CN114426583B - Chimeric antigen receptor for cell therapy of acute myeloid leukemia - Google Patents
Chimeric antigen receptor for cell therapy of acute myeloid leukemia Download PDFInfo
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Abstract
The present invention relates to a polypeptide comprising a sequence as set forth in SEQ ID No:2, a human Chimeric Antigen Receptor (CAR) of the amino acid sequence shown in fig. 2, CAR T cells containing the same, and methods of constructing the cells, and cell therapies for acute myeloid leukemia using the CAR T cells. CAR T cells prepared using the expression sequences of the Chimeric Antigen Receptor (CAR) can be used to treat acute myeloid leukemia (Acute myeloid leukemia, AML).
Description
Technical Field
The present invention relates to a Chimeric Antigen Receptor (CAR), the invention further relates to a CAR T cell comprising the CAR, and cell therapy using the CAR T cell.
Background
Acute myeloid leukemia (Acute myeloid leukemia, AML) is a bone marrow derived tumor that recurs in a vast majority of patients, although chemotherapy can induce remission rates of up to 70%. ((Bishop, 1997)).
Chimeric antigen receptor T cell (CAR T) therapy, a novel cell therapy recently applied in clinic, is an accurate targeted tumor therapy by designing immune cells of a patient by using chimeric antigen receptor T cells to combat cancer cells. The target is killed by designing the CAR and using it to activate T cells. Chimeric antigen receptors, CARs, consist essentially of two domains: an extracellular domain and an intracellular signal transduction region. The mechanism is to modify T cells into CAR T cells for therapy by transferring T cells with a viral vector that recognizes tumor cells and activates T cells.
Treatment of acute lymphoblastic leukemia using CD 19-targeted chimeric antigen receptor T cells has been reported (Acute lymphoblastic leukemia, ALL), with surprising results (Maude et al, 2014), showing the great potential of chimeric antigen receptor T cell therapies for the treatment of hematological disorders.
Currently, targets such as CD123, CD33, CLL1 have been clinically validated as potential targets for AML (Kenderian et al 2015;Mardiros et al, 2013; wang et al, 2018). However, therapies directed against these targets suffer from the disadvantage that, as these targets are expressed not only on AML cells, but also in hematopoietic stem cells, they result in bone marrow toxicity when CAR T therapy is performed, compromising hematopoietic cell formation, which may be difficult for patients to tolerate.
Therefore, there is a need for a safe therapeutic target that does not cause bone marrow toxicity and does not affect hematopoietic cell formation for CAR T treatment in relapsed refractory AML, CAR T cells for the target, and cell therapies for treatment using such cells.
Disclosure of Invention
Epithelial cell adhesion factor (Epithelial cell adhesion factor, epCAM) belongs to the class i transmembrane protein and is expressed in most normal upper cells. EpCAM has also been reported to have high expression on numerous solid tumors. EpCAM has also been reported to be useful as a marker for tumor stem cells (Munz, baeulere et al 2009).
The inventors have found in earlier work that EpCAM is also expressed on part of leukemic cells, especially on AML refractory to relapse (Zheng et al, 2017). Furthermore, we found that EpCAM is not expressed on hematopoietic stem cells and therefore does not cause myelotoxicity.
In addition, it is known that cetuximab (catumab) is a mouse bispecific antibody against CD3 and the epithelial cell adhesion factor EpCAM, and that catumab can target both tumor surface antigen EpCAM and T cell surface receptor CD3, activate and recruit killer T cells, thereby achieving the purpose of treating tumors. And EpCAM CAR T is a CAR structure which is over expressed on T cells and can directly identify EpCAM, and the two have certain similarity. This also suggests the safety of EpCAM CAR T when applied to human therapy, given that the european union has clinically approved Catumaxomab for the treatment of malignant ascites patients with EpCAM positive tumors.
The inventors have experimentally demonstrated that EpCAM can be a target for AML treatment. In one embodiment, the invention provides EpCAM CARs useful for treating EpCAM positive replication refractory AML, CAR T cells containing the EpCAM CARs, methods of making the cells, and cell therapies using the cells.
