufacturing time of 9-14 days, which is critical for patients with rapidly progressing disease. More importantly, CD19-directed FasT CAR-T has been shown to have superior expansion capability, younger and less exhausted phenotype, and higher potency in eliminating B-ALL both in vitro and in vivo. Based on the preclinical study, we initiated a multi-center clinical study to determine the safety, feasibility and efficacy of CD19-FasT CAR-T in treating patients with CD19+ relapsed/refractory B-ALL. Methods CD19-directed CAR-T was manufactured using the FasT CAR-T platform. Peripheral blood (PB) mononuclear cells were obtained by leukapheresis and T cells were separated. CD19-FasT CAR-T manufacturing were all successful. Conventional CAR-T (C-CAR-T) from healthy donor were also made in parallel for comparison in preclinical study. From Dec. 2018 to July 2019, 10 adult CD19+ R/R ALL patients were recruited and all patients received fludarabine and cyclophosphamide as pre-conditioning followed by a single CAR-T infusion 48-72 hours later. Doses used in this study were: 3 DL1 (5 x 104 CAR+ T/kg), 4 DL2 (1 x 105 CAR+ T/kg), and 3 DL3 (1.5 x 105 CAR+ T/kg). The endpoints of the study were clinical toxicity, feasibility, PK of CAR-T and efficacy. Results: In comparison to conventional CAR-T cells, CD19-FasT CAR-T cells had several key features (Table 1). 1) More robust expansion. Upon antigen stimulation, the FasT CAR-T proliferated 5-30 times stronger than that of C-CAR-T. 2) Higher percentage of CD62L+CD45RO- (Tscm) and CD62L+CD45+ (Tcm) population in FasT CAR-T. 3) Lower expression of PD-1+, LAG3+ and Tim3+ in FasT CAR-T. 4). More potent in eliminating Raji tumor in an in vivo xenograft mouse model. 5) More efficient migration to bone marrow which is likely due to the higher expression of CXCR4 on the FasT CAR-T cells. The trial was conducted during Dec. 2018 to July 2019. The pre-treatment bone marrow (BM) blasts were < 5% in 5 cases, 5%-50% in 3 cases, and >50% in 2 cases (Table 2). All 10 patients achieved complete remission (CR) 4 weeks after FasT CAR-T infusion, and 9 were with negative minimal residual disease (MRD-). CAR-T cells proliferation and persistence in peripheral blood (PB) were monitored by qPCR and flow cytometry. CAR-T cells peaked at Day 10 (range Day 8-13) after infusion. The median persistence period of CAR-T in PB was 56 days ((range 28-212 days) after infusion, and the longest persistence is 7 months and still being monitored at the last follow-up. The median peak of CAR copy number is 90,446/mg DNA (range 4,670-247,507/mg DNA) (Figure). The major adverse event was cytokine release syndrome (CRS) which was observed in 9 patients, including 1 patient with grade IV in DL3 group, 3 grade III, 4 grade II and 1 grade I. The clinical manifestation of CRS mainly included fever and hypotension. The median time to the development of CRS was 5 days (2-10 days), and the peak body temperature was at Day 7 (Day 5- 11) and fever lasted for an average of 5 days (3-8 days). Serum IL-6 level increased and peaked on Day 7 post-infusion, which coincided with fever but slightly preceded the CAR-T expansion peak. Three patients experienced CAR-related encephalopathy syndrome (CRES) after CAR-T infusion, in which 1 was grade III CRES. All patients who developed CRS or CRES recovered after intervention. Conclusion FasT CAR-T have superior expansion capacity with younger and less exhausted phenotype, and more potent cytotoxicity against B-ALL. This first-in-human clinical study in China showed CD19-directed FasT CAR-T therapy is highly effective in treating R/R B-ALL with manageable toxicity. The safety, efficacy and potential long-term clinical benefit of FasT CAR-T therapy will be further evaluated in large multi-center trial. (http://www.chictr.org.cn/listbycreater.aspx:ChiCTR1900023212) Disclosures No relevant conflicts of interest to declare.

