The online version of this article (https://doi.org/10.1186/s13045-017-0553-5) contains supplementary material, which is available to authorized users.
Jinghua Wang, Siyu Chen and Wei Xiao are co-first authors.
Xing Zhang, Yue Lu and Penghui Zhou are co-last authors.
Acute myeloid leukemia (AML) is one of the most common types of adult acute leukemia. Standard chemotherapies can induce complete remission in selected patients; however, a majority of patients eventually relapse and succumb to the disease. Thus, the development of novel therapeutics for AML is urgently needed. Human C-type lectin-like molecule-1 (CLL-1) is a type II transmembrane glycoprotein, and its expression is restricted to myeloid cells and the majority of AML blasts. Moreover, CLL-1 is expressed in leukemia stem cells (LSCs), but absent in hematopoietic stem cells (HSCs), which may provide a potential therapeutic target for AML treatment.
We tested the expression of CLL-1 antigen on peripheral blood cells and bone marrow cells in healthy donor and AML patients. Then, we developed a chimeric antigen receptor (CAR) containing a CLL1-specific single-chain variable fragment, in combination with CD28, 4-1BB costimulatory domains, and CD3-ζ signaling domain. We further investigate the function of CLL-1 CAR-T cells.
The CLL-1 CAR-T cells specifically lysed CLL-1+ cell lines as well as primary AML patient samples in vitro. Strong anti-leukemic activity was observed in vivo by using a xenograft model of disseminated AML. Importantly, CLL-1+ myeloid progenitor cells and mature myeloid cells were specifically eliminated by CLL-1 CAR-T cells, while normal HSCs were not targeted due to the lack of CLL-1 expression.
CLL-1 CAR-T represents a promising immunotherapy for the treatment of AML.
Additional file 1: Table S1. Patient characteristics and CLL-1 expression of primary AML patient sample. F, female; M, male; BM, bone marrow; PB, peripheral blood. (DOCX 25 kb)
Additional file 2: Figure S1. Co-expression CLL-1 and CD33 in primary AML samples. (A) Initially, cells were gated based on forward and side scatter properties. Subsequently, AML blasts were selected based on low side scatter versus CD45dim expression. (B) CLL-1 and CD33 expression on four representative gated AML blast cell populations are depicted. Percentages in each quadrant are indicated. (TIFF 1776 kb)
Additional file 3: Figure S2. The gating strategy of CD34+ AML blasts. Cells were initially gated based on forward and side scatter properties. Subsequently, AML blasts were selected based on low side scatter versus CD45dim expression. Then, CD34+ cells were gated. Finally, CD38+/CD38− cells were gated and used for CLL-1 expression analysis. (TIFF 1656 kb)
Additional file 4: Figure S3. CLL-1 CAR-T cells lyse CLL1-expressing AML cells. (A) Expression of CLL-1 on the cell lines HL-60 and K562. (B) CLL-1 CAR-T cells lysed CLL-1+ cell line HL-60. CLL-1− cell line K562 was used as negative control. NT cells were used to evaluate unspecific lysis. Data represent mean values of triplicate wells ± SD. (TIFF 481 kb)
Additional file 5: Figure S4. Proliferation of CLL-1 CAR-T cells in response to CLL-1+ cells. Pair-matched CFSE-labeled CLL-1 CAR-T cells or NT cells were co-cultured with the indicated stimulator cell lines for 96 h at an E:T of 1:1. CFSE dilution was analyzed by flow cytometry. Unstimulated T cells (gray histograms) were used as baseline T cell proliferation controls. (TIFF 866 kb)
Additional file 6: Figure S5. CLL-1 CAR expression in T cells derived from AML patients. T cells from three AML patients were transduced with CLL-1 CAR. Shown are CLL-1 CAR-T and NT cells from the three AML patients 14 days post transduction. Percentages in each quadrant are indicated. (TIFF 557 kb)
Dohner H, Estey EH, Amadori S, Appelbaum FR, Buchner T, Burnett AK, Dombret H, Fenaux P, Grimwade D, Larson RA, Lo-Coco F, Naoe T, Niederwieser D, Ossenkoppele GJ, Sanz MA, Sierra J, Tallman MS, Löwenberg B, Bloomfield CD, European LeukemiaNet. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood. 2010;115(3):453–74. CrossRefPubMed
