Selection and Characterization of Antibody Clones Are Critical for Accurate Flow Cytometry-Based Monitoring of CD123 in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1693-1693
Author(s):  
Nicole M. Cruz ◽  
Linda Lam ◽  
Mayumi Sugita ◽  
Roman Galetto ◽  
Agnès Gouble ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Surface Expression ◽  
T Cell Therapy ◽  
Transcript Levels ◽  
Car T Cell Therapy ◽  
Car T

Abstract Background: CD123, the trans-membrane alpha chain of the interleukin-3 receptor (IL-3RA) is overexpressed in acute myeloid leukemia (AML) and distinguishes leukemia stem cells from their normal counterparts. There are several novel therapeutics under development to target CD123 in AML, including CD123 fused to Diphtheria toxin, a recombinant chimeric anti-CD123 MoAb, CD3/CD123 bi-specific T cell engagers, and engineered T cells that express chimeric antigen receptors (CARs). Thus, accurate detection and quantification of CD123 is critical for newly diagnosed and relapsed patients, and to follow minimal residual disease for patients in remission. Our data suggest that the evaluation of CD123 by flow cytometry varies significantly with different antibody clones. Objective: To identify the most accurate flow cytometry method for evaluation of CD123 expression in patients with AML to evaluate CD123 targeting therapies. Methods: 51 AML patient samples and 7 normal cord blood or bone marrow samples were stained with five different commercially available monoclonal antibodies to detect CD123 (7G3, 6H6, 9F5, AC145 and FAB301P), as well as CD45 and CD5, for evaluation by multiparameter flow cytometry. CD123 gene expression was also compared between these primary AML samples and bone marrow samples from healthy donors. Cell surface expression (by percentage and MFI) was evaluated relative to transcriptional expression and sensitivity to known therapeutics (cytarabine, parthenolide, and HSP90 inhibitors). Results: We observed CD123 surface expression patterns varied between the antibody clones tested. For the 9F5 and 6H6 clones, 93% and 82% of the samples, respectively, showed >60% CD123+ cells whereas for the 7G3, FAB 301P and AC145 clones, 71 to 76% of the samples showed >60% positivity. Also, surface expression of CD123 using 7G3, AC 145 and FAB 301P did not correlate with transcript levels for IL3RA assessed using qPCR, while surface expression of CD123 using 9F5 and 6H6 did correlate with transcript levels of IL3RA, using both mean fluorescence intensity (MFI) and percentage. For example, the correlation between CD123 surface expression as measured by percentage and IL3RA transcripts was most significant using the 9F5 and 6H6 clone (R2=0.1084, p=0.0183, R2=0.1588, p=0.0038 respectively) whereas the correlation for 7G3 (R2=0.0004, p=0.8945), FAB301P (R2=0.0027, p=0.7151) and AC145 (R2=0.0392, p=0.1638) were not significant. Surface expression of CD123 evaluated with 7G3 antibody did not correlate with overall sensitivity to in vitro treatment with cytarabine (R2=0.03767, p= 0.6451). However, using the 9F5 antibody, we found that higher levels of surface CD123 were associated with resistance to cytarabine in vitro (R2= 0.5502, p= 0.0351). Differences were noted for other experimental therapeutics including parthenolide and PU-H71. Most importantly, when we tested the ability of a novel allogeneic anti-CD123 CAR T-cell therapy (UCART123) to eliminate CD123+ AML cells, we found that CD123 positivity as measured by the 7G3 clone was not predictive of sensitivity to UCART123 in vitro or in vivo AML patient derived xenotransplants. Conclusions: Several novel therapeutic modalities targeting CD123 in AML are under development, including allogeneic anti-CD123 CAR T-cell therapy. Accurate, quantitative assessment of CD123 expression is thus of utmost importance for patient selection in clinical trials as well as disease monitoring. We found discrepancies between antibody clones, and such discrepancies may alter patient selection and data interpretation regarding patient response to CD123 based therapies. For therapies targeting CD123, protocol design and antibody selection should be done considering the results in this study. Based on our findings we recommend 9F5 or 6H6 antibody clones as well as the utilization of qPCR along side flow cytometry for adequate detection. Flow cytometry findings should be reported as percent positive cells. If utilizing the 9F5 clone, samples with > 60% CD123+ should be considered positive for CD123. A comparison in a large cohort may be warranted to determine the impact of multiple CD123 measurements on disease outcome. Disclosures Galetto: Cellectis SA: Employment. Gouble:Cellectis: Employment. Smith:Cellectis SA: Employment. Roboz:Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy; Cellectis: Research Funding. Guzman:Cellectis: Research Funding.

