scholarly journals 871 Construction and evaluation of interleukin 3 (IL3)-zetakine redirected cytolytic T Cells for the treatment of CD123 expressing acute myeloid leukemia

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A912-A912
Author(s):  
Rebecca Moeller ◽  
Julian Scherer ◽  
Sadik Kassim
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Jurkat Cells ◽  
Leukemic Stem Cells ◽  
Cd69 Expression ◽  
Acute Myeloid

BackgroundAcute Myeloid Leukemia (AML) is an aggressive bone marrow malignancy, characterized by the presence of leukemic blasts in the peripheral blood of patients. Poor AML prognoses1 are largely attributable to high rates of disease relapse, of which CD123+ leukemic stem cells (LSCs) are the primary cause.2 3 CD123, the alpha-chain of the IL3 cytokine receptor,6 has been identified as a favorable therapeutic AML target, overexpressed in both LSCs and blasts.4 5 We sought to direct T cells to CD123+ AML cells via cell surface tethered IL3 (termed ”IL3-zetakine”).7 The use of a zetakine instead of a chimeric antigen receptor (CAR) construct enables structure-guided site-directed mutagenesis to increase binding affinity and alter target cell signaling without detrimental T cell hyperactivation.MethodsZetakine constructs were designed using IL3 sequences bound to a transmembrane domain and intracellular costimulatory and CD3z signaling domains. The constructs were transduced into Jurkat cells with lentiviral vectors (LVV). T cell activation via CD69 expression was assessed via flow cytometry of sorted IL3 zetakine-positive Jurkat cells after co-culture with MOLM13 AML cells. Lead constructs were selected based on initial transduction percentage and activation response. In vitro functionality of each IL3 zetakine was tested with LVV transduced primary T cells by flow cytometry.ResultsZetakine constructs yielded a wide range of transduction percentages in Jurkat cells (0 – 98%) prior to sorting. In co-cultures with CD123+ MOLM13 AML cells, Jurkat cells expressing wildtype IL3 constructs lacking a costimulatory domain induced the highest level of CD69 expression (18.7% CD69+ T cells) in an antigen-specific manner (5.3-fold increase of CD69+ T cells over those cultured with MOLM13 CD123KO cells). The K110E mutant IL3 was reported to exhibit a 40-fold increased affinity over wildtype,8 but it showed no detectable zetakine function. However, additional mutant IL3 zetakines increased Jurkat cell activation up to 5.8-fold. Antigen-specific increases in CD69, as well as CD25, surface expression were also observed with zetakine-transduced primary T cells co-cultured with MOLM13 cells, in addition to target cell killing comparable to antibody-based CD123CAR T-cells.ConclusionsThis work establishes IL3 zetakines as a viable alternative to traditional CD123-targeted CAR constructs. Structure-guided IL3 zetakine mutants with altered affinity and activation profiles will further our understanding of CD123-specific cytotoxicity modulation without inducing acute T cell hyperactivation and exhaustion. These results indicate the ability of IL3 zetakine-expressing T cells to kill CD123-expressing AML cells and illustrate the potential of this novel class of therapeutics.ReferencesGanzel C, et al. Very poor long-term survival in past and more recent studies for relapsed AML patients: the ECOG-ACRIN experience. American journal of hematology 2018:10.1002/ajh.25162.Shlush LI, et al. Tracing the origins of relapse in acute myeloid leukaemia to stem cells. Nature 2017;547(7661):104–108.Hanekamp D, Cloos J, Schuurhuis GJ. Leukemic stem cells: identification and clinical application. International Journal of Hematology 2017;105(5):549–557.Bras AE, et al. CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping. Cytometry part B: Clinical Cytometry 2019;96(2):134–142.Sugita M, Guzman ML. CD123 as a therapeutic target against malignant stem cells. Hematology/Oncology clinics of North America 2020;34(3):553–564.Mingyue S, et al. CD123: a novel biomarker for diagnosis and treatment of leukemia. Cardiovascular & Hematological Disorders-Drug Targets 2019;19(3):195–204.Kahlon KS, et al. Specific recognition and killing of glioblastoma multiforme by interleukin 13-zetakine redirected cytolytic T cells. Cancer Res 2004;64(24):9160–6.Bagley CJ, et al. A discontinuous eight-amino acid epitope in human interleukin-3 binds the alpha-chain of its receptor. J Biol Chem 1996;271(50):31922–8.

