scholarly journals Selective recruitment of γδ T cells by a bispecific antibody for the treatment of acute myeloid leukemia

Leukemia ◽  
2021 ◽  
Author(s):  
Rajkumar Ganesan ◽  
Vijaykumar Chennupati ◽  
Balaji Ramachandran ◽  
Michael Riis Hansen ◽  
Sanjaya Singh ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Tumor Cells ◽  
Myeloid Leukemia ◽  
Bispecific Antibody ◽  
Γδ T Cells ◽  
Severe Toxicity ◽  
Γδ T Cell ◽  
Acute Myeloid

AbstractDespite significant progress over the last few decades in the treatment of acute myeloid leukemia (AML), there still remains a major unmet medical need for this disease. Immunotherapy approaches for redirecting pan CD3+ T cells to target leukemia blasts have shown limited efficacy in clinical trials and often accompanied with severe toxicity in AML patients. We designed an alternative engager molecule (Anti-TRGV9/anti-CD123), a bispecific antibody that can simultaneously bind to the Vγ9 chain of the Vγ9Vδ2+ γδ T cell receptor and to AML target antigen, CD123, to selectively recruit Vγ9+ γδ T cells rather than pan T cells to target AML blasts. Our results suggest that prototypic bispecific antibodies (a) selectively activate Vγ9+ γδ T cells as judged by CD69 and CD25 surface expression, and intracellular Granzyme B expression, (b) selectively recruit Vγ9+ γδ T cells into cell–cell conjugate formation of γδ T cells with tumor cells indicating selective and effective engagement of effector and target tumor cells, and (c) mediate γδ T cell cytotoxicity (in vitro and in vivo) against tumor antigen-expressing cells. Collectively, these findings suggest that selectively redirecting Vγ9+ γδ T cells to target AML blasts has a potential for immunotherapy for AML patients and favors further exploration of this concept.

scholarly journals Identification and Isolation of Tumor-Specific Cytotoxic T Lymphocytes in Acute Myeloid Leukemia Patients through the Identification of T-Cells Capable to Establish Stable Interactions with Leukemic Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3336-3336
Author(s):  
Estefania Garcia-Guerrero ◽  
Luis I. Sanchez-Abarca ◽  
Esther Domingo ◽  
Teresa Ramos ◽  
Jose Antonio Bejarano-García ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
T Lymphocytes ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Target Cells ◽  
Acute Myeloid

Abstract Introduction Autologous adoptive T cell therapies, based on the use of tumor infiltrating lymphocytes (TILs), have made great progress in recent years for the treatment of solid tumors, especially melanoma. However, further work is needed to isolate tumor-reactive T cells among patients diagnosed with hematologic malignancies. The dynamics of the interaction between T cells and antigen presenting cells (APC) dictate the quality of the immune responses. While stable joints between target cells and T lymphocytes lead to the induction of T cell activation and immune response, brief contacts contribute to the induction of immune-tolerance. Taking advantage of the strong interaction between target cell and activated T-cells, we show the feasibility to identify and isolate tumor-specific cytotoxic T lymphocytes (CTLs) from acute myeloid leukemia (AML) patients. Using this approach, CTLs stably bound through T cell receptor to tumor cells (doublet forming T-cells) can be identified in peripheral blood and bone marrow and subsequently selected and isolated by FACS-based cell sorting. Methods Co-cultures between PBMC from AML patients in complete remission and AML tumor cells (PKH-stained) from the same patient were performed to study the percentage of doublet-forming T cells (CD3+PKH+) (T cell bound to a tumor cell). After 15 hours of co-culture, cells were stained and sorted. Secondary co-cultures with autologous tumor cells (used in primary co-culture) were performed to study the cytotoxic activity and cytokine production of T-cells capable or not to form stable joints with the leukemic cells (doublet population vs non-doublet population). Results Doublet-forming T cells from AML patients were identified in a range of 2% to 6% (mean=3.83%, n=5). Immunophenotyping analysis showed differences between doublet-forming T cells (CD3+PKH+) and those T cells which did not form stable and strong interactions with target cells (CD3+PKH-). Doublet T cells displayed a higher percentage of CD8+ T cells and higher percentage of effector CD4+ and CD8+ T cells compared to non-doublet T cells. Next, we explored, among effector CD4+ and CD8+ cells, those with cytotoxic phenotype. As expected, a high percentage of effector CD8+ doublet T cells showed Granzyme B and perforin expression, thus corresponding with a cytotoxic immune-phenotype (n=3, mean 65.51%). Within effector CD4+ doublet T cells, a mean of 9.053 % showed expression of both Granzyme B and perforin corresponding with CD4+ CTL (n=3). Regarding CD57 and CD16 markers, a mean of 18.62% of effector CD4+ doublet T cells were positive for both markers, compared to 65.84% of effector CD8+ doublet T cells (n=3). Further, we performed secondary co-cultures to analyze the CD69 activation marker after 24h of co-culture. A high percentage of CD69+ cells was observed in co-cultures with doublet-forming T cells against target cells as compared to non-doublet T cells (n=3, p=0.0053). Finally, analysis of supernatants of co-culture of doublet T cells and non-doublet T cells with target cells revealed specific secretion of IFNγ and IL-2 (n=3, p=0.0001; p=0.0005, respectively). The cytolytic activity was evaluated comparing the viability of tumor cells cultured alone or with doublet-forming T cells or non-doublet T cells from the same patient. A significant increase of the specific lysis of AML cells was observed when doublet T cells were co-cultured as compared to non-doublet T cells (p=0.0424, n=5). This encouraged us to examine whether we were able to identify doublet-forming T cells from bone marrow of AML patients at diagnosis. Analyses of bone marrow by flow cytometry reveled a small percentage of CD3+CD34+ population corresponding with bone marrow-doublet-forming T cells (n=3, mean=2.9%). Interestingly, bone marrow-doublet-forming T cells show a higher percentage of CD4+ T cells, whereas bone marrow-non-doublet T cells show a higher percentage of CD8+ T cells. Conclusions Our data demonstrate that when T cells from AML patients are co-cultured with tumor cells, a "doublet T cell" population appears. This population consists of T cells capable to bind tumor cells. These CTLs display higher percentage of effector cells and a marked cytotoxic activity against AML blasts. In conclusion, we have developed a new procedure to identify and select specific cytotoxic T cells in both bone marrow and peripheral blood from patients diagnosed with acute myeloid leukemia. Figure. Figure. Disclosures Sanchez-Abarca: Virgen del Rocio University Hospital: Patents & Royalties. Ramos:Takeda Oncology: Research Funding.

