scholarly journals Priming Death Receptor Mediated Apoptosis with Arginine Starvation Sensitises Arginine Auxotrophic B-ALL to CAR-T

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2787-2787
Author(s):  
Michael J Austin ◽  
Leena Halim ◽  
Farideh Miraki-Moud ◽  
David Taussig ◽  
John Bomalaski ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Death Receptor ◽  
Primary Resistance ◽  
Arginine Deprivation ◽  
Car T ◽  
Extrinsic Apoptosis

Abstract Background Chimeric antigen receptor (CAR)-T cell therapy has revolutionised the treatment of relapsed or refractory B-ALL in children and young adults with unprecedented response rates. However, primary resistance and relapse are unresolved challenges that limit long term benefit in a significant proportion of patients. Death receptor mediated extrinsic apoptosis is a key component of CAR-T cytotoxicity and impairment of this system, of which CAR-T derived TRAIL (tumour necrosis factor related apoptosis inducing ligand) is a key initiator, is a principal driver of primary resistance. Arginine deprivation with the therapeutic enzyme ADI-PEG20 (pegylated arginine deiminase) sensitises cancers deficient in the enzyme argininosuccinate synthase (ASS1) to the apoptosis initiating activity of TRAIL through tumour cell surface upregulation of death receptors DR4 and DR5. Whether this effect could potentiate the TRAIL-DR activity in CAR-T therapy has not been explored. Aim We tested the hypothesis that ADI-PEG20 treatment can sensitise susceptible B-ALL to anti-CD19 CAR-T through priming of death receptor mediated apoptosis signalling. Methods The effect of ADI-PEG20 on cell survival and death receptor expression in B-ALL cell lines and primary samples was analysed by flow cytometry. Second generation anti-CD19 CAR-T cells with a CD28 costimulatory domain were generated by retroviral transduction of activated peripheral blood mononuclear cells (PBMC) from healthy donors. For CAR-T co-culture experiments, B-ALL cell lines were pre-treated with ADI-PEG20 before washing and re-suspending in arginine replete media prior to CAR-T cell addition. Results To establish potential susceptibility of B-ALL to ADI-PEG20 we measured expression of ASS1, which inversely correlates with sensitivity to the drug, using combined in situ immunohistochemistry (n=6) and RT-qPCR (n=7). ASS1 deficiency was consistently seen in this series of primary samples suggesting the potential utility of ADI-PEG20 in B-ALL, with comparable expression levels to those seen in a cohort of primary AML samples proven to be sensitive to the drug (figure 1a). Next, to examine variation in ASS1 expression between genetically defined subtypes of B-ALL we re-analysed transcriptome data from a cohort of 215 patients treated on the ECOG E2993 trial, filtered into a network of 58 genes generated according to known or predicted interaction with ASS1. We found an enrichment of Philadelphia chromosome positive (Ph+) and Philadelphia-like (Ph-L) samples in the cluster characterised by lowest ASS1 expression along with high HIF1A expression, matching a recurrent pattern reported in other ADI-PEG20 sensitive tumours. This therefore predicts that among B-ALL subtypes, Ph+ and Ph-L are likely to be most sensitive to therapeutic arginine deprivation. We then functionally confirmed, using in vitro cell line (n=3) and in vivo patient derived xenograft models of B-ALL (n=2), that ASS1 deficiency is required for ADI-PEG20 sensitivity. Using the ASS1-low, Ph-L cell line MUTZ-5, we established that ADI-PEG20 induced apoptosis accompanies cell surface upregulation of both DR4 and DR5 expression. Upregulation of DR4 was observed to follow an upwards trend after treated cells were washed and re-suspended in arginine replete media, suggesting that transient arginine starvation can commit ASS1-low B-ALL to a state of apoptotic priming (figure 1b). With confirmed engagement of arginine starvation and death receptor upregulation we tested the synergy potential of ADI-PEG20 pre-treatment of MUTZ-5 followed by CAR-T, utilising calculated combination drug indices (CDIs). Across independent PBMC donors (n=3) we observed greater potency killing of CD19 + leukaemia cells in the combination treated co-cultures when compared to the single agent treated conditions, with CDIs consistently less than 1 confirming a synergistic effect (figure 1c). Conclusion Our study proposes a synergistic interaction between the arginine depleting enzyme ADI-PEG20 and anti-CD19 CAR-T for the treatment of ASS1 deficient B-ALL, whereby priming of death receptor signalling may underlie enhanced CAR-T cytotoxicity against CD19 + tumour cells. These data support an emerging framework for CAR-T optimisation based on targeting of the death receptor mediated extrinsic apoptosis pathway and can inform future refinements in the development of cellular immunotherapy. Figure 1 Figure 1. Disclosures Bomalaski: Polaris Pharmaceuticals Inc.: Current Employment. Maher: Leucid Bio: Other: Chief Scientific Officer. Gribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria.

scholarly journals Targeting B-Cell Maturation Antigen (BCMA) with CC-93269, a 2+1 T Cell Engager, Elicits Significant Apoptosis in Diffuse Large B-Cell Lymphoma Preclinical Models

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1580-1580 ◽  
Author(s):  
Patrick R. Hagner ◽  
Michelle Waldman ◽  
Falon D. Gray ◽  
Renee Yura ◽  
Sarah Hersey ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cells ◽  
Surface Expression ◽  
Employment Equity ◽  
Car T ◽  
Equity Ownership