CD8 T-cell responses to Wilms tumor gene product WT1 and proteinase 3 in patients with acute myeloid leukemia

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2132-2137 ◽  
Author(s):  
Carmen Scheibenbogen ◽  
Anne Letsch ◽  
Eckhard Thiel ◽  
Alexander Schmittel ◽  
Volker Mailaender ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Wilms Tumor ◽  
Proteinase 3 ◽  
Ifn Γ ◽  
Tumor Gene ◽  
Acute Myeloid

Abstract Wilms tumor gene product WT1 and proteinase 3 are overexpressed antigens in acute myeloid leukemia (AML), against which cytotoxic T lymphocytes can be elicited in vitro and in murine models. We performed this study to investigate whether WT1- and proteinase 3-specific CD8 T cells spontaneously occur in AML patients. T cells recognizing HLA-A2.1-binding epitopes from WT1 or proteinase 3 could be detected ex vivo in 5 of 15 HLA-A2–positive AML patients by interferon-γ (IFN-γ) ELISPOT assay and flow cytometry for intracellular IFN-γ and in 3 additional patients by flow cytometry only. T cells producing IFN-γ in response to proteinase 3 were further characterized in one patient by 4-color flow cytometry, identifying them as CD3+CD8+CD45RA+ CCR7−T cells, resembling cytotoxic effector T cells. In line with this phenotype, most of the WT1- and proteinase-reactive T cells were granzyme B+. These results provide for the first time evidence for spontaneous T-cell reactivity against defined antigens in AML patients. These data therefore support the immunogenicity of WT1 and proteinase 3 in acute leukemia patients and the potential usefulness of these antigens for leukemia vaccines.

scholarly journals Characterization of the DNAM-1, TIGIT and TACTILE Axis on Circulating NK, NKT-Like and T Cell Subsets in Patients with Acute Myeloid Leukemia

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2171
Author(s):  
Isabel Valhondo ◽  
Fakhri Hassouneh ◽  
Nelson Lopez-Sejas ◽  
Alejandra Pera ◽  
Beatriz Sanchez-Correa ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Healthy Volunteers ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Nk Cell ◽  
T Cell Subsets ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) remains a major clinical challenge due to poor overall survival, which is even more dramatic in elderly patients. TIGIT, an inhibitory receptor that interacts with CD155 and CD112 molecules, is considered as a checkpoint in T and NK cell activation. This receptor shares ligands with the co-stimulatory receptor DNAM-1 and with TACTILE. The aim of this work was to analyze the expression of DNAM-1, TIGIT and TACTILE in NK cells and T cell subsets in AML patients. Methods: We have studied 36 patients at the time of diagnosis of AML and 20 healthy volunteers. The expression of DNAM-1, TIGIT and TACTILE in NK cells and T cells, according to the expression of CD3 and CD56, was performed by flow cytometry. Results: NK cells, CD56− T cells and CD56+ T (NKT-like) cells from AML patients presented a reduced expression of DNAM-1 compared with healthy volunteers. An increased expression of TIGIT was observed in mainstream CD56− T cells. No differences were observed in the expression of TACTILE. Simplified presentation of incredibly complex evaluations (SPICE) analysis of the co-expression of DNAM-1, TIGIT and TACTILE showed an increase in NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE. Low percentages of DNAM-1−TIGIT+TACTILE+ NK cells and DNAM-1− TIGIT+TACTILE+ CD56− T cells were associated with a better survival of AML patients. Conclusions: The expression of DNAM-1 is reduced in NK cells and in CD4+ and CD8+ T cells from AML patients compared with those from healthy volunteers. An increased percentage of NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE is associated with patient survival, supporting the role of TIGIT as a novel candidate for checkpoint blockade.

scholarly journals L4 Synthetic agonistic receptor-activating BiTEs – a modular platform for the efficient targeting of acute myeloid leukemia

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A2.2-A3
Author(s):  
M Benmebarek ◽  
BL Cadilha ◽  
M Hermann ◽  
S Lesch ◽  
C Augsburger ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
T Cell Activation ◽  
Tumour Cell ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Cell Lysis ◽  
Target Cells ◽  
Acute Myeloid