scholarly journals 133 CRISPR/Cas9 gene-edited allogeneic CAR-T cells targeting CD33 show high preclinical efficacy against AML without long-term hematopoietic toxicity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A143-A143
Author(s):  
Jonathan Terrett ◽  
Brigid Mcewan ◽  
Daniel Hostetter ◽  
Luis Gamboa ◽  
Meghna Kuppuraju ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antibody Drug Conjugate ◽  
T Cell Therapy ◽  
In Vivo Models ◽  
Car T Cells ◽  
Car T

BackgroundCD33 is the most consistently expressed antigen in AML, with high levels and homogeneous expression observed in malignant AML cells from most patients, including those with relapsed disease. Normal myelomonocytic cell lineages and a percentage of hematopoietic progenitors also express CD33, and the extreme myeloablation caused by the CD33-targeted antibody-drug conjugate (ADC) gemtuzumab ozogamicin reinforced concerns about targeting this antigen with more potent agents such as T-cell engaging bispecific antibodies and CAR-T cells. We have shown previously that allogeneic CRISPR/Cas9 gene-edited CAR-T cells targeting CD33 with TRAC disruption to reduce GvHD and B2M disruption to reduce allogeneic host rejection could eliminate tumors in xenograft models of AMLMethodsGiven that off-target activity of the toxin could contribute to the myeloablation seen with CD33-targeted ADCs, we created in vitro and in vivo models to examine reconstitution of the myeloid compartment following treatment of CD33-targeted allogeneic CAR-T cells.ResultsAlthough co-culture of CD34+ stem cells in vitro with our CD33-targeted allogeneic CAR-T cells did significantly deplete the cell population, colonies still formed after removal of the CAR-T cells as the presumably CD33-negative stem/progenitor cells expanded and differentiated. A similar phenomenon was observed in vivo with CD34 humanized mice bearing an AML tumor (THP-1 cells) and treated with the CD33-targeted allogeneic CAR-T cells. The CAR-T cells completely eradicated the THP-1 tumor but did not lead to long-term myelosuppression or B cell aplasia.ConclusionsThus, allogeneic CRISPR/Cas9 multiplex gene-edited CD33-targeted CAR-T cell therapy may be both efficacious and tolerable in AML.