B-cell maturation antigen (BCMA), a member of the TNF receptor superfamily, serves as the cell surface receptor for B-cell activating factor (BAFF). Upon binding of BAFF to BCMA, an intracellular signaling cascade is initiated resulting in upregulation of JNK pathway signaling events and NFkB mediated transcription. The canonical expression pattern of BCMA begins on germinal center B-cells and becomes maximally expressed on mature cells such as plasma cells. Given the high degree of expression of BCMA on multiple myeloma (MM) cells, the malignant counterpart to plasma cells, it has become a target of interest for CAR T and antibody mediated modalities such as antibody drug conjugates and bispecific molecules. Recent clinical data from clinical trials employing BCMA targeted CAR T cells or T cell engager (TCE) antibodies have demonstrated significant responses in heavily pretreated myeloma patients with overall response rates ranging from 70% to 80%. As BCMA is known to be expressed in earlier B-cell lineages, we sought to explore the expression of BCMA in non-Hodgkin lymphoma (NHL) and its sensitivity to CC-93269, a 2+1 TCE currently being clinically investigated in MM. NHL is a heterogeneous collection of lymphomas that can be classified into two major subgroups; aggressive lymphomas of which diffuse large B-cell (DLBCL) is the most prevalent subtype and indolent lymphomas of which follicular lymphoma is the largest constituent. We first sought to directly quantitate cell surface expression of BCMA utilizing a flow cytometry system based on a logarithmic dilution of phycoerythrin beads of a known quantity. In a panel of 43 NHL cell lines, we determined that BCMA expression ranged from 43 to 17,048 molecules per cell (median, 420). An isogenic pair of K562 that is null for BCMA expression and K562 constitutively overexpressing BCMA (K562-BCMA) (15,866 molecules/cell) served as negative and positive controls, respectively. Additionally, the MM cell line H929 was profiled to serve as an additional control with a BCMA expression level of 7,065 molecules/cell. Next, utilizing quantitative PCR we found that relative BCMA mRNA expression in the lymphoma cell lines ranged from 0.001 to 0.17-fold when normalized to the H929 MM cell line. Furthermore, we were able to determine that in the lymphoma cells there is a poor correlation between protein expression (mean fluorescent intensity) and mRNA expression (r2, 0.33). We next examined if there was any correlation between BCMA surface expression and T-cell mediated cytotoxicity after administration of CC-93269 in a co-culture assay. We selected 11 DLBCL cell lines with a surface expression ranging from 45 molecules to 17,000 molecules per cells and incubated them in a co-culture system with a defined 1:5 target:effector ratio with CC-93269 (0-200 ng/ml) for five days. Significant apoptosis as measured by annexin V and ToPro-3 staining of CFSE positive target cells was observed in 10 of the 11 cell lines profiled with an IC50 of 0.1 to 38 ng/ml for CC-93269. As controls, the K562 isogenic pair were also profiled with the K562-BCMA cell line exhibiting an IC50 of 0.5 ng/ml and no activity observed against the parental K562 cell line. Additionally, a bispecific antibody where the two binding domains for BCMA was altered to target HEL (hen egg lysozyme) demonstrated no activity against any of the cell lines profiled at a defined dose of 200 ng/ml. No association between CC-93269 activity and BCMA expression or cell of origin was found. To determine the expression of BCMA in primary DLBCL biopsy samples, we developed a novel monoclonal BCMA immunohistochemistry antibody (clone: G12). The antibody and IHC staining protocol were validated to have good on-target specificity in both cell lines and tissues, including MM and DLBCL biopsies, with a range of stain intensity (1-3+) observed in both the golgi and on the plasma membrane. A proof of concept study on a cohort of 110 commercial DLBCL samples is currently underway. Cumulatively, our data demonstrate that BCMA is expressed on the cell surface of a broad panel of NHL cell lines and in primary DLBCL lymph node biopsies. Additionally, the expression levels of BCMA in these preclinical cell line models was sufficient to elicit significant CC-93269 mediated cytotoxicity. These data highlight the potential for the treatment of DLBCL patients with a 2+1 T-cell engager targeting BCMA. Disclosures Hagner: Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Waldman:Celgene: Employment, Equity Ownership, Patents & Royalties. Gray:Celgene: Employment, Equity Ownership. Yura:Celgene: Employment, Equity Ownership. Hersey:Celgene: Employment, Equity Ownership. Chan:Celgene: Employment, Equity Ownership. Zhang:Celgene: Employment, Equity Ownership. Boss:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Tolinapant (ASTX660), a Non-Peptidomimetic Antagonist of cIAP1/2 and XIAP, and the HDAC Inhibitor Romidepsin Are Synergistic in in Vitro Models of T Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4356-4356
Author(s):  
John S Manavalan ◽  
Ipsita Pal ◽  
Aidan Pursley ◽  
George A. Ward ◽  
Tomoko Smyth ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Tnf Alpha ◽  
Sensitive Cell ◽  
Advisory Committees