BackgroundTargeted immunotherapies have shown limited success in the context of acute myeloid leukemia (AML). Due to the mutational landscape and heterogeneity attributed to this malignancy and toxicities associated with the targeting of myeloid lineage antigens, it has become apparent that a modular and controllable cell therapy approach with the potential to target multiple antigens is required. We propose a controlled ACT approach, where T cells are armed with synthetic agonistic receptors (SARs) that are conditionally activated only in the presence of a target AML-associated antigen, and a cross-linking bispecific T cell engager (BiTE) specific for both (SAR) T cell and tumour cell.Materials and MethodsA SAR composed of an extracellular EGFRvIII, trans-membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap-extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. SAR-specific bispecific T cell engagers (BiTE) that target AML-associated antigens were designed and expressed in Expi293FTMcells and purified by nickel affinity and size exclusion chromatography (SEC). We validated our approach in three human cancer models and patient-derived AML blasts expressing our AML-associated target antigen CD33.ResultsCD33-EGFRvIII BiTE, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation and differentiation of SAR-T cells. Further, SAR T cells bridged to their target cells by BiTE could form functional immunological synapses, resulting in efficient tumor cell lysis with specificity towards CD33-expressing AML cells. SAR.BiTE combination could also mediate specific cytotoxicity against patient-derived AML blasts whilst driving SAR T cell activation. In vivo, treatment with SAR.BiTE combination could efficiently eradicate leukemia and enhance survival in an AML xenograft model. Furthermore, we could show selective activation of SAR T cells, as well as a controllable reversibility of said activation upon depletion of the T cell engaging molecule.ConclusionsHere we apply the SAR x BiAb approach in efforts to deliver specific and conditional activation of agonistic receptor-transduced T cells, and targeted tumour cell lysis. The modularity of our platform will allow for a multi-targeting ACT approach with the potential to translate the ACT successes of B cell malignancies to AML. With a lack of truly specific AML antigens, it is invaluable that this approach possesses an intrinsic safety switch via its BiTE facet. Moreover, we are able to circumvent pan-T cell activation due to the specific targeting and activation of SAR T cells.Disclosure InformationM. Benmebarek: None. B.L. Cadilha: None. M. Hermann: None. S. Lesch: None. C. Augsburger: None. B. Brauchle: None. S. Stoiber: None. A. Darwich: None. F. Rataj: None. C. Klein: A. Employment (full or part-time); Significant; Roche. K. Hopfner: None. M. Subklewe: None. S. Endres: None. S. Kobold: None.

The Genotype of T-Cell Costimulatory Molecule Is Associated with Relapse Incidence In Patients with Acute Myeloid Leukemia After Allogeneic Hematopoietic Cell Transplantation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 684-684
Author(s):  
Haowen Xiao ◽  
Xiaoyu Lai ◽  
Yi Luo ◽  
Jimin Shi ◽  
Yamin Tan ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Costimulatory Molecule ◽  
Leukemia Relapse ◽  
The Media ◽  
Risk Of Relapse ◽  
Acute Myeloid