P04.17 Antigen heterogeneity in glioblastoma cell lines, patient-derived cells, and patients’ glioblastoma tissue is an obstacle for CAR-T cell therapy development

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii21-ii22
Author(s):  
V Dufner ◽  
E Schulz ◽  
C Monoranu ◽  
M Hudecek ◽  
R Ernestus ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Therapy ◽  
Cell Lines ◽  
Surface Expression ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T ◽  
Low Expression

Abstract BACKGROUND Immunotherapy targeting surface antigens, e.g. CAR-T-cell therapy has become a promising therapeutic approach for glioblastoma (GBM) treatment. Antigen heterogeneity constitutes a major obstacle not only for preclinical in vitro studies but also for clinical translation. Here, we provide information about the surface expression of the eight in GBM CAR-T-cell therapy most targeted antigens (GD2, CSPG4, CD133, CD70, HER2, Il13Rα2, EGFRvIII and EphA2) by analyzing GBM cell lines (GCL), patient-derived cells (PDCL) and patients’ tumor tissue (PT) and comparing the expression profiles. MATERIAL AND METHODS We measured expression of the above-mentioned antigens in 7 GCL (GaMG, U87, U373, U343, U251, U138, DKMG), 7 PDCL and PT of 9 patients by flow cytometry (FACSCanto II). After evaluation with FlowJo software (TreeStar), we scored antigen expression (0–1.0: low expression, 1.1–2.0: medium expression, 2.1–3.0: high expression) and calculated the mean expression and range. RESULTS GD2 showed a medium expression in GCL (x̅=1.3) but was highly expressed in PDCL (x̅=2.6) and PT (x̅=2.4). In contrast, CSPG4 displayed low expression in GCL (x̅=0.3) and PT (x̅=0.4), but medium expression in PDCL (x̅=1.9). CD133, a stem cell marker, showed low expression in GCL (x̅=0.8) and PDCL (x̅=0.4), whereas the expression in PT was medium (x̅=1.4). Both CD70 and Il13Rα2 were weakly expressed in GCL, PDCL and PT (x̅=0.2, 0.4, 0.0 and 0.5, 0.6, 0.4, respectively). HER2 displayed medium expression in GCL (x̅=1.4) and PDCL (x̅=1.4) and low expression in PT (x̅=0.6). For EGFRvIII medium expression was detectable in all three entities (x̅=1.4, 1.1 and 1.1). EphA2 was mildly expressed in GCL (x̅=0.1), medium expressed in PT (x̅=2.0) and highly expressed in PDCL (x̅=2.6). Overall, there was high variability of antigen surface expression even within each of the groups. CONCLUSION GCL, PDCL and PT display heterogenic antigen surface expression with high variability within each group, thereby complicating clinical translation of in vitro results obtained using cell lines. This aspect should be taken into account in GBM target antigen research.