Abstract Background: The PTCL are a heterogeneous group of non-Hodgkin lymphomas originating from mature T-lymphocytes. They are aggressive diseases, often resistant to conventional chemotherapy. Despite the fact that a number of new agents have been approved, treatment paradigms tailored to the biology of the disease have yet to emerge. Tolinapant (ASTX660) is a potent antagonist of both cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), and is presently in phase I/II trials in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists enhance tumor necrosis factor (TNF) receptor superfamily mediated apoptosis (Ward GA, et al. Mol Cancer Ther. 2018), are potent anti-tumor immune enhancers and induce markers of immunogenic cell death such as damage associated molecular patterns (DAMPs; Ye W, et al, Oncoimmunology, 2020). Objectives: We explored the sensitivity of a range of T-cell lymphoma (TCL) cell lines to tolinapant. We establish the synergy coefficient between tolinapant and the HDAC inhibitor, romidepsin, and interrogated the molecular basis of their synergistic interaction. Methods: A panel of human T-cell lymphoma cell lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10ng/ml of TNF alpha. For combination studies, with tolinapant and romidepsin, each drug was tested at the IC10 and IC40 concentrations in the presence or absence of TNF alpha. Synergy scores using the Excess over Bliss (EOB) model were calculated using SynergyFinder (Aleksandr Ianevski et al; Nucleic Acids Research, 2020). Additionally, the effects of tolinapant and romidepsin on the IAPs and caspases were analyzed by western blots. TNFR1 receptor expression and induction of DAMPs were also analyzed by flow cytometry. Results: TCL Lines demonstrated varying sensitivities to tolinapant in the presence or absence of TNF alpha. The most sensitive cell lines, ALK+ ALCL and SUP-M2, had IC50 concentrations ranging from 200nM ± 100nM to 20nM ± 1nM in the absence or presence of TNF alpha, respectively, at 24, 48 and 72hrs, while a resistant CTCL cell line HH had an IC50 concentration of over 20mM, even in the presence of TNF alpha. Interestingly, using western blot analysis, we found that the presence of TNF alpha increased the levels of cIAP1 in the tolinapant sensitive SUP-M2 cell line, but not in the resistant HH cell line. However, there was a concentration dependent decrease in cIAP1 but not in XIAP in both cell lines treated with tolinapant. Flow cytometry analysis demonstrated that tolinapant increases the expression of TNFR1 and DAMPs in a dose dependent manner on the sensitive SUP-M2, but not in the resistant HH cells. In combination experiments, using the EOB model, tolinapant plus romidepsin was found to be synergistic in the absence of TNF alpha, at 36hrs, in both the sensitive cell line SUP-M2 and the resistant cell line HH. In the presence of TNF alpha, synergism was seen only in the sensitive cell line SUP-M2 and antagonistic in the HH cell line (Fig. 3). In the tolinapant plus romidepsin treated samples, cIAP1 levels decreased in the SUP-M2 cell line, in the absence of TNF alpha, however, addition of TNF alpha did not alter the levels of cIAP1 in the SUP-M2 cells. The cIAP1 levels decreased in the HH cells treated with the combination, in both the presence or absence of TNF alpha (Figure). Our findings indicate that the synergy of the tolinapant plus romidepsin is not dependent on the presence of TNF alpha. Conclusion: Tolinapant has demonstrated potent cytotoxic effects against a broad range of TCL lines both as a monotherapy and in combination with the HDAC Inhibitor, romidepsin. In in vitro studies, T cell lymphoma cell lines demonstrated varying sensitivity to tolinapant with certain cell lines being more resistant, even in the presence of TNF alpha. Interestingly, the addition of romidepsin appeared to overcome the intrinsic resistance to tolinapant in the absence of TNF alpha. These data provide the rationale to continue to explore the combination of tolinapant and romidepsin in vivo and to investigate additional combinations with T-cell specific agents (e.g. pralatrexate, belinostat, azacitidine and decitabine). Figure 1 Figure 1. Disclosures Smyth: Astex Pharmaceuticals: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Marchi: Kyowa Kirin: Honoraria; Myeloid Therapeutics: Honoraria; Astex: Research Funding; BMS: Research Funding; Merck: Research Funding; Kymera Therapeutics: Other: Scientific Advisor.

scholarly journals Multi-Modal Targeting of FLT3 with Chimeric Antigen Receptor T Cell Immunotherapy and Tyrosine Kinase Inhibition in High-Risk Pediatric Leukemias

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 404-404
Author(s):  
Lisa M Niswander ◽  
Zachary Graff ◽  
Asen Bagashev ◽  
Lillie Leach ◽  
Terry J. Fry ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Car T Cell ◽  
Cell Immunotherapy ◽  
Car T ◽  
T Cell Immunotherapy