In one embodiment, the invention provides a Chimeric Antigen Receptor (CAR), wherein an EpCAM binding domain, a transmembrane domain, and an intracellular signaling domain are included in the CAR.
The present invention specifically includes the following.
1. A chimeric antigen receptor comprising sequences encoding a binding domain, a transmembrane domain, and an intracellular signaling domain, respectively, of a human epithelial cell adhesion molecule (EpCAM) protein, comprising a sequence as set forth in SEQ ID No:2, and a polypeptide having the amino acid sequence shown in 2.
2. The chimeric antigen receptor of item 1, comprising the amino acid sequence set forth in SEQ ID No:1, and a nucleotide sequence shown in the specification.
A car T cell comprising the chimeric antigen receptor of item 1 or 2.
4. A pharmaceutical composition comprising the recombinant T cell of claim 4.
5. Use of the chimeric antigen receptor of item 1 or 2, or the CAR T cell of item 3, in the manufacture of a medicament for treating EpCAM positive leukemia, wherein the leukemia is preferably Acute Myeloid Leukemia (AML), e.g. leukemia cell line HL60, K562.
A method of making a car T cell comprising contacting a car T cell as set forth in SEQ ID No:1 or encodes a nucleotide sequence as set forth in SEQ ID No:2 into T cells,
preferably the method is transfection with lentiviral vectors, more preferably transfection using PCDH, ps.pAX2, pMD G co-transfected 293T cells.
Brief Description of Drawings
Figure 1, T flow assay for CAR positive rate of cells.
FIG. 2 is a flow chart of two myeloid leukemia cell lines EpCAM positive.
Figure 3 is a graph of EpCAM CAR T cell killing activity in vitro.
Figure 4 is a graph of the results of cytokines produced by EpCAM CAR T cells.
Figure 5EpCAM CAR T cells were EpCAM + Graph of results of AML stimulation of proliferation.
Figure 6 is a graph of the effect of EpCAM CAR T cells on the differentiation of hematopoietic stem cells.
Figure 7EpCAM CAR T cell clearance in vivo for AML, (a) schematic of HL60 tumor-bearing murine in vivo treatment protocol, (b) tumor burden representation of fluorescent imaging of mice from one experiment (n=8), (c) statistical plot of total fluorescence values, (d) Kaplan-Meier survival analysis.
Detailed Description
Example 1 preparation of EpCAM CAR T cells
Preparation and production of chimeric antigen receptor T cells (CAR T cells)
The murine scFv against human EpCAM (sequence shown as Seq ID: 3) was then linked to the CD8 transmembrane region (sequence shown as Seq ID: 4), and the 41bb co-stimulatory domain (sequence shown as Seq ID: 5) and CD3 zeta (sequence shown as Seq ID: 6) were inserted into PCDH-MSCV-MCS-EF 1-copGGFP (available from Addgene Corp.).
Or CD19CAR (sequence shown as Seq ID: 7) (Ying et al, 2019) was inserted into PCDH-MSCV-MCS-EF 1-copGGFP (available from Addgene Corp.).
The 3 plasmids of the following table were then transfected into 293T cells (purchased from the national academy of sciences Shanghai cell bank) using PEI (Polyscience, 23966) according to the protocol, respectively, and cell culture supernatants were collected for 48h and 72h to transduce T cells.
Mononuclear cells were isolated from fresh human peripheral blood (from Anhui provincial blood center) by Ficoll density gradient centrifugation, and T cells were isolated using CD 3T cell cationic isolation kit (Miltenyi, 30-097-043). The initially isolated T cells were split into two parts, one for flow detection and the other for construction of CAR T cells.
On day 0, 5X 10X-VIVO 15 medium (lonza, BE 02-060F) supplemented with 5% human type AB serum (GEMINI, 100-512), 2mmol/L glutamine 5 T cells were resuspended at a concentration of/ml and anti-CD 3/CD28 Dynabeads (thermo, 11161D) were added at a 1:1 ratio, with simultaneous addition of IL-2 (gold, 100 ten thousand units) at the time of anti-CD 3/CD28 Dynabeads addition to a final concentration of 100U/ml, supplemented with IL-2 every two days.