scholarly journals Tumor Inflammation and Myeloid Derived Suppressor Cells Reduce the Efficacy of CD19 CAR T Cell Therapy in Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2885-2885 ◽  
Author(s):  
Michael D. Jain ◽  
Hua Zhao ◽  
Reginald Atkins ◽  
Meghan A Menges ◽  
Crystal R Pope ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Research Funding ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Introduction: Approximately 60% of Large B cell Lymphoma (LBCL) patients that receive CD19 CAR T cell therapy with axicabtagene ciloleucel (axi-cel) experience lymphoma progression (Locke et al. Lancet Oncol. 2019) and the likelihood of response to subsequent therapy is low (Spiegel, Dahiya et al. ASCO 2019). Target loss of CD19 is observed in less than a third of patients experiencing relapse. Alternative mechanisms of resistance to axi-cel are poorly understood. Lymphoma patients with elevated serum markers of systemic inflammation, such as ferritin and IL-6, have worse outcomes following axi-cel (Locke, Neelapu et al. Mol.Ther.2017; Faramand et al. ASH 2018). We hypothesized that suppressive monocytic myeloid derived suppressor cells (M-MDSCs), which are associated with worse chemotherapy outcomes in LBCL (Azzaoui et al. Blood 2016), and tumor driven inflammation may be present and responsible for decreased efficacy of axi-cel in LBCL. Methods: LBCL patients undergoing axi-cel treatment were enrolled onto prospective sample collection protocols. Patients were stratified for analysis into ongoing responders (complete response or partial response) or relapsed (progressive disease) after a minimum of 3 months follow-up (range 3 - 15 months). M-MDSCs, defined as a Lin-, CD11b+, CD33+, CD15-, CD14+, HLA-DRlow population, were sorted from leftover apheresis material after collection for axi-cel manufacture. M-MDSC ability to suppress proliferation of autologous T cells stimulated with CD3/CD28 coated beads was measured by 3H thymidine incorporation. Circulating peripheral blood M-MDSCs, quantified by % of live cells by flow cytometry, were measured at the time of apheresis and serially after axi-cel infusion until day 30. In vitro mouse experiments utilized a CD19-CD28 CAR and cytokine-induced bone marrow MDSCs (Thevenot et al. Immunity 2014). Cytokines were measured by ELISA and cytotoxicity against CD19 bearing cell lines used xCELLigence real-time cell analysis, as we have done previously (Li et al. JCI Insight 2018).Tumor biopsies were taken within 1 month prior to infusion of axi-cel. Limited gene expression profiling of tumor microenvironment (TME) genes used the Nanostring IO360 panel (770 genes). Analysis used nSolver to identify cell types, GSEA and differential gene expression between groups. Results: First, we demonstrated that M-MDSCs sorted from patient apheresis material suppressed the proliferation of autologous T cells (n=6). We next enumerated M-MDSCs in the peripheral blood (n = 32). M-MDSC numbers initially decreased after lymphodepleting chemotherapy but recovered to baseline levels by day +10. The level of M-MDSCs following CAR T cell therapy strongly correlated with pre-CAR T baseline levels (R = 0.871, p <0.0001), suggesting that the number of M-MDSCs present during CAR T cell expansion is dependent on factors already present before therapy began. M-MDSC levels were significantly higher in patients who subsequently relapsed, both at baseline (p= 0.01) and after axi-cel (p=0.04), as compared to patients with durable response. Mouse MDSCs were able to suppress CAR T cell IFN-gamma excretion (p<0.0001) and cytotoxicity (p<0.0001) in vitro. To evaluate the role of the TME we interrogated limited set gene expression profiling on patient (n=27) pre-axi-cel tumor biopsies. By cell type scoring, the macrophage gene score was significantly higher in patients who relapsed after CAR T therapy (p <0.001). By differential gene expression and gene set enrichment, patients who relapsed had a significantly higher expression (p <0.01) of multiple genes indicative of chronic interferon (IFN) signaling including higher levels of OAS2, OAS3, IFI6 and IFIT1, as well as the IFN-stimulated macrophage gene SIGLEC-1/CD169. Conclusions: Systemic inflammatory myeloid cytokines, circulating M-MDSCs in the blood and chronic IFN in the TME all associate with LBCL relapse after axi-cel CAR T cell therapy. Our observations support that CAR T cells can be suppressed by baseline patient and tumor-related factors and strategies to overcome these factors should be targeted to improve patient outcomes. MDJ and HZ contributed equally. Disclosures Jain: Kite/Gilead: Consultancy. Bachmeier:Kite/Gilead: Speakers Bureau. Chavez:Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Kite Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals, Inc.: Speakers Bureau. Shah:Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding; Kite/Gilead: Honoraria; Celgene/Juno: Honoraria; Pharmacyclics: Honoraria; Adaptive Biotechnologies: Honoraria; Spectrum/Astrotech: Honoraria; Novartis: Honoraria; AstraZeneca: Honoraria. Mullinax:Iovance: Research Funding. Davila:Celgene: Research Funding; GlaxoSmithKline: Consultancy; Precision Biosciences: Consultancy; Novartis: Research Funding; Atara: Research Funding; Bellicum: Consultancy; Adaptive: Consultancy; Anixa: Consultancy. Locke:Kite: Other: Scientific Advisor; Novartis: Other: Scientific Advisor; Cellular BioMedicine Group Inc.: Consultancy.