Abstract Background: Clinical outcomes for children with FLT3-mutant AML and infants with KMT2A-rearranged (KMT2A-R) B-ALL remain dismal. These leukemias share a common feature of aberrant activation of FLT3 kinase signaling, which occurs by activating FLT3 mutations in AML and by overexpression of wild-type FLT3 in KMT2A-R ALL. Several FLT3 tyrosine kinase inhibitors (FLT3i) are approved for adults with FLT3-mutant AML, but potential efficacy against KMT2A-R ALL remains incompletely characterized and may differ from responses in AML. We previously developed and preclinically validated chimeric antigen receptor (CAR) T cells directed against FLT3 (FLT3CART), which importantly showed potent anti-leukemia activity in preclinical models of both childhood FLT3-mutant AML and infant KMT2A-R ALL (Chien CD et al. ASH 2016). In the current studies, we hypothesized that combinatorial targeting of these two high-risk leukemia subtypes with FLT3CART and the selective next-generation FLT3i gilteritinib would have superior activity and potentially mitigate therapeutic resistance now known to occur with kinase inhibitors or CAR T cell immunotherapy. Methods and Results: We first assessed in vitro sensitivity of human FLT3-mutant AML and KMT2A-R ALL cell lines to gilteritinib, a second-generation selective FLT3i with established clinical activity in FLT3-mutant AML and unknown activity in KMT2A-R ALL. As detrimental effects of kinase inhibitors (e.g., dasatinib, ruxolitinib) upon CAR T cells have been reported, we evaluated for similar effects with gilteritinib co-incubated in vitro with CD3/CD28-bead activated healthy human donor T cells. However, we observed minimal deleterious effects of gilteritinib on normal T cell viability, immunophenotype, and IL-2 and interferon-gamma (IFNg) production. We validated combinatorial effects of gilteritinib and FLT3CART-induced cytotoxicity against FLT3-mutant AML and KMT2A-R ALL cell lines in vitro without impairment of IL-2/IFNg production. We then assessed this dual therapy approach in luciferase+ FLT3-mutant AML (MOLM14) and KMT2A-R ALL (SEM) cell line murine xenograft models. As predicted, both FLT3CART and gilteritinib monotherapies transiently inhibited in vivo leukemia proliferation, although leukemia progression eventually occurred. Conversely, FLT3CART and gilteritinib combination therapy strikingly induced enhanced and sustained leukemia clearance in all assessed AML and ALL cell line xenograft models (Figure 1). Confirmatory studies in our established childhood FLT3-mutant AML and KMT2A-R ALL patient-derived xenograft (PDX) models have also demonstrated potent anti-leukemia efficacy of combined FLT3CART and gilteritinib therapy. Earlier-generation FLT3i have been reported to increase cell surface FLT3 expression on FLT3-mutant AML cells. Given the known importance of target antigen site density for CAR T cell efficacy, we reasoned that a sequential approach to dual therapy with FLT3i 'priming' followed by FLT3CART may be superior to a simultaneous treatment strategy. In vitro studies with leukemia cell lines and in vivo studies with PDX models indeed confirmed gilteritinib-induced increases in FLT3 surface antigen density in FLT3-mutant AML cells. Intriguingly, we observed contrasting effects in KMT2A-R ALL cell lines and PDX with decreased surface FLT3 expression upon gilteritinib exposure. Ongoing studies are currently validating gilteritinib priming for FLT3CART given these initial data suggesting potentially divergent sequencing approaches in FLT3-mutant AML versus KMT2A-R ALL. Conclusions: Taken together, our preclinical studies demonstrate that dual targeting with FLT3CART immunotherapy and gilteritinib is a promising therapeutic strategy in FLT3-mutant AML and, importantly, also in KMT2A-R ALL. Notably, we also report minimal negative effects of gilteritinib on FLT3CART, suggesting that FLT3i may be used to enhance CAR T cell immunotherapy without inhibiting T cell function. Phase 1 clinical trials of FLT3CART will open soon for adults and children with FLT3-mutant AML and/or KMT2A-R ALL. Figure 1 Figure 1. Disclosures Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company; ElevateBio: Research Funding. Tasian: Kura Oncology: Consultancy; Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

A CXCL10/CXCR3 Driven Thymic Epithelium-Leukemia Cell Crosstalk Augments T Cell Acute Lymphoblastic Leukemia Notch1 Signalling

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2537-2537
Author(s):  
Ashwini M Patil ◽  
Stefanie Kesper ◽  
Vishal Khairnar ◽  
Marco Luciani ◽  
Michael Möllmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Thymic Epithelial Cells ◽  
Hematopoietic Organ ◽  
Notch1 Mutations

Introduction: The thymus is a specialized hematopoietic organ, which is responsible for the generation of T cells. The central thymic cell type controlling T cell development are thymic epithelial cells (TECs). Based on their specific function and anatomic location TECs are separated into cortical and medullary subsets (cTECs and mTECs). cTECs express pivotal NOTCH-ligands such as DLL4 controlling T cell lineage commitment while mTECs play a central role in negative selection of developing T cells. Acquisition of NOTCH1 gain-of-function mutations play a central role in acute T cell lymphoblastic leukemia (T-ALL) development. During T-ALL leukemogenesis aberrant expression of transcription factors such as SCL/TAL1 and LMO1 block T cell differentiation and increase self-renewal while NOTCH1 mutations promote survival and proliferation. Since most acquired NOTCH1 mutations still require ligand binding to exert augmented signaling we propose DLL4-expressing TECs playing a critical role during T-ALL leukemogenesis. Methods: In the present study, we used a Scl/Lmo1 T-ALL transgenic mouse model, murine ANV and TE71 TEC cell lines and human T-ALL cell lines (Jurkat, ALL-SIL, DND-41, and HPB-ALL) to investigate TEC dynamics and function in the T-ALL context. Results: First, we demonstrated T-ALL supporting potential of TEC cell lines in vitro, which was comparable to the mesenchymal cell line OP9. Next, we showed in the Scl/Lmo1 T-ALL mouse model which had a mean survival rate of 90 days that preleukemic thymocytes displayed a striking upregulation of Notch1 target genes. Interestingly, fluorescence microscopy revealed a relative expansion of cortical and a relative reduction of the medullary thymic areas in Scl/Lmo1 thymi (Fig. 1A). Correspondingly, absolute numbers of cTECs expanded while mTEC numbers declined (Fig. 1B). Gene expression profiling of sorted preleukemic Scl/Lmo1 cTECs revealed upregulation of the chemokine CXCL10 (Fig. 1C). Moreover, increased CXCL10 chemokine concentrations were detected in Scl/Lmo1 thymic interstitial fluid (Fig.1D). Strikingly, we demonstrated T-ALL dependence of TEC Cxcl10 upregulation. We showed that Cxcl10 upregulation in TEC cell lines was only induced by direct cellular contact with Scl/Lmo1 thymocytes while wild-type control thymocytes did not alter TEC cell line Cxcl10 expression (Fig. 1E). Next, a high proportion of the CXCL10 receptor CXCR3 expressing cells was revealed in Scl/Lmo1 thymi (Fig. 1F) and by human T-ALL cell lines. Finally, we demonstrated a CXCL10 dependent pro-survival effect within cultured SCL/LMO1 thymocytes (Fig. 1G), which was associated with the activation of NOTCH1 signaling (Fig. 1H). Conclusions: In summary, the data support a novel T-ALL-promoting regulatory circuit in which emerging T-ALL lymphoblasts induce CXCL10 in expanding TECs which positively feeds back to T-ALL cells via the CXCL10 receptor CXCR3. Disclosures Dührsen: Celgene: Research Funding; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Teva: Honoraria; Novartis: Consultancy, Honoraria; Alexion: Honoraria; Roche: Honoraria, Research Funding; CPT: Consultancy, Honoraria; Janssen: Honoraria. Göthert:Proteros Biostructures: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel support; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria, Other: Travel support; Bristol-Myers Squibb: Consultancy, Honoraria, Other: Travel support; AOP Orphan Pharmaceuticals: Honoraria, Other: Travel support.