scholarly journals Therapeutic Targeting of Mesothelin in Acute Myeloid Leukemia with Chimeric Antigen Receptor T Cell Therapy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-11 ◽  
Author(s):  
Quy Le ◽  
Sommer Castro ◽  
Thao T. Tang ◽  
Anisha Loeb ◽  
Amanda R. Leonti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Cell Surface ◽  
Myeloid Leukemia ◽  
Car T Cells ◽  
Car T ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) is one of the most highly refractory hematologic malignancies despite intensive combination chemotherapy and bone marrow stem cell transplantation. Lack of curative treatments is in large part due to our poor understanding of the disease biology and paucity of therapeutic targets. In an effort to identify actionable targets, we recently completed the largest genome, epigenome and transcriptome profiling of AML in nearly 3000 children and young adults. This discovery effort has led to the identification of a library of novel AML-restricted targets (high expression in AML, minimal-to-no expression in normal hematopoiesis) for therapeutic development. One such target was MSLN which encodes for mesothelin, a cell surface adhesion molecule that is highly expressed in 30-50% of AML cases in pediatric (Children Oncology Group) and adult (MD Anderson) cohorts and is entirely absent in normal bone marrow and peripheral blood CD34+ cells. MSLN expression in normal tissues is confined to mesothelial cells lining the pleura, pericardium, and peritoneum. Previous studies targeting MSLN in solid tumors have demonstrated clinical efficacy with minimal toxicities. Given that T cells genetically modified to express chimeric antigen receptors (CARs) are extremely effective at eradicating relapsed and refractory malignancy, we developed MSLN-directed CAR T cells for pre-clinical evaluation in AML. Methods: From primary patient samples, we verified MSLN expression by RT-PCR and confirmed mesothelin surface protein expression on leukemic blasts by flow-cytometry as well as detected soluble mesothelin in the plasma by ELISA. The VH and VL sequences from Amatuximab were used to create the scFv domain of the standard CAR (41-BB and CD3Zeta). For in vivo CAR T study, Nomo-1 cells, which express endogenous level of MSLN, and Kasumi-1 cells engineered to express MSLN with a lentivirus construct (Kasumi-1 MSLN+) were transplanted into NSG mice. Mock transduced MSLN-directed CAR T cells were infused 1 week (Nomo-1) and 2 weeks (Kasumi-1 MSLN+) following leukemic cell injection. Leukemic burden was measured by bioluminescence IVIS imaging weekly. For in vitro study, Nomo-1 cells were treated with GM6001 (50uM), a metalloprotease inhibitor, or DMSO control for 48 hr prior to evaluation of surface mesothelin by flow cytometry and soluble mesothelin in the culture supernatant by ELISA. Results: In vivo cytotoxicity of CAR T cells against Nomo-1 and Kasumi-1MSLN+ AML models demonstrated potent, target-dependent tumor killing. After 1- and 2-weeks post CAR T infusion, leukemic cells were eradicated in both Nomo-1 (p<0.0005, week 2, Figure 1A) and Kasumi-1 MSLN+ xenografts (p<0.005 at week 2, Figure 1B). Mesothelin undergoes shedding at the cell membrane as a result of ADAM17-mediated cleavage. Blocking ADAM17 activity with GM6001 in Nomo-1 cells led to increased cell surface mesothelin (Figure 1C) with a corresponding reduction in the shed form (Figure 1D), suggesting that GM6001 treatment stabilizes mesothelin on the cell surface. Furthermore, GM6001 treatment during co-culture of Nomo-1 and CAR T cells enhanced cytolytic activity of CAR T cells (Figure 1E). GM6001 treatment did not significantly impact cell viability of Nomo-1 cells in the absence of CAR T cells (data not shown). Conclusion: In this study, we demonstrate that mesothelin is a viable therapeutic target and a potential diagnostic biomarker in AML. We show that MSLN CAR T cells were highly effective in eliminating MSLN-positive AML cells in vitro and in vivo. Shedding contributes to the loss of mesothelin antigen and provides a source of soluble mesothelin that may interfere with antibody-based therapies, including CAR T cells. Modulating MSLN shedding by inhibiting ADAM17-mediated cleavage resulted in stabilized mesothelin and improved CAR T cell functionality. This work warrants further evaluation of MSLN CAR T cells to be tested in clinical trials for AML and demonstrates that inhibiting MSLN shedding is a promising approach to improve CAR T efficacy. Disclosures No relevant conflicts of interest to declare.

scholarly journals PL2.1 Exploiting the DNAM-1 system for chimeric antigen receptor (CAR) T cell therapy of glioblastoma

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii2-iii2
Author(s):  
T Weiss ◽  
H Meister ◽  
M Weller ◽  
C Sentman ◽  
P Roth
Keyword(s):  
T Cells ◽  
Cell Lines ◽  
Glioma Cell ◽  
Knock Out ◽  
Car T Cells ◽  
Car T ◽  
Glioma Cell Lines ◽  
Mouse Glioma

Abstract BACKGROUND Cancer immunotherapy with genetically engineered T cells that express a chimeric antigen receptor (CAR) has led to impressive responses in extracranial malignancies and is also explored against glioblastoma. However, CAR T cell strategies that are currently being explored against glioblastoma target single tumor antigens, which are non-homogeneously expressed and are prone to antigen escape. Furthermore, the immunosuppressive brain tumor microenvironment hampers anti-tumor efficacy. METHODS By immunohistochemistry and flow cytometry, we investigated the expression of CD155 and CD112, which are ligands to the activating immune cell receptor DNAX accessory molecule-1 (DNAM-1), in human and mouse glioma cell lines as well as in human glioblastoma samples. To understand their functional role, we generated CD155 or CD112 knock-out glioma cell lines using CRISPR/Cas9 and studied proliferation, sensitivity to irradiation or temozolomide as well as migration. To exploit the promiscuous binding features of DNAM-1, we generated different first or second-generation CAR T cells that use DNAM-1 as a tumor-binding domain. Subsequently, we investigated their anti-tumor activity in vitro in co-culture assays and in vivo in syngeneic orthotopic murine glioma models. RESULTS CD155 and CD112 are homogenously expressed in human and mouse glioma cell lines and human glioblastoma tissues. Knock-out of these ligands affected the migration of tumor cells, but did not affect proliferation or sensitivity to irradition or temozolomide. DNAM-1-based CAR T cells demonstrated high cytolytic activity and effector cytokine secretion in vitro. In vivo, DNAM-1 based CAR T cells reached to the tumor site in the brain upon intravenous administration, prolonged survival of orthotopic glioma-bearing mice and led to a durable anti-tumor response in a fraction of mice. The treatment was tolerated without toxicities. CONCLUSION We elucidated the tumor-intrinisic role of CD155 and CD112 and provide the first systematical preclincal assessment of DNAM-1 CAR T cells against glioma. These findings provide a rationale to test this immunotherapeutic strategy also in human glioma patients.