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

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

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

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

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

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

scholarly journals B-CLL Mediated Resistance to CAR T Cell Therapy Via Insufficient Activation Is CAR-Independent

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 44-44
Author(s):  
McKensie Collins ◽  
Weimin Kong ◽  
Inyoung Jung ◽  
Stefan M Lundh ◽  
J. Joseph Melenhorst
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cytokine Production ◽  
Cell Function ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chronic Lymphocytic Leukemia (CLL) is a B cell malignancy that accounts for nearly 1/3rd of adult leukemia diagnoses in the Western world. Conventional chemo-immunotherapies initially control progression, but in the absence of curative options patients ultimately succumb to their disease. Chimeric Antigen Receptor (CAR) T cell therapy is potentially curative, but only 26% of CLL patients have a complete response. CLL-stimulated T cells have reduced effector functions and B-CLL cells themselves are believed to be immunosuppressive. Our work demonstrates that insufficient activation of CAR T cells by CLL cells mediates some of these effects and that the results are conserved between ROR1- and CD19-targeting CARs. Results: In this study we used an in vitro system to model the in vivo anti-tumor response in which CAR T cells serially engage with CLL cells. Multiple stimulations of CD19 or ROR1-targeting CAR T cells with primary CLL cells recapitulated many aspects of known T cell dysfunction including reduced proliferation, cytokine production, and activation. While the initial stimulation induced low level proliferation, subsequent stimulations failed to elicit additional effector functions. We further found that these functional defects were not permanent, and that CAR T cell function could be restored by switching to a stimulus with an aAPC (artificial Antigen Presenting Cell) control cell line. The aAPCs are well-characterized as potent stimulators of CAR T cell effector responses. Flow cytometry revealed that CLL-stimulated CAR T cells retained a non-activated, baseline differentiation profile, suggesting that CLL cells fail to stimulate CAR T cells rather than rendering them non-functional. One mechanism that could dampen activation is immune suppression. We assessed this at a high level by stimulating CAR T cells with CLL cells and aAPCs mixed at known ratios. However, even cultures containing 75% CLL cells stimulated proliferation and cytokine production. Extensive immune-phenotyping revealed high level expression of the IL-2 Receptor on 90% (18/20) of the B-CLL cells tested. Since cytokine sinking via IL-2 receptor expression is a well-known mechanism of regulatory T cell suppression, we hypothesized that CLL cells similarly sink IL-2, blunting T cell activation. To test this, we supplemented IL-2 into CLL/CAR T cell co-cultures and showed that this rescued proliferation but only partially restored cytokine production. In contrast to our hypothesis, analysis of cytokine production by flow cytometry showed that CLL-stimulated CAR T cells did not produce IL-2 following a 6- or 12-hour stimulus, but TNFα was expressed after 12-hours. Similarly, CAR T cell degranulation, a prerequisite for target cell lysis was triggered after CLL recognition. These data again suggested that CLL cells insufficiently stimulate CAR T cell cytokine production, but also showed that cytolytic activity against CLL cells is intact. We further proposed that CLL cells express insufficient levels of co-stimulatory and adhesion molecules to activate CAR T cells. Flow cytometry showed that most CLL cells expressed co-stimulatory and adhesion molecules at low levels; we hypothesized that up-regulating these molecules would enhance CAR T cell targeting of CLL cells. CLL cells were activated with CD40L and IL-4, which increased expression of CD54, CD58, CD80, and CD86. Stimulating CAR T cells with activated CLL cells enhanced CAR T cell proliferation and induced cell conjugate formation, indicating cell activation. Therefore, improving CLL stimulatory capacity can rescue T cell dysfunctions. To assess whether IL-2 addition and CD40 ligation were synergistic, we combined the two assays; however, we saw no additional improvement over IL-2 addition alone, suggesting that the two interventions may act upon the same pathway. Importantly, we also showed that rescue of CAR T cell function via IL-2 addition or CD40 ligation was not CAR-specific, as we observed the functional defects and subsequent rescue with both a ROR1-targeting CAR and the gold standard CD19-targeting CAR. Conclusions: Together, these data show that CAR T cell "defects" in CLL are actually insufficient activation, and improving the stimulatory capacity of CLL cells may enable better clinical responses. Further, this effect is not CAR-specific and these results may therefore be broadly applicable to multiple therapies for this disease. Disclosures Melenhorst: IASO Biotherapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Novartis: Other: Speaker, Research Funding; Johnson & Johnson: Consultancy, Other: Speaker; Simcere of America: Consultancy; Poseida Therapeutics: Consultancy.