scholarly journals Metabolic Changes in Venetoclax Resistance Are Determined By Differentiation State in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3401-3401
Author(s):  
Alessandra Di Grande ◽  
Andrew Roe ◽  
Thomas Lefeivre ◽  
Theresa E. Leon ◽  
Marc R. Mansour ◽  
...  
Keyword(s):  
T Cell ◽  
Oxidative Phosphorylation ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Metabolic Profile ◽  
Metabolic Phenotype ◽  
Maturation Stage ◽  
Mitochondrial Structure ◽  
Altered Metabolism

Abstract T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy arising from the transformation of immune T-cell lymphocytes. Early T-cell progenitor (ETP-ALL) is a subgroup particularly associated with chemoresistance and a high risk for relapse. Recently, it was shown that ETP-ALL is dependent on the expression of the anti-apoptotic protein BCL-2, and is sensitive to inhibition with ABT-199, a BCL-2 specific BH3 mimetic 1,2. However, one issue with a targeted agent, such as ABT-199, is the development of acquired resistance. Interestingly, there have been numerous high impact papers connecting ABT-199 resistance to altered oxidative phosphorylation (OXPHOS) 3,4. While there are relatively few studies into T-ALL metabolism, there is evidence that aerobic glycolysis, the conversion of glucose to lactate, is greater in proliferating T-cells than in T-ALL and that NOTCH signalling can drive mitochondrial OXPHOS 5. A recent study showed that the transcription factor RUNX2 altered T-ALL metabolism, increasing both glycolysis and OXPHOS and enhancing leukemic cell migration 6. However, there has been relatively little research into the metabolic profile of T-ALL at distinct stages of differentiation. The aim of this study was to determine the role of ABT-199 resistance in altering metabolism and determine if that was due to the differentiation state of the T-ALL. ABT-199 R LOUCY cells were generated by chronic exposure to increasing concentrations of ABT-199 administered every two days. The EZH2 KO Jurkat cell lines were previously generated through CRISPR-Cas9 engineering 7. In order to assess the metabolic profile, cells were attached to a 96 well plate using CellTak and the extracellular acidification rate (ECAR) and oxidative phosphorylation (OXPHOS) was measured on a Seahorse Bioscience XF96 Extracellular Flux Analyzer. Anti-apoptotic dependence was measured using BH3 profiling and cell death by Annexin V/propidium iodide staining. The mitochondrial structure was visualized using transmission electron microscopy. Previously, we generated ABT-199 resistant ETP-ALL LOUCY cells (ABT-199 R) following continuous exposure to ABT-199 over a prolonged period of several months 8. The ABT-199 R cells showed dependence on BCL-XL for survival and sensitivity to the BCL-XL inhibitor WEHI 539. The ABT-199 R cells showed evidence of differentiation to a more mature T-cell. The ABT-199 R cells had increased surface CD3 (sCD3) expression and CD1A expression, along with increased expression of TAL1 and LMO2 genes compared to parental LOUCY cells. Interestingly, the ABT-199 R cells showed enhanced basal respiration, ATP production and max respiration compared to the parental cells. Indeed, analysis of the expression of OXPHOS complexes showed increased expression of complexes I-IV in the ABT-199 R cells, compared to the parental controls. Indeed, the parental LOUCY cells appeared to have reduced cristae number and length compared to the ABT-199R cells. Next, we assessed if inhibiting OXPHOS with a series of inhibitors (oligomycin, rotenone, antimycin) could sensitize the ABT-199 R LOUCY cells to ABT-199. However, we did not detect any changes to sensitivity of ABT-199. This led us to hypothesize that perhaps the changes in OXPHOS were due differentiation state of the LOUCY cells. We confirmed that more typical T-ALL cell lines (JURKAT and CEM-CCRF) had higher OXPHOS than the ETP-ALL cell line LOUCY and had higher expression of the OXPHOS complexes I-IV by Western blotting. To assess if de-differentiation of a more typical T-ALL cell line would cause a reduction in OXPHOS we turned to the EZH2 knockout (K/O) Jurkat cells 7. We found that EZH2 KO2 showed a reduction in the differentiation markers CD1A and CD3 on the cell surface and TAL1 gene expression, compared to WT control Jurkats. Next, we assessed the OXPHOS and found that the de-differentiated EZH2 cells had reduced OXPHOS compared to the parental controls, with altered mitochondrial structure. Suggesting, that de-differentiation of typical T-ALL cell line reduces OXPHOS. In this study we show that metabolic phenotype is linked to the maturation stage of T-ALL. We believe that the altered metabolism identified in ABT-199 resistance is linked to the selection of a more mature cell type. Highlighting, that altered metabolism may not be a driver of resistance to ABT-199 but a consequence of the maturation stage of the resistant cell. Disclosures Di Grande: Novartis: Current Employment. Leon: BenevolentAI: Current Employment. Mansour: Astellas: Consultancy, Honoraria; Janssen: Consultancy. Bond: Haematology Association of Ireland Award funded by Novartis: Research Funding. Ni Chonghaile: AbbVie: Research Funding.