Abstract Abstract 684 Background: Disease relapse is one of the leading causes of death following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Donor T lymphocytes play a critical role in alloimmune recognition and their ability to detect non-self-antigens can lead to graft-versus-host disease (GVHD) or contribute to relapse prevention through recognition and elimination of minimal residual disease. CD28 is the primary T-cell costimulatory molecule constitutively expressed on the majority of T cells. Upon interaction with its ligands B7, CD28 transduces a signal that enhances the activation and proliferation of T cells. Another member of the CD28 family is inducible co-stimulator (ICOS). Although not constitutively expressed, ICOS is rapidly upregulated on T lymphocytes upon activation. Cytotoxic T-lymphocyte antigen 4 (CTLA-4), a homologous molecule of CD28, is a key factor in regulating and maintaining self-tolerance, providing a negative signal to T cells. Although several single-nucleotide polymorphisms (SNPs) within those costimulatory molecule genes have been identified to be associated with the risk of autoimmune disease, their association with outcome after allo-HSCT has yet to be explored. This present study was designed to investigate the influence of CD28, ICOS and CTLA-4 genes polymorphisms on the outcome of patients with acute myeloid leukemia (AML) after allo-HSCT. Methods: The entire study population consisted of 86 consecutive AML patients and their donors who were transplanted from 2001 to 2009 in our Unit. Genomic DNA was extracted from peripheral blood samples obtained from recipients and donors before transplantation. Five SNPs of CD28 -594(A/G), ICOS -693(G/A) and CTLA-4 -1722(A/G), +49(A/G) and CT60(A/G) were analyzed. Results: (1) The media age of the patients was 27 years (range, 12–49 years). At the time of transplantation, 70 patients were in first complete remission (CR), 10 patients in second CR, 3 patients in third CR and 3 patients in progressive status. 33 patients underwent HLA-matched sibling HSCT and 53 patients underwent unrelated HSCT. All patients received myeloablative conditioning based on busulfan/cyclophosphamide (BuCy) without total body irradiation (TBI). The GVHD prophylaxis is consisted of cyclosporin A, a short-term methotrexate and mycophenolate mofetil. (2) Only one patient experienced engraftment failure. 18(20.9%), 20(23.3%) and 3(3.5%) patients respectively developed grade I, II and severe acute GVHD (aGVHD). 16(18.6%) and 15(17.4%) patients developed limited and extensive chronic GVHD (cGVHD). (3) 17 patients (19.8%) experienced relapse, the media time was 15.6 months after allo-HSCT (range, 2–24 months). (4) Patients receiving stem cells from a donor with AA genotype in position +49 or CT60 of CTLA-4 gene relapsed more frequently than those with AG/GG genotypes (for +49: 50% vs 14.1%, P=0.012; for CT60: 80% vs 16%, P<0.0001). (5) Patients receiving stem cells from a donor with CD28 -594 AA genotype had a lower incidence of relapse than those with other genotypes (0 vs 25%, P=0.04), due to a higher incidence of aGVHD (83.3% vs 38.2%, P=0.004). (6) In multivariate analysis, donor with CTLA-4 CT60 AA genotype (RR=13.411, 95%CI: 3.808–47.233, P<0.0001), and absence of cGVHD (RR=2.12, 95%CI: 1.112–4.042, P=0.022) were found to significantly contribute to the risk of relapse after allo-HSCT. Furthermore, patients receiving CTLA-4 CT60 AA genotype donor had worse overall survival at 7 years (20% vs 77.8%, RR=10, 95%CI: 3.761–16.239, P<0.0001). The polymorphic sites CTLA-4 -1722 and ICOS -693 did not correlate with the risk of relapse. Nor did patient 5 polymorphic sites genotype. Conclusions: These results, which is the first report of T-cell costimulatory molecule genes polymorphic features with the risk of leukemia relapse in AML patients after allo-HSCT, suggest an interaction between donor CTLA-4 CT60 AA genotype and the risk of relapse. The CTLA-4 CT60 AA genotype has been reported to increase the production of soluble form of CTLA-4, suggesting that increased expression of CTLA-4 would be associated with a decrease ability of the immune system to detect and eliminate tumor associated antigens. According to our results, anti-CTLA-4 antibodies may be a promising therapeutic strategy in leukemia relapse after allo-HSCT at least in patients receiving CTLA-4 CT60 AA genotype donor. Disclosures: No relevant conflicts of interest to declare.

Conditional T-Cell Activation for Tumor Under Hypoxia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3906-3906 ◽  
Author(s):  
Sonny O Ang ◽  
Simon Olivares ◽  
Elizabeth J. Shpall ◽  
Dean A. Lee ◽  
Richard Champlin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
T Cell Activation ◽  
Transgene Expression ◽  
Cell Activation ◽  
Oxygen Sensitive ◽  
Hypoxic Tumor

Abstract Solid tumors are characterized by cell clusters experiencing chronic and intermittent hypoxia due to haphazard growth. Importantly, highly migratory tumorigenic stem cells are localized within hypoxic niches, shielded by hypoxia-induced metabolites that inhibit tumor-infiltrating T cells, restricting their survival, cytotoxicity, and beneficial immunomodulatory cytokine responses. To eliminate bulky solid tumors including stem cells, T cells must overcome physiological changes distinctive for the tumor microenvironment, such as hypoxia, depleted nutrient levels, and low extracellular pH. To exploit the hypoxic tumor microenivornment, we introduce a new approach that exploits hypoxia as a condition for T-cell activation. We demonstrate a chimeric antigen receptor (CAR), which is specific for CD19 and capable of activating T cells through chimeric CD28 and/or CD3-zeta signaling endodomain, that can be conditionally expresed in a strictly oxygen-sensitive manner. Using the Sleeping Beauty transposon/transposase system, we can achieve stable, persistent CAR transgene expression without the expense and complexity of retroviral production. Cell surface expression of our CAR is high at 1% O and not detectable at 20% O (Figure). Anticipating the circulatory nature of T cells in vivo, our oxygen-sensitive CAR is designed to deactivate upon exiting from hypoxic tumor microenvironment to minimize deleterious off-target effects. When supplemented with one or more survival factors and/or homing receptors, this approach to CAR expression has the advantage of transforming hypoxia from an adverse factor to a triggering mechanism, efficiency, and high levels of transgene expression. Figure: Flow cytometry expression of CAR under normoxia and hypoxia. Figure:. Flow cytometry expression of CAR under normoxia and hypoxia.