scholarly journals Costs of Postinfusion Monitoring By Site of Care for Patients with Relapsed/Refractory (R/R) Large B-Cell Lymphoma (LBCL) Who Received Third-Line or Later Treatment with Lisocabtagene Maraleucel (liso-cel) in the Transcend NHL 001 and Outreach Trials

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 16-17
Author(s):  
M. Lia Palomba ◽  
Monika P. Jun ◽  
Jacob Garcia ◽  
James Lymp ◽  
November McGarvey ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Therapy ◽  
Research Funding ◽  
Cost Ratio ◽  
Publicly Traded ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T ◽  
Third Line

Background: Chimeric antigen receptor (CAR) T cell therapy is generally limited to inpatient settings; yet, exploration of outpatient infusion and monitoring is ongoing. Information on health care resource utilization (HCRU) and costs associated with CAR T cell therapy administration is limited and may differ by postinfusion monitoring site. Liso-cel is an investigational, CD19-directed, defined composition, 4-1BB CAR T cell product administered at equal target doses of CD8+ and CD4+ CAR+ T cells. An interim analysis from the OUTREACH study (NCT03744676) observed lower HCRU with outpatient vs inpatient administration (Bachier et al. J Clin Oncol 2020;38:8037). The patient journey after CAR T cell therapy administration may differ for patients with outpatient vs inpatient monitoring and may result in varying costs of care. This study estimated the cost of postinfusion monitoring by site of care for patients with R/R LBCL who received third-line or later treatment with liso-cel in the TRANSCEND NHL 001 (TRANSCEND; NCT02631044) and OUTREACH clinical trials. Methods: This retrospective study analyzed HCRU reported in clinical trial databases from TRANSCEND and OUTREACH. A 2-step microcosting method was used to identify key HCRU and to estimate postinfusion costs: (1) HCRU was analyzed from the index date (day of liso-cel infusion) through the 6-month follow-up; and (2) costs were applied to each HCRU. HCRU included standard inpatient and intensive care unit (ICU) length of stay (LOS), diagnostics (laboratory work and imaging), procedures (dialysis and intubation), and medications (supportive care, prophylactic treatment, and adverse event management). Unit costs were obtained from the health care system (provider) perspective and adjusted to 2020 US dollars. Cost per standard inpatient day ($2,542) was estimated from Healthcare Cost and Utilization Project databases, and cost per ICU day ($7,556) was sourced from Dasta et al (Crit Care Med. 2005;33:1266-77). All medication costs were obtained from REDBOOK (IBM Micromedex) using wholesale acquisition costs. Diagnostic and procedure costs were obtained from the Centers for Medicare & Medicaid Services laboratory fee schedule, physician fee schedule, or outpatient prospective payment system. A payment-to-cost ratio was applied to Medicare payment rates to estimate unit costs. Costs were adjusted to reflect the site of care where the HCRU occurred. A cost ratio was applied to adjust costs from the physician's office/community oncology clinic to the hospital outpatient department (Winfield, Muhlestein, Leavitt Partners; 2017) and from outpatient to inpatient (Meisenberg et al. Bone Marrow Transplant. 1998;21:927-32). Costs were aggregated by HCRU category, specifically medications, diagnostics, procedures, and facility costs. An average total cost by post-liso-cel infusion month was calculated for patients with ongoing status in that month (patients censored due to data cutoff were not included). Analyses were stratified by site of postinfusion monitoring (inpatients vs outpatients). Results: A total of 303 patients with R/R LBCL across the 2 trials received liso-cel and postinfusion monitoring (inpatients, n = 256; outpatients, n = 47). HCRU and LOS, including standard inpatient and ICU days, are shown in the Table. Inpatients had higher rates of inpatient stays (&lt;100% vs 62%) and tocilizumab use (for CRS and/or NE; 20% vs 9%) than outpatients, respectively. Rates of ICU admission, corticosteroid use, vasopressor use, dialysis, and intubation were similar between groups. Median and average LOS in standard inpatient and ICU settings were higher among inpatients. Median (range) total LOS for inpatients and outpatients was 15 (0-88) and 4 (0-77) days, respectively. The estimated mean postinfusion cost of care was $89,535 for inpatients and $36,702 for outpatients. Over 6 months, most costs were incurred in the first month after infusion ($50,369 [56%] for inpatients and $19,837 [54%] for outpatients). Costs were largely driven by facility costs, namely standard inpatient and ICU stays (Figure). Conclusions: Lower overall HCRU was observed with outpatient liso-cel postinfusion monitoring, primarily due to hospitalizations, which resulted in a mean 6-month cost savings of $52,833 (59%) compared with inpatient monitoring. These results are based on national average costs and may not be generalizable to specific institutions. Disclosures Palomba: Regeneron: Research Funding; Juno Therapeutics, a Bristol-Meyers Squibb Company: Honoraria, Research Funding; Genentech: Research Funding; Merck: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Pharmacyclics: Honoraria. Jun:Bristol-Myers Squibb Company: Current Employment, Current equity holder in publicly-traded company. Garcia:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. Lymp:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. McGarvey:Pfizer, Inc.: Ended employment in the past 24 months; BluePath Solutions: Current Employment. Gitlin:BMS: Research Funding. Pelletier:BMS: Current Employment, Current equity holder in publicly-traded company. Nguyen:BluePath Solutions: Current Employment.