After 24 hours, concentrated lentiviruses obtained by centrifugation at 50000g at 4℃for 2 hours from the supernatant of the above-collected cell culture broth were added to activated T cells at MOI 50 with polybrene (available from sigma, H9268) at a final concentration of 8ng/ml according to the instructions of polybrene, centrifuged at 720g at 32℃for 1H, and the solution was changed after 6-8 hours.
Cell culture was monitored daily during transduction and the cell concentration was maintained at 0.5-1X 10 by addition of complete X-VIVO 15 medium 6 cells/mL. Dynabeads were removed on day 4 post transduction. Cells, epCAM CAR T cells, were harvested after day 5 for subsequent analysis and in vivo experiments, respectively.
Example 2 flow cytometer identification of EpCAM CAR T cells
The measurement group: epCAM CAR T cell line, myeloid leukemia cell line K562 and HL60 cultured in vitro.
Flow cytometry assays
Detection was performed using BD LSRII flow cytometer using antibodies described below, and analysis was performed using Flowjo V10.
Antibodies for use in flow cytometry in the present invention include:
anti-human antibody EpCAM (from Biolengend, 324208), anti-murine Fab antibody (from Jackson immunoresearch-606-006),
anti-human CD34 antibodies (purchased from BD, 555821),
anti-human CD38 antibodies (available from biolegend, 303522),
alexa Fluor 647 conjugated goat anti-mouse F (ab') 2 antibody (purchased from Jackson Immunoresearch).
The in vitro cultured EpCAM CAR T cell line, or myeloid leukemia cell line K562 or HL60, harvested in example 1 was washed once in phosphate buffer supplemented with 2% fetal bovine serum and stained 30min at 4 ℃ in the absence of light after blocking Fc receptors with mouse serum, washed twice with pbs and then checked on-machine (BD LSRII) to obtain T cell CAR positive rates for each set of cell flow assays.
Specifically, detection of EpCAM CAR surface expression was stained with Alexa Fluor 647 conjugated goat anti-mouse F (ab') 2 antibody, mouse scFv was labeled with anti-mouse anti-Fab antibody, and the results are shown in fig. 1,2.
Fig. 1 shows that the positive rate of the anti-Fab antibody-stained T cell CAR reaches 92.6%, which proves that the EpCAM CAR lentiviral vector effectively infects T cells, and that the CAR of the invention is successfully expressed on the cell membrane, and the obtained T cells are EpCAM CAR T cells of the invention.
As cells suitable for the cell therapy of the present invention, the inventors used EpCAM positive myeloid leukemia cell lines K562 and HL60 (purchased from the college of science Shanghai cell bank) in the subsequent examples, and fig. 2 shows: peaks in the APC-conjugated EpCAM antibody staining appeared in the myeloid leukemia cell lines K562 and HL60, confirming that both cells were EpCAM positive cells.
Example 3 cell killing assay of EpCAM CAR T cells
The in vitro killing activity of EpCAM CAR T cells of the invention was assessed by bioluminescence.
The measurement group: epCAM CAR T cell group, CD19CAR T cells (construction of this cell see example 1) served as negative control. The target cells were myeloid leukemia cell lines K562 and HL60 (stable expression of luciferase, purchased from Shanghai cell Bank of China academy of sciences).
Bioluminescence is a luciferase-based assay in which K562, HL60 cells (1 ten thousand per well) and EpCAM CAR T cells or CD19CAR T cells stably expressing luciferase are assayed as effector cells: the target cell ratio (effective target ratio E: T) was 5:1-1.25:1 co-incubated, and residual luciferase activity of the remaining tumor cells was detected using a multifunctional enzyme-labeled instrument (CLARIOstar).
The percent lysis coefficients were calculated as follows: percent lysis = 100- (((average signal from T cell treated wells)/(average signal from untreated target wells)) ×100).
Three effect target ratios (5:1, 2.5:1, 1.25:1) were set for comparison, respectively, and the results are shown in FIG. 3.