Brentjens RJ, Riviere I, Park JH, Davila ML, Wang X, Stefanski J, Taylor C, Yeh R, Bartido S, Borquez-Ojeda O, Olszewska M, Bernal Y, Pegram H, Przybylowski M, Hollyman D, Usachenko Y, Pirraglia D, Hosey J, Santos E, Halton E, Maslak P, Scheinberg D, Jurcic J, Heaney M, Heller G, Frattini M, Sadelain M. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood. 2011;118(18):4817–28. CrossRefPubMedPubMedCentral
Kochenderfer JN, Dudley ME, Feldman SA, Wilson WH, Spaner DE, Maric I, Stetler-Stevenson M, Phan GQ, Hughes MS, Sherry RM, Yang JC, Kammula US, Devillier L, Carpenter R, Nathan DA, Morgan RA, Laurencot C, Rosenberg SA. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood. 2012;119(12):2709–20. CrossRefPubMedPubMedCentral
Brentjens RJ, Davila ML, Riviere I, Park J, Wang X, Cowell LG, Bartido S, Stefanski J, Taylor C, Olszewska M, Borquez-Ojeda O, Qu J, Wasielewska T, He Q, Bernal Y, Rijo IV, Hedvat C, Kobos R, Curran K, Steinherz P, Jurcic J, Rosenblat T, Maslak P, Frattini M, Sadelain M. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med. 2013;5(177):177ra38. CrossRefPubMedPubMedCentral
Jin L, Lee EM, Ramshaw HS, Busfield SJ, Peoppl AG, Wilkinson L, Guthridge MA, Thomas D, Barry EF, Boyd A, Gearing DP, Vairo G, Lopez AF, Dick JE, Lock RB, Jurcic J, Rosenblat T, Maslak P, Frattini M, Sadelain M. Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cell. 2009;5(1):31–42. CrossRefPubMed
Saito Y, Kitamura H, Hijikata A, Tomizawa-Murasawa M, Tanaka S, Takagi S, Uchida N, Suzuki N, Sone A, Najima Y, Ozawa H, Wake A, Taniguchi S, Shultz LD, Ohara O, Ishikawa F. Identification of therapeutic targets for quiescent, chemotherapy-resistant human leukemia stem cells. Sci Transl Med. 2010;2(17):17ra9. CrossRefPubMedPubMedCentral
Nakazawa Y, Matsuda K, Kurata T, Sueki A, Tanaka M, Sakashita K, Imai C, Wilson MH, Koike K. Anti-proliferative effects of T cells expressing a ligand-based chimeric antigen receptor against CD116 on CD34(+) cells of juvenile myelomonocytic leukemia. J Hematol Oncol. 2016;9:27. CrossRefPubMedPubMedCentral
Taussig DC, Pearce DJ, Simpson C, Rohatiner AZ, Lister TA, Kelly G, Luongo JL, Danet-Desnoyers GA, Bonnet D. Hematopoietic stem cells express multiple myeloid markers: implications for the origin and targeted therapy of acute myeloid leukemia. Blood. 2005;106(13):4086–92. CrossRefPubMedPubMedCentral
Bakker AB, van den Oudenrijn S, Bakker AQ, Feller N, van Meijer M, Bia JA, Jongeneelen MA, Visser TJ, Bijl N, Geuijen CA, Marissen WE, Radosevic K, Throsby M, Schuurhuis GJ, Ossenkoppele GJ, de Kruif J, Goudsmit J, Kruisbeek AM. C-type lectin-like molecule-1: a novel myeloid cell surface marker associated with acute myeloid leukemia. Cancer Res. 2004;64(22):8443–50. CrossRefPubMed
Wiersma VR, de Bruyn M, Shi C, Gooden MJ, Wouters MC, Samplonius DF, Hendriks D, Nijman HW, Wei Y, Zhou J, Helfrich W, Bremer E. C-type lectin-like molecule-1 (CLL1)-targeted TRAIL augments the tumoricidal activity of granulocytes and potentiates therapeutic antibody-dependent cell-mediated cytotoxicity. MAbs. 2015;7(2):321–30. CrossRefPubMedPubMedCentral
van Rhenen A, Moshaver B, Kelder A, Feller N, Nieuwint AW, Zweegman S, Ossenkoppele GJ, Schuurhuis GJ. Aberrant marker expression patterns on the CD34+CD38-stem cell compartment in acute myeloid leukemia allows to distinguish the malignant from the normal stem cell compartment both at diagnosis and in remission. Leukemia. 2007;21(8):1700–7. CrossRefPubMed
Lu H, Zhou Q, Deshmukh V, Phull H, Ma J, Tardif V, Naik RR, Bouvard C, Zhang Y, Choi S, Lawson BR, Zhu S, Kim CH, Schultz PG. Targeting human C-type lectin-like molecule-1 (CLL1) with a bispecific antibody for immunotherapy of acute myeloid leukemia. Angew Chem Int Ed Engl. 2014;53(37):9841–5. CrossRefPubMedPubMedCentral
Di Stasi A, Tey SK, Dotti G, Fujita Y, Kennedy-Nasser A, Martinez C, Straathof K, Liu E, Durett AG, Grilley B, Liu H, Cruz CR, Savoldo B, Gee AP, Schindler J, Krance RA, Heslop HE, Spencer DM, Rooney CM, Brenner MK. Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med. 2011;365(18):1673–83. CrossRefPubMedPubMedCentral
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