scholarly journals Engineered T Cell Receptor-Mimic Antibody, (TCRm) Chimeric Antigen Receptor (CAR) T Cells Against the Intracellular Protein Wilms Tumor-1 (WT1) for Treatment of Hematologic and Solid Cancers

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2155-2155 ◽  
Author(s):  
Sarwish Rafiq ◽  
Tao Dao ◽  
Cheng Liu ◽  
David A. Scheinberg ◽  
Renier J Brentjens
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Primary Cells ◽  
Intracellular Protein ◽  
Car T Cells ◽  
Car T

Abstract Adoptive transfer therapy of T cells expressing chimeric antigen receptors (CAR) against tumor-associated antigens has been shown to be clinically successful in a limited set of leukemia. However, novel antigen targets for both hematological and solid malignancies are required. Most CARs described thus far are targeted against external antigens on particular cell types. We have designed and engineered the first CAR T cell against a human intracellular protein, WT1. WT1 is overexpressed in many cancers, including acute and chronic leukemias and numerous solid tumors. Our TCRm CAR, derived from the ESK1 TCRm mAb, termed WT1 28z, is reactive with the RMFPNAPYL peptide of the WT1 protein that is processed and presented on the surface of cells in the context of HLA-A*02:01. WT1 28z expressing T cells have high expression of the CAR on their surface. They are cytotoxic in standard 51Cr assays against a range of cancer cell lines, including the megakaryoblastic cell line SET2, the acute myeloid leukemia (AML) cell line AML14, the multiple myeloma cell line KARPAS, and the ovarian cancer line, OVCAR3, as compared to CAR T cells against an irrelevant antigen. The WT1 28z CAR T cells are also cytotoxic against primary AML bone marrow blasts in this assay. When co-cultured with these primary cells or cancer cell lines, the WT1 28z CAR T cells have enhanced production of proinflammatory cytokines such as IFN-g, IL-2, and GM-CSF, as compared to irrelevant CAR T cells. Importantly, WT1 28z T cells are specific for the WT1-HLA-A*02:01 complex. The cells do not show cytotoxicity against cell lines or primary cells that are not both HLA-A*02:01- positive and WT1 positive. WT1 28z T cells are currently being tested alongside irrelevant antigen CAR T cells in AML and ovarian cancer murine models in vivo to assess efficacy, with the ultimate goal of translating this novel approach into the clinical setting for both hematological and solid cancers. The data provide the proof-of-concept that CAR T cells also may be directed at intracellular antigens. Disclosures Dao: Novartis: Patents & Royalties. Liu:Eureka: Employment, Inventor Other. Scheinberg:Novartis: Patents & Royalties. Brentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

scholarly journals Interleukin-2-dependent T-cell lines established from paroxysmal nocturnal hemoglobinuria patients

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 309-314
Author(s):  
H Nakakuma ◽  
S Nagakura ◽  
K Horikawa ◽  
M Hidaka ◽  
T Kawaguchi ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
Cell Line ◽  
Cell Lines ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Control Cell ◽  
Interleukin 2 ◽  
Culture Cell ◽  
Adult T Cell Leukemia ◽  
T Cell Lines

Peripheral blood T lymphocytes obtained from two patients with paroxysmal nocturnal hemoglobinuria (PNH) were immortalized with human T-lymphotropic virus type 1 (HTLV-1). These cells showed interleukin-2 (IL-2)-dependent cell growth in culture. Cell surface analysis showed that they had the phenotype of a helper/inducer T subset that was positive for CD2, CD3, and CD4, but negative for CD8, similar to adult T-cell leukemia cells induced by HTLV-1. These cell lines lacked glycosylphosphatidylinositol (GPI)-anchored proteins, CDw52, CD55 (decay-accelerating factor; DAF), and CD59 on the cell surface, whereas intracellular DAF protein was detected. These T-subset cell lines with a PNH phenotype did not synthesize GPI anchor, whereas a control cell line, similarly prepared from the T cells of a healthy volunteer, produced the anchor. The control cells expressed CDw52, DAF, and CD59 on the cell surface and showed the phenotype of a helper/inducer subset. Southern blot analysis confirmed the clonality of each cell line. These CD4+ T-cell lines with a PNH phenotype and a subset-matched control counterpart could be a useful model for PNH investigation.

scholarly journals Repurposing Bi-Specific Chimeric Antigen Receptor (CAR) Approach to Enhance CAR T Cell Activity Against Low Antigen Density Tumors