CD22-Targeted Chimeric Antigen Receptor (CAR) T Cells Containing The 4-1BB Costimulatory Domain Demonstrate Enhanced Persistence and Superior Efficacy Against B-Cell Precursor Acute Lymphoblastic Leukemia (ALL) Compared To Those Containing CD28

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1431-1431 ◽  
Author(s):  
Waleed Haso ◽  
Haiying Qin ◽  
Ling Zhang ◽  
Rimas J Orentas ◽  
Terry J Fry
Keyword(s):  
T Cells ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Precursor ◽  
Car T Cells ◽  
Car T ◽  
Anti Tumor Activity

Abstract B cell precursor acute lymphoblastic leukemia (BCP-ALL) remains a leading cause of death from childhood cancers despite survival rates exceeding 80%. Antibody-based CAR-engineered T cells can recognize and eliminate tumors by binding directly to a surface antigen independent from MHC restriction. CAR immunotherapy against BCP-ALL has demonstrated impressive responses and sustained remission in clinical trials targeting CD19. However, some patients receiving the CD19 CAR T cells relapse with a CD19 negative leukemia. Thus, additional CAR targets are needed. CD22 is a Siglec family lectin consisting of 7 extracellular Ig domains that is expressed on the cell surface from the pre-B cell stage of development through mature B cells and is expressed on most B cell hematologic malignancies. We previously generated a second-generation (CD3-Zeta + CD28 costimulatory domain) anti-CD22 CAR derived from a membrane proximal epitope binding scFv (m971-28z) with potent activity in vivo (Haso W et al, Blood 2013). In clinical trials T cells expressing CD19-targeted CAR with 4-1BB costimulatory domains on CD19 CARs show prolonged persistence. To improve long-term persistence of the CD22 CAR, we re-engineered our CAR vector to include a 4-1BB signaling domain (m971-BBz). In vitro data using m971-BBz improved proliferation and expansion compared to m971-28z especially when lower concentrations of IL2 were included in the culture media. When no IL2 was added to the media only the 4-1BB containing CAR expanded. No difference in killing was detected in in vitro cytotoxicity assays. We next evaluated anti-tumor activity and persistence in the NSG mouse model engrafted with the NALM6-GL cell line on day 0 and treated with CAR T cells on day 3 to directly compare the efficacy of m971-28z and m971-BBz modified T cells activated with either OKT3 or anti-CD3/CD28 beads. m971-BBz outperformed m971-28z in terms of in vivo anti-tumor activity and long-term persistence. This effect was only detected when anti-CD3/CD28 beads were used for T cell expansion. OKT3-activated cells failed to persist and demonstrated inferior antitumor activity compared to bead-expanded T cells irrespective of the costimulatory domain and despite a higher percentage of CD8 T cells with significantly better cytotoxicity in vitro. Interestingly, early peripheral blood numbers of CAR T cells in recipients of bead-expanded products demonstrated a predominance of CD4+CAR T cells consistent with preinfusion CD4/CD8 ratios. At later time points this ratio decreased with a predominance of CD8+CAR T cells. In mice receiving m971-28z CAR the CD8+CAR T cells failed to persist resulting in leukemic relapse. Furthermore, direct comparison to the CD19 CAR (FMC63-BBz) in vivo showed that the anti-CD22 CAR (m971-BBz) has equivalent activity. We conclude that anti-CD3/CD28 bead-activated T cells modified to express an anti-CD22 CAR with a 4-1BB costimulatory domain demonstrates potent antitumor activity with long-term leukemic control and offers a promising therapeutic option for pediatric ALL. Disclosures: No relevant conflicts of interest to declare.

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