scholarly journals Qualitative Analysis of the Treatment Experience and Well-Being of Patients with Relapsed/Refractory (R/R) Large B-Cell Lymphoma (LBCL) Enrolled in 2 Trials of Lisocabtagene Maraleucel (liso-cel) during the Initial Stages of Therapy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23
Author(s):  
Tanya Siddiqi ◽  
Ulrich Jaeger ◽  
Olga Moshkovich ◽  
Jennifer Devlen ◽  
Matthew Miera ◽  
...  
Keyword(s):  
Clinical Trials ◽  
T Cell ◽  
Cell Therapy ◽  
Research Funding ◽  
Well Being ◽  
Publicly Traded ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Background: Chimeric antigen receptor (CAR) T cell therapy is a novel treatment modality for patients with R/R LBCL. Limited information exists regarding patients' views of CAR T cell therapy. Our research aimed to better understand patients' needs by capturing their expectations/concerns, current well-being, and treatment experiences during the beginning stages of CAR T cell therapy in the clinical trial setting. Methods: Patients with R/R LBCL from 2 ongoing trials of the investigational, CD19-directed CAR T cell therapy liso-cel (TRANSCEND WORLD [NCT03484702] or PLATFORM [NCT03310619]) were invited to participate in an optional interview component. Semistructured interviews were conducted to gain insight about patients' experience with CAR T cell therapy in the clinical trials. Interviews of ≤1 hour (in-person or over the phone) were conducted in parallel with screening procedures (interview 1), after leukapheresis (interview 2), and up to 3 days after liso-cel infusion (interview 3). Interviews were audio recorded and transcribed. MAXQDA (VERBI GmbH, Berlin, Germany) qualitative analysis software was used to manage and thematically organize interview transcript data to identify key concepts related to each research objective. Previously reported results of interview 1 showed a high perception of unmet needs, lack of alternative options, and expectations for positive outcomes. The analysis presented here primarily focused on interviews 2 and 3. Denominators shown in the Results vary by question as some patients skipped questions. Results: A total of 75 interviews were analyzed, including 35, 24, and 16 patients at interviews 1, 2, and 3, respectively, across sites in the US (n = 14), Europe (n = 26), and Japan (n = 2). Among 42 patients who completed ≥1 interview, the mean age was 62 years and 69% were male. Treatment Experience: Of 24 patients who completed interview 2, 22 (92%) reported positive experiences during leukapheresis and 16 (67%) reported the procedure was as expected. Patients thought the most difficult part of leukapheresis was the length of the procedure (n = 8/21 [38%]). Of 15 patients who provided feedback on lymphodepleting chemotherapy, a majority reported that it was as expected (n = 8 [53%]) or easier than expected (n = 3 [20%]); when asked about the most difficult part, many patients (n = 7/17 [41%]) discussed side effects (eg, nausea, fatigue, and lack of appetite). Of patients who described liso-cel infusion as different than expected, differences included easier (n = 12/13 [92%]) or quicker (n = 3/12 [25%]) than expected, and 5/12 (42%) reported few/no side effects within 3 days post-infusion. Over half of patients (n = 8/14 [57%]) reported that the infusion, as a whole, was not difficult. Changes over Time: At interviews 1, 2, and 3, respectively, 47% (n = 14/30), 47% (n = 9/19), and 69% (n = 9/13) of patients reported hoping for successful treatment. Similarly, patients generally