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.

Stimulation of autologous proliferative and cytotoxic T-cell responses by “leukemic dendritic cells” derived from blast cells in acute myeloid leukemia

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2764-2771 ◽  
Author(s):  
Beth D. Harrison ◽  
Julie A. Adams ◽  
Mark Briggs ◽  
Michelle L. Brereton ◽  
John A. Liu Yin
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Dendritic Cells ◽  
T Cell ◽  
Myeloid Leukemia ◽  
T Cell Responses ◽  
Cell Responses ◽  
Antigen Presenting ◽  
Acute Myeloid ◽  
Stimulation Of

Abstract Effective presentation of tumor antigens is fundamental to strategies aimed at enrolling the immune system in eradication of residual disease after conventional treatments. Myeloid malignancies provide a unique opportunity to derive dendritic cells (DCs), functioning antigen-presenting cells, from the malignant cells themselves. These may then co-express leukemic antigens together with appropriate secondary signals and be used to generate a specific, antileukemic immune response. In this study, blasts from 40 patients with acute myeloid leukemia (AML) were cultured with combinations of granulocyte-macrophage colony-stimulating factor, interleukin 4, and tumor necrosis factor α, and development to DCs was assessed. After culture, cells from 24 samples exhibited morphological and immunophenotypic features of DCs, including expression of major histocompatibility complex class II, CD1a, CD83, and CD86, and were potent stimulators in an allogeneic mixed lymphocyte reaction (MLR). Stimulation of autologous T-cell responses was assessed by the proliferative response of autologous T cells to the leukemic DCs and by demonstration of the induction of specific, autologous, antileukemic cytotoxicity. Of 17 samples, 11 were effective stimulators in the autologous MLR, and low, but consistent, autologous, antileukemic cytotoxicity was induced in 8 of 11 cases (mean, 27%; range, 17%-37%). This study indicates that cells with enhanced antigen-presenting ability can be generated from AML blasts, that these cells can effectively prime autologous cytotoxic T cells in vitro, and that they may be used as potential vaccines in the immunotherapy of AML.

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.

scholarly journals A potent tetravalent T-cell–engaging bispecific antibody against CD33 in acute myeloid leukemia

2018 ◽  
Vol 2 (11) ◽  
pp. 1250-1258 ◽  
Author(s):  
Sayed Shahabuddin Hoseini ◽  
Hongfen Guo ◽  
Zhihao Wu ◽  
Miho Nakajima Hatano ◽  
Nai-Kong V. Cheung
Keyword(s):  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Bispecific Antibody ◽  
Key Points ◽  
Acute Myeloid

Key PointsA tetravalent BsAb against CD33 treated AML human xenografts despite CD33 internalization. BsAb with bivalent versus monolavent binding to CD33 and CD3 had more than 10-fold greater potency against leukemia.