The results show that: when the killing activity of each group of CAR T cells on EpCAM positive AML cell lines was detected by a bioluminescence method, it was observed that the percentage of lysis of EpCAM CAR T cell groups in both leukemia cell lines K562 and HL60 was significantly higher than that of the negative control CD19CAR T cell group, i.e., the killing ability of EpCAM CAR T cell groups was higher than that of CD19CAR T cells, demonstrating that EpCAM CAR T cells of the invention were able to specifically kill EpCAM positive target cells.
Example 4 cytokine detection by EpCAM CAR T cells in vitro generated
The levels of cytokines IL-2, TNFa and IFN-gamma produced by EpCAM CAR T cells in vitro were evaluated.
The measurement group: epCAM CAR T cell group, CD19CAR T cells (construction of this cell see example 1) served as negative control. Target cells were the myeloid leukemia cell lines K562 and HL60, purchased from the college of sciences, the Shanghai cell bank).
Each set of CAR T cells and target cells were each expressed at 1:1 (100,000 cells per well), after 24 hours, the supernatant was collected and cytokine levels of IL-2, TNFα and IFN- γ in the supernatant were measured according to the instructions using ELISA kits (IL 2 ELISA kit, dake, DKW12-1020-096; TNFα ELISA kit, dake, DKW12-1720-096; IFNγ ELISA kit, dake, DKW 12-1000-096). The results are shown in FIG. 4.
The results show that, after 24h co-incubation with EpCAM positive target cells, epCAM CAR T cell groups produced significantly high levels of the killer cytokines IL-2, tnfα and IFN- γ in both K562 and HL60 (x represents P <0.05, x represents P <0.01, x represents P < 0.001). In contrast, in target cells K562 and HL60 treated with CD19CAR T cells, little or no expression of these cytokines was detected, further demonstrating that EpCAM CAR T can specifically kill EpCAM positive target cells.
Example 5 cell proliferation assay based on CFSE markers
CFSE (carboxyfluorescein diacetate, succinimidyl ester), which can react with lysine side chains or other amino groups, is covalently coupled to intracellular and cell surface proteins, and is suitable for cell labeling.
The measurement group: epCAM CAR T cells, T cells not lentivirally transduced after isolation (abbreviated UTD). Target cells: k562 cells (purchased from the Shanghai cell bank of the national academy of sciences).
CFSE (eBioscience, 85-65-0850-84) labeled EpCAM CAR T cells or T cells without lentiviral transduction (UTD) were incubated with K562 cells at a 1:1 ratio at the instruction concentrations and procedures, and after 3 days each group of cells was collected and analyzed for cell proliferation using a flow cytometer, the results of the flow cytometry are shown in FIG. 5.
The results show that: after co-incubation with EpCAM positive target cells, epCAM CAR T cells were 66.6% in proportion, demonstrating that they were stimulated to proliferate rapidly, and UTD cells were 37.6% in proportion, demonstrating slower proliferation of non-CAR transduced T cells. This further demonstrates that EpCAM CAR T cells of the invention can specifically recognize EpCAM positive cell lines and be stimulated to proliferate.
Example 6 safety test based on colony formation experiments
Colony-Forming Cell (CFC) detection, also known as Colony Forming unit detection, is a gold standard for in vitro detection of hematopoietic stem Cell function and is a method of identifying the function and quality of hematopoietic cells based on the type and number of Cell colonies.
To test the safety of CAR T cells of the invention. Its effect on the formation of the following colonies in hematopoietic cells was examined: CFU-E (erythroid colony forming unit), BFU-E (blast erythroid colony forming unit), CFU-GM (granulocyte, macrophage colony forming unit), CFU-GEMM (mixed cell colony forming unit).
The measurement group: epCAM CAR T cell-treated group, PBS group, CD19CAR T cell-treated group.
Isolation of CD34 + Cells
Isolation of CD34 from fresh human umbilical blood (from the attached first Hospital of the university of science and technology) using CD34 cationic magnetic bead separation kit (Meitian and Twaii, 130-097-047) + Cells are then treated with MethoCult TM H4434 Classic (stemcell, 04434) was cultured in vitro and operated with reference to the manufacturer's instructions.