scholarly journals Cyclin-A1 represents a new immunogenic targetable antigen expressed in acute myeloid leukemia stem cells with characteristics of a cancer-testis antigen

Blood ◽  
2012 ◽  
Vol 119 (23) ◽  
pp. 5492-5501 ◽  
Author(s):  
Sebastian Ochsenreither ◽  
Ravindra Majeti ◽  
Thomas Schmitt ◽  
Derek Stirewalt ◽  
Ulrich Keilholz ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Myeloid Leukemia ◽  
High Expression ◽  
Target Cells ◽  
Cyclin A1 ◽  
Peripheral Tissues ◽  
Acute Myeloid

Abstract Targeted T-cell therapy is a potentially less toxic strategy than allogeneic stem cell transplantation for providing a cytotoxic antileukemic response to eliminate leukemic stem cells (LSCs) in acute myeloid leukemia (AML). However, this strategy requires identification of leukemia-associated antigens that are immunogenic and exhibit selective high expression in AML LSCs. Using microarray expression analysis of LSCs, hematopoietic cell subpopulations, and peripheral tissues to screen for candidate antigens, cyclin-A1 was identified as a candidate gene. Cyclin-A1 promotes cell proliferation and survival, has been shown to be leukemogenic in mice, is detected in LSCs of more than 50% of AML patients, and is minimally expressed in normal tissues with exception of testis. Using dendritic cells pulsed with a cyclin-A1 peptide library, we generated T cells against several cyclin-A1 oligopeptides. Two HLA A*0201-restricted epitopes were further characterized, and specific CD8 T-cell clones recognized both peptide-pulsed target cells and the HLA A*0201-positive AML line THP-1, which expresses cyclin-A1. Furthermore, cyclin-A1–specific CD8 T cells lysed primary AML cells. Thus, cyclin-A1 is the first prototypic leukemia-testis-antigen to be expressed in AML LSCs. The pro-oncogenic activity, high expression levels, and multitude of immunogenic epitopes make it a viable target for pursuing T cell–based therapy approaches.

Targeting of Acute Myeloid Leukemia by Cytokine-Induced Killer Cells Redirected with a Novel CD123-Specific Chimeric Antigen Receptor.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3010-3010 ◽  
Author(s):  
Sarah Tettamanti ◽  
Virna Marin ◽  
Irene Pizzitola ◽  
Chiara Francesca Magnani ◽  
Greta Maria Paola Giordano Attianese ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Cytotoxicity Assay ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Cik Cells ◽  
Acute Myeloid