Targeting of a B7-1 (CD80) immunoglobulin G fusion protein to acute myeloid leukemia blasts increases their costimulatory activity for autologous remission T cells

Blood ◽  
2001 ◽  
Vol 97 (10) ◽  
pp. 3138-3145 ◽  
Author(s):  
Michael Notter ◽  
Tim Willinger ◽  
Ulrike Erben ◽  
Eckhard Thiel
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Fusion Protein ◽  
Immunoglobulin G ◽  
Myeloid Leukemia ◽  
Costimulatory Molecule ◽  
T Cell Response ◽  
Cytotoxic T Cell ◽  
Acute Myeloid

Abstract Transfection of tumor cells with the gene encoding the costimulatory molecule B7-1 (CD80), the ligand for CD28 and cytotoxic T lymphocye antigen-4 on T cells, has been shown to result in potent T-cell–mediated antitumor immunity. As an alternative approach, this study analyzed the costimulatory capacity of a human B7-1 immunoglobulin G (IgG) fusion protein targeted to the cell membrane of human acute myeloid leukemia (AML) blasts. Flow cytometric analysis revealed a low constitutive expression of B7-1 on human AML blasts (on average, 3.0 ± 4.3%; n = 50). In contrast, the expression of B7-2 (CD86) was highly heterogeneous and higher in AML blasts of French-American-British classification types M4 and M5 (P < .0001). The B7-1 IgG fusion protein used in this study efficiently costimulated the proliferation of resting and preactivated T cells when immobilized on plastic. After preincubation with B7-1 IgG, specific binding of the fusion protein to the high-affinity Fcγreceptor I (CD64) on leukemic cells was demonstrated and was found to increase the proliferation of both allogeneic and autologous T cells in costimulation experiments. Furthermore, targeting of B7-1 IgG to the tumor membrane resulted in increased proliferation of autologous remission T cells and had the potential to generate an enhanced redirected cytotoxic T-cell response against autologous AML blasts. In summary, the targeting of B7-1 IgG fusion protein described in this study represents a strategy alternative to gene therapy to restore the expression of the costimulatory molecule B7-1 on human AML blasts, thereby enhancing their immunogenicity for autologous T cells. This new approach may have implications for T-cell–mediated immunotherapy in AML.

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 A novel C2 domain binding CD33xCD3 bispecific antibody with potent T-cell redirection activity against acute myeloid leukemia

2020 ◽  
Vol 4 (5) ◽  
pp. 906-919 ◽  
Author(s):  
Priyanka Nair-Gupta ◽  
Michael Diem ◽  
Dara Reeves ◽  
Weirong Wang ◽  
Robert Schulingkamp ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
T Cell ◽  
T Cell Activation ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Bispecific Antibody ◽  
C2 Domain ◽  
Cytokine Release ◽  
Cynomolgus Monkeys ◽  
Acute Myeloid

Abstract CD33 is expressed in 90% of patients with acute myeloid leukemia (AML), and its extracellular portion consists of a V domain and a C2 domain. A recent study showed that a single nucleotide polymorphism (SNP), rs12459419 (C > T), results in the reduced expression of V domain–containing CD33 and limited efficacy of V domain–binding anti-CD33 antibodies. We developed JNJ-67571244, a novel human bispecific antibody capable of binding to the C2 domain of CD33 and to CD3, to induce T-cell recruitment and CD33+ tumor cell cytotoxicity independently of their SNP genotype status. JNJ-67571244 specifically binds to CD33-expressing target cells and induces cytotoxicity of CD33+ AML cell lines in vitro along with T-cell activation and cytokine release. JNJ-67571244 also exhibited statistically significant antitumor activity in vivo in established disseminated and subcutaneous mouse models of human AML. Furthermore, this antibody depletes CD33+ blasts in AML patient blood samples with concurrent T-cell activation. JNJ-67571244 also cross-reacts with cynomolgus monkey CD33 and CD3, and dosing of JNJ-67571244 in cynomolgus monkeys resulted in T-cell activation, transient cytokine release, and sustained reduction in CD33+ leukocyte populations. JNJ-67571244 was well tolerated in cynomolgus monkeys up to 30 mg/kg. Lastly, JNJ-67571244 mediated efficient cytotoxicity of cell lines and primary samples regardless of their SNP genotype status, suggesting a potential therapeutic benefit over other V-binding antibodies. JNJ-67571244 is currently in phase 1 clinical trials in patients with relapsed/refractory AML and high-risk myelodysplastic syndrome.

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