Formation of colonies
Will be 1X 10 3 CD34 of (c) + Cord blood stem cells and 1X 10 4 In vitro co-incubation of EpCAM CAR T cells, CD19CAR T cells or equal volume PBS for 4h, whole cell mixtures were transferred to MethoCult TM H4434 In Classic, triplicate plates. After 14 days, the number of CFU-E, BFU-E, CFU-GM, CFU-GEMM colony formations in each group was calculated (FIG. 6).
The results show that: (a) CD34 was not detected by the flow assay + CD38 and EpCAM positive cells, demonstrating CD34 + CD38 - Does not express EpCAM. (b) In contrast to PBS and CD19CAR T-treated groups, co-incubation with EpCAM CAR T cells did not affect the distribution of individual colonies of hematopoietic stem cells, i.e., epCAM CAR T cells did not affect the condition of hematopoietic stem cell differentiation. EpCAM CAR T cells proved not to cause myelotoxicity, and not to impair hematopoietic cell formation.
Example 7 in vivo tumor-bearing treatment experiments in mice
Tail vein infusion was modeled using immunodeficient NCG mice (Kenderian et al 2015) and CAR T cells were infused once weekly for treatment following tumor formation and fluorescence imaging measurements were performed weekly.
The measurement group: epCAM CAR T cells, CD19CAR T cells, PBS group. Sample collection time points: day 0, 13, 20, 27.
The specific scheme is as follows: female NSG mice (6-10 weeks old, available from JieXUKANG Co.) were injected 5X 10 by tail vein 6 After tumor formation was determined on day 6, mice were divided into three groups, epCAM CAR T cells, CD19CAR T cells, PBS groups of 8, and the average fluorescence was kept consistent for each group.
For three groups, 5X 10 tail vein infusion was performed 6 CD19CAR T cells or 5X 10 6 The EpCAM CAR T cells were treated with the same volume of PBS. 15mg/ml potassium fluorescein salt (Gold biotechnology, lock) was intraperitoneally injected on days 0, 13, 20, 27 and examined by fluorescence imaging. The treatment schedule and test results are shown in fig. 7, and Kaplan-Meier survival analysis was performed on the number of survival days for each group of mice.
The results show that: at each time point, epCAM CAR T cell groups were significantly less fluorescent than both PBS and CD19CAR T treatment groups, demonstrating that EpCAM CAR T cells were effective in clearing AML tumors in vivo (fig. 7b, c).
The Kaplan-Meier survival analysis showed that the survival rate of CD19CAR T-treated group was 50%, epCAM CAR T-treated group was 100% on day 40, CD19CAR T-treated group was 0% on day 60, epCAM CAR T-treated group was 37.5%, epCAM CAR T-cells were found to be more effective in prolonging survival time of tumor-bearing mice than PBS-treated group and CD19CAR T-cell-treated group (fig. 7 d).
Reference to the literature
Bishop,J.F.(1997).The treatment of adult acute myeloid leukemia.Seminars in oncology 24,57-69.
Kenderian,S.S.,Ruella,M.,Shestova,O.,Klichinsky,M.,Aikawa,V.,Morrissette,J.J.,Scholler,J.,Song,D.,Porter,D.L.,Carroll,M.,et al.(2015).CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia.Leukemia 29,1637-1647.
Mardiros,A.,Dos Santos,C.,McDonald,T.,Brown,C.E.,Wang,X.,Budde,L.E.,Hoffman,L.,Aguilar,B.,Chang,W.C.,Bretzlaff,W.,et al.(2013).T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.Blood 122,3138-3148.
Maude,S.L.,Frey,N.,Shaw,P.A.,Aplenc,R.,Barrett,D.M.,Bunin,N.J.,Chew,A.,Gonzalez,V.E.,Zheng,Z.,Lacey,S.F.,et al.(2014).Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia.New England Journal of Medicine 371,1507-1517.
Wang,J.,Chen,S.,Xiao,W.,Li,W.,Wang,L.,Yang,S.,Wang,W.,Xu,L.,Liao,S.,Liu,W.,et al.(2018).CAR T cells targeting CLL-1as an approach to treat acute myeloid leukemia.J Hematol Oncol 11,7.