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1727-1727
Author(s):  
Sherly Mardiana ◽  
Olga Shestova ◽  
Stephan A. Grupp ◽  
Marco Ruella ◽  
David M. Barrett ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Tumor Cells ◽  
Therapeutic Efficacy ◽  
Research Funding ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract BACKGROUND Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory B-cell malignancies, as highlighted by high complete remission rates and FDA approval of CD19-specific CAR T cell products. However, depth and duration of remission are limited by antigen loss/downregulation on tumors, as observed in clinical trials using CAR T cells targeting the CD19 or CD22 in leukemia and lymphoma, BCMA in multiple myeloma, and EGFRvIII in glioblastoma. This observation forms the basis of current efforts to develop multi-targeting CAR T cells to prevent antigen-negative escape. Antigen density is an important factor modulating CAR T cell response, since antigen expression below a certain threshold fails to trigger the full range of T cell functions. Given that signal strength induced upon antigen encounter determines CAR T cell activity, we hypothesized that simultaneous targeting of two dimly-expressed antigens will result in enhanced CAR T cell signaling and anti-tumor function, approaching that seen in response to one highly-expressed antigen. This is important given the heterogeneity of antigen expression in various cancers. Therefore, the bi-specific CAR T cells currently being developed to prevent antigen-negative escape could also be used to enhance efficacy against low antigen density (LAD) tumors. Results from this study will provide a novel rationale for using multi-specific CAR T cells and illuminate the mechanisms of successful CAR T cell therapy. METHODS Lentivirus transduction was performed to generate CAR T cells from healthy human T cells, using second generation 4-1BBz CARs specific for either human CD19 or CD22, or both in cis, herein referred to as CAR19, CAR22, or CAR19/22, respectively (Figure 1A). For in vitro functional characterization, we performed co-culture assay of T cells and B cell leukemia cell line NALM6, which is known to express high levels of both CD19 and CD22. To assess T cell function against LAD tumor cells, primary patients' B-ALL samples expressing low antigen density in comparison to the NALM6 cell line were used (Figure 1B). CAR T cell anti-tumor potency was determined by assessing CAR T cell cytotoxicity and cytokine production. For in vivo therapeutic study, primary patients' B-ALL samples with dimly expressed CD19 and CD22 were used to evaluate and compare the therapeutic efficacy of mono- versus bi-specific CAR T cells. Additionally, we generated a LAD tumor model by deleting the highly expressed CD19 and CD22 from the ALL cell line NALM6 using CRISPR/Cas9, transducing the now antigen-negative cell line with CD19 and CD22, followed by single cell cloning to generate a cell line expressing low antigen density for both the CD19 and CD22. We engrafted tumor cells in NSG mice, followed by administration of CAR19, CAR22, CAR19/22 or untransduced T cells. Therapeutic efficacy was assessed by measuring tumor burden using either flow cytometry or bioluminescent imaging. RESULTS Cytotoxicity assay revealed that the bi-specific CAR19/22 T cells killed tumor cells more rapidly than CAR19 or CAR22 T cells. Further, compared to mono-specific CAR T cells, the bi-specific CAR19/22 T cells produced significantly more pro-inflammatory cytokines including IL-2 and IFNg, in response to stimulation with LAD primary samples or NALM6 cells. This increased cytokine-producing capacity compared to mono-specific CAR T cells was maintained following repeated antigen stimulation when in vitro exhaustion assay was performed. In vivo, enhanced tumor elimination was observed in mice receiving bi-specific CAR19/22 T cells compared to either of the mono-specific CAR T cells, in both low antigen density primary ALL and NALM6 tumor models. This translated to increased survival rates seen in mice treated with the bi-specific CAR19/22 T cells (Figure 1C-D). CONCLUSIONS Here we showed that bi-specific CAR19/22 T cells are superior to mono-specific CAR19 or CAR22 T cells, not only against LAD tumors but also tumor cells expressing high antigen density, NALM6. This was demonstrated by their enhanced cytokine-producing function, cytotoxic capacity, and therapeutic efficacy in vivo. Results from this study provide a novel rationale for repurposing multi-specific CAR T cells as a strategy to improve efficacy against LAD tumors, in addition to the recognized benefit of reducing antigen-negative escape. Figure 1 Figure 1. Disclosures Shestova: Hemogenyx Pharmaceuticals LLC: Research Funding. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Kite, Vertex, and Servier: Research Funding; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Ruella: viTToria biotherapeutics: Research Funding; Novartis: Patents & Royalties; BMS, BAYER, GSK: Consultancy; AbClon: Consultancy, Research Funding; Tmunity: Patents & Royalties. Gill: Novartis: Other: licensed intellectual property, Research Funding; Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

scholarly journals CD7 CAR for the Treatment of Acute Myeloid and Lymphoid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4555-4555 ◽  
Author(s):  
Diogo Silva ◽  
Haruko Tashiro ◽  
Madhuwanti Srinivasan ◽  
Malcolm K. Brenner ◽  
Maksim Mamonkin
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoid Leukemia ◽  
Car T Cells ◽  
Car T ◽  
Target Ratio ◽  
Acute Myeloid ◽  
Myeloid Progenitors