Abstract Abstract 3010 Current therapeutic regimens for Acute Myeloid Leukemia (AML) are still associated with high rates of relapse. In the last years, great interest has been focused on the identification of surface molecules that are preferentially expressed by AML cells and leukemic stem cells (LSCs), in order to selectively target the tumor population, whilst sparing the normal counterpart of hematopoietic stem/progenitor cells (HSPC), and possibly impeding disease recurrence. Immunotherapy with T-cells genetically modified to express chimeric antigen receptors (CARs) represents a valid and innovative cell therapy approach for hematological malignancies. In this study we developed a new CAR molecule specific for the IL-3Rα (CD123) target antigen, which is overexpressed on AML blasts, CD34+ leukemic progenitors, and leukemic stem cells (AML-LSCs) compared to normal hematopoietic stem/progenitor cells (HSPCs), and whose overexpression is associated with poor prognosis. Cytokine Induced Killer (CIK) cells, ex-vivo expanded T cells with spontaneous antitumoral activity, were transduced with an SFG-retroviral vector encoding an anti-CD123.CAR and CAR functionality has been evaluated by short-term cytotoxicity assay. Transduced CIK cells strongly killed CD123+ THP-1 cell line (60%±5.4%, Effector:Target –E:T- ratio of 5:1, n=3), as well as primary AML blasts (59%±5.4%, E:T ratio of 3:1, n=4). With the aim to better characterize the ability of anti-CD123.CAR+CIK cells to kill leukaemia cells over time we performed long-term cytotoxicity assay, observing a leukemic cell recovery for THP-1 of 3.5%±1.5% (n=5) and for primary AML cells of 2.4%±1.4% (n=3) when co-cultured with CIK cells expressing anti-CD123.CAR, compared to an average target survival of up to 80%, when co-cultured with unmanipulated (NT) CIK cells. Interestingly, secondary colonies experiments after co-culture of healthy donor cord blood-derived HSPCs (Lin-) with anti-CD123.CAR+CIK cells demonstrated that this newly generated CAR molecule better preserved the normal haematopoietic reconstitution in contrast to a previously generated anti-CD33.CAR (total number of colonies of 146.8±6.6, 66.4±5.1, 117.6±4.6, for Lin- cells co-cultured with NT CIK cells, anti-CD33.CAR+CIK cells, anti-CD123.CAR+CIK cells respectively, n=4), while keeping identical cytotoxicity profile towards AML. Furthermore, a limited killing of normal CD123 expressing monocytes and CD123-low expressing endothelial cells was measured, accompanied by a lesser release of stimulatory cytokines such as IFN-gamma, TNF-alfa and TNF-beta when compared to the levels released after stimulation with CD123+ leukemic cells (THP-1 and AML), thus indicating a low toxicity profile of the anti-CD123.CAR. Taken together, our results indicate that CD123-specific CAR strongly enhances anti-leukemic CIK functions towards AML, while sparing HSPCs and normal CD123-expressing tissues, paving the way for the development of novel immunotherapy approaches for the treatment of resistant forms of AML, particularly for a precocious intervention in presence of minimal residual disease, in the context of early relapse after HSCT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Leukemic Stem Cells Persistence Measured By Multiparametric Flow Cytometry Is a Biomarker of Poor Prognosis in Adult Patients with Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2688-2688
Author(s):  
Francesco Buccisano ◽  
Raffaele Palmieri ◽  
Maria Irno Consalvo ◽  
Alfonso Piciocchi ◽  
Luca Maurillo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Poor Prognosis ◽  
Leukemic Stem Cells ◽  
Advisory Committees ◽  
Multiparametric Flow Cytometry ◽  
Significant Difference ◽  
Acute Myeloid