had fewer concerns later in the process, with 21 (64%) and 11 (33%) of 33 patients reporting side-effect and treatment efficacy concerns, respectively, during interview 1 vs 5 (33%) and 3 (20%) of 15 patients, respectively, during interview 3. At time of enrollment, most patients (n = 21/34 [62%]) were able to function normally or with minimal impact from their lymphoma, although most reported some symptoms like fatigue, pain, or stomach problems. At interview 1, 14 (40%) of 35 patients were employed; most patients reported no changes in their work life at interviews 2 (n = 19/20 [95%]) and 3 (n = 11/12 [92%]). From enrollment to immediately post-infusion, the physical health of most patients remained stable (n = 4/16 [25%]) or deteriorated (n = 9/16 [56%]). However, most patients (n = 14/15 [93%]) reported feeling positive at interview 3. Conclusions: This study provided the unique opportunity to gather feedback directly from patients participating in clinical trials of liso-cel therapy, specifically during the initial treatment stages. The overall impression of the treatment was positive, with most patients reporting that study procedures were easier than expected. The results of this qualitative research provide useful insight into the motivations, expectations, and experiences of patients with R/R LBCL receiving liso-cel therapy, which can inform the design of health care support systems and future clinical trials to better meet patients' needs. Disclosures Siddiqi: AstraZeneca: Consultancy, Research Funding, Speakers Bureau; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding; Juno: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Oncternal: Research Funding; TG Therapeutics: Research Funding; Janssen: Speakers Bureau; Seattle Genetics: Speakers Bureau. Jaeger:F. Hoffmann-La Roche: Honoraria, Research Funding; AbbVie: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding; Karyopharm: Honoraria; CDR Life AG: Consultancy, Research Funding; Miltenyi: Consultancy, Honoraria. Moshkovich:Icon Plc: Current Employment. Devlen:Icon Plc: Current Employment, Current equity holder in publicly-traded company. Miera:Icon Plc: Current Employment. Williams:Icon Plc: Current Employment. Hasskarl:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Liu:Bristol-Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Braverman:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Salles:MorphoSys: Consultancy, Honoraria, Other; Kite: Consultancy, Honoraria, Other; Debiopharm: Consultancy; Novartis: Consultancy, Honoraria, Other; Janssen: Consultancy, Honoraria, Other: Participation in educational events; Gilead: Consultancy, Honoraria, Other: Participation in educational events; F. Hoffman-La Roche Ltd: Consultancy, Honoraria, Other; Epizyme: Consultancy; Takeda: Consultancy, Honoraria, Other; Bristol Myers Squibb: Consultancy, Other; Karyopharm: Consultancy; Amgen: Honoraria, Other: Participation in educational events; Celgene: Consultancy, Honoraria, Other: Participation in educational events; Abbvie: Consultancy, Honoraria, Other: Participation in educational events; Autolus: Consultancy; Genmab: Consultancy.

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

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

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

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

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

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

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