Ying,Z.,Huang,X.F.,Xiang,X.,Liu,Y.,Kang,X.,Song,Y.,Guo,X.,Liu,H.,Ding,N.,Zhang,T.,et al.(2019).A safe and potent anti-CD19 CAR Tcell therapy.Nat Med 25,947-953.
Zheng,X.,Fan,X.,Fu,B.,Zheng,M.,Zhang,A.,Zhong,K.,Yan,J.,Sun,R.,Tian,Z.,and Wei,H.(2017).EpCAM Inhibition Sensitizes ChemoresistantLeukemia to Immune Surveillance.Cancer Research 77,482-493.
Claims (10)
1. A chimeric antigen receptor having the sequence of SEQ ID No:2, and a polypeptide having the amino acid sequence shown in 2.
2. The chimeric antigen receptor of claim 1, which consists of SEQ ID No:1, and the nucleotide sequence shown in 1.
A car T cell comprising the chimeric antigen receptor of claim 1 or 2.
4. A pharmaceutical composition comprising the CAR T cell of claim 3.
5. Use of the chimeric antigen receptor of claim 1 or 2 or the CAR T cell of claim 3 in the manufacture of a medicament for treating EpCAM positive leukemia.
6. The use of claim 5, wherein the leukemia is Acute Myeloid Leukemia (AML).
A method of making a car T cell comprising combining SEQ ID No:1 into T cells.
A method of making a car T cell comprising encoding SEQ ID No:2 into T cells.
9. The method of claim 7 or 8, wherein the method is a method of transfection with a lentiviral vector.
10. The method of claim 9, wherein the method is a method of transfection using PCDH, ps.pAX2, pMD G co-transfected 293T cells.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877032A (en) * | 2015-06-26 | 2015-09-02 | 中国医学科学院血液病医院（血液学研究所） | CD33 specificity chimeric antigen receptor and application thereof |
| CN106103475A (en) * | 2014-03-11 | 2016-11-09 | 塞勒克提斯公司 | Produce the method for the compatible T cell of allograft |
| CN106163547A (en) * | 2014-03-15 | 2016-11-23 | 诺华股份有限公司 | Use Chimeric antigen receptor treatment cancer |
| CN108276497A (en) * | 2018-01-26 | 2018-07-13 | 英普乐孚生物技术(上海)有限公司 | Target Chimeric antigen receptor T cell and its application of CD19 |
| CN110248669A (en) * | 2016-12-09 | 2019-09-17 | 昂克免疫有限公司 | It is engineered natural killer cell and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3634474A1 (en) * | 2017-06-09 | 2020-04-15 | GEMoaB Monoclonals GmbH | Targeting modules for universal chimeric antigen receptor expressing immune cells and use in the treatment of cancer infections and autoimmune disorders |
-
2020
- 2020-10-29 CN CN202011178806.1A patent/CN114426583B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106103475A (en) * | 2014-03-11 | 2016-11-09 | 塞勒克提斯公司 | Produce the method for the compatible T cell of allograft |
| CN106163547A (en) * | 2014-03-15 | 2016-11-23 | 诺华股份有限公司 | Use Chimeric antigen receptor treatment cancer |
| CN104877032A (en) * | 2015-06-26 | 2015-09-02 | 中国医学科学院血液病医院（血液学研究所） | CD33 specificity chimeric antigen receptor and application thereof |
| CN110248669A (en) * | 2016-12-09 | 2019-09-17 | 昂克免疫有限公司 | It is engineered natural killer cell and application thereof |
| CN108276497A (en) * | 2018-01-26 | 2018-07-13 | 英普乐孚生物技术(上海)有限公司 | Target Chimeric antigen receptor T cell and its application of CD19 |
Non-Patent Citations (2)
| Title |
|---|
| Construction of chimeric antigen receptor‑modified T cells targeting EpCAM and assessment of their anti‑tumor effect on cancer cells;YAN ZHOU et al;《Molecular Medicine RE POR TS》;第第20卷卷;第2355-2364页 * |
| 靶向EpCAM抗原的CAR-T对结直肠癌的杀伤作用研究;曾思衡;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》(第第10期期);摘要 * |
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