Abstract CD7 is a T- and NK-cell marker highly expressed in T cell leukemia and in 30% of AML cases, where its presence correlates with chemoresistance and worse prognosis. We created CD7-directed chimeric antigen receptors (CARs) using single chain variable fragment (scFv) sequences derived from three different CD7-specific mAb clones and evaluated the feasibility and potency of using CD7 CAR T cells for the treatment of the CD7+ hematologic malignancies. Since normal T cells themselves express CD7, their transduction with CD7 CARs limited their expansion in vitro, resulting in approximately 30-fold fewer T cells after 14 days of culture compared to control CAR-transduced cells. To circumvent this problem of fratricide, we used the CRISPR/Cas9 system to disrupt the CD7 gene in T cells prior to retroviral transduction with CD7 CAR. The CD7 gene was successfully disrupted in 90% of T cells, minimizing fratricide of CD7 CAR T cells, and leading to a robust T cell expansion, similar to that of control T cells transduced with an irrelevant CAR (310-fold vs 380-fold expansion after 14 days, respectively). Lack of CD7 did not compromise CAR T cell effector function, as we observed equal cytotoxicity and expansion in CD7+ and CD7- CD19 CAR T cells upon coculture with a CD19+ cell line Raji (P=0.99 and P=0.86, respectively). Expanded CD7-deficient CD7 CAR T cells demonstrated robust cytotoxicity against CD7+ AML cell lines KG-1a and Kasumi-3 and T-ALL cell line CCRF-CEM, resulting on average in a 19-fold expansion of CAR T cells and elimination of malignant cells (92-99.9%) after 7 days of co-culture at a 1:4 initial effector-to-target ratio. This cytotoxicity was CD7-specific, as CD7 CAR T cells showed no activity against a CD7-negative cell line Raji. We also observed potent activity of CD7 CAR T cells against primary AML cells, leading to an 80% reduction in blast counts after 48 hours of co-culture at a 1:1 effector-to-target ratio, compared with control CAR T cells. Moreover, a 5-hour co-culture of CD7 CAR T cells with primary AML blasts and subsequent culture on a methylcellulose medium for 12 days resulted on average in a 26-fold reduction in leukemic colonies; these data suggest that the CD7 CAR T cells can eliminate primitive leukemic progenitors. Because CD7 is expressed on some myeloid progenitors, we assessed the activity of CD7 CAR T cells against normal myeloid precursors. After coculturing cord blood cells with CD7 CAR T cells at a 10:1 effector-to-target ratio and subsequently expanding myeloid colonies on a methylcellulose medium for 14 days, we observed no significant differences in the numbers of monocytic, erythrocytic and granulocytic colonies compared to a coculture with control CAR T cells. Hence, CD7 CAR T cells appear non-toxic to normal myeloid progenitor cells. In summary, we show that CD7 CAR T cells can be efficiently generated and expanded following genomic disruption of the CD7 gene by CRISPR/Cas9. Expanded CD7 CAR T cells produce robust cytotoxic activity against AML and T-ALL cell lines as well as primary AML blasts, and show no toxicity against normal myeloid progenitors. These results demonstrate the potency and support the feasibility of using CD7 CAR T cells for the targeted therapy of acute myeloid and lymphoid leukemia. Disclosures Brenner: Viracyte: Equity Ownership; Cell Medica: Patents & Royalties.

Multi-Omic Based Antigen Discovery for the Immunotherapy of Pediatric Acute T Cell Lymphoblastic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
Nikhil Hebbar ◽  
Chunxu Qu ◽  
Hong Wang ◽  
Ying Shao ◽  
Phuong Nguyen ◽  
...  
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
T Cell ◽  
Cell Surface ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Differentially Expressed ◽  
Activated T Cells ◽  
Car T Cells ◽  
Car T

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk disease due to treatment related complications and poor prognosis of patients with relapsed disease. Immunotherapy with monoclonal antibodies (MAbs) and/or chimeric antigen receptor (CAR) T-cells for T-ALL is limited by identification of tumor specific target antigens. Differential expression is necessary to prevent on-target/off-tumor toxicities and fratricide of activated T-cells. Targeting multiple antigens can bypass immune escape and result in improved T-cell effector function, since antigen density correlates with T-cell activation. Here we designed a pipeline (Figure 1) to identify unique surface antigens expressed in T-ALL using proteomic and transcriptomic analyses followed by flow cytometry validation, and functional studies with CAR T cells targeting the identified antigens. We generated an Illumina total stranded RNAseq library from healthy donor myeloid and lymphoid cells of bone marrow, peripheral blood and cord blood (N= 116). We compared data to 265 St. Jude pediatric T-ALL samples and against 53 normal tissue expression data from the GTEx (Genotype-Tissue Expression) project. To analyze the T-cell surface proteome, we isolated plasma membrane fractions from 11 samples including healthy T-cells and T-ALL cell lines using a differential centrifugation-based method. The purity of the plasma membrane fraction was confirmed by western blot. Na+/K+ ATPase and GAPDH were used as controls for the plasma membrane and cytosolic fractions respectively. Following plasma membrane enrichment, the membrane proteins were applied for proteomic analysis using an advanced TMT-L/LC-MS/MS pipeline, and the acquired proteomic data were further processed via the JUMP software suite. 997 unique proteins were quantified from the membrane fractions. Integrated analysis the transcriptomic and proteomic datasets showed significant correlation and yielded a list of candidate genes, which were validated by flow cytometry on a panel of T-ALL cell lines (CCRF, RPMI8402, and MOLT3) and resting and activated T-cells from healthy donors. We identified GRP78 as one of the differentially expressed cell surface antigens and further confirmed its expression on additional T-ALL cell lines (KE37, PF382, PEER, CEMC7) and 3 PDX samples. Finally, we generated GRP78-CAR T cells and demonstrate that GRP78-CAR T cells recognize and kill GRP78+ T-ALL cells and have potent antitumor activity in xenograft and PDX models. We have established an unbiased pipeline to identify differentially expressed antigens on the cell surface of T-ALL blasts and created a healthy tissue RNAseq library. The results from our analyses are encouraging and interrogation of our pipeline has yielded differentially expressed immunotherapy targets for the treatment of relapsed refractory T-ALL. Our results highlight the importance of integrated surface proteomics and transcriptomics analysis. Figure 1: Outline of strategy for target selection: Figure Disclosures Hebbar: St. Jude: Patents & Royalties. Epperly:St. Jude: Patents & Royalties. Gottschalk:Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; TESSA Therapeutics: Other: research collaboration; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Merck and ViraCyte: Consultancy. Mullighan:AbbVie, Inc.: Research Funding; Illumina: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties.

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