Introduction: Despite the recent advances in chemotherapy regimens, relapse still substantially affects prognosis of intensively treated adult acute myeloid leukemia (AML) patients. There is growing evidence that a residual populations of leukemic cells may survive chemotherapy and outgrow, eventually causing relapse. These chemo-resistant cells are particularly abundant in the fraction of leukemic stem cells (LSC), which are endowed with pronounced self-renewal properties allowing to initiate and maintain leukemic clone. These cells can be detected, by high sensitivity multiparametric flow-cytometry (MFC), in the CD34+/CD38- fraction of the leukemic populations and can be distinguished from normal hematopoietic stem cells by the expression of specific markers. In recent clinical trials, LSC have been demonstrated to represent a biomarker of poor prognosis when detected at diagnosis but also during treatment course. Moreover, the combined estimate of measurable residual disease (MRD) and LSC refines the prognostic assessment as determined by the sole application of MRD detection. Aim: We analyzed a series of patients (pts) treated in the context of GIMEMA trials, in whom the LSC frequency was assessed by MFC at diagnosis. Pts with measurable levels of LSC were tested again after the consolidation cycle. At the same timepoint "standard" MRD was also determined. The purpose of the study was to demonstrate a correlation between LSC burden at baseline and prognosis in terms of overall (OS) and disease-free survival (DFS). Furthermore, we wanted to investigate the relationship between LSC and "standard" MRD persistence (>0.035%) after consolidation, and possible correlation with outcome. Methods: LSC were evaluated by MFC as described elsewhere (Terwijn, PLoS 2014). LSC were quantified exploiting the expression of the C-type lectin-like molecule-1 (CLL1) and applying a sequential gating strategy that contained the CD34+/CD38- population. Pts were defined as LSC negative (LSCneg) in case of zero LSC count, LSClow or LSChigh when LSC were >0<0,03% or >0.03%, respectively. After consolidation, any level >0 was considered as a LSC persistence. Methods of analysis and thresholds were set according to previous publications (Zeijlemaker, Leukemia 2019). Results: We analyzed 130 pts with de novo AML, in whom LSC determination was available at the baseline. Fifty-nine (45,4%) pts were LSCneg, 49 (37,7%) LSClow, 22 (16,9%) LSChigh. We did not observe any correlation between baseline LSC level and genetic/cytogenetic risk at diagnosis. There was not a significant difference in terms of OS duration according to the 3 LSC levels, however, pts who were LSChigh had the shortest OS (36-month estimate OS of 71.5% vs. 65.4 % vs 52.4 % for the LSCneg, LSClow and LSChigh categories respectively; p=0.21). A statistically significant difference, regardless of the belonging to the LSClow or LSChigh category was observed when we focus on the subgroup of 30 pts with intermediate-risk AML, with a 36-month estimate OS of 76% vs. 77.8% vs 25% for the LSCneg, LSClowand LSChigh categories respectively (p=0.023) (Figure 1A). In 19 patients, LSC persistence was assessed at the post-consolidation time-point. Nine LSChigh pts who failed to eradicate residual LSC at this timepoint had a worse outcome as compared to those belonging to the same category but achieving a LSC clearance or those who were LSClow (36-month OS of 62.5% vs. 59.2% vs. 66.7% vs. 25% for the LSClow converted into LSCneg, LSClow not converted into LSCneg, LSChigh converted into LSCneg and LSChigh not converted into LSCneg categories, respectively; P=0.062) (Figure 1B). In 27 pts LSC and "standard" MRD determination was available. LSC persistence determined a worse 3-years OS both in MRD negative (66.7% vs 85.7%, p=0.44) and MRD positive pts (<20% vs 75.0%, p=0.041). Conclusions: In line with the experience of other European groups, we demonstrated that MFC monitoring of LSC is feasible and provides prognostic information when performed at diagnosis and during treatment course. MFC assessment of LSC also offers the opportunity to monitor pts who lack aberrant phenotypes suitable for "standard" MRD investigation. When the 2 approaches - standard "MRD" and LSC assessment - are combined together, the prognosis prediction of AML can be further refined. Finally, LSC assessment can potentially represent an effective tool to monitor the effect of LSC targeting agents. Disclosures Buccisano: Astellas: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Venditti:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy.

scholarly journals A modular and controllable T cell therapy platform for acute myeloid leukemia

Leukemia ◽  
2021 ◽  
Author(s):  
Mohamed-Reda Benmebarek ◽  
Bruno L. Cadilha ◽  
Monika Herrmann ◽  
Stefanie Lesch ◽  
Saskia Schmitt ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Cell Therapy ◽  
Myeloid Leukemia ◽  
Tumor Antigens ◽  
Adoptive T Cell Therapy ◽  
Single Chain ◽  
T Cell Therapy ◽  
Acute Myeloid

AbstractTargeted T cell therapy is highly effective in disease settings where tumor antigens are uniformly expressed on malignant cells and where off-tumor on-target-associated toxicity is manageable. Although acute myeloid leukemia (AML) has in principle been shown to be a T cell-sensitive disease by the graft-versus-leukemia activity of allogeneic stem cell transplantation, T cell therapy has so far failed in this setting. This is largely due to the lack of target structures both sufficiently selective and uniformly expressed on AML, causing unacceptable myeloid cell toxicity. To address this, we developed a modular and controllable MHC-unrestricted adoptive T cell therapy platform tailored to AML. This platform combines synthetic agonistic receptor (SAR) -transduced T cells with AML-targeting tandem single chain variable fragment (scFv) constructs. Construct exchange allows SAR T cells to be redirected toward alternative targets, a process enabled by the short half-life and controllability of these antibody fragments. Combining SAR-transduced T cells with the scFv constructs resulted in selective killing of CD33+ and CD123+ AML cell lines, as well as of patient-derived AML blasts. Durable responses and persistence of SAR-transduced T cells could also be demonstrated in AML xenograft models. Together these results warrant further translation of this novel platform for AML treatment.

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